Citation
Role of Shear Stress Mediated Inflammation, ELR+ CXC Chemokines and Peroxisomal Proliferator Activated Receptor (PPAR) Pathway in Cerebral Aneurysm Formation

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Title:
Role of Shear Stress Mediated Inflammation, ELR+ CXC Chemokines and Peroxisomal Proliferator Activated Receptor (PPAR) Pathway in Cerebral Aneurysm Formation
Creator:
Nowicki, Kamil W
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (231 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences
Molecular Cell Biology (IDP)
Committee Chair:
HOH,BRIAN LIM
Committee Co-Chair:
SEGAL,MARK STUART
Committee Members:
SCOTT,EDWARD W
BOULTON,MICHAEL EDWIN
MCFETRIDGE,PETER S
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Agonists ( jstor )
Aneurysms ( jstor )
Cytokines ( jstor )
Endothelial cells ( jstor )
Inflammation ( jstor )
Intracranial aneurysm ( jstor )
Macrophages ( jstor )
Neutrophils ( jstor )
Receptors ( jstor )
Shear stress ( jstor )
Molecular Cell Biology (IDP) -- Dissertations, Academic -- UF
aneurysm -- cerebral -- cxcl1 -- endothelial -- flow -- hemodynamics -- il-8 -- inflammation -- peroxisomal -- ppar -- shear -- stress
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Medical Sciences thesis, Ph.D.

Notes

Abstract:
Cerebral aneurysms develop by an inflammatory-mediated process related to hemodynamic shear stress, but the molecular mechanism has not been elucidated. A recently discovered family of transcription factors known as peroxisome proliferator-activated receptor (PPAR) factors has been shown to be involved in immunomodulation of cell phenotype under conditions of shear stress. We hypothesized that unique shear stress conditions within the aneurysm micro-environment lead to a release of factors or induction of pro-inflammatory pathways, that, when blocked, could be used as a target to prevent aneurysm formation. Specific Aim I was designed to overcome the issue of a lack of a suitable in vitro model. We developed a novel in vitro model of a bifurcation aneurysm, and compared its endothelial phenotype to similar models of flow in a straight artery and a bifurcation. We characterized the model using computational fluid dynamics (CFD), enzyme immunoassay (EIA), real-time reverse transcriptase PCR (RT-PCR) for cyclooxygenase-1 (COX-1) and -2 (COX-2), and monocyte chemotactic protein-1 (MCP-1), and relative fluorescence immunocytochemistry (ICC). The bifurcation aneurysm model was characterized by significantly higher circulating PGE2 levels by EIA, and the aneurysm sac micro-environment had significantly higher COX-2 and MCP-1 expression by relative fluorescence ICC. These in vitro findings were confirmed by immunohistochemistry in murine and human cerebral aneurysms. Specific Aim 2 was designed to find a specific target for immunomodulation and study the targets role in a mouse model of aneurysm formation. We conducted a screen using a cytokine array and found that the bifurcation aneurysm model was characterized by significantly higher IL-8 concentrations when compared to other models. This was verified with IL-8 ELISA. Since mice do not express IL-8, we decided to study its closely related functional homologue CXCL1. IL-8 and CXCL1 expression in human- and CXCL1 in murine- cerebral aneurysms was demonstrated by immunohistochemistry. CXCL1 antibody blockade in mouse intracranial aneurysm model resulted in significantly less aneurysm than in IgG-treated control mice by decreasing neutrophil infiltration and VCAM-1 expression. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: HOH,BRIAN LIM.
Local:
Co-adviser: SEGAL,MARK STUART.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2016-08-31
Statement of Responsibility:
by Kamil W Nowicki.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
8/31/2016
Classification:
LD1780 2014 ( lcc )

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ROLE OF SHEAR STRESS MEDIATED INFLAMMATION , ELR+ CXC CHEMOKINES AND PEROXISOMAL PROLIFERATOR ACTIVATED RECEPTOR (PPAR) PATHWAY IN CEREBRAL ANEURYSM FORMATION By KAMIL W. NOWICKI A DISSERTATION PRESENTED TO THE GRADUATE S CHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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© 2014 Kamil W. Nowicki

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To my f amily

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Invictus Out of the night that covers me, Black as the Pit from pole to pole, I thank whatever gods may be For my unconquerable soul. In the fell clutch of circumstance I have not winced nor cried aloud. Under the bludgeonings of chance My head is bloody, but unbowed. B eyond this place of wrath and tears Looms but the Horror of the shade, And yet the menace of the years Finds, and shall find, me unafraid. It matters not how strait the gate, How charged with punishments the scroll. I am the master of my fate: I am the ca ptain of my soul. William Ernest Henley, Book of Verses , Vol. 1 , 1888

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5 ACKNOWLEDGMENTS First, I would like to thank my mentor, Brian L. Hoh, MD, for allowing me to be part of his research team and for all of his patience and support over the last four ye ar s . His dedication to his patients, both through direct care and research, has been a contin uing inspiration to me during the long days and nights spent in the lab. He has always fostered my interest in neurosurgical care and research and I am extremely t hankful for the opportunity to make a difference and a contribution to this field. thank my co mentor, Edward W. Scott, PhD for his continuous and unyielding support , and uplifting anecdotes throughout the years. His unique teaching style alway s kept my motivation and he maintained my interest in research when the answers from my experiments were less than clear. I would like to thank Koji Hosaka, PhD, who has served as both a friend and a teacher during my research years. He instilled in me the value of conducting high quality , simple and honest research as well as patience and perseverance in science . Koji taught me almost all of the laboratory techniques and animal surgery skills I currently possess, and I have no doubt that without him this j ourney into the world of biomedical research would have been a much lonelier and difficult undertaking. Next, I would like to thank my collaborator and committee member, Peter S. McFetridge, PhD and the graduate students in his lab, especially Joseph Uzar ski, PhD, for teaching me the fundamentals of running shear stress experiments and isolating the endothelial cells, which were a critical component of my research work. I would like to thank the remaining members of my supervisory committee without whom t his project and wor k would not have been possible; Michael Boulton, PhD, for his no nonsense approach to science and experiment planning, and an

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6 insistence on attention to detail; Mark Segal, MD, PhD for his always insightful comments and unique clinical perspective on my project. I would also like to thank Alan Miles, MS, from Oklahoma University for manufacturing the designed flow chambers and his patience while working with me on making sure that the final product adhered to the required specifications . I would like to acknowledge Yong He, PhD, from the Department of Surgery at the University of Florida for performing detailed computational fluid dynamics of the designed flow chambers, which allowed us to verify the heterogeneity of hemodynamic microenv ironments in our model. In addition, I would like to thank Marda Jorgensen for her help with concepts and fine tuning of my immunohistochemistry studies. She was always ready to share her wealth of knowledge and skillful techniques regardless of her load o f work. Sincere thanks and acknowledgements go to the Brain Aneurysm Foundation, which has kindly founded two of my research grants, thus allowing me to continue my work with confidence and without having to worry about budgetary constraints. I would like to express my gratitude to the research and support staff in the Department in Neurosurgery: Dan Neal, MS, for his detailed and timely statistical analyses and modeling; Adrianne Fagan and Julie Ludlow for their help with research grant submissions; and Do nna Davis for her help with scheduling and coordinating skills. Also, I would like to thank Kim Hodges from the Department of Anatomy and Cell Biology for helping me navigate the graduate school requirements, looking out for me as a student, and helping me schedule my committee meetings, qualifying exam, and final defense.

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7 I would like to thank Robbie Regenhardt, MD, PhD for acting as my mentor in times when I needed a more senior perspective on the life of a physician scientist in training. His unwavering support and the get it kept me on track and sustained my motivation. Special thanks go to Stephen I. Hsu, MD, PhD, Wayne McCormack, PhD, Stratford May, MD, PhD, Paul Gulig, PhD, and Skip Harris, who recru ited, selected, and hel ped me be come part of the MD PhD training program at the University of Florida College of Medicine. I would also like to thank my family , especially my dad, stepmom, grandmother and my energetic siblings Michael and Ela, for their love, patience , support and Lastly, I would like to thank all of my friends who have shown tremendous support and love during my years in the MD PhD program. First and foremost, I would like to Sop agna Kheang , Tanner Amdur Clark, and Brittany Dobson for being my best friends during medical school and keeping in touch with me even after graduating or moving to different cities . I would like to thank my fellow colleagues in the program and close frie nds Raphael Bosse, David Lopez, Steven Chrzanowski and Amanda Sacino for being there for me when I needed someone to laugh with or just listen to my concerns . Finally, I would like t o thank all my other friends, colleagues , teachers and mentors who I have who have helped me throughout this journey.

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8 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 5 LIST OF FIGURES ................................ ................................ ................................ ........ 13 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 CEREBRAL ANEURYSM FORMATION AND THE ROLE OF INFLAMMATION ... 17 Cerebral Aneurysm Epidemiology ................................ ................................ .......... 17 Current Treatment Modalities for Cerebral Aneurysms ................................ ........... 18 Surgical Clipping ................................ ................................ .............................. 19 Endovascular Coiling ................................ ................................ ........................ 19 Clinical Outcomes and Costs of Clipping versus Coiling ................................ .. 20 Pipeline Embolization Devices and Flow Diverters ................................ ........... 21 Surgical Bypass Procedures ................................ ................................ ............ 22 Pharmacotherapy ................................ ................................ ............................. 22 Cerebral Aneurysm Pathophysiology ................................ ................................ ..... 23 Structure and Composition of Arterial Blood Vessels ................................ ....... 23 Pathophysiology of Cerebral Aneurysms of Congenital and Genetic Etiology . 24 Pathophysiology of Aneurysm Formation of Atherosclerotic or Hypertensive Etiology ................................ ................................ ................................ ......... 26 Molecular Basis of Chronic Inflammation in Cerebral Aneurysm Formation ..... 27 Role of Hemodynamic Shear Stress in Inflammation ................................ ............. 33 Application of Hemodynamic Shear Stress to in vitro Systems ........................ 34 Molecular Basis of Mechano transduction and Cellular Response to Shear Stress ................................ ................................ ................................ ............ 39 Inflammatory Effects of Abnormal Shear Stress in Cerebral Aneurysm Format ion ................................ ................................ ................................ ...... 47 Inflammatory Cascade and Components of the Innate Immune System ................ 5 0 Phase I: Coagulation Cascade ................................ ................................ ......... 51 Phase II: Inflammation ................................ ................................ ...................... 52 Phase III: Resolution ................................ ................................ ........................ 65 Role of Peroxisomal Proliferator Activat ed Receptor (PPAR) Pathway in Inflammation ................................ ................................ ................................ ......... 65 PPAR Overview ................................ ................................ ................................ 66 Role of PPARs in Metabolism and Cell Differentiation ................................ ..... 68 Role of PPARs in Inflammation ................................ ................................ ........ 70 Summary ................................ ................................ ................................ ................ 73

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9 2 MODELING SHEAR STRESS INFLAMMAT ORY MICRO ENVIRONMENT OF CEREBRAL ANEURYSMS IN VITRO ................................ ................................ .... 77 Introduction ................................ ................................ ................................ ............. 77 Methods ................................ ................................ ................................ .................. 78 Flow Chamber Design ................................ ................................ ...................... 78 Parallel Plate Flow Chamber and Flow Circuit Setup ................................ ....... 79 Modeling Shear Stress ................................ ................................ ..................... 80 Human Umbilical Vein Endothelial Cell Isolation ................................ .............. 81 Cell Culture Purity Verification by Immunohistochemistry ................................ 82 Cell Culture Purity Verification by Flow Cytometry ................................ ........... 83 Shear Stress Experiments ................................ ................................ ................ 83 Cell Density Qua ntification ................................ ................................ ............... 84 Dissolved Oxygen Quantification ................................ ................................ ...... 84 Prostaglandin E2 and Prostacyclin Quantification ................................ ............ 84 RNA Isolation, Reverse Transcription, and Quantitative Real Time Polymerase Chain Reaction ................................ ................................ .......... 85 Relative Fluorescence Immunocytochemistry and Confocal Microscop y ......... 85 Animals ................................ ................................ ................................ ............. 86 Murine Intracranial Aneurysm Model ................................ ................................ 86 Human Aneurys m and Superficial Temporal Artery Specimens ....................... 87 Immunohistochemistry of Mouse and Human Aneurysm Specimens ............... 87 Statistical Analysis ................................ ................................ ............................ 88 Results ................................ ................................ ................................ ................... 88 Bifurcation and Bifurcation Aneurysm Parallel Plate Flow Chamber Models Simulate Wall Shear Stress Patterns Found in Bifurcations and Cerebral Aneurysms ................................ ................................ ................................ .... 88 Isolated Cells Are Genuine Human Umbilical Vein Endothelial Cells ............... 89 Endothelial Cell Dens ity in Different Flow Chamber Systems .......................... 89 Dissolved Oxygen in Unconditioned and Conditioned Media ........................... 90 Hemodynamic Shear Stress in Bi furcation Aneurysm Flow Chamber Activates Increased Endothelial Expression of Inflammatory Mediator PGE2 ................................ ................................ ................................ ............ 90 Hemodynamic Shear Stress at Bifurcation and Bifurcation Aneurysm Increases Gene Expre ssion of COX 2 and MCP 1 ................................ ....... 91 Hemodynamic Shear Stress at Bifurcation and Bifurcation Aneurysm Promotes Increased Protein Expression of COX 2 and MCP 1 .................... 92 Increased COX 2 and MCP 1 Protein Expression in Murine Intracranial Aneurysms ................................ ................................ ................................ .... 93 Increased COX 2, mPGES 1, and MCP 1 Expression in Human Aneurysm Specimens ................................ ................................ ................................ .... 94 Discussion ................................ ................................ ................................ .............. 94 Role of shear stress in propagating aneurysm growth ................................ ..... 95 Modeling shear stress induced endothelial inflammation in vitro ................... 95 Limitations ................................ ................................ ................................ ...... 100

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10 3 PRO INLFAMMATORY ROLE OF SHEAR STRESS INDUCED ELR+ CH EMOKINES CXCL1/GRO a AND CXCL8/IL 8 IN CEREBRAL ANEURYSM FORMATION ................................ ................................ ................................ ........ 121 Introduction ................................ ................................ ................................ ........... 121 Methods ................................ ................................ ................................ ................ 122 Cytokine Array and ELISAs ................................ ................................ ............ 122 Concentration of Perfusing Medium for CXCL1/GRO ELISA ...................... 122 Relative Fluorescence Immunocytochemistry (RF ICC) and Confocal Microscopy ................................ ................................ ................................ .. 123 Animals ................................ ................................ ................................ ........... 124 Murine Intracranial Aneurysm Model ................................ .............................. 124 Quantification of Aneurysm Formation, Inflammatory Cell Infiltration and VCAM 1 Expression in CXCL1 Blockade ................................ .................... 125 Human Aneurysm and Superficial Temporal Artery Specimens ..................... 127 Immunohistochemistry of Mouse and Human Aneurysm Specimens ............. 127 Quantification of Aneurysm Formation, Inflammatory Cell Infiltration and VCAM 1 Expression in CXCL1 Blockade ................................ .................... 128 Results ................................ ................................ ................................ ................. 130 Low Hemodynamic Shear Stress and Shear Stress Gradients in Bifurcation and Bifurcation Aneurysm Flow Chamber Lead to Increased Endothelial Expression of Inflammatory Mediators ................................ ........................ 130 Low Hemodynamic Shear Stress and Shear Stress Gradients at the Aneurysm Region Promotes Increased Protein Expression of CXCL8/IL 8 130 Low Hemodynamic Shear St ress and Shear Stress Gradients at the Bifurcation and Aneurysm Regions Promote Increased Protein Expression of CXCL1/GRO ................................ ................................ ..... 132 ELR+ CXC Chemokines IL 8 and CXCL1 Are Expressed in Human Aneurysms ................................ ................................ ................................ .. 133 CXCL1/GRO is the Primary ELR+ CXC Chemokine Expres sed in Murine Cerebral Aneurysms ................................ ................................ ................... 133 CXCL1 Blockade Reduces Aneurysm Formation in Mouse Intracranial Aneurysm Model by Preventing Inflammatory Cell Infiltration ..................... 134 Hemodynamic Shear Stress at the Aneurysm Region Promotes Increased Protein Expression of VCAM 1 ................................ ................................ .... 135 Discussion ................................ ................................ ................................ ............ 136 Role of shear stress in aneurysm formation and growth ................................ 136 Role of ELR+ CXC Chemokines in Inflammation and Cerebral Aneurysm Formation ................................ ................................ ................................ .... 137 4 PROTECTIVE EFFECTS OF PPAR AGONISTS IN CEREBRAL ANEURYSM FORMATION ................................ ................................ ................................ ........ 153 Introduction ................................ ................................ ................................ ........... 153 Methods ................................ ................................ ................................ ................ 155 PPAR Agonist Screen Using Cytokine Array and ELISAs .............................. 155 Immunohistochemistry of PPAR Expression on CXCL1 Challenge ............ 155

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11 Relative Fluorescence Immunocytochemistry of PPAR Expression on CXCL1 and IL 8 Challenge ................................ ................................ ......... 156 Immunohistochemistry of PPAR Expression in Bifurcation Aneurysm Flow Chamber Model ................................ ................................ ........................... 156 In Vitro CXCR1/CXCR2 and CXCR2 Blockade and PPAR Expression ..... 156 Animals ................................ ................................ ................................ ........... 157 Immunohistochemistry of Mouse Aneurysm Specimens ................................ 157 Murine Intracranial Aneurysm Model ................................ .............................. 158 Quantification of Aneurysm Formation ................................ ........................... 159 Results ................................ ................................ ................................ ................. 159 PPAR Agonists Decrease Endothelial Expression of Inflammat ory Mediators ................................ ................................ ................................ .... 159 PPAR Agonists Decrease Endothelial Expression of IL 8 .............................. 159 Dose Dependent Relationship Between PPAR Expression and CXCL1 Levels ................................ ................................ ................................ .......... 160 Relative Fluorescence Immunocytochemistry of PPAR Expression on CXCL1 and IL 8 Challenge ................................ ................................ ......... 160 Immunohistoche mistry of PPAR Expression in Bifurcation Aneurysm Flow Chamber Model ................................ ................................ ........................... 161 Activation of CXCR1 and CXCR2 Has Opposing Effects on PPAR Expression ................................ ................................ ................................ .. 161 PPAR Shows Robust Expression in Murine Cerebral Aneurysms .............. 162 PPAR Treatment Does Not Reduce Aneurysm Formation in Mouse Intracranial Aneurysm Model ................................ ................................ ....... 163 PPAR Treatment Does Not Reduce Aneurysm Formation in Mouse Intracranial Aneurysm Model ................................ ................................ ....... 163 Discussion ................................ ................................ ................................ ............ 164 Role of PPAR Agonists in Lowering the Endothelial Shear Stress Mediated Cytokine Inflammatory Profile ................................ ................................ ..... 164 Feedback Loop Between PPAR and ELR+ Chemokines CXCL1 and IL 8 . 165 Role of PPAR and PPAR Agonists in Preventing Cerebral Aneurysm Formation ................................ ................................ ................................ .... 166 5 SUMMARY AND CONCLUSIONS ................................ ................................ ........ 181 Summary ................................ ................................ ................................ .............. 181 Specific Aim 1: Model Shear Stress Inflammatory Micro environment of Cerebral Aneurysm In Vitro ................................ ................................ ......... 181 Specific Aim 2: Study the Role of ELR+ CXC Chemokines in Cerebral Aneurysm Formation ................................ ................................ ................... 181 Specific Aim 3: Determine the Role of PPAR Pathway in Prevention of Cer ebral Aneurysm Formation. ................................ ................................ ... 182 Discussion ................................ ................................ ................................ ............ 183 LIST OF REFERENCES ................................ ................................ ............................. 187

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12 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 23 0

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13 LIST OF FIGURES Figure page 2 1 Bifurcation and bifurcation aneurysm flow chamber concept design and components of the flo w field.. ................................ ................................ ........... 101 2 2 Polycarbonate bioreactor base design for the bifurcation and bifurcation aneurysm flow chambers. ................................ ................................ ................. 103 2 3 Represe ntative photographs of the manufactured flow chambers. ................... 104 2 4 Parallel Plate Flow Chamber and Flow Circuit Setup. ................................ ...... 105 2 5 Measure d flow rate in one cycle for the designed flow chamber.. .................... 106 2 6 Computational fluid dynamics of the designed flow chambers. ........................ 107 2 7 HUVEC verification at P1 using immunohistochemistry ................................ ... 108 2 8 HUVEC verifica tion at P3 using flow cytometry . ................................ ............... 109 2 9 Endothelial cell density in different flow chamber systems ............................... 110 2 10 Dissolved oxygen content in different culture and flow circuits ......................... 111 2 11 P GE2 and 6k PGF 1a analysis in the straight, bifurcation, and aneurysm flow chambers ................................ ................................ ................................ .......... 112 2 12 Inflammatory gene expression in straight, bifurcation and aneurysm flow chambers ................................ ................................ ................................ .......... 113 2 13 COX 2 and MCP 1 protein expression in the bifurcation and aneurysm flow chambers ................................ ................................ ................................ .......... 115 2 14 COX 2, mPGES 1, and MCP 1 are expressed in walls o f murine aneurysms .. 116 2 15 COX 2, mPGES 1, and MCP 1 are expressed by endothelial cells in human aneurysm walls ................................ ................................ ................................ . 118 2 16 Proposed mechanism by which hemodynamic changes lead to initial aneurysm formation, progression and rupture ................................ .................. 120 3 1 Cytokine array from shear stress experiments ................................ ................. 140 3 2 ELR+ chemokines IL 8 and CXCL1 are increased in the bifurcation aneurysm flow chamber ................................ ................................ ................... 141 3 3 ELR+ chemokine expression in human aneurysm specimens ......................... 142

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14 3 4 ELR+ chemokine expression in murine aneurysm specimens ......................... 143 3 5 CXCL1 expression is associated with inflammatory cell infiltration.. ................. 144 3 6 CXCL1 blockade prevents mouse intracranial aneurysm formation ................. 145 3 7 CXCL1 blockade prevents mouse intracranial aneurysm prog ression ............. 146 3 8 Aneurysm rupture in anti CXCL1 and IgG2A treated mice ............................... 147 3 9 Effects of CXCL1 blockade on inflammatory cell infil tration .............................. 148 3 10 Macrophage phenotype in CXCL1 blockade in mouse 2 week intracranial aneurysms ................................ ................................ ................................ ........ 150 3 11 CXCL1 blockade decreases e ndothelial VCAM 1 expression .......................... 151 3 12 Proposed mechanism by which hemodynamic changes lead to aneurysm formation, progression and rupture ................................ ................................ ... 152 4 1 Effects of PPAR , and expression ................................ ................................ ................................ ........ 169 4 2 IL 8 secretion under different PPAR agonist treatments ................................ ... 170 4 3 PPAR expression increases on CXCL1 challenge ................................ ........ 171 4 4 PPAR expression on CXCL1 or IL 8 challenge ................................ ............. 172 4 5 PPA R expression within the bifurcation flow chamber ................................ .. 173 4 6 CXCR1 and CXCR2 have opposing effects on PPAR expression ................ 174 4 7 PPAR staining of mouse aneurysm specimens and aneurysmal arteries ..... 175 4 8 Mouse Intracranial Aneurysm Model: Surgical and PPAR agonist Treatment Scheme. ................................ ................................ ................................ ........... 176 4 9 Body mass recovery post aneurysm induction surgery during PPAR agonist treatment ................................ ................................ ................................ .......... 177 4 10 Treatment with PPAR agonist Wy 14,643 does not appear to prevent mouse i ntracranial aneurysm formation ................................ ............................ 178 4 11 Treatment with PPAR agonist rosiglitazone does not prevent mouse intracranial aneurysm formation ................................ ................................ ....... 179 4 12 Proposed Role of PPAR Agonists in Prevention of Cerebral Aneurysm Formation ................................ ................................ ................................ ......... 180

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF SHEAR STRESS MEDIATED INFLAMMATION , ELR+ CXC CHEMOKINES AND PEROXISOMAL PROLIFERATOR ACTIVATED RECEPTOR (PPAR) PATHWAY IN CEREBRAL ANEURYSM FORMATION By Kamil W. Nowicki August 2014 C hair: Brian L. Hoh Major: Medical Sciences Molecular Cell Biology Cerebral aneurysms develop by an inflammatory mediated process relate d to hemodynamic shear stress, but t he molecular mechanism has not been elucidated. A recently discovered family of tr anscription factors known as peroxisome proliferator activated receptor (PPAR) factors has been shown to be involved in immunomodulation under conditions of shear stress. We hypothesized that shear stress within the a neur ysm micro environment lead s to an i nduction of pro inflammatory pathways, that, when blocked, could be used to prevent aneurysm formation . Specific Aim I was designed to overcome the lack of a suitable in vitro model. We developed a novel in vitro model of a bifurcation aneurysm, and compa red its endothelial phenotype to similar models of flow in a straight artery and a bifurcation . We characterized the model using computational fluid dynamics (CFD), enzyme immunoassay (EIA), real time reverse transcriptase PCR (RT PCR), and relative fluore sce nce immunocytochemistry (ICC) for cyclooxygenase 1 (COX 1) and 2 (COX 2), and monocyte chemotactic protein 1 (MCP 1) . Findings were confirmed by immunohistochemistry in murine and human cerebral aneurysms.

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16 Specific Aim 2 was designed to find a speci f ic target for immunomodulation and further study in a mouse model of aneurysm formatio n. We conducted a screen using a cytokine array and found that t he bifurcation aneurysm model was characterized by significantly higher IL 8 concentrations when compared to other models. This was verified with IL 8 ELISA . Since mice do not express IL 8, we decided to study its closely related functional homologue CXCL1 . A ntibody blockade of CXCL1 in mouse intracranial aneurysm model resulted in significantly less aneurysm s than in control mice by decreasing neutrophil inf iltration and VCAM 1 expression. Specific Aim 3 was designed to study the relationship between the ELR+ CXC cytokines IL 8 and CXCL1 , and PPAR in cerebral aneurysm formation. Based on a cytokine screen, two PPAR agonists, Wy14643 (PPAR agonist) and rosiglitazone ( agonist), were found to have protective effects in vitro . This protective mechanism was primarily mediated via CXCR1, while CXCR2 countered these effects. We then studied the role of PPAR and in aneurysm formation in mice using respective agonists, but the results were inconclusive for PPAR , and PPAR had no protective role .

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17 CHAPTER 1 CEREBRAL ANEURYSM FORMATION AND THE ROLE OF INFLAMMATION Cerebral Aneurysm Epidemiology Cerebral aneury sms are focal dilations of intracranial blood vessels that occur in 3 5% of the general population. 1 , 2 Cerebral a neurysms can rupture and result in subarachnoid hemorrhage 2 , and are the cause of 5 to 15% of all strokes 3 , 4 leading to significant mortality and morbidity. About half of all patients with a ruptured aneurysm never reach the hospital in time to receive proper treatment. 1 , 5 Of the patients who survive a ruptured aneurysm, a third will develop moderate to severe lifetime disabilities . 2 , 3 Total direct costs for diagnosis and treatment of post aneurysm rupture subarachnoid hemorrhage are estimat ed to be about $25,000 per patient. 6 Cerebral aneurysm rupture is a significant risk although it is estimated that 65% of aneurysms do not rupture. 5 Common risk factors for cerebral aneurysms include hypertension, smoking 7 , female sex, and post menopause s tatus. 8 Women are 50% more likely to suffer from cerebral aneurysms than men. 1 , 8 Moreover, women are almost 25% more likely to suffer from subarachnoid hemorrhage 9 and have higher mortality rates 10 than men. Cerebral aneurysms in younger popula tion are uncommon with childhood presentations accounting for only 2% of all cases. 8 The majority of cerebral aneurysms are thought to be sporadically acquired lesions with atherosclerotic or hypertensive etiology with only a small percentage being related to genetic diseases such as autosomal dominant polycystic kidney disease (ADPKD), moyamoya disease or connective tissue disorders . 8 , 11 , 12 Other less frequent etiologies include congenital or genetic predisposition, embolic, infectious, and traumatic. 8 Subtypes include saccular or berry aneurysms, fusiform and dissecting or

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18 false aneurysms. Cerebral aneurysms typically occur within the circle of Willis, a vascular network on the ventral side of the brain. 1 , 5 , 8 , 11 Saccular aneurysms accoun t for 80 90% of all intracranial aneurysms and are characterized by their round or balloon like shape. 2 Saccular aneurysms are most commonly found in the carotid sy stem (85 95%) with the anterior communicating artery (ACoA) accounting for 30%, posterior communicating artery for 25% and middle cerebral artery for 20% of all cases. 8 About 5 15% of all saccular aneurysms are found in the posterior or vertebr o basilar circulation with 10% of that total occurring on basilar artery. 8 The most frequent presentation is major rupture resulting in subarachnoid hemorrhage, which accounts for 80% of all sp ontaneous subarachnoid hemorrhage. 8 Once subarachnoid hemorrhage has occurred, patients are under risk for secondary stroke due to arterial vasospasm. 1 , 4 , 5 , 8 , 13 On the other hand, unruptured aneurysms can cause no symptoms at all or result in hemiparesis or cranial nerve palsies, most of which present as eye movement abnormalities. 1 , 5 , 8 Other presentations include headaches, seizures, and minor infarcts or transient ischemia due to small hemorrhages and resulting vasospasm . Knowledge of the causes and mechanisms of aneurysm formation and progression is limited preventing efficient development of new treatment modalities . 12 Current Treatment Modalities for Cerebral Aneurysms The first surgical treatments of cerebral aneurysms were reported in the late 19 th cent ury and utilized simple flow redirection via ligation of carotid arteries. 14 A more sophisticated repair technique using muscle wrapping was reported by Norman Dott in 1931 although it is possible he learned this approach from Harvey Cushing. 14 Since those early approaches, the neurosurgical techniques have been refined , although t he

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19 available treatments for intracranial aneurysms are still limited to surgical procedures and primarily rely on clipping and coiling. Both of these modern procedures aim to isolate the aneurysm from circulation , reduce hemodynamic blood pressure on the outer wall of the aneurysm sac and prevent possible rupture. 1 , 5 , 8 Surgical Clipping Surgical clipping of cerebral aneurysms was introduced by Walter Dandy in 1937 14 , 15 14 Since then, the technique has seen many refinements and improvements including MRI safe titanium clips 16 and endoscopic/endonasal approaches 17 19 for difficult to reach aneurysms. Clipping is more advantageous for tr eatment of fusiform aneurysms or aneurysms with a wide or not readily identifiable neck than coiling. However, as in the case of coiling, clipping also carries with it the risk of causing aneurysm rupture during procedure in a of its proper application. 20 22 Coiling is generally preferred to clipping due to its endovascular, and thus minimally invasive approach, while clipping requires an open craniotomy procedure. 8 , 13 , 23 Recently, minimally invasive craniotomies have been introduced to compete with endovascular approaches, but overall efficacy and safety when compared with traditional craniotomies have been questioned. 24 Endovascular Coiling Coiling was introduced by Guido Guglielmi in 1991 and m akes use of platinum Guglielmi detachable c oils (GDC s) which are inserted into the aneurysm dome with a catheter via an endovascular approach with the goal of causing intra aneurysmal thrombosis and tissue ingrowth. 25 Since their original and efficient design, newer and more sophisticated coils have been introduced that contain collagen coatings 26 ,

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20 biodegradeable pol ymers 27 , gene delivery systems, 28 or chemokine releasing coatings that attract cells of th e immune system and bone marrow progenitor cells 29 , 30 . Coiling is increasing in use nowadays due to its minimally inv asive approach and b etter perioperative outcomes 1 , 23 with the exception of the very small (<3 mm) aneurysms 31 . A combination technique of stent coiling, or using both a stent and a coil, to treat aneurysms with difficult shapes or wide necks, is increasingly more popular and has demonstrated comparable outcomes. 32 Clinical Outcomes and Costs of Clipping versus Coiling Clipping has been shown to be significantly more expensive than co iling in terms of length of hospital stay and direct patient costs for both unruptured and ruptured aneurysms. 33 Costs of treating unruptured aneurysms, however, are similar between coiling and clipping, although the cost of materials in coiling is offset by longer hospital stays in clipping. 33 , 34 In fact, for cases with no complications, clipping was on average $2188 less expensive than coiling. 23 , 34 Both , coiling and clipping approaches carry significant operative and postoperative risks such as rupture, thromboembolism, and thrombosis of the parent vessel, 1 , 5 , 13 , 31 as well as risks associated with anesthesia and contra st induce d nephrotoxicity 35 . However , a recent study has demonstrated that about 40% of patients who undergo coiling show long term changes in aneurysm obliteration patte rn and 8% have to undergo re coiling or re clipping due t o extensive recanalization. 36 It has been suggested that all endovascular approaches need to be explored before proceeding with clipping to treat a re canalized aneurysm due to risks inherent with working with an intraluminal coil mass and difficulties with visualization. 37 Undergoing re coiling or re clipping carries with it the same risks as the original procedure. Nevertheless, recurrent aneury sms are rare and represent about 2% of

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21 cerebral aneurysm patient population. 38 Next generation coils are currently in development or seeing limited use, and they make use of biodegradable polymers, gene delivery systems, and protein releasing coatings to modulate the immune system response. The end goal is to improve tissue ingrowth and prevent recanalization, as described earlier. 26 30 Pipeline Embolization Devices and Flow Diverters In the early 2000s, p ipeline embolization devices (PEDs), or flow diverters, have been introduced to permit treatment of difficult aneurysms such as fu siform, wide necked, or large and giant aneurysms. 39 42 The se devices are deployable stents with high mesh density that allow for diversion and maintenan ce of flow in the parent artery, while causing pro thrombotic low flow conditions within the aneurysm sac. 42 Their efficacy has been well demonstrated in unruptur ed aneurysms allowing for robust aneurysm occlusion at 6 months post procedure. 24 , 39 42 Interestingly enough, th ese devices are capable of causing re endothelialization across the neck of the aneurysm once intra sac thrombosis has occurred, resulting in restoration of parent artery structure and an aneurysm that is effectively shut off from circulation. 24 , 39 42 Therefore, PEDs present a viable surgical alternative to treatment of aneurysms with stent coil technique. 43 Their use in cases of ruptured aneurysms , however, carries inherent risks due to requirement for anti platelet and/or anti coagulation t herapy to prevent parent or distal artery thrombosis after deploying the device . 24 , 39 42 The danger of rebleedin g from a ruptured aneurysm while on anti platelet and/or anti coagulation therapy is not trivial and is the primary reason for their scant use in such cases. 24 , 39 42

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22 Surgical Bypass Procedures Another set of surgical approaches that utilize flow diversion include intracranial extracranial and intracranial intracranial bypass procedures, although those are used only in complex aneurysm cases. 24 , 44 46 Most recent approaches utilize intracranial vessels only , sparing the patient additional procedures and ensuring the safety of the bypass which is confined to the cranium. 24 , 44 Bypass proce dures do not require complex devices or techniques, but the demand on the experience and skill of the surgeon performing the procedure is high. 24 , 44 Moreover, risks associated with complications due to the procedure are considerable and include parent artery thrombosis and flow reversal, intracranial hemorrhage due to antiplatelet and/or anticoagulation therapy, and occlusion of minor perforating arteries. 44 Pharmacotherapy Currently, no widely used pharmacological approach exists to treat cerebral aneurysms with the exceptio n of lowering associated risks to prevent future rupture. 1 , 5 , 8 , 12 , 47 As outlined in the 2012 Guidelines for the Management of Aneurysmal Subarachnoid Hemorrhage , i t is widely recognized that lowering blood pressure in patients with systolic hypertension is beneficial to prevent cerebral aneurysm rupture. 47 However, since molecular mechanisms of cerebral aneurysm f ormation and rupture have not been fully elucidated, no guidelines exist as to which anti hypertensive agents should be used to lower blood pressure in such patients. 5 , 8 , 47 , 48 Recently, pharmacotherapy with anti inflammatory agents such as acetylsalicylic acid or aspirin has been suggested as a potential avenue to prevent new aneurysm formation and aneurysm rupture in patients diagnosed with cerebral aneurysms. 49 In the International Study of Unruptured Intracranial Aneurysms (ISUIA) published in 2011 by David Hasan

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23 et al, patients who took aspirin on a weekly to daily schedule were less likely to suffer from intracranial aneurysm rupture than subjects who never took an anti inflammatory agent during that time period. 49 This study represents a significant change in aneurysm treatment strategy due recognition of cerebral aneurysm s as a complex inflammatory disease. The biggest shortcoming of all currently used modalities is that they aim to treat aneurysms after the lesion is already established and has either ruptured or is causing significant decrease in quality of life. Clearly , the development of inexpensive and viable pharmacotherapy is being hindered by the lack of knowledge of the biological mechanisms involved in aneurysm formation and rupture. 1 , 8 , 11 , 12 , 47 Cerebral Aneurysm Pathophysiology Cerebral aneurysms typically occur at points of arterial bifurcations or points of curvature and sharp angles. 1 , 5 , 8 , 11 , 12 The current , widely accepted hypothesis is that most cerebral aneur ysms form through an inflammatory mediated process that leads to the degeneration of the components of the vessel wall. 1 , 11 , 12 , 50 Structure and Composition of Arterial Blood Vessels Arteries are composed of three well defined layers: the intima, tunica media, a nd the adventitia. The intima is lined with endothelial cells which both respond to and regulate the blo od flow going through the artery . 51 In addition to having an active role in vascular inflammation and providing a gateway or a barrier to immune cell infiltration, t hey also support and have direct effects on the underlying smooth muscle cells . The tunica media, which provide s strength to the vessel wall , is pri marily composed of collagen and smooth muscle cells which respond to cues from endothelial cells and

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24 engage in a complex cross talk that regulates both physiological functions such as relaxation 51 to pathological processes such as inflammation 52 . These two layers are separated by the internal elastic lamina (IEL) which is t echnically part of the tunica intima and is composed of elastin and fibrillin. The IEL provides the majority of the elastic properties to the vessel wall and also prevents direct physical contact between the endothelial and smooth muscle cells , which has p rofound effects on the resulting cell phenotype . In higher caliber arteries such as the aorta, additional bands of elastin can be found in the medial layer providing structural support. The adventitia is a layer that is more prominent in larger arteries an d is actually thinner in cerebral blood vessels than other arteries in the body. This layer is composed of various connective tissue support cells such as fibroblasts 53 , pericytes, cells of the immune systems such as macrophages, mast cells, dendritic cells, and resident T and B cells. 54 The adventit ia also contains penetrating perivascular nerves and smaller feeder vessels known as the vasa vasorum. 54 Recently, it was discovered that the adventitia is much more dynamic than originally thought as it contains a reservoir of progenitor cells that can be activated to provide local or systemic repair . 54 56 Pathophysio logy of Cerebral Aneurysms of Congenital and Genetic Etiology Congenital or genetic defects in any of the vascular wall layers increase the risk for cerebral aneurysm formation. However, such causes are thought to account for only a small number of intracr anial aneurysm cases as familial intracranial aneurysms make up only 7 20% of all SAH cases. 57 , 58 C on genital aneurysms are thought to arise as a defect in migration or function of neural crest cells, which contribute to the embryonic development of the aortic arch, specifically the carotid system. 1 , 8 , 11 In addition, genetic defects in any of the components of the vascular wall predispose patients to cerebral

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25 a neurysm formation. Autosomal dominant po lycystic kidney disease (ADPKD) is a result of a mutation in PKD1 or PKD2 genes, and is seen in about 0.2% of the population. 8 , 59 It leads to progressive chronic renal failure and cysts developing in several organs. 8 Aneurysms occur in 10 30% of all ADPKD patients and about 64% of them rupture. Aneurysms are also commonly seen in patients with arteriovenous malformations (AVMs) such as in Moyamoya disease. 8 In all, about 7% of all AVM patients present with cerebral aneurysms. 8 In Ehlers Danlos syndromes , especially type IV, a mutated collagen gene COL3A1 results in deficient collagen metabolism. 60 62 This leads to a ph enotype with hypermovable joints, extendable skin, and propensity to bruising. 60 , 61 Moreover, patients are more prone to aneurysm formation throughout the circulatory system and about 2.1% of all patients suffer from major cerebrovascular events such as subarachnoid hemorrhage due to aneurysm rupture. 61 , 63 Marfan syndrome is a connective tissue diseas e caused by a m utation in fibrillin 1 gene known as FBN 1 seen in about 0.01% of the entire population . 64 Fibrillin is responsible for a sheath around elastin proteins, which toget her form the elastin layer in the IEL. Defect s in elastin lead to decreased structural integrity of cardiovascular system, abnormal growth of the skeletal system, and vision problems. In addition, fibrillin 1 defects lead to abnormal transforming growth fa ctor (TGF ) signaling. 65 Patients with Marfan syndrome commonly experience cardiovascular complications, which are the cause of 58% of all deaths in this pati ent population. 64 , 66 Other conditions that typically have higher rates of aneurysm formation include hereditary h emorrhagic talangactesia (HHT) or Osler Weber Rendu syndrome 67 , fibromuscular dysplasia and coarctation of the aorta. 8

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26 However, chronic inflammation of hypertensive or atherosclerotic etiology is thought to be the primary cause of cerebral a neurysm formation. Pathophysiology of Aneurysm Formation of Atherosclerotic or Hypertensive Etiology Inflammatory changes in cerebral aneurysm specimens were first reported in late 19 th century. 12 Since then, t he inflammation hypothesis has been expanded to include the role of hemodynamic stress as the initiating factor in aneurysm formation. 1 , 2 , 5 , 11 , 12 , 48 Although inflammation is a response normally exhibited against microbial pathogens and mycotic aneurysms do occur, it is thought that the majority of cerebral aneurysms form through sterile chronic inflammation. 9 , 12 , 14 , 23 , 30 , 48 In fact, infectious aneurysms are thought to represent only 1 2% of all cases. 68 Chronic inflammation is pathological response of tissues t hat initially react to the stressor in a physiologically expected process of wound healing . This process is supposed to pro tect the local micro environment. Wound healing is a continuous process that is thought to occur in four classical, overlapping phases: hemostasis, inflammation, proliferation, and resolution. Hemostasis refers to the initial process of coagulation cascad e that aims to prevent fluid loss and exposure to the outside environment by restoring the barriers between tissues. Another way to think about the wound healing process is to divide it into an early and cellular phase. 69 Early phase refers to the molecular signaling cascades that result in cellular activation, inflammatory protein expression, and formation of an initial extracellular matrix. The cellular phase describes the events that occur during the inflammatory cell infiltration. Once a make shift matrix of cells and extracellular matrix is established, cells of the immune system, especially the innate component, infiltrate the wound to remove

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27 any potential pathogens and then direct local proliferation and remodeling. 69 However, failure to remove the offending stressors and stimuli or lack of resolution lead s t o a vicious cycle of continuous inflammation that results in tissue degradation and loss of function. In the case of aneurysm formation, this tissue degradation and loss of function of proper structural support has devastating consequences that can result in aneurysm rupture and subarachnoid hemorrhage. 1 , 11 , 12 , 58 , 70 The initiating stressor in aneurysm formation is thought to be abnormal hemodynamic shear stress, a force that is experienced by all endothel ial cells lining the arteries in the direction parallel to the blood flow. Experi mental animal aneurysm models 71 74 and computationa l fluid dynamic (CFD) studies 75 81 have implicated high wall shear stress and shear stress gradients in initiating aneurysm development through effects on the endothelium and remodeling of the loc al vessel wall, specifically, the damage to the internal elastic lamina (IEL). Aneurysm growth is believed to occur through a chronic inflammatory process characterized by a poptosis of endothelial cells, 76 increased secretion of the matrix metallo proteinases (MMPs), 82 and activity of innate immune ce lls, primarily macrophages. 72 , 83 , 84 Molecular Basis of Chronic Inflammation in Cer e br al Aneurysm Formation Tulamo et al have described four immuno histological grades of aneurysm walls as these lesions progress to rupture. 12 Type A histology is thought to occur in early aneurysms and is characte rized by intact endothelium and a normal, well organized layer of smooth muscle cells in the media. 12 It is believed that at this step the IEL could be damaged and that the endothelial cells attain a pro inflam matory phenotype 72 , 85 . Those early changes are most likely mediated by upregulation of cyclo oxygenase 2 (COX 2), m icrosomal prostaglandin E synthase 1 (mPGES 1) and prostaglandin

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28 receptor type 2 (EP(2)) by high shear stress acting on the endothelium. 85 , 86 COX 2 and mPGES 1 are components of the canonical eicosanoid inflammatory system that acts within second to minute s of detecting stress; they cause production of pro inflammatory prostaglandins, the major product being prostag landin E2 (PGE2). 85 92 Type B histology is characterized by myointimal hyperplasia, or migration and proliferation of smooth muscle cells in to the intima. 12 Some studies have shown that the migrating smooth muscle cells originate from progenitor cells that reside in the adventitia in atherosclerosis 93 or in response to angioplasty 94 . This myointimal hyperplasia is thought to occur through increased signaling of m itogen activated protein kinase (MAPK) . 95 Once myointimal hyperplasia occurs, inflammation is sustained through hypoxia due to higher oxygen demand of the lo cal vascular tissues 96 , both collagen deposition 12 and breakdown 97 , 98 , reactive oxygen species (ROS) formation 99 , oxidized lipid accumulat ion 12 , 100 and fibrosis 12 , 101 . At the same time, these inflammatory changes are mediated by persistent nuclear factor kappa beta (NF ) activation 97 , which leads to increased expression of inflammatory genes such as inducible nitric oxide synthase (iNOS) 97 , MMPs 98 , VCAM 1 102 , 103 and interleukin 1 (IL 1) 97 , 104 . Inhibition of NF activation by gene knockdown 97 , deoxynucleotide decoys 97 , statin ther apy 105 , 106 or nifedipine 107 inhibits cerebral aneurysm formation in rodent models. In fact, NF and Ets inhibition with deoxynucleotide decoys has been reported to induce cerebral aneurysm regression, but the aneurysms formed in the model used were small and did not fully represent histological patterns seen in human aneurysms . 108 It is important to note that only iNOS expression correlates with increased aneurysm formation due to its role in ROS production by macrophages in inflammation. 97 , 99 , 109 Other NOS isoforms

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29 such as neuronal (nNOS) and endothelial (eNOS) have been found to have a protective role in cerebral aneurysm formation. 109 These protective effects are thought to occur through relaxation of the hemodynamic stress on the vessel wall via vasodilation. 109 In any case, this massive activation of inflammatory pathways leads to progressive damage and thinning of the blood vessel wall through apoptosis of the smooth muscle cells, which is the defining feature of Type C wall in intracranial aneurysms. 12 In particular, tumor necrosis factor alpha (TNF ) and its target Fas associated death domain protein cause increased apoptosis of vascular mural cells and progression of aneurysm formation 110 , 111 and rupture 111 . TNF also causes a phenotypic change in vascular smooth muscle cells to a non contractile phenotype characterize d by increased secretion of MMP 3 and 9, IL 1 , and VCAM 1, which leads to vessel wall degradation. 112 Such phenotypic change is typically seen in patients who are smokers due to increased levels of circulating TNF and ROS. 7 IL 1 upregulation has been reported to decrease secretion of collagen within aneurysmal walls. 104 This process of complete decellularization and thinning with intraluminal thrombus formation is thought to result in Type D wall, which is especially prone to rupture. 12 Structural ves sel wall weakness has been shown to be associated with higher incidence of aneurysm rupture. 12 , 101 , 113 Hypertension is one of the major factors associated with aneurysm formation and rupture 5 , 47 and virtually all animal intracranial aneurysm models use hypertension either via artery ligation, pharmacotherapy, high salt diet, or a combination of these approaches to induce aneurysm formation. 48 , 73 , 114 116 117 Return to physiological range of blood pressure after aneurysm formation and hypertension has been shown to reduce aneurysm rupture in a mouse model . 48 The physical effects of

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30 high blood pressure are not the only reasons for increased tendency of aneurysms to progress to rupture. Recent study has shown that angiotensin converting enzym e (ACE) inhibitor captopril and angiotensin II receptor blocker losartan are capable of decreasing aneurysm rupture by inhibiting the local inflammatory effects of the renin angiotensin system (RAS) . 48 Thi pressure. Upon stimulation of cells in the juxtaglomerular apparatus in the kidneys by low blood perfusion, decrease in sodium chloride levels, or beta 1 adrenergic stimulation of the sympathet ic system , renin is released into the circulation. Then, renin converts angiotensinogen (ATG) produced in the liver to angiotensin I (Ang I). Ang I is then converted to angiotensin II (Ang II) by angiotensin converting enzyme (ACE) produced by the pulmonar y endothelium. Ang II acts on a family of receptors, the most well described being angiotensin II receptor 1 and 2 (ATR1 and ATR2), which cause vascular smooth muscle cell constriction, aldoresterone release from the adrenal glands, and finally vasopressi n release, which all cause increase in systemic blood pressure. In addition, Aoki at el have previously shown that ACE inhibitor imidapril acts in ACE independent manner and exerts its effects by decreasing activity of MMP 9 118 , confirming the findings by Tada et al 48 . However, differences in the role of RAS in local inflammation may exist for different systemi c arteries. In another study, Aoki et al found that angiotensin II receptor type I (Ang II type R(1)) is not upregulated in cerebral aneurysms in rats in contrast to abdominal aneurysms . 119 Angiotensin II has also been found to cause systemic fibrosis in models of systemic sclerosis. 120 There is no doubt, however, that hist ological and structural changes seen in cerebral aneurysm formation in response to remodeling only tell half the story. The molecular cross talk between

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31 different cells of the vascular wall and cells of the immune system in aneurysm formation is not only c omplex, but also of paramount im portance to development of this disease. 1 , 11 , 12 , 50 , 77 , 79 , 100 , 102 As inflammatory pathways are being activated during cerebral aneurysm progression, from formation to rupture , the endothelial cells increase their expression of chemokines, cytokines with chemotactic properties, 72 , 121 and cell adhesion molecules such as selectins 122 and ICAM 1 123 . 72 , 97 , 102 , 124 This allows for inflammatory infiltration by cells of the innate immune system such as neutrophils 125 127 , monocytes and macrophages 72 , 98 , and mast cells 128 , and amplification of these inflammatory cascades 1 , 11 , 12 , 50 , 58 . The critical r ole of monocyte and macrophage attracting CCL chemokines such as MCP 1 has been described in several models and increased macrophage accumulation has been shown to lead to aneurysm formation and rupture. 48 , 72 , 82 84 , 98 , 129 MCP 1 blockade or blockade of macrophage derived pro inflammatory ROS with free radical scavengers 99 and MMPs with tetracycline and its analogs 130 effectively prevents aneurysm formation and rupture . Tissue inhibitors of matrix metallo proteinases 1 and 2 (TIMP 1 and 2) also play a key role in protecting against aneury sm formation and their inbalance has deleterious effects in this disease. 82 Proteolytic activity of other enzymes such as cathepsins , a family of serine proteases, secreted by vascular mural cells has also been shown to cause aneurysm formation. 131 Macrophage depletion using clodronate liposomes in mouse intracranial aneurysm model prevents aneurysm formation, which is the definitive proof for the role of these cells in this cerebrovascular disease. 83 Macrophages are especially plas tic cells and are capable of both tissue destruction and wound healing. In fact, a recent study of human

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32 aneurysm specimens by Hasan et al has revealed that macrophages found in ruptured aneurysms are predominantly of the M1, pro inflammatory rather than M 2, anti inflammatory phenotype. 84 At present, no studies have been done to confirm the role of M1 macrophages in experimental models of aneurysm formation. 50 Despite a number of studies on monocytes and macrophages, no experimental data exists on the other phagocytic cells o f the myeloid system. 11 , 12 , 50 No role or function of neut rophil attracting ELR+ CXCL chemokines such as CXCL1 or IL 8 in cerebral aneurysm formation has been reported. 12 , 72 , 83 , 121 Neutrophils act as the first responder cells of the innate immune system yet their role in cerebral aneurysm formation has n ot been examined i n detail. 12 , 127 This is a significant oversight as neutrophils are the earliest cells of the innate immun e system to arrive at sites of inflammation. 127 Increased numbers of mast cells have been found in human ruptured aneurysm specimens 84 , 132 and these cells have been found to contribute to cerebral aneurysm formation in a rat model. 128 De granulated mast cells were found to upregulate MMP 2 and 9, IL 1 , and NF in vascular smooth muscle cells. 128 Sporadic eosinophils and basophils have been found in cerebral aneurysms on immunohistochemistry, but their r ole is not currently known. 12 , 133 Eosinophils are actually more likely to be found in aneurysms formed in pa tients with autoimmune diseases such as Churg Strauss syndrome 133 . Other cells of the immune system such as cells of the adaptive immune system, T cells and B cells, have been reported in cerebral aneurysm specimens but their role has not been extensively studied. 12 It is believed that if adaptive immunity is involved, it is predominantly through the Th 1 rather than Th 2 T helper cell response due to the increased levels of TNF and possibly interferon gamma (IFN ) in cerebral aneurysms. 12

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33 Additional gaps in knowledge exist due to lack of an effective in vitro screening tool that would allow for modeling of hemodynamic conditions within cerebral aneurysms. The link between hemodynamic forces, such as shear stress, and biologi cal mechanisms, such as inflammation, that contribute to aneurysm development is still not fully understood. 12 , 7 7 , 79 , 80 , 134 , 135 Role of Hemodynamic Shear Str ess in Inflammation As discussed earlier, hemodynamic stress is the driving force behind aneurysm initiation and formation. Shear stress is a frictional force that is experienced by most cells in the body in addition to endothelial cells and is mechano tra nsduced to exert a complex biological response within the cell. 136 There are two principal stresses experienced by endothelial cells within blood vessels: shear stress and tensile stress. Hemodynamic shea r stress is the force parallel to the vessel wall and the direction of blood flow and is p rimarily experienced by endothelial cells in a normal , healthy blood vessel. 136 Tensile stress is the force perpendicular to the vessel wall and is experienced by all vascular cells including smooth muscle and adventitial cells during systole . Most of the shear stress experienced by vasculatur e in the body is in the form of non uniform laminar shear stress, which means that the absolute shear stress can vary at a given point in space or time, but the movements of blood are not random and the fluid moves in parallel sheaths or layers. Turbulent flow can occur in areas distal to arterial stenosis and around heart valves, but has not been described in cerebral vasculature. 136 First study reporting endothelial cell response to flow was published in 1985 by Frangos et al. 137 Short ly after , another group reported the release of endothelial cell factor that caused vascular relaxation 138 , which later would be identified as nitric oxide. 51

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34 These early studies showcase how important shear stress is for norm al physiological function of end othelial cells and blood vessels. 136 Important differences between in vivo and in vitro systems nee d to be descr ibed when discussing hemodynamic shear stress. Application of Hemodynamic Shear Stress to in vitro Systems B lood flow in vivo is pulsatile and the value of shear stress changes over time in response to cellular and tissue needs of the local en vironment . This results in a non uniform endothelial cell phenotype that is highly distinct and heterogeneous throughout the body based on gene 139 and miRNA 140 expression patterns. This phenotypic heterogeneity can even occur in the same segments of large caliber arteries and cause predisposition to cardiovascular disease . 141 Heritable features that contribute to aortic arch geometry can heavily influence predisposition to atherosclerosis as seen in differences between C57BL/6 and 129/SvEv mice. 142 , 143 In fact, quantitative trait loci (QTL) found on chromosomes 1 and 15 that highly correlate with increased aortic arch geomet ry have been implicated in development of atherosclerosis in 129/SvEv mice. 144 Therefore, heritable traits that do not directly influence inflammatory gene expre ssion can be a factor in geometry resulting in a specific set of hemodynamic conditions that dictate endothelial cell phenotype. 142 145 F or ex a mple , it has been sho wn that differences in inflammatory gene expression exist between endothelial cells lining the inner and outer curvature of the aorta. 141 Cells lining the inner curvature are typically exposed to re circulating flow and eddies resulting in decreased alignment with flow 146 and increased levels of inflammatory gene expression than the cells on the outside curve. 141 , 147 Endothelial cells at bifurcation points are especially susceptible to pro inflammatory changes as zones of low wall shear stress, flow reattachment and recirculating flow exist. 147 In these zones, endothelial cells are not aligned with flow 148 ,

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35 display increased plasma cell membrane permeability 136 , 149 , and increased levels of p roliferation 150 , 151 . Moreover, at bifurcations, both temporal and spatial gradients in shear stress occur 136 , 151 Some studies have shown that in absence of t emporal gradients, spatial gradients in shear stress do not affect endothelial cell proliferation 151 , but that does not hold true at very high levels of shear stress that is typically seen in cerebral vessels. 71 , 76 As the cardiovascular system goes through the cardiac cycle, those zones of flow reattachment move back and forth resulting i n an area exposed to low mean temporal shear stress. 136 Atherosclerotic plaque formation has been correlated with those low wall shear stress regions. Because of these subtle nuances in physiology and geometry of the cardiovascular system, in vitro experiments have to be carefully designed in order to provide useful data. Most early in vitro shear stress experiments were performed under u niform conditions and non pulsatile steady flow. In fact, some modern shear stress experiments still utilize this continuous flow scheme , for example when stu dying immune cell rolling since it allows for easier observation and acquisition of data. 152 It sy stems expo sed to steady non pulsatile , pulsatile , and dynamic pulsatile flow. 153 Just as there are temporal differences in shear stress, spatial differences as exhibited by cell behavior at arterial bifurcations can have considerable effects. Endothelial cells are not only capable of sensing shear stress gradients, or changes in shear stress over distance 136 , but their response differs depending on whether the gradient is in , or opposite, the direction of hemodynamic flow 76 .

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36 Gene expression is not only tightly linked to absolute values of shear stress, but some genes show variations in express ion with gradient at lower or higher values of mean shear stress. 145 LaMack et al reported that endothelial nitric oxide synthase expression was sensitive to magnitude of shear stress but had no dependence on gradient. 145 On the other hand, MCP 1 expression was inversely proportional with shear stress magnitude and was also found to be increased at lower values of shear stress gradient. 145 The authors reported that c jun, a protein that is part of the AP 1 early transcription factor with multiple role s in cell cycle, proliferation and cancer progre ssion, had a weak dependence on shear stress magnitude and gradient without a combination effect. 145 Finally, increase in ICAM 1, an adhesion molecule important i n inflammatory cell influx, expression was associated with increased shear stress magnitude and gradient. 145 Furthermore, important differences exist in cell resp onse depending on how hemodynamic shear stress is applied during the experiment. 154 In their 1999 study, Bao et al showed that when shear st ress is applied to a sys tem in ramp flow, meaning that flow is slowly and continuously increased until it reaches a certain value, only minor differences are seen in inflammatory gene expression such as platelet derived growth factor (PDGF ) or there are no differences as seen wit h MCP 1 expression. 154 When step flow was applied, meaning flow was increased in an abrupt manner in a ste p wise fashion over time until reaching certain value, 2 to 3 fold increases in gene expression of PDGF and MCP 1 , respectively, were noted. When the same mean value of shear stress was applied in an impulse for 3 seconds, PDGF and MCP 1 expression was upregulated 6 and 7 fold for up to 4 hours. These changes i n gene expression were found to be regulated by NO, although there was a difference in how this regulation

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37 acted for each flow scheme. In the case of ramp and step flow, NO was able to inhibit pro inflammatory PDGF and MCP 1 expression, while in impulse fl ow NO was responsible for increase in expression of those genes. These changes in PDGF and MCP 1 expression mediated by NO were found to be dependent on transcription factors early growth response protein 1 (egr 1) and NF , respectively. 154 Using multiple markers such as NO secretion, gene expression arrays, and leukocyte rolling experiments, Uzarski et al elegantly showed that under dynamic pulsatile flow, or phys iological flow characterized by changes in shear stress magnitude and duration, endothelial cells are more quiescent than when exposed to simple, pulsatile or steady flow. 153 Endothelial cells exposed to physiological flow exhibited significantly lower expression of pro inflammatory cell adhesion molecules such as E selectin, ICAM 1 and PECAM 1 and cytokines such as MCP 1 when compared to cells exposed to steady pulsatile flow. 153 Zhang et al reported that endothelial cells that have been preconditioned under shear stress conditions respond differently to increases in shear stress than static cultures. 155 Interestingly, they found that pre conditioned endothelial cells downregulated pro inflammatory genes that would otherwise be upregulated in shear stress naïve cultures upon increases in shear stress. 155 An inverse response was noted with anti oxidative genes that could provide a protective effect. The authors also discovered that endothelial cell permeability was incr eased following acute increases in shear stress magnitude and that the overall adaptation response to flow has three phases: induction, early adaptation and remodeling. They suggested that the early adaptation phase lasts for about 45 minutes and cells in this phase are phenotypically distinct from adapted cells based on expression of 85 differentially identified genes. 155 In

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38 another closely related study, Zhang et al showed that changes in frequency of pulsatile flow at the same mean values of shear stress lead to differential gene expression during the adaptive phase. 156 Upon increase in pulse flow frequency, cyclin A2 (CCNA2) and B1 (CCNB1) and cyclin dependent kinase 1 (CD C2) genes, which are related to cell cycle and proliferation, were increased along with expression of several genes related to angiogenesis such as VEGF. The authors suggested that pulse frequency increases, as seen in increased heart rate during exercise, could increase the potential for endothelial repair and attributed their findings as anti inflammatory and anti oxidative. 156 However, their studies were in vitro and it is not known whether the same results would hold true in an animal model. Nevertheless, t he exp erimental results reported by Uzarski and Zhang in their independent studies are clinically significant as they provide a basis for protective effects of cardiovascular exercise in daily life. 153 , 155 , 156 It has been demonstrated that changes in shear stress during exercise induce endothelial and vascular functi onal adaptations and remodeling. 157 , 158 The protective effects of dynamic or physiological changes in she ar stress are not solely dependent on endothelial cell phenotype. 136 , 159 As mentioned previously, endothelial cell released cytokines and other factors, especially NO can influence the phenotype and health of the underlying smooth muscle cells. 51 , 52 , 138 , 160 In addition, protective effects of exercise related increases in shear stress on the cardiovascular system h ave also been suggested to occur through downstream effects on differentiation of the progenitor cells residing in the vascular adventitia. 56 , 159 Despite a wealth of knowledge about effects of hemodynamic shear stress on cardiovascular health and its role in vascular disease, the basis for biological response to shear stress on molecular level remains unanswere d. In other words, the

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39 answer to how shear stress is actually transmitted from the plasma cell membrane to regulate immediate cell function and gene expression remains an enigma although several theories and lines of evidence exist . 136 Molecular Basis of Mechano transduction and Cellular Respo nse to Shear Stress As of the time of writing this work , several mechanisms have been described to be involved in endothelial ability to transduce and respond to changes in shear stress. 136 , 161 , 162 It is known that steady hemodynamic shear stress is necessary for proper endothelial cell, and therefore blood vessel, function and health and has protective effects against atherosclerosis or vascular stenosis. 136 , 145 , 163 On the other hand, low or oscillatory shear stress results in pro inflammatory endothelial phenotype 75 leading to a secretion of inflammatory cytokines and endothelial dysfunction. Clearly, endothelial cells and other cells in the body such as fibroblasts 164 are capable of distinguishing fine details of physical forces that act on them and in their micro environment. 145 , 153 , 155 , 156 , 164 Several lines of evidence suggest that distinct cellular structures and pro cesses are involved in shear stress sensing and transduction. It is known that endothelial cells and their nuclei elongate and realign in the direction of flow 146 , 148 , 165 , plasma membrane permeability increases with acute application of shear stress 149 , several signaling pathways are activated , and that NO secretion is dependent on hemodynamic conditions. 136 , 161 These observations implicate cytoskeletal reorganization, nitric oxide production, the glycocalyx, plasma cell membrane and ion channels in shear stress sensing and mechano transduction. Hemodynamic shear stress a cts directly on the plasma cell membrane and it has been proposed that the lipid bilayer may act as a mechano sensor that transmits the hemodynamic force to cytoskeletal or adhesion proteins. 136 , 166 168 Several proteins have been implicated as

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40 the transducers attached to the plasma cell membrane such as the focal adhesion kinase (FAK), platelet endothelial cell a dhesion molecule 1 (PECAM 1) als o known as CD31, and G protein activation . In two separate studies, Katoh et al and Kano et al reported that t he apical cell membrane plaques are composed of vinculin, talin, paxilin, fibronectin receptor protein, and fibro nectin, but not integrin alpha v subunit or focal adhesion kinase indicating that they are important attachment points of stress fibers with function other than simple cell adhesion. 166 , 169 In a follow up study, the authors suggested that the cell cell adhesion sites and the baso l ateral surface are the points where shear stress is transduced from the apical membr ane and the apical plaques based on tyrosine residue phosphorylation paterns . 170 The cellular cytoskeleton is composed of three main classes of filaments or fibers and several types of adhes ion complexes. Microfilaments are composed of polymerized actin subunits with diameter of 6 nm and can generate force by either walking of myosin anchored structures or elongation at one end. Their primary role is in cell migration and stress fiber formati on. They are associated with small GTP binding pr oteins such as Rho, Rac, and CDC 42, which regulate their growth. Signaling through actin based stress fibers leads to a stiffening response within the cell and viscosity changes within the cytoplasm. 164 They also have a role in cell division and form microvilli. Intermediate filaments are a family of heterogeneous proteins such as keratin, lamin, neurofilaments, and vi mentin that give the cell its overall shape and help anchor intracellular structures. Microtubules are large filaments composed of alpha and beta tubulin resulting in proto filaments with a diameter of 23 nm. They are typically organized in a 9+2 structure of 13 protofilaments. They play a role in intracellular

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41 movement of organelles such as vesicles via dynein and kinesin motors, are an integral part of the mitotic spindle, cilia, and flagella. Cell migration and movement illustrate best how the cellular c ytoskeleton could be used to sense changes in shear stress. During cell movement, the advancing filopodia and lamellipodium form nascent adhesions that mature into focal complexes through actin growth and actinomyosin contractile action. 171 Actin bundles are polymerized via Rho GTPases Rac and CDC42 catalyzed action of ARP2/3 complex. 172 174 In the meantime, at the rear of the moving cell mass, actin is organized into stress fibers which connect to the focal adhesions at the basal cell membrane. Focal adhesions are macromolecular complexes that contain actin filaments, actinin, paxilin, talin, FAK, vinculin, and various integrins. 171 , 173 Their role is to anchor the cell via the cellular cytoskeleton, particularly stress fibers, to the extracellular matrix. During cell movement, t hese stress fibers are continuously acted upon by the contractile action of myosin II, which transmits stress into the advancing cell front causing conformational changes in nascent adhesions leading to adhesion maturation by action of FAK and paxilin on Rac . 173 , 175 , 176 Phosphorylation of Thr18 and Ser19 on the regulatory light chain 177 as well as other sites on the heavy chain dictates the amount of myosin II activity. 174 , 178 Tensile stress transmitted via the action of myosin II on the stress fibers in the center or lamellum of the cell mass leads to maturation of focal complexes into focal adhesions through Rho and Rho associated prot ein kinase (ROCK) as well. The focal adhesions are then disassembled at the rear through various proteases , especially calpain driven talin degradation . 179 181 The process of cell locomotion illustrates an important point: tensile stress transmitted through action of myosin II on stress fibers can cause conformational changes in protein complexes

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42 leading to nascent adhesion maturation, anchoring, and movement. Th erefore, it is not difficult to imagine that if focal adhesions on the basal layer of the cell membrane can mature when acted upon by stress, then they could also be used to transduce mechanical stress. 182 In fact, it has been shown that transduction of shear stress through focal adhesions on fibronectin leads to upregulation of PGE2 and COX 2 in osteoblasts. 183 , 184 When components of the cytoskeleton were disrupted or blocked, such as actin microfilaments, intermediate filaments, and microtubules, no effect was obse rved on prostaglandin production or COX 2 enzyme upregulation. 183 Focal adhesion kinase (FAK) has also been shown to be involved in focal adhesion mechanotransd uction of shear stress in osteoblasts. Similar processes with both MAP and FAK activation have been described in endothelial cells. 185 , 186 FAK signaling has been shown to be critical in shear stress mediated activation of extracellular signal related kinase ( ERK ) and c Jun N terminal kinase ( JNK ) signaling pathways on the intracellular basal cell surface. 187 ERK1/2 signaling has been shown to be particularly important in shear stress induced endothelial cell migration via integrin signaling and the fibronectin receptor subun its alpha(5) and beta(1) . 188 This abluminal, or basal, mechanotransduction of shear stress appears to be complemented by baso lateral signaling via PECAM 1 and N O. 189 PECAM 1 or CD31 is a 128 kDa glycoprotein that is present on the surface of endothelial cells, platelets, megakaryocytes, and cells of the myeloid system and lymphocytes. 189 It has been proposed to be the mechanotransducer of shear stress in endothelial cells at the cell cell lateral adhesions. Under shear stress conditions PECAM 1 becomes extensively phosphorylated in endothelial ce lls at cell cell

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43 junctions. 189 It is thought that conformational changes induced via stress transmitted to cell cell junctions is responsible for this process. 136 Another group has shown that SHP2, a tyrosine phosphatase, is associate d with phosphorylated PECAM 1 and is crucial in downstream signaling v ia ERK . 190 In their experiment, the authors used magnetic beads conjugated to an antibody against PECAM 1 and when magnetic force was applied, extensive phosphorylation of ERK and PECAM 1 was observed. 190 In addition, PECAM 1 is highly distributed along the periphery in cell cell junctions in confluent endothel ial monolayers, but shows weak expression in subconfluent cultures. 191 Furthermore, heterotrimeric G proteins, especially G q/11, and their activation may play an intricate role in PECAM 1 mediated mechanotransduction. 136 Heterotrimeric G proteins are proteins that typically get activated when their coupled receptors undergo a conformational change. G proteins are composed of three subunits, , , and , and, upon activation, the complex is released following GDP GTP exchange. The complex can then act as a signaling molecule by activating phospholipase A2 or ion channels. Several families of G proteins exist with distinct signaling pathw ay activation profiles. G protein G q/11 and PECAM 1 have been found to co immunoprecipitate together. 150 PECAM 1/ G q/11 complex has been found to dissociate on application of temporal gradient in shear stress. 192 Interestingly, this compl ex seems to be absent from endothelial cells in atheroprone areas and blockade of G protein activation prevents complex disassociation. 192 Using ramp flow instead of impulse flow prevented complex disassociation as well. However, Sumpio et al reported that MAPKs, ERK1/2 and p38, and AKT are phosphorylated following application of shear stress in HUVECs independently of PECAM 1. 193 This suggests that PECAM 1 ,

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44 and other components of the cytoskeleton for that matter, may not be the major or the only mechanotransducer of shear stress. This is supported by several studies that used targeted deletions of desmin 194 and dystrophin 195 . In fac t, there appears to be a cross talk between mechanotransduction and other biological pathways in sensing shear stress as exhibited by NO and PECAM 1 . In a study that utilized PECAM 1 knockout mice, Bagi et al found that PECAM 1 regulate s NO mediated arteriolar dilation in environments with sudden increases in shear stress. 196 Although Wt and PECAM 1 / endothelial cells responded in a similar man ner to NO under steady shear, during high temporal gradients of shear stress, the dilation response in KO arterioles was reduced. 196 Some studies have implicated N O as a molecule involved in shear stress signal mechanotransduction. Endothelial NOS has been found to be associated with PECAM 1 at cell cell junctions . When endothelial cells are subjected to impulse flow, the eNOS PECAM 1 complex dissociates with concur rent increase in eNOS activity as measured by cGMP production, but those changes are not seen when ramped flow is applied. 197 Although NO may not act as a sin gle signaling molecule to transduce the effects of shear stress, it is thought that in concert with ROS it presents a major mechanism through which many of the intracellular changes occur in response to shear stress. 161 Shear stress induces NOS activation and NO pro duction via several mechanisms such as PI3K/AKT activation 198 , phosphorylation of Ser and Thr residues on NOS by AMP activated protein kinase (AMPK) 199 , 200 , increased NOS expression via action of Krüppel like factor (KLF2) 201 , 202 , and increased bioavailabili ty of NO via Nrf2 transcription of anti oxidative genes 203 . Downstream auto and paracrine actions of NO can be summarized as general anti inflammatory and anti atherogenic

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45 effects. 136 , 161 Nitric oxide has been shown to decrease expression of pro inflammatory cytokines such as IL 6 and IL 8 and adhesion m olecules such as ICAM 1 by inhibiting NF . 204 This NF inhibition of DNA binding 205 is thought t o occur through ability to cause S nitrosylation of a critical group on the p50 subunit of NF 206 and quench pro inflammatory superoxide anion and other ROS 207 . NO can also act as a scavenger and form nitrogen containing reactive species such as p eroxynitrite. Oscillatory flow or flow reversa l has pro inflammatory effects 208 by causing production of the superoxide anion via upregulation of Nox4 209 , a component of NADPH oxidase. NAD PH oxidase (Nox) uses NADPH in a classical respiratory burst to cause damage to pathogens by producing superoxide anion. 210 213 Under normal conditions Nox enz ymes are used to regulate cell growth, proliferation and wound repair. Several Nox isoforms and homologues exist within the cardiovascular system. Long term , steady shear stress and pulsatile shear stress downregulate Nox expression, and therefore activity and superoxide production, by action of Nrf2 and Oct 1 transcription factors. Another set of evidence exists that the apical part of the cell is involved in shear stress sensing either via changes in the glycocalyx or plasma membrane fluidity. The endoth elial glycocalyx is a carbohydrate coating on the apical membrane of the endothelial cells that extends up to 1 m into the vascular lumen. 214 It is composed of glycoproteins (glycated proteins with terminal groups of sialic acid ), negatively charged proteoglycans (carbohydrate complexes with some protein content) such as heparin sulfate , and glycosoaminoglycans (GAGs) such as hyaluronan. 214 The glycocalyx is attached to the apical membrane via various anchoring proteins such as syndecans and CD44. 215 Some anchoring proteins such as glypican 1 attach the glycocalyx to

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46 glycosylphosphatidylinositol (GPI) anchor s in c aveola containing eNOS. 216 NO signaling has been described to have a role in shear stress signaling as described earlier. This glycocalyx coating has a role in homeostasis, shear stress sensing, inflammatory response, and the maintenance of vascular permeability. 217 During endothelial activation, inflammatory cyt okines are deposited on the glycocalyx providing a chemotactic gradient for leukocytes. Degradation of this coating by MMPs allows increased inflammatory infiltration. Furthermore, both in vitro and animal studies have shown that sialic acid and hyaluronan are necessary components for mechanotransduction of shear stress via NO mediated vasodilation. 218 221 Thi et al were the first ones to show that the glycocalyx me diated shear stress transduction is distinct from focal adhesion and apical surface/stress fiber mediate d transduction. 222 The authors implicated the torque acting through the glycocalyx on the actin cortical web and dense peripheral actin bands (DPABs) concurrent with vinculin migration in shear stress mechanotransduction. 222 Pahakis et al confirmed that the apical shear stress sensing by the endothelial glycocalyx is mediated by signaling pathways different from mechanotransduction exhibited at the baso lateral cell membrane. 216 When heparan sulfate and hyaluronic acid were depleted in a bovine endothelial cell perfusion system with heparinase and hyaluronidase , shear stress mediated NO production was blocked. 216 However, production of prostacyclin (PGI 2 ) transduced in the basal membrane was unaffected by the presence or lack of the glycocalyx. 216 , 219 R ecent studies suggest that the glycocalyx can transduce its effects by affecting plasma cell membrane fluidity in addition to its effects on lo ose cytoskeletal networks . 151 , 223

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47 Alternative mechanism for transduction of shear stress is that the cell plasma membrane acts a large sensor capable of differentiating small changes in shear over the surface of the cell. 223 It is thought that conformational changes in membrane bound G proteins 224 , 225 and ion channels transduce detected variations in shear stress t hrough local differences in membrane fluidity. 136 Gudi et al were able to show that membrane composition and fluidity can regulate shear stress mediated GTPase activity in G protein reconstituted phospholipid vesicles in the absence of any cytoskeletal elements or cellular machinery. 226 Studies with fluorescent probes and dyes such as 9 (d icyanovinyl) julolidine (DCVJ) have shown that application of shear stress changes quantum yield of the dye with con current changes in viscosity of the environment. 223 Furthermore, other cell types such as NIH 3T3 fibroblasts have been shown to exhibit a ROCK dependent stif fening response with increase of intracellular viscosity upon application of shear stress. 164 This suggests that shear stress transduction through changes in plasma membrane fluidity may not represent a whole picture and that overall changes in cell viscosity through cytoskelet al elements are also involved. Inflammatory Effects of Abnormal Shear Stress in Cerebral Aneurysm Formation C h ronic pro inflammatory hemodyna m ic shear stress exerts its deleterious effects through several molecular pathways that lead to lesion formation. Both high and low wall shear stress have been implicated in cerebral aneurysm formation, which ha s led to the hypothesis that there may be two distinct types of aneurysms that form. An alternative and more easily supported hypothesis is that high shear stress causes aneurysm initiation and then low wall shear stress leads to aneurysm progression. Recent data published by several groups makes a s trong case for this scenario .

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48 Experimental animal aneurysm models 71 74 and c omputational fluid dynamic (CFD) studies have shown that hemodynamic conditions within cerebral vasculature and aneurysms is quite complex and includes areas of high and low shear stress, non uniform shear stress gradients, impingement zones and helical flow patterns. 75 81 H igh wall shear stress and shear stress gradients have been shown to lead to damage to the internal elastic lamina (IEL). At high shear stress gradients (>40 dynes/cm 2 ) that occur near impingement zones, endothelial cells becom e dis organized and migrate away from stagnation zones leading to areas of reduced cell density. 227 Although the average spatial wall shear stress gradient is nega tive in magnitude at bifurcations, local zones of both positive and negative wall shear stress occur. 76 , 78 , 160 Dolan et al used a flow chamber with multiple shear stress microenvironments to study effects of negative and positive shear stress gradients. Consequently, endothelial cells exposed to positive wall shea r stress gradient are more prone to undergo both proliferation and apoptosis and extracellular matrix remodeling . 76 In contrast, endothelial cells exposed to negat ive wall shear stress gradients are more prone to inflammation 76 and exhibit increased secretion of cytokines 75 , 145 , 160 and matrix metallo proteinases (MMPs) . 77 , 82 , 135 Negative shear stress gradients also opposed inhibitory effects of high shear stress on inflammatory gene expres sion related to cytokines and chemotaxis. 76 Low or oscillatory shear stress such as the one found in arterial bifurcations, stenosed vessels, or atherosclerotic ve ssels results in pro inflammatory phenotype 75 through upregulation of NF and IL 8 via MAPK 229 . Low wall shear stress increases levels of pro inflammatory mediators such as MCP 1 leading to vascular atherosclerosis. 145 , 163 In addition, under such pro inflammatory shear stress condit ions

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49 endothelial cells secrete decreased amounts of nitric oxide (NO), 230 prostacyclin (PGI2), 52 and miR 126, 231 factors known for keeping the neighboring vascular smooth muscle cel ls (VSMCs) in a quiescent state . Loss of endothelial function can result in rapid inflammation and proliferation of smooth muscle cells as seen in animal m odels of vascular stenosis . 232 In addition to secretion of pro inflammatory mediators, inflamed endothelium displays higher propensity for binding of platelets and circulating leukocytes through upregulation of intercellular adhesion proteins such as ICAM and VCAM 1 and sele ctins, allowing for infla mmatory cells infiltration . 233 Therefore, it is reasonable to assume that early on during aneurysm formation, the endothelium plays a critical role in an ever changing rela tionship between inflammation, remodeling, and local hemodynamic conditions. Arterial bifurcations where the majority of cerebral aneurysms develop are especially prone to inflammation due to presence of non uniform shear st ress 71 , 78 , 227 . In a recent in vitro study, it has been shown that endothelial cells at bifurcations acquire a distinct c hemokine secretory profile . 145 , 160 The authors exposed activated endothelia l cells to non uniform shear stress in bifurcating parallel plate flow chamber and found increases in sICAM 1, MCP 1, and IL 8 when compared to cells exposed to flow in straight channel slides. Even though the cell population exposed to non uniform shear s tress was small, the amount of secreted pro inflammatory chemokines was not only enough to be detected but w as also above the baseline . 160 A similar scenario cou ld occur in the endothelial cell population lining cerebral aneurysm domes. Shear stress has long been thought to play a key role in cerebral aneurysm formation, but, until recently, there has been disagreement as to the exact hemodynamic conditions that c ause progression of these lesions. Once an

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50 aneurysm forms, a change occurs in the local blood flow and shear stress pattern resulting in a low shear stress micro environment within the dome. 77 , 80 , 135 Further changes to this WSS pattern occur during aneurysm growth causing pathological remodeling. 77 , 135 Recently, aneurysm growth has been shown to occur in regions of low WSS of the aneurysm dome. 77 , 135 However, the endothelium is not the only component of the vascular wall that can sense changes in shear stress and blood p ressure. As mentioned earlier, a dventitial fibroblasts have been found to sense inflammatory and hypertensive stress and often become activated and proliferate in far greater numbers than cells in the tunica intima or media. 53 56 , 94 An adventitial signaling cascade involving IL 6 and MCP 1 has been described in aortic dissection. 234 The authors found that infiltrating monocytes and macrophages interacted with local IL 6 secreting fibroblasts. IL 6 produced within the adventitia led to increased recruitment and activation of macrophages resulting in higher levels of MCP 1 and MMPs. 234 Any kind of inflammatory signaling transmitted to the adventitia from the intimal layer could resul t in massive recruitment of adventitial fibroblasts and progenitor cells. 54 , 55 Whether this recruitment of adven titial support cells is deleterious 53 , 55 , 93 , 94 , 234 or protective 53 , 54 , 56 in cerebral aneurysm formation is not currently known. As of this time, no research has been done exploring the role of the adventitia and its cellular components in cerebral aneurysm formation and rupture. Inflammatory Cascade and Components of the Innate Immune System The components of the inflammatory cascade and the innate immune system are best described in the context of vascular injury and subsequent healing response. In a hypothetical sc enario, the endothelial layer becomes damaged by mechanical injury or infiltrating pathogens. This sets in a complex wound healing response that can be

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51 divided into easily identifiable, but overlapping components such as hemostasis or coagulation cascade, inflammati on with cell proliferation, and remodeling with resolution. Phase I: Coagulation Cascade The initial mechanical injury leads to the release of tissue factor (TF) from endothelial cells or exposure of van Willebrand factor (vWF) and collagen on th e basal lamina. 235 These two events allow the coagulation cascade to proceed which is the first step in the classical process of wound healing. In the extrinsic pathway the endothelial cells release TF, which combines with Factor VII to cleave Factor X to Factor Xa, allowing for thrombin formation. The contact activation or intrinsic pathway relies on initial coagulation complex formation on exposed collagen. Thi s initial complex is composed of prekallikrein, high molecular weight kininogen (HMWK) and Factor XII. There is a strict requirement for Ca 2+ , phospholipids, and a negatively charged surface for this pathway to proceed. Once an activated Factor XIIa is for med, additional proteolytic events occur with the final formation of Factor Va/Xa complex which has the capability to form thrombin. 235 C oagulation cascade is a series of enzymatic reactions that activate zymogens and lead to polymerization fibrin from soluble monomers by actions of thrombin and Factor IIIa. In conjunction with platelets, this polymer fibrin network results in thrombus formation which includes re d blood cells and any other cells of the circulatory system caught in the hemostatic net. 235 A thrombus does not only prevent further blood loss, but also patho gen spread and invasion. Although the intrinsic pathway has only a minor role in hemostasis, it appears to be important in inflammation. In particular, polyphosphate, a natural and extremely efficient activato r of the intrinsic pathway, has potent pro infl ammatory functions and the ability to stop functions of

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52 tissue factor pathway inhibitor. 235 , 236 In addi tion, platelets trapped within the fibrin network release lysin, a cationic protein with bactericidal properties 237 , 238 Furthermore, activated platelets release cytokines which lead to mobilization and infiltration by the cellular effectors of the innate immune system. Thrombus formation and platelet aggregation and lysis leads to release of pro inflammatory factors such as PDGF which further cause local inflamma tion. Among many different inflammatory mediators, locally trapped platelets release connective tissue activating peptide III (CTAP III), which is enzymatically converted to CXCL7, also known as thromboglobulin neutrophil activating peptide 2 (NAP 2). 239 , 240 CXCL7 acts as a chemoattractant for ne utrophils. Hemostatic events occurring within the vascular lumen have the ability to attract cells of the immune system, but the majority of inflammatory processes occur on the endothelium. Phase II: Inflammation Acute inflammation is triggered by activat ion of the NF and MAPK pathways through binding of bacterial or other pathogen components to the genetically conserved pattern recognition receptors (PRRs) such as TLRs, Nod like receptors (NLRs), C type lectin receptors (CLRs), and others. 241 Although the endothe lial cells are considered a barrier against a further spread of infection, the circulatory system contains another physical barrier composed of a protein cascade known as the complement system. The complement is a series of proteins that are enzymatically cleaved and deposited on pathogens following binding to conserved molecular patterns, lack of protective proteins such as complement receptor 1 (CR 1/CD35), membrane co factor protein (MCP/CD46), or decay accelerating factor (DAF/CD55) on the surface, or

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53 i mmunoglobulin M (IgM) or G (IgG) binding. 242 The classical complement pathway is activated by binding of the C1 complement protein to IgG or IgM antigen complexes. The alternative pathway occurs continuously at a low level due to spontaneous hydrolysis of C3 complement protein, which is inactivated by Factor H or I in the body. 242 The lectin pathway involves binding of the mannose binding lectin (MBL) on the pathogen surface. The importance of these pathways is that they all result in the formation of neutrophil chemotactic factors C5a and C3a , as well as formation of the membrane attack complex (MAC) on the pathogen surface. 242 The MAC allows for destruction of a pathogen by pore formation and osmolysis. The classical pathway in complement activation has been shown to be involved in cerebral aneurysm formation and rupture. 100 , 243 This is thought to occur through sterile inflammation and lack of complement inhibitors. 244 This illustrates that inflammatory cascades can occur despite lack of nearby or systemic pathogens and induce sterile inflammation in combination with prolonged stressors such as ROS or hypertension. Regardless of whether inflammation is initiated via sterile or non sterile triggers, endothelial cells express cell adhesion molecules on their surface within seconds to minutes of activation . 245 This early process allows for inflammatory cell adhesion which is defined by initial chemotaxis and subsequent rolling, adhesion, crawling and transmigration on the activated endothelial cells. In addition, the activated endothelial cells release chemokines, cytokines which cause migration of cells, leading to an influx of inflammatory cells. One of the first chemokines to be released is IL 8 in the humans and CXCL1 in the mouse species. IL 8 and CXCL1 are chemokines of the CXC family with glutamic acid leucine arginine (ELR) motif . ELR+ c hemokines within this family are

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54 known for their ability to attra ct neutrophils via the receptor CXCR2 and include GRO /CXCL1, GRO /CXCL2, GRO /CXCL3, CXCL5/ENA 78 , CXCL6/GCP 2, CXCL5 6 (mouse only) , CXCL7, IL 8/CXCL8, and CXCL15 . 239 CXCL1, CXCL7, and IL 8 appear to be the most highly expressed ELR+ chemokines during early tissue injury. 69 , 239 IL 8 and CXCL1 can be release d immediately from activated endothelial cell s via Weibel Palade bodies (WBPs) and type 2 chemokine containing organelle. 246 However, the storage efficiency of pro inflammatory cytokines in WBPs is quite low and vWF and P selectin are the primary constituents of these organelles. 246 , 247 Once secreted via stored organelles, IL 8 and CXCL1 expression is gradually increased for sustained release. 239 , 246 This leads to an increase in chemokine gradient and neutrophil recruitment. The chemokines are deposited on the endothelial ECM, which prevents them from being washed out by the blood, and allowing the inflammatory cells an uninterrupted chemotactic gradient. It has been suggested that the early neutrophil recruitment by CXCL1 gradually diminishes through overstimulation of CX CR2, and that IL 8 can then overcome this effect through stimulation of CXCR1. 69 , 239 , 247 249 Although such effect of differential IL 8 and CXCL1 expression and neutrophil recruitment has been shown in skin conditions such as psoriasis 250 and occluded blood vessel vasculitis 251 , evidence suggests that CXCR2 is the receptor through which most ELR+ CXC chemokines exert t heir chemotactic effects 252 , 253 . The initial mechanism of capture and rolling is mediated through E , L , and P selectins, which interact with sialyl LewisX groups on glycoproteins such as P selectin glycoprotein ligand 1 (PSGL 1). 254 P selectin is expressed immediately via Weibel Palade bodies (WBPs) while E selectin is synthesized following a trigger. 127 L sele ctin is expressed on neutrophils and

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55 monocytes, T and B cells, and specific subsets of more specialized inflammatory cells. 254 Neutrophils , or polymorphonuclear granulocytes (PMN s ) , are the first cells of the innate immune system to arrive at the location of injury. Their concentrations usually peak between days 4 7 following injury. Neutrophils are th e most numerous leukocytes or white blood cells in mammals. 127 At any point, about 50% of all neutrophils within the body are rolling along the vasculature patrolling for any signs of infection , while the other half is free floating or free circulating . Neutrophils attach to the activated endothelium and migrate deeper into tissues in a process characterized by well defined steps of rolling, adhesion, crawlin g and transmigration. 227 Rolling refers to the mechanism of tethering in which the bonds between the endothelium and the neutrophil are continuously broken a nd reformed as the neutrophil moves forward. At low values of shear stress, neutrophil rolling is rather straightforward and it involves the L selectin and P selectin glycoprotein ligand 1 (PSGL 1) rich microvilli . 255 However, at higher values of shear stress, neutrophils utilize a complex tether and sling mechanism. 255 258 Neutrophil slings have high content of PSGL 1 patches, but display homogeneous expression of LFA 1. 257 , 258 As the neutrophil rolls across the substrate, the sling is thrown forward in front of the rolling cell allowing for an autonomous cell adhesive substrate which can be utilized at high shear. 257 , 258 This sling tether formation actually allows neutrophils to roll at shear stress values ten times higher (10 20 dynes/cm 2 ) than other leukocytes. 258 Slow rolling is mediated through an interaction between l ymphoc yte function associated antigen 1 (LFA 1) on neutrophi ls and intercellular adhesion molecule 1 (ICAM 1) on the endothelium. Once the

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56 neutrophils slow down and the number of bonding interactions exceeds peeling, these cells interact with the substrate in a much stronger manner known as adhesion. 127 , 245 The cell surface proteins involved in adhesion on neutrophils are primarily LFA 1 and very late antigen 4 (VLA 4) . 127 , 245 This much stronger interaction is stimulated by local cytokines and selectin engagement via MAPK 259 and is mediated by ICAM 1 LFA 1 and VCAM 1 VLA4 interactions on the endothelium 127 . ELR+ chemokines IL 8 and CXCL1 are critical for neutrophil chemotaxis and firm adhesion. 127 , 248 , 260 Once firmly adhered, the neutrophils probe the local environment using pseudopods and undergo crawling to find the nearest endothelial cell cell junction. 127 , 250 This process occurs in the direction perpendicular to the direction of blood flow and is mediated by ICAM 1 on the endothelial cells and macrophage antigen 1 (MAC 1) on neutrophils. 127 The final step of diapedesis and transmigration is mediated via multiple cell surface molecules such as PECAM 1, ICAM 1, ICAM 2, and VCAM 1 on the endothelium and LFA 1, VLA 4, PECAM 1, and MAC 1 on the neutrophils. 127 , 245 The most common route that n eutrophils use to infiltrate the local micro environment is by extravasating at cell cell junctions between endothelial cells. 127 , 245 However, neutrophils are also capable of using the direct transcellular route through the endothelial cell, but this process takes longer than paracellular transmigration. 261 Once within the vascular wall, they patrol for pathogens causing collateral damage . Neutrophils primarily exert their effects through secretion of cytokines and chemokines, granule components, and formation of ROS, which all contribute to the inflammatory state of local environment and tissue remodeling. 262

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57 Activated neutrophils secrete MMPs, cathepsins, ROS, and neutrophil elastase which have the potential to cause grave injury to both the local host tissue as well as pathogens. 127 Neutrophil elastase is an especially potent protease with capability of breaking down the elastin layer and weakening the vessel wall structure. Recent evidence suggests that distinct sub populations of neutrophils may exist within the body, and especially during infections, in addition to the normal non activated (PMN N) phenotype. 127 , 263 Pro inflammatory neutrophils (PMN I) are thought to express IL 12 and macrophage inflammatory protein 1 ( MIP 1 /CCL3) , while anti inflammatory neutrophils (PMN II) are IL 10 and MCP 1 double positive. 263 MIP 1 is chemotactic for neutrophils, macrophages, and can activate nearby inflammatory cells and fibroblasts. Next, PMN I neutrophils were found to polarize macrophages into M1 pro inflammatory phenotype while PMN 263 The same authors reported that PMN like receptors (TLRs) 5 and 8, while PMN and 9, in addition to TLR2 and TLR4. 263 Activated neutrophils possess the capability to release neutrophil extracellular traps (NETs) to prevent spread of microbial pathogens. 264 These NETs are composed of chromatin DNA and histones as well as granular proteins such as lactoferrin, cathepsin G, defensins, LL37, bacterial permeability increasing protein, neutrophil elastase, proteinase 3 (PR3), gelatinase and myeloperoxidase (MPO) . 264 NETs have been found to be absolutely necessary to prevent sepsis and dissemination of pathogens to distant organs, and microbial DNases diminish their eff icacy. 265 , 266 During the process of NET formation, the neutrophil undergoes cell death due to loss of chromatin DNA, 267 but this is more than offset by its ability to stop pathogen spread 3 4 fold more effectively than other cells such as Kupffer

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58 cells 127 . This process of NET formation and neutrophil death is mediated through a non classical cell death program and requires ROS generation by NADPH oxidase. 268 Mast cells and basophils, the other two members of the granulocyte family, have also been reported to form NETs. 269 Several studies have shown that NET formation occurs in multiple , sterile or auto immune diseases such as atherosclerosis 270 , systemic lupus erythematosus (SLE) 271 , vasculitis 272 , thrombosis 273 , and transfusion related acute lung injury (TRALI) 274 . Whether NET format ion is involved in aneurysm formation is not currently known. Shortly following neutrophil infiltration cued by IL 8 and CXL1, monocytes arrive to the site of inflammation. 69 , 239 Monocytes, guided by MIP 1 MIP MCP 1 signaling, bind to cell adhesion molecules and penetrate into the inflamed tissues in an adhesion process similar to that o f neutrophils . 245 Several other cytokines that are strongly che motactic for monocytes/macrophages include regulated on activation, normal T expressed and secreted (RANTES/ CCL5), I309 (CCL1), and monocyte chemo attractant protein 3 (MCP 3). 239 However, MCP 1 is the primary monocyte/macrophage chemoattracting cytokine during the first week of wound healing and during progression of many inflammatory diseases including atherosclerosis 163 , cerebral aneurysm formation 29 , 72 , 121 , and skin injury 239 . Depending on the cytokine milieu, infiltrating monocytes are capable of differentiating into dendritic cells (via granulocyte macrophage colony stimulating factor [GM CSF] and IL 4) or tissue macrophages (via macrophage colony stimulating factor [M CSF]) . 242 Dendritic cells are specialized antigen presenting cells (APCs) with the capability to present the broadest range of antigens and ar e important in inducing T and B cell responses. 275 , 276 Specific

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59 subsets of monocytes have been described and it has been suggested that they have different capabilities when it comes to developing into macrophages with unique functions. 277 In the mouse species, Gr1 + /Ly6C high CCR2 + CX3CR1 low m onocyte are thought to be the source of M1 macrophages and are referred to as inflammatory monocytes. 278 , 2 79 In the human, the equivalent monocytes are thought to be CD14 high CD16 l ow . Their expression of receptor for MCP 1, CCR2, helps explain why they predomina te in acutely inflamed tissues. Their primary role is to phagocytose and digest tissues. On the ot her hand, Gr1 /Ly6C low high mouse monocytes are thought to predominate under homeostatic conditions , induce granulation tissue formation and angiogenesis promoting wound healing , and give rise to M2 macrophages. 278 , 279 In the human CD14 low CD16 high monocytes are thought to be the murine equivalent. It is thought that in the absence of inflammatio n , pro inflammatory monocytes can switch to the anti inflammatory phenotype, but this process is still controversial and under intense scrutiny. The lack of CCR2 receptor in anti inflammatory monocytes helps explain why they typically do not accumulate ear ly on during inflammation. 280 Once within tissues, monocytes can differentiate into tissue macrophages with diverse functions and roles. Macrophages within the body have distinct subsets and perform multiple homeostatic functions depending on the their anatomical location, and include alveolar cells (lungs), osteoclasts (bone), Kuppfer cells (liver), histiocytes (connective tissue), and tumor a ssociated macrophag es (TAMs) (in cancerous lesions). 281 Some macrophages are also located with in secondary lymp node organs and tissues such as gut associated lymphoid tissues (GALT) like associated

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60 lymphoid tissues (MALT) such as the tonsils. 242 In several immune protect ed areas such as the central nervous system, macrophages have a distinct subtype referred to as the microglia that is replenished on a regular basis through both local proliferation of microglia and monocyte recruitment from the circulation . 282 , 283 In pathogen associated inflammation, t he role of macrophages is to clean up cellular debris, locate and phagoc ytose pathogens, and induce sufficient inflammatory response to stop the spread of infect i on. Macrophages also direct granulation tissue formation, tissue remodeling and resolution phase of wound healing and their role is malleable depending on phenotypic polarizatio n. However, if the inflammatory process is not resolved, as in the case of sterile inflammation, macrophages can cause deleterious functional and structural damage to the tissues that can progress to chronic inflammation. 241 , 281 Progression to resolution or chronic inflammation is dependent on the predominating macrophage phenotype within the lesion. Several different macrophage phenotypes have been described in the human and mouse species with pro and anti inflammatory roles in vascular disease . In the human circulation, M1 macrophages are pro inflammatory, Arg II+ + i NOS+, and are formed following ac tivation via M CSF or lipo polysaccharide (LPS) and interferon (IFN ) through STAT1 and STAT3 signaling . COX 2, IL 12 and IL 23 are usually highly expressed by this macrophage subpopulation. This macrophage phenotype is usually associated with T H 1 cell specific response in a forward feedback loop in which secreted IL 12 will lead to T H 1 expression of IFN and activation of neighboring macrophages. M2 macrophages are anti inflammatory, Arg I+ + i NOS , and are formed following activation via IL 4 and IL 13 , immune antibody complexes or IL 10 through STAT6 -

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61 dependent phosphorylation . They secrete CXCL13, CCL12, and CCL24, chemokines known for T H 2 cell specific response. Furthermore, M1 macrophages can induce fibrosis of the local tissues during inflammation a nd resolution, while M2 macrophages are capable of suppressing this process. Pro inflammatory macrophages can induce apoptosis of smooth muscle cells (SMCs) through IL 1 and TNF signaling, fibrosis through transforming growth factor (TGF , and matrix degradation through MMPs. 12 Secretion of ROS by macrophages can lead to oxidation of matrix components and accumulated l ipids leading to increased apoptosis and necrosis of local tissues. It has been suggested that early atherosclerotic lesions, which are predominated by M2 macrophages, undergo a conversion to M1 phenotype rather than a direct recruitment of pro inflammator y monocytes and macrophages. 284 Kadl et al have reported that M1 and M2 macrophages exposed to oxidized lipids acquire the so called Mox phenotype, d istinct from M1 or M2, which has decreased chemotactic and phagocytic capabilities. 285 In addition, this novel phenotype displays increased expression of Nrf2 mediated genes, but its role in atherosclerosis and disease is not known. 285 A nother phenotype, a recently reported M4 macrophage phenotype , in atherosclerosis has been characterized by lack of hemoglobin haptoglobin (Hb Hp) scavenger receptor CD163 and expression of CD36/SR A, IL 6, TN F , TRAIL, CCL18 and CCL22. 286 This phenotype has been shown to be induced by CXCL4 also known as platelet factor 4 (PF 4) and knockdown of CXCL4 reduce s atherosclerosis suggesting that M4 macrophages may be important in progression of this disease . 286 As summarized above, the roles of monocytes and ma crophages in wound healing can be complex and are highly dependent on the surrounding cytokine micro environment. However, one of

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62 the most important roles in wound healing is deposition of ECM material and orchestration of granulation tissue formation. Gra nulation tissue is a new tissue deposited during the wound healing process that is composed of connective tissue and includes fibroblasts, macrophages, and blood vessel formation through a ngiogenesis. 242 The purpose of granulation tissue is to allow sufficient amount of nutrients, oxygen, substrate, and progenitor cells to repair the local damage. Formation of this tissue is primarily directed by macrophages 278 , 279 and is highly dependent on several cytokines and growth factors such as vascular endothelial growth factor (VEGF) 287 , TGF , NO 291 , and stromal derived growth factor 1 (SDF 1 ) 292 294 . In VEGF mediated angiogenesis, macrophages act as chaperones and link distant tip endothelial cells. 295 Macrophage TGF accelerates wound healing 290 and failure of induction of extracellular matrix compone nt genes by macrophages has been reported to result in lack of limb regeneration in salamanders 296 . SDF 1 mediated angiogenesis and progenitor cell homing associated with granulation tissue has been shown to be beneficial in myocardial infarcation 297 299 , atheroscle rosis 300 302 , and stroke 303 305 . Formation of neovasculature directed by SDF 1 within the walls of cerebral aneurysms has been reported but it is not known whether its role is protective or pro inflammatory. 30 Before the resolution phase of wound healing occur s, various other granulocyte cells of the innate immune system are recruited into the inflamed tissues such as mast cells, eosinophils , and basophils . Mast cells are granulocytes that are important in allergies and anaphylaxis. Upon stimulation by direct i njury, complement, or antigen binding to a cell surface IgE, mast cells release histamine, serotonin, heparin, and

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63 serine proteases. They also begin to upregulate expression of leukotriene C4 and prostaglandin D2 as well as eosinophil chemotactic factor (E CF). Histamine is the most important effector as it causes endothelial cell activation and increased vascular permeability leading to local edema. Mast cells have been detected in ruptured cerebral aneurysms but their role is currently unknown. 84 Eosinophils are granulocytes associated with allergic reactions, asthma, and are involved in combating multicellular parasites and fungi. Upon activation they release catio nic granule proteins, ROS, leukotrienes and prostaglandins, elastase, TGF , VEGF, PDGF, as well as several pro inflammatory cytokines. Basophils are the least common of white blood cells and are thought to regulate T cell functions through secretion of he parin, histamine, leukotrienes, elastase, and IL 4. The cytokine IL 4 is thought to be important in allergic reactions and can induce increased production of IgE by plasma cells. In addition, lymphocytes have also been implicated in the wound healing respo nse despite lack of antigen specific T cell response. In a classical response, lymphocytes such as the effector and helper T cells and antibody producing B cells are involved in adaptive immune response. However, i n multiple models, especially skin wound m odels, lymphocytes are guided via MCP 1, IP 10 and monokine induced by gamma interferon ( MIG ) and predominate in later stages of wound healing. 69 Many residen t T and B cells are found in the adventitia and thus can participate in vascular inflammatory response by affecting the local cytokine milieu. 53 56 , 94 In general, T cells can be divided into CD4+ helper T cells that potentiate functions of other cells and effector CD8+ T cells. CD4 T H 1 cells activate macrophages and cytotoxic CD8+ T cells via INF . 306 T H 2 cells, which secrete IL 4, IL 5, and IL 13 are involved in activating

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64 allergy associated cells such as mast ce lls and eosinophils. 306 However, several studies have discovered multiple cells of the innate and adaptive immune systems that provide a bridge and allow cross talk between the two responses. Natural killer (NK) cells 307 and macrophages 308 have been reported to bind to Fc regions of antibody coated tumor cells and induce effector cell mediated antibody dependent cell mediated cytotoxicity (ADCC) via ROS and phagocytosis 309 . It appears that ADCC mediated via NK cells has a role in atherosclerosis as NK cell depletion decreases lesion size by 70%. 310 Recently, a subset of four new types of innate immune cells known as natural helper cells (NHCs), multi potent progenitor type 2 (MPP(type2)) cells 311 , nuocytes 312 , and innate type 2 helper (Ih2) has been discovered. 313 These cells have been described to have a pro T H 2 response and found to activate macrophage s, basophils, and mast cells via IL 5 and IL 13 to secrete IL 4. 313 However, as these cells have been found to be associated with mucosal immunity and secondary lymphoid organs, their role in vascular inflammation is not known and probably unlikely. A subset of T cells known as T helper 17 (T H 17) cells have been shown to a mplify innate inflammatory cascades. T H 17 cells are involved in autoimmune diseases such as multiple sclerosis and psoriasis . They exert their primary effects through IL 17, upon induction by IL 23, which acts with TNF and IL 1 to cause synergistic pro inflammatory effects and tissue damage. IL 17 can act on endothelial cells, smooth muscle cells, fibroblasts and macrophages, and cause increase s expression of pro inflammatory prostagl andins, MCP 1, IL 8, and CXCL1 via p38 MA PK and ERK1/2 dependent NF activation. 314 M2 macrophages express lower levels of IL 17 than the M1 phenotype, and thus promote wound healing. 315 Blockade of IL 17 results in lesser scarring in myocardial infarction 316 , dec reased

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65 atherosclerosis 317 , and has protective effects in stroke 31 8 . The so called gamma delta T cells ( T cells) have also been found to secrete IL 17, suppress regulatory T cells and have been implicated in several auto immune diseases. 319 It is interesting to note that this more primitive T cell subtype has the ability to phagocytose pathogens, a function previously thou ght to be unique to the myeloid cells. 319 CD4+CD25+ regul atory T cells (T Regs ) are a specialized T cell subtype that has the capability of suppressing immune response through cytokines TGF and IL 10, as well as direct contact inhibition of T cell functions and other mechanisms. 319 , 320 Another type of T cells, CD8+ T suppressor cells have been found to downregulate the healing signaling cascades in skin wounds bu t their role in vascular inflammation has not been examined extensively . 321 Phase III: Resolution Clearance or deactivation of M1 pro inflammatory macrophages is believed to be a critical step in decreasing inflammation. K ey cytokine s in inducing the resolution phase are TGF and IL 10 which tilt the balance towards anti inflammatory M2 macrophage activation and eventual deactivation. In fact, IL 10, TGF , CD200 CD200R interactions, or steroids can lead to eventual deactivation of macrophage to a more quiescent phenotype. 319 321 Role of Peroxisomal Proliferator Activated Receptor (PPAR) Pathway in Inflammation PPARs are a family of nuclear receptors ( , / , and ) that regulate transcription of genes and play a role in cell differentiation, metabolism, indirect response to shear stress and inflammation. 52 , 322 326 The PPARs are extremely versatile and they were initially describe d in studies of metabolism and metabolic disease.

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66 PPAR Overview Issemann et al reported the presence of a transcription factor that mediated hypolipidaemic effects associated with peroxisome expan sion following administration of hepatocarcinogenic compounds. 327 Peroxisomes are cellular organelles that are intricately involved in and oxidation of lo ng fatty acid chains in mammals and serve several additional functions in other species . 328 Under normal, physiologic conditions, oxidation in peroxisomes occurs as a minor pathway, and the majority is performed in the mitochondria. 329 When not active, PPARs are bound with co rep ressors. 330 332 PPAR is activated by fatty acids, eicosanoids, and leukotriene B4 333 ; / by unsaturated and saturated fatty acids, and prostacyclin 334 ; and by fatty acids, oxidized and nitrated fatty acids, multiple eicosan oids and 15 d eoxy 12,14 prostaglandin J2 (PGJ2) 326 , 335 . When activated, PPAR receptors heterodimerize wi th retinoid X receptors (RXRs), translocate to the nucleus , and bind to peroxisome proliferator response element s (PPRE s ) using a zinc finger like DNA binding domain (DBD) . 336 , 337 Co activators such as PPAR binding protein (PBP), steroid receptor co activator 1 (SRC 1) , 338 and PPAR co activator 1 (PGC 1) proteins , 339 , 340 help facilitate transcription. PGC 1 has been shown to be upregulated in exercise and important in prevention of insulin resistance. 341 Phosphorylation of specific residues can either increase (via p38) or decrease (via ERK) PPAR transcriptional activity. 332 S everal other ligands are necessary for PPAR activation such as 9 cis retinoic aci d for RXR activation. 336 Upon activation by fatty acid or eicosanoid derivatives, the transcriptional activit y of PPAR, for example PPAR c an be modified further by addition of a s mall ubiquitin like m odifier

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67 (SUMO) protein SUMO1 through a process known as sumoylation. 342 SUMO proteins function in cytosolic nuclear transport, protein stabilization, and modification of transcriptional activity. 343 , 344 The three subfamilies of PPARs display a slightly different tissue distribution 345 , 346 and functions as shown by differential effects in gene double knockout studies. PPAR is located in tissues and cells with high mitochondrial oxidation rates of fatty acids such as the endothelial cells and smooth muscle cells in the vasculature, liver, kidney, and he art muscle . 345 , 346 PPAR / is primarily expressed by smooth muscle cells of the vasculature, skin, t he brain, and adipose tissue. 345 , 346 PPAR is expressed in almost all tissues at various levels with subtle variations among the three different isoforms. 345 , 346 PPAR 1 is expressed in cardiac and s keletal muscle, colo n, kidney, pancreas, and spleen; 2 is found in the adipose tissue; and 3 in the macrophages, intestinal tract, and adipose tissue. The alternative isoforms are formed through differential splicing and promoter usage. 347 PPAR double knockout mice are viable and fertile but display abnormalities in metabolism and lipid storage. 348 , 349 PPAR / double knockouts are viable but only 10% survive gestation, and resulting pups are smaller than wild type mice and display epidermal hyperplasia. 350 PPAR double knockouts are embryonically lethal. 351 Targeted deletion of PPAR in vascular smooth muscle cells leads to loss of SMC phenotype and hypotensive phenotype. 352 On the other hand, targeted deletion in pulmonary SMCs leads to pu lmonary hypertension. 353 The PPARs are localized to cell cytoplasm, and upon activation, translocate to the nucleus where they can exert the ir effects on gene expression. 322

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68 Recently, the three peroxisomal proliferator activating receptor (PPAR) pathways ( , / , and ) have been implicated in having a role in cerebrovascular developmental abnormalities 354 , and shown to have profound effects on the inflammatory sta te of macrophages 326 , 354 , 355 , which are the primary im mune cells found in aneurysms. 12 Before discussing the role of PPARs in inflammation, it is important to understand their function in metabolism to fully appreciate the cross talk between these pathways. Role of PPAR s in Metabolism and Cell Differentiation The discovery of PPAR s was significant as it provided a direct link between dietary and nutritional intake and changes in related gene expression. 356 The PPARs are particularly important in liver and adipose tissue mediated changes in blood levels of lipids. Hypolipedemic, and especially triglyceride lower ing, effects of PPAR agonists are mediated though increased formation of acyl CoA derivatives from fatty acids, increased levels and action of acyl CoA synthetase, increased peroxisomal and mitochondrial oxidation of long chain fatty acids, increased lev els of lipoprotein lipase (LPL) and decreased apoC III expression and decreased production of lipids, and lipid carrying lipoproteins such as very low density lipoprotein (VLDL) . 347 , 356 PPAR has a protective role in lipid homeostasis as increases in liver fat conten t lead to PPAR driven maintenance of blood glucose, lipid, and cholesterol levels. 357 PPAR expression protects against development of non alcoholic fatty liver development in high fat diet in mice 358 and alcoholic liver damage 359 . In the presence of glucocorticoids, activation of PPAR in the liver leads to increased insulin resistance. 360 During fasting, PPAR is important in regulating beta islet insulin release through regulation of oxidation. 361 Furthermore, n 3 fatty acids help maintain insulin

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69 sensitivity through PPAR . 362 Although involved in lipid homeostasis, PPAR role in obesity is controversial and evidence exists suggesting both lack of its involvement 363 and a specific role in conjunction with LDL receptor 364 . PPAR has been described as a general regulator of fat burning and PPAR deficient mice are prone to obesity. 365 Targeted deletion of PPAR in cardiomyocytes leads to increased lipid deposition in the heart, cardiac hypertrophy, congestive heart failure, and increased death from cardiovascular complications. 366 PPAR knockouts are prone to insulin resistance in the adipose tissue and liver. 351 PPAR has also been shown to be important in bone formation through Wnt signaling and its activation restores normal bone density in osteoporosis model. 367 PPARs are also important in cell differentiation. PPAR has been shown to regulate c ell cycle arrest at the G0 to G1 phase transition and prevent TGF induced SMC phenotype induction in mesenchymal stem cells. 368 Cir culating microparticles containing PPAR are important in differentiation of bone marrow derived endothelial progenitor cells and angiogenesis. 369 PPAR mous e knockouts also display delayed liver regeneration. 370 PPAR has been shown to arrest VSMCs at the G1 to S phase transition through induction of p21 and p5 2. 371 PPAR regulates skeletal muscle progenitor cell proliferation and thus skeletal muscle regeneration in a Forkhead box class O transcription factor 1 (FoxO1 ) dependent manner. 372 Interestingly, PPAR has been shown to be important in hematopoietic stem cell maintenance. 373 Together, PPAR and PGE2 reg ulate conversion of pre adipocytes to fat storing white adipose tissue or thermoregulatory brown adipose tissue. 374 In PPAR CD4 Cre targeted

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70 deletions, lack of PPAR function induced CD4 T cells to differentiate into Th17 T cells 375 , known for their pro inflammatory functions. Role of PPARs in Inflammation In mult iple studies, a link between metabolism and inflammation has been establi shed in diseases such as dyslipi demias, diabetes, and atherosclerosis. 376 Diabetic dyslipidemia is characterized by high triglyceride and LDL , and reduced HDL conditions, which lea d to decreased eNOS activity. 376 As discussed earlier, NO has general anti inflammatory effects on the cardiovascular system through its effects on NF . 161 , 203 , 205 , 208 , 209 , 291 High levels of circulating lipids in a setting of inflammation can lead to formation of oxidized lipid products with highly pr o inflammatory properties. 377 , 378 Dyslipidemia and hyperglyceamia in type 2 diabetes mellitus (T2DM ) c ause both macro and microvascular complications through actions of vasoactive peptides such as endothelin 1 (ET 1), ROS, and oxidative stress . 332 , 379 Glineur et al reported that PPAR agonists protect aga inst microvascular ET 1 release through PPAR mediated Kruppel like factor 11 (KLF 11) increase and repression of TGF expression. 380 PPAR activation decreases expression of TNF and IL 1 in acute inflammation preventing local edema, exudate , mononuclear cell infiltration, and remodeling . 381 PPAR activation and PPAR agonists are especially efficacious in modulating inflammatory response through their trans repression of NF , which results in general pan anti in flammatory effects. 382 PPAR suppresses NF by upregulating expression of I , a co factor that prevent s NF from being translocated to the nucleus. 383 PPAR also stabilizes co repressor complexes at promoters of inflammatory genes. 332 , 379 Ang II driven cardiac

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71 fibrosis and remodeling is exacerbated in adiponectin knockout mice and this process is mediated through lack of PPAR activation. 384 Study of PPAR function is often complicated, as many of the agonists for PPARs also bind to cell surface G protein coupled receptors (GPCRs), often with opposing effects. 379 One such PPAR agonist is leukotriene B4 which binds to both PPAR and leukotriene B4 receptor 1 and 2 (LTB4R1 and LTB4R2). 333 Blockade of LTB4 actions at LTB4R1 has been found to decrease atherosclerotic lesion formation. 385 , 386 In obesity, white adipose tissue is frequently infiltrated by macrophages that secrete high levels of TNF and IL 6, which can decrease insulin sensitivity and lead to insulin resistance. 387 This heightened inflammatory profile is thought to be regulated by Kruppel like factor 15 (KLF15), which mediates endoplasmic reticulum (ER) stress induced insulin resistance. 388 Furthermore, this effect was found to be co mediated by ma mmalian target of rapamycin complex 1 (mTORC1) and dependent on overloading the capacity of the ER for protein folding. 388 The adipose tissue and the liver inflamm ation appear to be under control of macrophage PPAR axis. In particular, macrophages can be activated via TLR2 and TLR4 by saturated fatty acids. Stimulation of those TLRs results in NF activation and transcription of pro inflammatory genes. 332 , 379 On the other hand, binding of saturated fatty acids to PPAR prevents an inflammatory response. 389 Alternatively activated macrophages (M2) in the adipose tissue and Kupffer cells in the liver, when stimulated by fatty acids and other PPAR agonists, decrease inflammatory cytokine production through suppression of NF . 379 PPAR / is involved in multiple roles and contexts in inflammation. Its activation in macrophages during apoptotic cell clearance is necessary to induce a nti -

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72 inflammatory cytokine production and prevent expression of autoantibodies. 390 PPAR / pathway has been shown to regulate the inflammatory state of smooth muscle cells via the ligands secreted by endothelial cells due to shear stress. 52 Tsai et al have shown that prostacyclin secreted from the endothelial cells allows smooth muscle cells to retain their contractile phenotype as defined by caplonin, caldesmin, and MMP levels, and quiescence through activation of PPAR / . PPAR / activation is capable of driving macrophages into the M2 anti inflammatory phenotype directly 391 or through locally released cytokines 392 , similar to PPAR . In addition, PPAR has also been shown to have protective effects in ischemic brain injury by decreasing VSMC secreted MMP9. 367 These effects on VSMC phenotype have been shown to be mediated through decreased secretion of IL 1 and induction of p21 and p52. 371 PPAR activation has been shown to drive macrophages into the M 2 anti inflammatory phenotype. 326 However, any a ctivation of TLR4 causes NF mediated decrease in PPAR expression abrogating any potential anti inflammatory effects. 393 PPAR activation has also been foun d to synergize with liver X receptor (LXR ) stimulation to cause decreased NF activation and increased binding of PPAR to DNA. 394 In endothelial cells, targeted disruption of PPAR has been shown to result in endothelial dysfunction and hypertensive mouse phenotype driv en by decreased secretion of NO, increased oxidative stress markers, and increased activation of NF . 395 Targeted deletions in vascular smooth muscle cells exacerbate Ang II mediated vascular remodeling through oxidative stress and inflammation as measured by ICAM 1 and PECAM 1 expression. 396 PPAR dysfunction has also been found to mediate autoimmunity through increased formation of Th17 T cells. 375 PPAR activation in

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73 myeloid cells is deleterious in lun g cancer and metastasis due to induction of anti inflammatory M2 macrophage phenotype. 397 Conversely, deletion of PPAR in epidermal skin cells leads to heightened sensitivity to UV B and progression to cancerous lesions due to increased apoptosis and inflammation. 398 In addition, several studies have shown that PPAR dysfunct ion plays a role in progression of atherosclerosis and development of abdominal aortic aneurysms. 326 , 354 , 355 Activation of this PPAR isoform in cerebral ischemia is protective. 399 Together, PPAR and have been found to remove cholesterol from macrophage foam cells through increased expression of ABCA1, a transporter that mediates apo AI cholesterol efflux. 400 Summary Cerebral aneurysms represent a complex cardiovascular disease with multiple biological and mechanical components contributing to the overall pathophysiology. Intracranial aneurysms pose significant immediate and long term risks, and, when ruptured, result in subarachnoid hemor rhage and cerebral vasospasm . 1 , 10 Ruptured aneurysms are the caus e of 5 to 15% of all str okes 62 , 194 leading to high mortality and comorbidity 1 3 , 10 , 12 , 13 , 70 . Of the patients who survive, almost a third will develop moderate to se vere lifetime disabilities . 1 , 10 The current treatments for intracranial aneurysms are limited to surgical procedures and include clipping and coiling. Coiling is increasing in use nowadays due to its minimally invasive approach and b etter perioperative outcomes 1 with the exception of the very small (<3mm) aneurysms . 31 Both approaches carry significant operative and postoperative risks such as rupture, thromboembolism, and thrombosis of the parent vessel, as well as risks associated with anesthesia and contra st induced nephrotoxicity . 1 , 5 , 13 , 31 , 34 , 35 , 47 Moreover, a recent

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74 study has demonstrated that about 40% of patients who undergo coiling show long term changes in aneurysm obliteration pattern and 8% have to undergo re coiling or re clipping due to extensive recanaliz ation. 36 Undergoing re coiling or re clipping carries with it the same risks as the original procedure. Better management of intracranial aneurysms is needed, but the development of more ef fective therapies is limited by our incomplete knowledge of aneurysm formation and progression. It is not known what causes chronic and persistent inflammation within the aneurysm or why the formation of these lesions does not induce the native repair proc esses to prevent further tissue destruction. 1 , 2 , 12 Clearly, th e molecular details of chronic inflammation and apparent lack of tissue healing in these intracranial vessel lesions need to be studied in order to provide specific targets for pharmaceutical therapy of unruptured aneurysms. Until recently, there has not been much focus on shear stress induced endothelial inflammation in aneurysm progression from a molecular pathway perspective due to the limitations of studying this mechanism in animal models. To overcome the above challenges, we designed a novel in vitr o parallel plate flow chamber bifurcation aneurysm model characterized by reproducible hemodynamic conditions and the presence of multiple, co existent yet distinct micro environments to study the effect of shear stress on endothelial phenotype, and compar ed it to similar models of a straight artery and an arterial bifurcation. There is evidence that low shear stress and shear stress gradients at bifurcations and bifurcation aneurysms induce endothelial expression of specific inflammatory factors, which the n promote aneurysm formation and growth . Furthermore, there is evidence that the three PPAR pathways are involved in metabolism, inflammation, and sensing of shear stress. The question that we

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75 posed was whether PPAR agonists could be used to prevent cerebr al aneurysm formation by blocking a specific inflammatory factor released during early inflammation. We aimed to uncover the specific inflammatory factors expressed during early cerebral aneurysm formation and determine the involvement of PPAR pathway. To answer the above questions we developed three specific aims with a combination of in silico, in vitro , and in vivo approaches. Specific Aim 1: Model Shear Stress Inflammatory Micro environment of Cerebral Aneurysm In Vitro . Hypothesis: It is possible to re create the shear stress mediated inflammatory aneurysm micro environment in vitro using parallel plate flow chamber model. Furthermore, we will demonstrate that our in vitro model recreates key inflammatory features and markers found in experimental mouse aneurysms and aneurysm specimens from human patients. Specific Aim 2: Determine the Role of ELR+ CXC Chemokines in Cerebral Aneurysm Formation . Hypothesis: Since the first cytokines to be expressed in inflammation are ELR+ chemokines IL 8 and CXCL1, we exp ect these cytokines to be increased in our in vitro model, experimental mouse aneurysms, and human aneurysm specimens. Furthermore, we hypothesize that blockade of the major ELR+ chemokine in the mouse model will prevent aneurysm formation. Specific Aim 3 : Determine the Role of PPAR Pathway in Prevent ion of Cerebral Aneurysm Formation. Hypothesis: PPARs are a family of nuclear receptors that regulate transcription of genes and play a role in cell differentiation, metabolism, indirect response to shear stre ss and inflammation. Since several studies have shown that PPAR dysfunction plays a role in progression of atherosclerosis and development

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76 of abdominal aortic aneurysms , we hypothesize that PPAR agonists will dampen the inflammatory cytokine profile expres sed by the aneurysm micro environment and prevent cerebral aneurysm formation .

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77 CHAPTER 2 MODELING SHEAR STRESS INFLAMMATORY MICRO ENVIRONMENT OF CEREBRAL ANEURYSMS IN VITRO Introduction Cerebral aneurysms are focal dilations of intracranial vessels t hat can rupture and result in subarachnoid hemorrhage 1 , 2 and are the cau se of 5 to 15% of all strokes 3 , 13 , 70 leading to sign ificant mortality and morbidity 10 . Of the patients wh o survive a ruptured aneurysm, almost a third will develop moderate to severe lifetime disabilities . 2 , 3 , 10 Knowledge of the causes and mechanisms of aneurysm format ion and progression is limited. 12 Experim ental animal aneurysm models 48 , 72 74 , 102 , 129 , 401 and compu tational flow dynamic (CFD) studies 79 81 , 134 , 135 have implicated high wall shear stress in initiating aneurysm development through its effects on the endothelium and remodeling of the local vessel wall. It is believed that once endothelial dysfunction occurs, aneurysm growth is medi ated through influx of inflammatory cells leading to chronic vascular inflamm ation and further remodeling. 12 , 72 The link between hemodynamic forces, such as shear stress, and biological mechanisms, such as inflammation, that contribute to aneurysm development is still not fully understood. 12 , 72 , 79 , 80 The parallel plate flow chamber (PPFC) has been used successfully for more tha n two decades to study the behavior of cells, especially the endothelium, under defined conditions of flow and shear stress. 76 , 153 , 402 , 403 We designed a novel in vitro parallel plate flow chamber bifurcation aneurysm model to replicate the ge ometry and flow conditions 79 , 135 of an idealized artery bifurcation with a saccular aneurysm . Some of the considerations for flow chamber design included small size of the device 153 , low reagent requirements 403 , and physiologicall y relevant dimensions. The final model is

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78 characterized by reproducible hemodynamic conditions and the presence of multiple, co existent yet distinct micr o environments. We used the model to st udy the effect s of shear stress on endothelial phenotype, and compared it to similar models of a straight arter y and an arterial bifurcation. Our hypothesis is that low shear stress at bifurcation aneurysms induces endothelial expression of inflammatory fa ctors. We chose to study the effects of shear stres s on cyclooxygenase 1 (COX 1), 87 cyclooxygenase 2 (COX 2), 88 , 89 prostaglandin E2 (PGE2), 88 92 prostacyclin (PGI2), 92 and monocyte chemotactic protein 1 (MCP 1) 29 , 72 , 102 , 121 , 163 because of their usefulness as vascular inflammatory markers. We co nfirmed our in vitro findings by demonstrating expression of these same inflammatory markers in murine and human cerebral aneurysms. Methods Flow Chamber Design Arterial bifurcations are prone to inflammation due to lower average shear stress, shear stress gradients and regions of high wall shear stress near the flow divider. 76 , 78 , 160 CFD studies of human and rabbit aneurysms have demonstrated that average WSS within the aneurysm dome is much lower than in the parent vessel. 77 , 79 81 , 134 , 135 Ruptured aneurysms have been shown to have a much lower average WSS than unruptu red aneurysms. 80 Hemodynamic shear stress in intracranial vessels has been reported to average in value between 10 to above 100 dynes/cm 2 . 79 , 404 We designed our parallel plate flow chamber models using Google SketchUp 8 (Google, Mountain View, CA) (Figures 2 1, 2 2, 2 3, and 2 4) . We replicated WSS patterns of low shear stress to high shear stress ratio in the aneurysm region to parent vessel, respectively, by designing the bifurcation and bifurcation aneurysm parallel plate flow chambers to contain a region of uniform she ar stress of 10 dyne/cm 2 in the proximal straight

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79 segment, 5 dyne/cm 2 in the arms of the bifurcation, and an average WSS of 1.5 dyne/cm 2 within the aneurysm sac . The dimensions of the polycarbona te base are provided in Figure 2 2. For our design we used th e equation , where is wall shear stress, µ is fluid viscosity, Q is fluid flow, b is channel width, and h is channel height to arrive at the dimensions of the chamber . The idealized parallel plate flow chamber for flow models of a bifurcation and bi furcation aneurysm were created based on pr eviously published CFD studies 58 , 71 , 74 , 79 , 80 , 227 and anatomical dimensi ons of human cerebral arteries 405 . In particular, we aimed for the flow field in the device to have a cell surface area comparable to bifurcations at human carotid, anterior communicating and middle cerebral arteries. 405 The parallel plate flow chamber used for a straight artery flow model was a design recent ly published by Uzarski et al. 153 Parallel Plate Flow Chamber and Flow Circuit Setu p The flow circuit was set up using a previously described experimental scheme 153 and is shown in Figure 2 4. Gas permeable silicone tubing 3/32"ID x 5/32"OD ( EW 95802 03 , Cole Parmer) was used to conn ect a 250 mL Pyrex storage bottle ( 1395 250, Corning Life Sciences, Kennebunk, ME PharMed BPT tubing, 3/32 " ID x 7/32" OD x 1/16", (AY242005 , Murdock Industrial ) , another section of silicone tubing, bubble trap, and then the flow chamber. T he outlets from the flow chamber were connected via silicone tubing back to the storage bottle. Caps from Corning 250 mL round media bottles (Corning Life Scienes) were modified by drilling 3 burr holes using (Dremel, Racine, WI). Silicone tubing was used to ensure gas exchange with the circulating medium. PharMed BPT tubing was used at the cartridge/pump interface to provide long term durability and prevent

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80 changes in delivered volume of the medium across the system. Sterile A 100 Medical Silicone Adhesive (A 100, Factor II, Lakeside, AZ ) was used as a sealant for connections between storage bottle, tubing, and bubble trap device. The perfusion circuit was set up under sterile conditions in a Biological Safety Hood (Figure 2 4). Once the individual gaskets (Figure 2 1 and 2 3) were aligne d with each polycarbonate base, culture medium was perfused into the system up to the flow chamber inlet . Special care was taken to prevent the fluid from getting underneath the gasket . Plastic coverslips with grown HUVECs were then dropped onto the gasket with cells facing the inside of the flow field within the perfusion chamber and culture medium was allowed to fully fill the flow field within the device by capillary action . V acuum line was then opened to allow negative pressure to seal the device with the silicone gasket between the polycarbonate base and plastic coverslip with cells (Figure 2 1) . Once the whole system was sealed, it was transferred to an incubator and attache d to a perfusion pump (Figure 2 4) . Modeling Shear Stress Flow characteristics in the flow chambers were obtained using Computational Fluid Dynamics (CFD) analyses. High resolution mesh generation was performed in Gambit (ANSYS, Canonsburg, PA). Boundary l ayers were created near the wall. The height of the first layer was 0.01 mm. Only one fourth of the chamber volume was simulated using the symmetric nature of the chamber. Extensions were added to the inlet and outlet to eliminate the interference of the i nlet and outlet. Approximately 1.1 million cells were created. The density and viscosity of the culture medium were set to 1000 kg/m 3 and 0.001 Pa·s, respectively. Laminar flow was assumed. Lumen average velocity waveform was applied to the inlet boundary. Flow rates and pressures were

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81 measured using a perivascular ultrasonic transducer (model 2SB) and a flow meter (model T106, Transonic Systems, Ithaca, NY ). The measured flow rates in o ne cycle are shown in Figure 2 5A and the measured pressures are shown for five cycles in Figure 1 5B . A pressure boundary condition was set at the outlet. Time dependent terms were discretized in an implicit scheme with second order accuracy. The momentum equations were discretized using a second order upwind scheme. A segre gated solver was used to solve the momentum and continuity equations. Pressure velocity coupling was realized through the Semi Implicit Method for Pressure Linked Equations (SIMPLE) algorithm. The time step size was 0.005 s. A residual of 10 5 was set as t he convergence criterion. Simulation was performed in Fluent (ANSYS, Canonsburg, PA). Flow was converged in the second cycle and data from the second cycle were analyzed. Post processing and visualization of velocity streamlines and wall shear stress (WSS) were performed in Tecplot 360 (Tecplot, Bellevue, WA). Human Umbilical Vein Endothelial Cell Isolation Human Umbilical Vein Endothelial Cells (HUVECs) were isolated from Human Umbilical Cords (HUCs) by Joseph Uzarski as a gift from the McFetridge lab as d escribed previously 406 . Labor and Delivery u nit and stored on ice at 4 o C, and processed within 12 hours . All cord processing post collection was performed under sterile conditions. First, the cords were cannulated with a 14 G needle at the umbilical vein and perfused with 100 mL of phosphate buffered saline (PBS) to flush out the blood. Then, 10 mL of StemPro Acc utase Cell Dissociation Reagent ( A1110501 , Life Technologies, Grand Island, NY) was infused into the cord and the ends were clamped. HUVECs were left to dissociate from the vein over 10 mins. After incubation, the cords were flushed with 30 mL of PBS,

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82 the perfusate was centrifuged at 1250 RPM, and cells were collected. HUVECs were pooled from 3 donors, and cultured in Vasculife VEGF medium prepared as per manufacturer's instructions ( LL 0003, Lifeline Cell Technology, F rederick, MD) at 5% CO2 and 37 o C in T 25 flasks ( Techno Plastic Products, Trasadingen, Switzerland ) . The Vasculife VEGF medium was prepared with 2% FBS, 10 mM L glutamine, 0.75 U/mL heparin sulfate, 5 ng/mL rh EGF, 5 ng/mL rh basic FGF, 15 ng/mL rh IGF 1, 50 g/mL ascorbic acid, and 1.0 g/mL instructions. The medium was supplemented with 10,000 U /mL penicillin and g/mL streptomycin ( Life Technologies) at a final concentration of 1% . Collection of HUC specimens was performed in accordance to a research protocol approved by our Institutional Review Board. Cell Culture Purity Verification by Immunohistochemistry HUVECs at P1 were cultured for 2 3 days in Nunc Lab Tek II Tissue Culture slides ( 154534 , Thermo Fisher Scientific, Roch ester, NY) until confluence and fixed in 4% PFA. Cells were then permeabilized by 15 min treatment with 0.2% Triton X 100 (Sigma Aldrich, St. Louis, MO), blocked in 2% normal horse serum (S 2000, Vector Labs, Burlingame, CA) for 1 hour and stained with mon oclonal mouse anti human CD31 (M082301 2, DAKO) or monoclonal mouse anti human von Willebrand Factor (vWF) antibody (M061601 2, DAKO) for 2 hours, washed, and incubated with donkey secondary anti mouse antibody Alexa Fluor 488 (Life Technologies) for 1 hou r. Cells were then washed and counterstained with DAPI. The whole field of each chamber was manually scanned to look for non staining cells.

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83 Cell Culture Purity Verification by Flow Cytometry HUVECs at P1 were cultured for 2 3 days in T25 flask ( Techno Pl astic Products ) until confluence and then collected . Cells were washed 3x in PBS, centrifuged at 1250 RPM in 5 mL BD Falcon culture tubes (BD Biosciences, San Jose, CA). Cells were then analyzed by FACS with BD FACSCanto II Analyzer (BD Biosciences, San Jo se, CA). Shear Stress Experiments HUVECs (passage 2 5) were seeded on coverslips cut from 10 cm tissue culture plastic dishes (Techno Plastic Products ) at a density of 20,000 cells/cm 2 and grown to confluence. Cells were grown in Vasculife VEGF medium ( Lif eline Cell Technology ) with medium prepared as described earlier. Endothelial cells were exposed to pulsatile shear stress over a total of 27 hours in Vasculife VEGF medium. Recent studies have shown that shear stress pre conditioned endothelial cells re spond to hemodynamics changes in a more physiological manner than when shear stress naïve static cultures are exposed to shear stress. 155 , 156 The system was pre sheared at increasing rate until reaching the final average magnitude of shear stress of 10 dyne/cm 2 in order to achieve a more physiologically relevant cell phenotype. First, cells were pre conditioned at 1 dyne/cm 2 for one hour, followed by 5 dyne/cm 2 for two hours, and finally exposed to shear stress at 10 dyne/cm 2 , over a period of 24 hours in straight, bifurcation, and bifurcation aneurysm parallel plate flow chambers at 5% CO 2 and 37 o C. The volume of perfusing medium used was normalized with respect to the total cell growth area for each chamber type. Fluid flow was delivered in a pulsatile manner at 58 pulses/min with a Masterflex L/S Digital Drive , 600 RPM, 115/230 VAC ( HV 07523 80 , Cole Parmer, Vernon Hills, IL) with a Masterflex L/S® 8 channel, 3 roller cartridge pump head ( HV 07519 05 , Cole Parmer) . Up to three flow chambers were perfused at the

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84 same time for each experiment and the tubing was secured to the pump head with Masterflex L/S® larg e cartridges for 07519 05 and 06 pump heads ( EW 07519 70 , Cole Parmer). At the end of each experiment the chambers were disassembled and coverslips were removed under sterile conditions. Cells were fixed in ice cold methanol (Sigma Aldrich, St. Louis, MO ) for 5 minutes, washed twice in PBS and then stored in PBS at 4 o C for immunocytochemistry. Conditioned medium from each system was collected and frozen at 80 o C for analysis at a later time. Cell Density Quantification Cell density across the three flo w chamber systems (straight, bifurcation, and bifurcation aneurysm) was quantified by taking images at 40x using a Leica dissection microscope with Volocity 3D analysis software and counting viable cells in each field. Dissolved Oxygen Quantification Disso lved oxygen levels in unconditioned and conditioned media from static culture, straight, bifurcation, and bifurcation aneurysm flow chambers were measured using Milwaukee MW600 Portable Dissolved Oxygen Meter (Milwaukee Instruments, Rocky Mount, NC ). The o btained values were corrected for temperature and elevation. Prostaglandin E2 and Prostacyclin Quantification PGE2 levels in the perfusing medium were measured using the Prostaglandin E2 EIA Kit Monoclonal (Cayman 514010, Cayman Chemical, Ann Arbor, MI). PGI2 secretion was measured by quantifying its hydration breakdown product 6 keto Cayman Chemical, Ann Arbor, MI).

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85 RNA Isolation, Reverse Transcription, and Quantitative Real Ti me Polymerase Chain Reaction After the experimental period, the chambers were disassembled and coverslips were removed and placed on an inverted phase contrast microscope. Regions 6.5 mm x 6.5 mm were scraped off and cells were collected with a pipette un der sterile conditions from regions of interest and stored in Ambion RNAlater (Life Technologies, Carlsbad, CA). Total RNA was extracted using Ambion RNAqueous 4PCR Kit (Life Technologies, Carlsbad, CA). RNA purity and yield were assessed using the Infin ite 200 Pro plate reader with NanoQuant plate (Tecan Group, Männedorf, Switzerland), and only samples with sufficient purity (A260/A280 = 1.8 2.1) were used for further analysis. First strand cDNA was reverse transcribed using iScript cDNA Synthesis Kit ( Bio Rad Laboratories, Irvine, CA). Gene expression of COX 1, COX 2, and MCP 1 was measured relative to GAPDH using quantitative real time RT PCR performed on CFX384 Touch Real Time PCR Detection System (Bio Rad Laboratories, Irvine, CA) with iQ SYBR Green Supermix (Bio Rad Laboratories, Irvine, CA). Primer sequences were obtained from PrimerBank (Massachusetts General Hospital, Boston, MA) 407 and synthesized by IDT (Integrated DNA Technologies, Coralville, IO). After each run a melting curve was generated by heating the amplifi cation product from 55 o C to 95 o C at 0.5 o C/s to determine specificity of the PCR reaction products. Expression level for each gene of interest relative to GAPDH and reference cDNA from static controls was determined using the Pfaffl method 408 and presented as fold change. Relative Fluorescence Immunocytochemistry and Confocal Microscopy Briefly, immunocytochemic al (ICC) staining for COX 2 and MCP 1 was performed on cells from shear stress experiments and intensity values were quantified

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86 using confocal microscopy. Cells previously fixed in methanol were incubated with rabbit anti COX 2 antibody (160126, Cayman Ch emical, Ann Arbor, MI) or rabbit anti human MCP 1 antibody (ab9669, Abcam, Cambridge, MA) for 2 hours, washed, and incubated with Alexa Fluor 568 donkey anti rabbit antibody (A 21206, Life Technologies, Grand Island, NY) for 1 hour at room temperature, and finally counter stained with DAPI (H 1200, Vector Labs, Burlingame, CA). Relative protein expression of COX 2 and MCP 1 was quantified using a DSU Spinning Disk Confocal Scanner mounted on an Olympus IX81 inverted fluorescent microscope (Olympus, Center Valley, PA) with a 40x dry objective and C4742 80 12AG Monochrome CCD camera (Hamamatsu Photonics, Bridgewater, NJ). Animals All animal experimentation was performed in accordance with a protocol approved by our institution's Institutional Animal Care an d Use Committee. At all times, the principles governing the care and treatment of animals, as outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication no. 85 323, revised 1996) and ado pted by the American Physiological Society, were strictly adhered to during the course of this study. Mice had ad libitum access to water and standard mouse chow (or special diet as indicated) and were housed in a well ventilated, specific pathogen free, t emperatu re controlled environment (24± 1 °C; 12 h 12 h light dark cycle). Murine Intracranial Aneurysm Model Murine intracranial aneurysms were created in female C57BL/6 mice (Charles River Laboratories, Wilmington, MA) using a method modified from a previo usly described model. 10 Briefly, the left common carotid artery and the posterior branch of

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87 the right renal artery are ligated to induce hypertension. One week later, an Alzet micro osmotic pump model 1004 (DURECT Corp, Cupertino, CA) was implanted subder mally to deliver Angiotensin II at 1000ng/kg/min; and 20 uL of 8% porcine elastase (Worthington Biochemica l Corp, Lakewood, NJ ) in PBS was injected into the right basal cistern using stereotactic coordinates: 1.2 mm rostral of bregma, 1.2 mm lateral of mi dline and 5.3 mm ventral of the dorsal aspect of the skull . The animals were fed a hypertensive diet with 8% NaCl and 0.12% BAPN (Harlan Laboratories, Indianapolis, IN). Mice were euthanized one week after stereotactic elastase injection. Human Aneurysm and Superficial Temporal Artery Specimens Collection of human cerebral aneurysm and superficial temporal artery specimens was performed in accordance to a research protocol approved by our Institutional Review Board (IRB). Patients signed informed IRB res earch consent before aneurysm and superficial temporal artery specimens were harvested at the time of aneurysm clipping surgery. The collected specimens were then immediately fixed in 4% paraformaldehyde. Immunohistochemistry of Mouse and Human Aneurysm Specimens Murine aneurysm specimens were first fixed in 4% PFA for 24 hours, and then dehydrated in 18% sucrose solution. Tissues were mounted in Tissue Tek OCT antigen retr ieval in Dako Target Retrieval Solution (Dako, Carpinteria, CA) was performed. Following a block in 2% normal horse serum (S 2000, Vector Labs) for 1 hour, the specimens were incubated with rat anti MECA 32 antibody (BD 550563, BD Biosciences, San Jose, C A) and either rabbit anti COX 2 antibody (160126, Cayman Chemical) or rabbit anti microsomal prostaglandin E synthase 1 (mPGES 1) antibody

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88 (Cayman Chemical) or rabbit anti mouse MCP 1 antibody (ab7202, Abcam) overnight at 4 o C, washed, and incubated with A lexa Fluor 488 donkey anti rat antibody (A 21208, Life Technologies) and Alexa Fluor 568 donkey anti rabbit antibody (A 21206, Life Technologies) for 1 hour at room temperature. For immunohistochemistry of human aneurysm samples, the tissues were fixed in 4% PFA and embedded in paraffin. After paraffinized by xylene and ethanol. Heat mediated antigen retrieval in 10 M Sodium Citrate buffer pH 6.5 was performed. The staining protocol for COX 2, mPGES 1 and MCP 1 was then followed exactly as for immunohistochemistry of mouse aneurysm specimens except that mouse anti human CD31 antibody (IR61061 2, Dako) was used in place of MECA 32 to visualize the endothelial cell layer and rabbit anti human MCP 1 antibody ( ab9669, Abcam) was used to visualize MCP 1. Statistical Analysis Statistical analyses were performed by a biostatistician (D an Neal from Acknowledgments section) . For analysis of PGE2, 6 k PGF , and COX 1, COX 2, a nd MCP 1 gene expression ANOVA with Tukey HSD were performed. For protein expression analysis of COX 2 and MCP 1 a random effects linear model was used to determine whether intensity differed across the different locat ions within the flow chambers. Results Bifurcation and Bifurcation Aneurysm Parallel Plate Flow Chamber Models Simulate Wall Shear Stress Patterns Found in Bifurcations and Cerebral Aneurysms We performed CFD analysis using Ansys FLUENT to obtain velocity magnitude, WSS, and WSSG maps of the flow field in th e designed flow chambers (Fig ure 2 6 ).

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89 The time averaged WSS for the bifurcation flow chamber in the regions of interest where cells would be imaged was found to be as follows: 8.6 dyne/cm2 in the proximal straight segment (PSS), 5.2 dyne/cm2 at the bifurcation (B), and 4.3 dyne/cm2 in the branching arms (BA). For the bifurcation aneurysm flow chamber, WSS was found to be 8.6 dyne/cm2 in the proximal straight segment (PSS), 4.7 dyne/ cm2 at the bifurcation (B), 4.2 dyne/cm2 in the branching arms (BA), and 0.8 dyne/cm2 within the aneurysm sac (AS). Isolated Cells Are Genuine Human Umbilical Vein Endothelial Cells Cells isolated from human umbilical veins were analyzed by immunohistochem istry and flow cytometry. Immunohistochemistry analysis revealed that the cultured cells had cuboidal and flat morphology typical of endothelial cell s (Figure 2 7 ). Specifically, the cells at P1 were positive for PECAM 1 also known as CD31 (Figure 2 7 A) an d vWF (Figure 2 7 B), which are endothelial cell markers. Furthermore, flow cytometry showed that at P3, 99.7% of cells were double positive for VE Cadherin (FITC) and E Selectin (PE) (Figure 2 8 A ) and 95.2% were double positive for CD31 (FITC) and VCAM 1 ( PE) (Figure 2 8 B) indicating that genuine endothelial cells were successfully isolated and culture from human umbilical veins . Endothelial Cell D ensity in Different Flow Chamber Systems Average cell density was analyzed in different micro environments fr om straight, bifurcation, and bifurcation a neurysm flow chambers at 40x (Figure 2 9). N o differences in cell density across the micro environments in different flow chambers were found suggesting that any differences detected in protein expression would no t be due to changes in cell number ( n=27 34 each. p=0.509 for bifurcation, p=0.936 for bifurcation aneurysm ) .

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90 Dissolved Oxygen in Unconditioned and Conditioned Media Dissolved oxygen levels in unconditioned and conditioned media from static culture, straig ht, bifurcation, and bifurcation aneurysm flow chambers were found to be statistically significant (p=0.0099) (Figure 2 10) . The conditioned medium from the bifurcation aneurysm and bifurcation flow systems was found to have significantly lower dissolved oxygen content than unconditioned medium ( 5.58 vs 5.54 vs 6.0 mg/L, respectively, p <0.05 for both ). No statistically significant differences were found between conditioned media across straight, bifurcation, and bifurcation aneurysm flow chamber systems . Hemodynamic Shea r Stress in Bifurcation Aneurysm Flow Chamber Activates Increased Endothelial Expression of Inflammatory Mediator PGE2 We evaluated the inflammatory conditions within three different flow chamber models (bifurcation aneurysm, straight artery, and arterial bifurcation) by quantifying the levels of secreted PGI2 and PGE2 by HUVECs subjected to pulsatile flow. Significantly more PGE2 was secreted by endothelial cells in the bifurcation aneurysm flow chamber (27.11 pg/mL, n=10) than the bifurcation (13.78 pg/mL , n=7, p=0.028) or straight flow chamber (11.33 pg/mL, n=7, p=0.005) systems (Fig ure 2 11 A). The PGE2 levels between bifurcation and straight flow chambers were not found to be significantly different (p=0.772). No significant differences (p=0.297) in le vels of PGI2 breakdown product 6 bifurcation (90.81 pg/mL, n=7) or straight (73.13 pg/mL, n=7) flow cha mber s by ANOVA (p=0.297) (Fi gure 2 11 B ).

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91 Hemodynamic Shear Stress at Bifurcation and Bifurcation Aneurysm Increases Gene Expression of COX 2 and MCP 1 Quantitative real time RT PCR for COX 1, COX 2, and MCP 1 were performed on endothelial cells collected from micr o environments shown in Figure 2 12 for the straight, bifurcation, and b ifurcation aneurysm flow chamber systems. No significant differences were found in COX 1 gene expression between the micro environments analyzed within the bif urcat ion (n=6 9, p=0.588) (Figure 2 12 B ) or bifurcation aneurysm system s (n=5 for each, p=0.729) (Figure 2 12 C) by ANOVA. Gene expression analysis for the straight flow chamber revealed a non inflammatory phenotype with a 2.6 fold decrease in COX 2 expression and 7.9 fold dec rease in MCP 1 expression (Fig ure 2 12 B and C). COX 2 and MCP 1 relative e xpression in the bifurcation and aneurysm flow chambers were generally increased from the baseline with significant differences among the different micro environments. COX 2 gene expression by location was significantly different in the bifurcation flow c hamber by ANOVA (n=4 9, p=0.036) (Fig ure 2 12 B). Specifically, it was significantly higher at the bifurcation than in the proximal straight segment (+5.8 vs +2.9 fold relative change, p=0.028). MCP 1 gene expression was found to be significantly differen t by location in the bifurcation flow chamber by ANOVA (n=5 8, p=0.029). Relative MCP 1 expression was significantly increased at the bifurcation micro environment than the proximal straight segment ( 2.7 vs 4.4 fold change, p=0.026) (Fig ure 2 12 C). Al l other pairs were not found to be significantly different for the bifurcation flow chamber. Although increased from baseline, COX 2 gene expression in the aneurysm flow chamber was not, paradoxically, significantly different by location by ANOVA (n=3 4, p =0.671) (Fig ure 2 12 B), which could be attributed to small sample size which resulted in only 24% power. MCP 1

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92 gene expression was found to be significantly different by location in the bifurcation aneurysm flow chamber by ANOVA (n=3, p=0.005). Relative MCP 1 expression was significantly increased at the bifurcation micro environment than the branching arms ( 3.1 vs 9.5 fold change, p=0.040) (Fig ure 2 12 C). The proximal straight segment MCP 1 expression was also significantly increased compared to at t he branching arms ( 3.4 vs 9.5 fold change, p=0.048). All other pairs were not found to be significantly different. Hemodynamic Shear Stress at Bifurcation and Bifurcation Aneurysm Promotes Increased Protein Expression of COX 2 and MCP 1 Relative fluo rescence ICC and statistical analysis were performed using a random effects linear model (the lme() function in the R package nlme). Analysis demonstrated the overall effect of location on COX 2 protein expression in the bifurcation flow chamber was margi nally sign if i cant (n=20 28, p=0.52) (Figure 2 13 A). COX 2 signal intensity at the proximal straight segment was 12% lower than at the bifurcation (1.00 vs 1.12 RFU, p=0.016). The overall effect of location on MCP 1 protein expression was significant (n=1 8 20, p=0.020). MCP 1 relative signal intensity was estimated to be 22% lower at the proximal straight segment than at the bifurcation (1.00 vs 1.22 RFU, p=0.010) and 20% lower than at the distal branching arms ( 1.00 vs 1.20 RFU, p=0.22) (Fig 2 8 B). No s ignificant differences were detected between other pairs in the bifurcation flow chamber. In contrast, relative fluorescence ICC for COX 2 in the bifurcation aneurysm flow chamber revealed that the overall effect of location was highly signif icant (n=24 3 4, p<0.0001) (Fig ure 2 13 A). COX 2 protein expression in the aneurysm sac was estimated to be 43% higher than at the proximal straight segment (1.48 vs 1.00 RFU, p<0.0001), 17% higher than at the bifurcation (1.48 vs 1.30 RFU, p=0.005), and 17% higher tha n at the branching arms (1.48 vs 0.91 RFU, p<0.0001).

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93 COX 2 expression at the proximal straight segment was estimated to be 27% lower than at the bifurcation (1.00 vs 1.30, p<0.0001) and 15% higher than at the branching arms (1.00 vs 0.91 RFU, p=0.015). MCP 1 protein expression in the bifurcation aneurysm flow chamber by location was also highly significant (n=29 33 , p<0.0001) as shown in Figure 2 13 B. MCP 1 expression at the aneurysm sac was estimated to be 44% higher than at the proximal straight segme nt (1.42 vs 1.00 RFU, p<0.0001), 19% higher than at the bifurcation (1.42 vs 1.24, p=0.0006) and 20% higher than at the branching arms (1.42 vs 1.24 RFU, p=0.0003). MCP 1 expression at the proximal straight segment was estimated to be 25% lower than at t he bifurcation (1.00 vs 1.24 RFU, p<0.0001) and 24% lower than at the branching arms (1.00 vs 1.24 RFU, p<0.0001). No differences were found in MCP 1 expression between the bifurcation and the arms (p=0.835). Increased COX 2 and MCP 1 Protein Expression in Murine Intracranial Aneurysms One week after stereotactic elastas e injection, two thirds of the mice de velop ed aneurysms (n=4/6) (Figure 2 14 A). COX 2 was expressed in the intima a nd the media of aneurysms (Figure 2 14 B), and mPGES 1 was expressed in the endothelial cells of aneurysms although the smooth muscle cells in the media exhibited higher immunofluorescence (Fig ure 2 14 B). MCP 1 was mainly observed in the intima with some positive cells in the media (Fig ure 2 14 B). In both cases the endotheli al cells were visualized with MECA 32 (Fig ure 2 14 B). Both COX 2 and MCP 1 expression were limited to the aneurysmal wall and were not seen in artery sections proximal or distal to the lesion or in non aneurysmal vessels on the contralateral side of the ci rcle of Willis (results not shown). No endothelial cells were positive for mPGES 1 in the contralateral vessels in aneurysmal mice, although strong mPGES 1 signal was found

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94 in some portions of the media (results not shown). In control arteries from sham animals, no COX 2, mPGES 1 or MCP 1 expression was observed in the intima or the media (Fig ure 2 14 C). However, an occasional mPGES 1 positive cell could be found in the adventitia. Increased COX 2, mPGES 1, and MCP 1 Expression in Human Aneurysm Specime ns Of the human aneurysm specimens analyzed, one third (n=5/15) had intact endothelium lining the dome. Of those five, each specimen was positive for COX 2, mPGES 1, and MCP 1 (Figure 2 15 A). Compared to control superficial temporal arteries (STA), which exhibited no staining for COX 2 and MCP 1, and only weak mPGES 1 expression (Figure 2 15 B), weak to medium COX 2 staining with strong mPGES 1 and MCP 1 staining was seen in the endothelial cell layer in the aneurysm tissue. Strong staining for each of th ose inflammatory markers was observed in the media of aneurysms. Some mPGES 1 and MCP 1 staining was also found in the adventitia. Discussion The pathophysiology of cerebral aneurysm formation is not well understood. 7 It is believed that cerebral aneurysm s develop following damage to the internal elastic lamina (IEL) of the vessel wall due to high wall shear stress. 7 12 It is thought that once functional endothelium is lost, the vessel wall undergoes proliferation of smooth muscle cells, infiltration of im mune cells, and further remodeling of the wall leading to aneurysm progression. 7 Early during aneurysm formation, the endothelium plays a critical role in an ever changing relationship between inflammation, remodeling, and local hemodynamic conditions. 17,1 8 Shear stress is thought to play a key role in cerebral

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95 aneurysm formation, but, until recently, there has been disagreement as to the hemodynamic conditions that cause progression of these lesions. Role of shear stress in propagating aneurysm growth Once an aneurysm forms, a change occurs in the local blood flow and shear stress pattern resulting in a low shear stress micro environment within the dome. 14,17,18 Further changes to this wall shear stress pattern occur during aneurysm growth 17 causing further pathological remodeling. 17,18 Recently, remodeling has been shown to coexist in regions of low wall shear stress of the aneurysm dome. 17 A rabbit elastase induced carotid aneurysm model study revealed that the low shear stress found within the aneurysm do me (<5 dyne/cm 2 ) was associated with increased MMP 2 activity and decreased MMP inhibitor TIMP 1 and 2 expression. 18 These findings regarding low shear stress led to our hypothesis that low shear stress at arterial bifurcations and bifurcation aneurysms induces endothelial expression of inflammatory factors. Modeling shear stress induced endothelial inflammation in vitro In this work, we studied shear stress induced endothelial cell inflammation within a bifurcation aneurysm. We developed a novel in vitro parallel plate flow chamber model 33 35 of a bifurcation aneurysm (Fig ure s 2 1, 2 2, 2 3, and 2 4 ) characterized by reproducible hemodynamic conditions and presence of multiple, co existent yet distinct micro environments. Multiple PPFCs have been d esigned and used to study the behavior of cells, especially the endothelium, under defined hemodynamic conditions . 76 , 153 , 402 , 403 Several models have been designed to replicate certain features of conditions seen at bifurcations or in cerebral aneurysms. Szy manski et al designed a T shaped flow chamber mimicking the impingement zone near the flow divider at bifurcations to study the effects of high pressure and shear stress gradients on endothelial cells. 227

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96 Using this device, they showed that endothelial cells moved away from stagnation points to regions exposed to shear stress gradients. This effectively reduced local cell density, which could explain increased permeability of the vascular wall at aneurysm formation sites allowing for increased oxidized lipid and ROS deposition. 12 , 227 , 409 Urschel et al used commercially available double bifurcation PPFCs and showed that the secreted chemokine profile of endothelial cells was much different between endothelial cells exposed to homogenous versus heterogeneous shear stress. 160 In particular, they reported increased MCP 1 and IL 8 levels in TNF activated endothelial cells from bifurcating slides. Again, this suggests that endothelial cells at bifurcations are predisposed to increased inflammatory response when activated by a stressor. However, despite these elegant studies, no one has tried to replicate the hemodynamic features of bifurcation aneurysms to study the effects of shear stress in those micro environments. Our modified PPFCs (Fig ure s 2 1 , 2 2, 2 3, and 2 4 ) replicate the low to high wall shear stress ratio pattern found in murine 30 a nd human cerebral vessels and aneurysms 14,17 . We studied and validated the aneurysm flow chamber design by performing CFD analysis (Fig ure 2 6 ) to ensure that the WSS patterns present would match those from previously published human 14,17 and animal 18 CFD investigations of aneurysms. Cell numbers across the different micro environments within the three systems were not found to be different indicating that any detected changes in circulating levels would be due to differences in shear stress alone (Figure 2 9). The dissolved oxygen content levels were not different among the three flow chamber systems, although the bifurcation and bifurcation aneurysm systems had significantly lower oxygen content than non conditioned medium (Figure 2 10). The

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97 measured diss olved oxygen content values were also about 25% higher (5.68, 5.54, and 5.58 mg/L for the three systems) than the dissolved oxygen content typically found in human blood (~4.43 mg/L) 410 indicating that three systems are hyperoxic relative to in vivo conditions. H owever, the total oxygen content found in blood is much higher than the dissolved component. 410 We analyzed the perfusate from 3 different flow chamber systems and found that PGE2 secretion was increased in the bifurcation aneurysm flow chamber when compared to th e conventional straight or simple bifurcation flow chambers. We found no significant differences in PGI2 secretion across the systems. Historically, the expression of COX enzymes and their products have been used to gauge the extent of the endothelial inf lammatory phenotype. 20 25 COX 1 is expressed constituitively and is primarily responsible for production of intermediates that are converted to PGI2, an anti inflammatory prostaglandin 25 that prevents vascular smooth muscle proliferation 36 . COX 2 is upreg ulated in regions of acute inflammation 21 23 and leads to production of end products such as PGE2, a prostaglandin with pro inflammatory vascular functions 24 26 . It is believed that COX 2 is not expressed in vasculature under normal physiologic conditions . 37 We observed suppressed COX 2 gene and protein expression in our short term experiments in the conventional straight channel flow chamber exposed to uniform shear stress. In contrast, gene and protein expression profiles for COX 2 in the bifurcation an d bifurcation aneurysm flow chambers showed important differences. In both the bifurcation aneurysm and arterial bifurcation flow chamber systems, COX 2 gene expression was increased above the baseline. In the bifurcation flow chamber we observed increase d COX 2 gene expression at the

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98 bifurcation with respect to the proximal straight segment. No differences in protein expression by location were found by analysis by relative fluorescence ICC. We found no differences in COX 2 gene expression in the aneury sm flow chamber between different locations, however, significant differences were observed via relative fluorescence immunocytochemistry. Increased COX 2 protein expression was found at the bifurcation and the aneurysm micro environments in the aneurysm flow chamber providing evidence that the increase in PGE2 was in fact due to the inflammation present at the non uniform and low shear stress micro environments. Recently, COX 2, mPGES 1, and their endproduct PGE2 have been implicated in cerebral aneurysm pathophysiology. 38,39 Aoki et al have shown that excessive PGE2 signaling through EP2 receptor due to high hemodynamic stress can lead to aneurysm formation. 38 In their in vitro experiments they implicated high shear stress in PGE2 induction and cerebral aneurysm formation. 38 They noted that COX 2 and mPGES 1 persisted within the aneurysmal walls for up to 3 months. We found this surprising since wall shear stress in aneurysm domes has been shown to be much lower than in the parent vessel. 13 18 It is poss ible that further PGE2 activation could be due to involvement of other inflammatory pathways, but the authors did not examine the reason for persistent activation of EP2 receptor or synthesis of PGE2. COX 2 ha s been shown to be upregulated at low levels o f wall shear stress in atherosclerosis. 22 This led us to believe that a similar mechanism could occur in formed aneurysms. In this study, we recreated the hemodynamic micro environment experienced by endothelial cells within cerebral aneurysms by designin g a novel parallel plate flow chamber model. We also clarify the pathophysiological stimulus for PGE2 synthesis

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99 once an aneurysm forms and show that the resulting low shear stress micro environment that develops post aneurysm formation leads to endothelial cell inflammation (Figure 2 16) . Specifically, we show that the increased PGE2 secretion within the aneurysm dome can occur due to non uniform and low shear stress induced inflammation alone. We also show that COX 2 and mPGES 1 are expressed in endothe lial cells in both murine and human aneurysms. We could not easily explain the discrepancy between COX 2 gene expression and protein expression for bifurcation and aneurysm flow chamber systems. Several other groups have also observed a weak or paradoxic al correlation between COX 2 gene and protein expression. 40,41 COX 2 expression has been reported to be tightly regulated at post transcriptional modification level, especially by mRNA stabilizing proteins such as HuR 41,42 and COX 2 gene expression does no t always correlate with protein expression 40 42 . Another possibility is that other inflammatory mediators are being expressed in the bifurcation aneurysm flow chamber augmenting COX 2 protein expression. As an additional verification, we studied endotheli al expression of MCP 1, a chemokine known for its chemoattractant abilities for macrophages, 8,26,27 in our perfusion systems. MCP 1 and macrophage mediated inflammation have been implicated in cerebral aneurysm pathophysiology by Aoki et al. 8 Increased MC P 1 expression at low levels of shear stress and static cultures have been shown in previous studies. 43 In our in vitro aneurysm flow chamber model we found increased MCP 1 expression by both gene and protein expression in the bifurcation and aneurysm flow chamber systems. This suggests that the low shear micro environment that develops within the aneurysm dome leads to endothelial inflammation.

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100 Limitations There are limitations to the in vitro approach used in this work since additional phenomena such as radial stretch, complex 3 D matrices, or trans mural transport cannot be easily studied. The rigid parallel plate flow geometry provides a well defined system, however, that can be verified with in vivo studies and as such is a useful tool for studying c ell behavior and signaling pathway dysfunction due to changes in wall shear stress alone. Nevertheless, the current system lacks the multi cell layer characteristic of a true vessel wall and the interactions between the endothelium, the smooth muscle cell s, and the adventitia have been shown to be important in development of atherosclerosis and aneurysms. 8,9,26,30 32,38,44 Previous studies by others and our own immunohistochemistry of murine and human aneurysm tissues have revealed expression of COX 2, mPG ES 1 and MCP 1 within the media and the adventitia. Further improvements to the system could be made in the future.

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101 Figure 2 1 . Bifurcation and bifurcation aneurysm flow chamber concept design and components of the flow field. A) 3 D model of the parallel plate flow chamber components showing plastic coverslip, silicone gasket with the flow field, and polycarbonate bioreactor cell. B) Complete, sealed chambers with color coded maps showing components of the flow field.

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102

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103 Figure 2 2. Polycarbonate bioreactor base design for the bifurcation and bifurcation aneurysm flow chambers. A) 3 D model of polycarbonate bioreactor cell. B) Top, C) Back, and D) Side views of the polycarbonate base.

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104 Figure 2 3. Representative p hotographs of the manufactured flow chambers. A) Bifurcation silicone gasket with polycarbonate base. B) Indicator dye perfusion of a fully sealed bifurcation flow chamber indicating full and equal perfusion throughout the cham ber.

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105 Figure 2 4. Parallel Plate Flow Chamber and Flow Circuit Setup. A) Schematic of the flow circuit . B) Representative photograph of the experimental setup.

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106 Figure 2 5 . A) M easured flow rate in one cycle for the designed flow chamber. Flow rate was measured using a perivascular ultrasonic transducer (model 2SB) and a flow meter. B) Measured pressures for five cycles at the inflow, and two outflows for the designed flow chambers.

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107 Figure 2 6 . Computati onal fluid dynamics of the designed flow chambers . Maps of A) velocity, B) wall shear stress (WSS), and C) wall shear stress gradient (WSSG) within the designed flow chambers show that both the bifurcation an d bifurcation aneurysm flow chambers have multiple and distinct hemodynamic micro environments .

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108 Figure 2 7 . HUVEC verification at P 1 using immunohistochemistry . Representative immunohistochemistry images showing HUVEC culture morphology at P1 demonst rating the purity of cells isolated from human umbilical veins that stain positive for A) CD31 and B) vWF . Green : CD 31 or vWF, Blue : DAPI. Scalebar = 20 m A B

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109 Figure 2 8 . HUVEC verification at P3 using flow cytometry. Follo wing activation with 10 nM TNF , flow cytometry analysis showed that the presumed HUVECs were A) 99.7% double positive for VE Cadherin (FITC) and E Selectin (PE) and B ) 95.2% double positive for CD31 (FITC) and VCAM 1 (PE) indicating that they were genuine endothelial cells. 0.2% 99.7% 0.0% 0.1% 0.0% 95.2% 0.0% 4.8%

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110 Figure 2 9 . Endothelial cell density in different flow chamber systems. Average cell density was analyzed in different micro environments from straight, bifurcation, and bifurcation aneurysm flow chambers at 40x . A) The results indicate that there are no differences in cell density suggesting that any differences detected in protein expression would not be due to changes in cel l number. One way ANOVA with Tukey HSD. n=27 34 each . p=0.509 for bifurcation, p=0 .936 for bifurcation aneurysm. * p<0.05. Data presented as mean±s.e.m. Representative microscopic images of fields taken at 1 0x from B) the bifurcation, and C) bifurcation an eurysm flow chambers are shown. Scale bar = 100 m

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111 Figure 2 10. Dissolved oxygen content in different culture and flow circuits. The differences in dissolved oxygen content across five different medium groups was found to be statistically significant (p=0.0099). The conditioned medium from the bifurca tion aneurysm and bifurcation flow systems was found to have significantly lower dissolved oxygen content than unconditioned medium (p<0.05). No statistically significant differences were found between conditioned media. One way ANOVA with Tukey HSD. n=3 e ach. *p<0.05, **p<0.01.

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112 Figure 2 11 . PGE2 and 6k PGF 1a analysis in the straight, bifurcation, and aneurysm flow chambers. HUVECs exposed to pulsatile flow at 10 dyne/cm2 for 24 hours secrete A) more pro inflammatory PGE2 in the aneurysm flow chamber than in the straight channel or bifurcation flow chambers. B) No significant difference was found in secreted levels of the prostacyclin breakdown product 6 k PGF 1a between the system s. One way ANOVA with Tukey HSD. Results are shown as mean±s.e. m., *p<0.05, ** p<0.01 .

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113 F igure 2 1 2 . Inflammatory gene expression in straight, bifurcation and aneurysm flow chambers. After the perfusion experiments, coverslips with HUVECs were removed and cells from different microenvironments were collected and quan titative real time RT PCR was performed via the Pfaffl method for COX 1, COX 2, and MCP 1, and data was normalized with respect to static controls. A) No significant differences were found in COX 1 gene expression between the microenvironments analyzed wit hin the bifurcation (p=0.588) or aneurysm flow chambers (p=0.729). B) COX 2 gene expression was significantly different in the bifurcation flow chamber by location (p=0.036), but not in the bifurcation aneurysm flow chamber (p=0.671). C) MCP 1 gene express ion was found to be significantly different across the micro environments in both the bifurcation (p=0.029) and the bifurcation aneurysm flow chamber (p=0.005). One way ANOVA with Tukey HSD for each system. Results are shown as mean±s.e.m., *p<0.05, **p<0. 01, ***p<0.001, ****p<0.0001.

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114

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115 Figure 2 1 3 . COX 2 and MCP 1 protein expression in the bifurcation and aneurysm flow chambers. After the perfusion experiments, coverslips with HUVECs were removed, fixed, and stained for ei ther COX 2 and MCP 1. Relative fluorescence immunocytochemistry was performed using confocal microscopy and intensity values were normalized with respect to the proximal straight segment for each chamber. A) Differences in COX 2 protein expression in the b ifurcation flow chamber were marginally significant (p=0.052), while the differences in the bifurcation aneurysm flow chamber across the different micro environments were highly significant (p<0.0001). B) MCP 1 protein expression was found to be significan tly different across the micro environments in both the bifurcation (p=0.020) and the bifurcation aneurysm flow chamber (p<0.0001). Data was analyzed using random effects linear model (lme() function in the R package nlme). Results are presented as mean±s. e.m., *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

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116 Figure 2 1 4 . COX 2, mPGES 1, and MCP 1 are expressed in walls of murine aneurysms. C57BL/6 mice developed A) aneurysms (arrowhead) 1 week following aneurysm induction surgery, whereas sham mice did not. E ndothelial cells ( Green : MECA32+) lining B) the aneurysm express COX 2, mPGES 1, and MCP 1 ( Red ) (arrowheads) in the arteries of aneurysmal mice, but not in C) arteries of mice that underwent sham surgery. Blue : DAPI. Scalebar represents 10 µm.

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117

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118 Figure 2 1 5 . COX 2, mPGES 1, and MCP 1 are expressed by endothelial cells in human aneurysm walls. Human aneurysm specimens and control superficial temporal arteries were harvested during aneurysm clipping surgery and immuno fluorescent staining wa s performed. A) Endothelial cells ( Green : CD31+) lining the aneurysm dome are under a state of inflammation and express COX 2, mPGES 1, and MCP 1 ( Red ) (arrowheads) recapitulating the pattern of inflammation seen in the mouse intracranial model, which also supports the data obtained from the aneurysm parallel plate flow chamber model. B) Endothelial cells lining the control superficial temporal artery (STA) do not express COX 2 or MCP 1 ( Red ). Only weak mPGES 1 (Red) expression was noted. Blue : DAPI. Scaleb ar represents 10 µm.

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119

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120 Figure 2 1 6 . Proposed mechanism by which hemodynamic changes lead to initial aneurysm formation, progression and rupture. Hemodynamic shear stress and shear stress gradients cause initial endothelial dysfunc tion by increasing proliferation and apoptosis, which leads to degeneration of the IEL. Once an aneurysm develops, the hemodynamics change and the endothelial cells within the dome are then exposed to low shear stress leading to increased secretion of infl ammatory mediators such PGE2 and MCP 1 . This leads to inflammatory cell infiltration in the low shear stress region of the dome, further degeneration of the vascular wall and eventual rupture.

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121 CHAPTER 3 PRO INLFAMMATORY ROLE OF SHEAR STRESS INDUCED ELR+ CHEMOKINES CXCL1/GRO a AND CXCL8/IL 8 IN CEREBRAL ANEURYSM FORMATION Introduction Aneurysm growth is believed to occur through a chronic inflammation characterized by a poptosis of endothelial cells, 76 increased secretion of the matr ix metallo proteinases (MMPs), 82 and activity of innate immune ce lls, primarily macrophages. 72 , 83 The critical role of monocyte and macrophage attracting CCL chemokines such as MCP 1 has been described, how ever, the role of neutrophil attracting ELR+ CXCL chemokines such as CXCL1 or IL 8 has not been reported. 12 , 72 , 83 Neutrophils act as the first responder cells of the innate immune system yet their role in cerebral aneurysm formation has n ot been examined in detail. 12 , 127 Significant gaps in knowledge exist due to lack of an effective in vitro screening tool that would allow for modeling of hemodynamic conditions withi n cerebral aneurysms. The link between hemodynamic forces, such as shear stress, and biological mechanisms, such as inflammation, that contribute to aneurysm development is still not fully understood. 77 , 79 81 , 134 , 135 To overcome the above challenges, under Spec ific Aim 1 , we designed a novel in vitro parallel plate flow chamber bifurcation aneurysm model characterized by reproducible hemodynamic conditions and the presence of multiple, co existent yet distinct micro environments to study the effect of shear stre ss on endothelial phenotype, and compared it to similar models of a straight artery and an arterial bifurcation. We then set out to screen for changes in the inflammatory profile of 40 different cytokines in the perfusing medium in three perfusion system s . Our hypothesis is low shear stress and shear stress gradients at bifurcations and bifurcation aneurysms induce endothelial

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122 expression of specific inflammatory factors, which then promote aneurysm formation and growth. Methods Cytokine Array and ELISAs Le vels of 40 different human inflammatory factors in the perfusing medium from the straight, bifurcation and bifurcation aneurysm flow chambers were measured using the Human Inflammation Array C3 (AAH INF 3, RayBiotech, Norcross, GA). Quantification of in tensity (integrated density) values was performed using the MicroArray Profile plugin for ImageJ. IL 8 levels in the perfusing medium were quantified using the Human CXCL8/IL 8 Quantikine ELISA Kit (D800C, R&D Systems, Minneapolis, MN). CXCL1/GRO levels were quantified using the Human CXCL1/GRO alpha Quantikine ELISA Kit (DGR00, R&D Systems, Minneapolis, MN). Concentration of Perfusing Medium for CXCL1/GRO ELISA Concentration and purification of perfusate from shear stress experiments was carr ied out using Centriprep Centrifugal Filter Unit with Ultracel 3 membrane filter device s (#4302, EMD Milipore, Billerica, MA). The Centriprep 3K device contains an Ulracel membrane with nominal molecular weight limit (NMWL) of 3,000 Da, which allows for re tention of solutes with molecular weights above that limit. Since CXCL1 has a molecular weight of 9 11 kDa 411 , 412 this allows for concentration of this protein within the device. Exactly 10 mL of conditioned medium from ea ch flow chamber was added to per cent rifugal device and concentrated using three separate spins at 4 o C. The first spin time was 98 minutes at 3,000 x g, and following centrifugation filtrate was decanted leaving a concentrate with a volume of about 2.0 mL. The second spin was carried out for 15 minutes at 3,000 x g, and the remaining concentrate had a volume of 1.0 mL.

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123 After the third centri fugation of 15 minutes at 3,000 x g, the final volume of the concentrate was 0.5 mL. The concentrate was frozen at 80 o C for use at a later time. Relative Fluorescence Immunocytochemistry (RF ICC) and Confocal Microscopy Cells previously fixed in ice cold methanol were permeabilized with 0.2% Triton X 100 (Sigma Aldrich, St. Louis, MO) for 15 minutes, blocked in 2% normal horse serum (S 2000, Vector Labs, Burlingame, CA) in PBS for 1 hour and then incubated with mouse anti human IL 8 antibody (ab18672, Abc am, Cambridge, MA) or rabbit anti CXCL1 antibody (ab86436, Abcam) or rabbit anti VCAM 1 antibody (sc 8304, Santa Cruz Biotechnology, Dallas, TX) at 1:200 overnight, washed, and incubated with the donkey secondary anti rabbit antibody Alexa Fluor 568 (Life Technologies, Grand Island, NY) at 1:500 for 1 hour at room temperature. Finally, nuclei were counterstained with DAPI (H 1200, Vector Labs, Burlingame, CA). As a negative control, and to quantify background fluorescence, coverslips with cells from shear s tress experiments were stained with rabbit IgG antibody (I 1000, Vector Labs) in place of IL 8, CXCL 1 or VCAM 1 antibody. Stained slides were imaged immediately or stored at 4 o C up to 5 days. To quantify relative protein expression of IL 8, CXCL 1 or VC AM 1 via relative immunofluorescence we used a DSU Spinning Disk Confocal Scanner mounted on an Olympus IX81 inverted fluorescent microscope (Olympus, Center Valley, PA) with a x40 dry objective and C4742 80 12AG Monochrome CCD camera (Hamamatsu Photonics, Bridgewater, NJ). Eight random images were taken from each micro environment of interest for each chamber. Each image was an average of a stack of 7 images taken at 1 µm apart in the z direction. Z stack images were analyzed using SlideBook software (Inte lligent Imaging Innovations, Inc., Denver, CO) and only raw image file were used to quantify intensity values. For each of the slides stained from each flow chamber

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124 experiment, an additional static culture slide was stained to act as a reference point in c ase slides could not be imaged immediately. Any of the raw intensity values from flow chamber experiment slides were normalized with respect to a reference static culture slide. This protocol, once established, was strictly adhered to for each experiment. Lastly, the final recalculated intensity values from each measurement were normalized with respect to the intensity values from the proximal straight segment for each sample. Animals All animal experimentation was performed in accordance with a protocol ap proved by our institution's Institutional Animal Care and Use Committee. At all times, the principles governing the care and treatment of animals, as outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication no. 85 323, revised 1996) and adopted by the American Physiological Society, were strictly adhered to during the course of this study. Mice had ad libitum access to water and standard mouse chow (or special diet as indicated) and wer e housed in a well ventilated, specific pathogen free, temperatu re controlled environment (24± 1 °C; 12 h 12 h light dark cycle). Murine Intracranial Aneurysm Model Murine intracranial aneurysms were created in female 8 12 week old C57BL/6 mice (Charles Riv er Laboratories, Wilmington, MA) using a method described previously. 114 Anesthesia was induced using ketamine/xylazine. Briefly, the left common carotid artery and the right renal artery are ligated to induce hypertension. One week l ater, an Alzet micro osmotic pump model 1004 (DURECT Corp, Cupertino, CA) is implanted subdermally to deliver Angiotensin II (Bachem AG, Switzerland) at 1000ng/kg/min; and 10 uL of 0.8% porcine elastase (Worthington Biochemical Corp,

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125 Lakewood, NJ ) in norm al saline is injected into the right basal cistern using stereotactic coordinates: 1.2 mm rostral of bregma, 0.7 mm lateral of midline and 5.3 mm ventral of the dorsal aspect of the skull. The animals are fed a hypertensive diet with 8% NaCl and 0.12% BAP N (Harlan Laboratories, Indianapolis, IN). For the antibody blockade, 100 g/mL mouse anti CXCL1/GRO alpha/KC/CINC 1 antibody (MAB453, R&D Systems, Minneapolis, MN) was injected retro orbitally two days before, on the day of, and every two days after aneur ysm induction. In control animals, 100 g/mL rat IgG2A Isotype Control antibody (MAB006, R&D Systems, Minneapolis, MN) was injected retro orbitally on the same schedule. For sham surgeries, the arteries were isolated but not ligated during the ligation pro cedure, and normal saline solution was injected instead of 0.8% elastase during aneurysm induction. Instead of Angiotensin II pump implant, normal saline was injected subdermally. Mice were euthanized 3 days, one week or two weeks after aneurysm induction. Quantification of Aneurysm Formation, Inflammatory Cell Infiltration and VCAM 1 Expression in CXCL1 Blockade At the end of the experimental period mice treated with IgG2A or anti CXCL1 were euthanized by 3 mL 4% PFA in PBS cardiac perfusion into the left ventricle followed by injection of 1 mL brilliant blue in 20% gelatin in PBS. The brilliant blue in gelatin injection was used for visualization of blood vessels to allow a blinded observer to determine saccular aneurysm formation in Circle of Willis (COW) from each mouse. The blinded observer did not perform any surgeries or treatments for this experiment. Representative images were taken using Leica dissection microscope with Volocity 3D analysis software . After recording aneurysm formation intact brains with COWs were fixed in 4% PFA for 24 hrs, and then transferred to 18% sucrose solution for 24 hrs.

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126 Tissues were then embedded in OCT and sectioned for staining and quantification of neutrophil and macrophage infiltration and VCAM 1 expression in endotheli al cells. The sections were mounted on numbered slides that did not directly identify the treatment regimen. The master sheet identifying the slides and the treatment regimen for each specimen was kept in a separate file in the laboratory. The following st ereology rules were used: for each aneurysm positive specimen, 3 sections 100 m apart through the aneurysm were taken; for aneurysm negative specimens, 3 sections 100 m apart were taken at the anterior cerebral artery (ACA) starting at the anterior commi ssure, the middle cerebral artery (MCA), and posterior cerebral artery (PCA). For both, aneurysm positive and negative specimens, the outcome measure was averaged from all sections taken for that specimen (either only aneurysm or total average of ACA and M CA and PCA sections) and representative images were taken. Sections were visualized using For each section, the number of inflammatory cells and VCAM 1 positive endothelial cells were counted. The observer performing inflammatory cell infiltration and VCAM 1 expression by endothelial cells did not have access to the master sheet during quantification. The outcome measure was quantified relative to vessel wall or aneurysm wall area. The visualization of immunohistochemistry slides and quantification of vesse l wall or aneurysm wall area was performed on an Olympus IX71 inverted fluorescent scope with QImaging Retiga 2000R CCD camera using Image Pro Plus software (MediaCybernetics, Silver Spring, MD) .

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127 Human Aneurysm and Superficial Temporal Artery Specimens C ollection of human cerebral aneurysm and superficial temporal artery specimens was performed in accordance to a research protocol approved by our Institutional Review Board (IRB). Patients signed informed IRB research consent before aneurysm and superfici al temporal artery specimens were harvested at the time of aneurysm clipping surgery. The collected specimens were then immediately fixed in 4% paraformaldehyde. Immunohistochemistry of Mouse and Human Aneurysm Specimens Murine aneurysm specimens were fi rst fixed in 4% PFA for 24 hours, and then dehydrated in 18% sucrose solution. Tissues were mounted in Tissue Tek OCT antigen retrieval in Dako Target Retrieval Solution (Da ko, Carpinteria, CA) was performed for all murine immunohistochemistry studies. Following a block in 2% normal horse serum (S 2000, Vector Labs) for 1 hour, the specimens were incubated with rat anti MECA 32 antibody (BD 550563, BD Biosciences, San Jose, CA) to visualize endothelial cells, rabbit anti CXCL1/GRO antibody (ab86436, Abcam), goat anti mouse CXCL2/GRO /MIP 2 antibody (AF 452 NA, R&D Systems), rabbit anti mouse CXCL5 6/LIX antibody (500 P146, Peprotech, Rocky Hill, NJ), rat anti neutrophil antibody [NIMP R14] (ab2557, Abcam), rat anti mouse F4/80 (MCA497R, AbD Serotec, Düsseldorf, Germany) to visualize macrophages, rabbit a nti iNOS antibody (ab15323 , Abcam ) , goat anti Arginase I antibody ( ab60176 , Abcam), rabbit anti CXCR2 (ab14935, Abcam) or rabbit VCAM 1 antibody (sc 8304, Santa Cruz, Dallas, TX ) overnight at 4 o C, and washed. For immunohistochemistry of human aneurysm samples, the tissues were

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128 fixed in 4% PFA and embedded in paraffin. After were de paraffinized by xylene and ethanol. Heat mediated antigen retrieval in 10 M Sodium Citrate buffer pH 6.5 for all samples was performed. The staining protocol for CXCL1/GRO , CXCR2 and VCAM 1 was then foll owed exactly as for immunohistochemistry of mouse aneurysm specimens except that mouse anti human CD31 antibody (IR61061 2, Dako) was used in place of MECA 32 to visualize the endothelial cell layer, rabbit anti neutrophil elastase antibody (ab68672, Abcam ) was used to visualize neutrophils, and mouse anti CD68 antibody [KP1] (ab955, Abcam) was used to visualize macrophages. To visualize IL 8, mouse anti human IL 8 antibody (ab18672, Abcam) was used. The secondary antibodies used were Alexa Fluor 488 donke y anti rat antibody (A 21208, Life Technologies), Alexa Fluor 488 donkey anti rabbit antibody (A 21206, Life Technologies), Alexa Fluor 568 donkey anti rabbit antibody (A 21206, Life Technologies), and Alexa Fluor 594 donkey anti mouse antibody (A 21203, Life Technologies) and were incubated for 1 hour at room temperature. Finally, for both murine and human specimens, nuclei were counterstained with DAPI (H 1200, Vector Labs, Burlingame, CA). Quantification of Aneurysm Formation, Inflammatory Cell Infiltr ation and VCAM 1 Expression in CXCL1 Blockade At the end of 2 week period mice treated with IgG2A or anti CXCL1 were euthanized by 3 mL 4% PFA in PBS cardiac perfusion into the left ventricle followed by injection of 1 mL brilliant blue in 20% gelatin in PBS. The brilliant blue in gelatin injection was used for visualization of blood vessels to allow a blinded observer to determine saccular aneurysm formation in Circle of Willis (COW) from each mouse. The blinded observer did not perform any surgeries or t reatments for this experiment.

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129 Representative images were taken using Leica dissection microscope with Volocity 3D analysis software. After recording aneurysm formation intact brains with COWs were fixed in 4% PFA for 24 hrs, and then transferred to 18% su crose solution for 24 hrs. Tissues were then embedded in OCT and sectioned for staining and quantification of neutrophil and macrophage infiltration and VCAM 1 expression in endothelial cells. The sections were mounted on numbered slides that did not direc tly identify the treatment regimen. The master sheet identifying the slides and the treatment regimen for each specimen was kept in a separate file in the laboratory. The following stereology rules were used: for each aneurysm positive specimen, 3 sections 100 m apart through the aneurysm were taken; for aneurysm negative specimens, 3 sections 100 m apart were taken at the anterior cerebral artery (ACA) starting at the anterior commissure, the middle cerebral artery (MCA), and posterior cerebral artery (PCA). For both, aneurysm positive and negative specimens, the outcome measure was averaged from all sections taken for that specimen (either only aneurysm or total average of ACA and MCA and PCA sections) and representative images were taken. Sections were visua lized using For each section, the number of inflammatory cells and VCAM 1 positive endothelial cells were counted. The observer performing inflammatory cell infiltration and VCAM 1 expression by endothelial cells did not have access to the master sheet dur ing quantification. The outcome measure was quantified relative to vessel wall or aneurysm wall area. The visualization of immunohistochemistry slides and quantification of vessel wall or aneurysm wall area was performed on an Olympus IX71 inverted fluores cent scope with QImaging Retiga 2000R CCD camera using Image Pro Plus software (MediaCybernetics, Silver Spring, MD).

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130 Results Low Hemodynamic Shear Stress and Shear Stress Gradients in Bifurcation and Bifurcation Aneurysm Flow Chamber Lead to Increased End othelial Expression of Inflammatory Mediators We evaluated the inflammatory conditions within three different flow chamber models (bifurcation aneurysm, straight artery, and arterial bifurcation, n=3 each) by using cytokine arrays and quantifying the level s of 40 different cytokines secreted by HUVECs subjected to pulsatile flow. Multiple markers were found to be upregulated in the bifurcation and bifurcation aneurysm flow chamber models such as sICAM 1, IFN , IL 1 , IL 8, IL 10, IL 16, MCP 1, MCP 2, M CSF, MIP 1 , RANTES, and TNF , when compared to the straight artery flow chamber model (Fig ure 3 1 ). IL 8 was the most differentially expressed cytokine across the three flow models (Fig ure 3 1 ). Low Hemodynam ic Shear Stress and Shear Stress Gradients at the Aneurysm Region Promotes Increased Protein Expression of CXCL8/IL 8 Analysis of perfusing medium from shear stress experiments by ELISA revealed that IL 8 secretion was significantly different among the th ree systems studied (p=0.0238 by ANOVA). Significantly more IL 8 was secreted in the bifurcation aneurysm flow chamber (269.5 pg/mL, n=5) than the bifurcation (146.8 pg/mL, n=5, p=0.0395) or straight flow chamber (147.5 pg/mL, n=5, p=0.0409) systems (by pa irwise t test with ure 3 2A ). The levels between bifurcation and straight flow chambers were not found to be significantly different (p=0.9998). We compared IL 8 RF ICC at different microenvironment sites within the bifurcation and th e bifurcation aneurysm flow models to determine whether the specific bifurcation or aneurysm microenvironment locales exposed to shear stress gradients are prone to inflammation.

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131 Within the bifurcation flow model, IL 8 expression was not significantly di fferent among the bifurcation, the proximal straight segment (1.12 vs 1.00 RFU, p=0.55), or the branching limbs (1.12 vs 1.08, p=0.195) microenvironements (n=24 for each group, p=0.65 by mixe d effects linear model, Figure 3 2B). We confirmed that the end othelial cells in the aneurysm sac region of the bifurcation aneurysm flow chamber were responsible for elevated levels of IL 8. Relative fluorescence ICC (RF ICC) and statistical analysis were performed using a mixed effects linear model (the lme() functi on in the R package nlme). Analysis demonstrated the overall effect of location on CXCL8/IL 8 protein expression in the bifurcation aneurysm flow chamber was highly significant (n=24 for each group, p<0.0001) (Fig ure 3 2 C). IL 8 protein expression in the aneurysm sac was estimated to be 33 % higher than at the proximal straight segment (1.28 vs 1.00 RFU, p<0.0001), 15 % higher than at the branching arms (1.28 vs 1.13 RFU, p=0.001) and not significantly different than at the bifurcation (1.28 vs 1.13 RFU, p=0.144). IL 8 expression at the proximal straight segment was estimated to be 26 % lower than at the bifurcation (1.00 vs 1.12, p<0.001) and 18 % lower than at the branching arms (1.00 vs 1.12 RFU, p<0.001). Finally, it was estimated that IL 8 was 8% hig her at the bifurcation than at the branching arms (1.13 vs 1.12 RFU, p=0.009). These findings confirmed that the aneurysm sac micro environment was the primary contributor of increased IL 8 in the circulating medium in the bifurcation aneurysm flow chamber model.

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132 Low Hemodynamic Shear Stress and Shear Stress Gradients at the Bifurcation and Aneurysm Regions Promote Increased Protein Expression of CXCL1/GRO Analysis of 20x concentrated perfusing medium from three different flow systems by ELISA revealed t hat CXCL1/GRO secretion was significantly different across the three groups (p=0.0003 by Kruskal Wallis test). Significantly more CXCL1/GRO was secreted by endothelial cells in the bifurcation flow chamber (34.3 pg/mL, n=7) than in the straight flow ch amber (14.4 pg/mL, n=9, p=0.003 by Mann Whitney pairwise comparison). CXCL1/GRO levels in the bifurcation aneurysm flow chamber (64.8 pg/mL, n=7) were found to be significantly higher than in the straight flow chamber (p=0.0006) system. The levels betwee n bifurcation and bifurcation aneurysm chambers were not found to be significantly different (p=0.477) (Fig ure 3 2 D). Differential CXCL1 protein expression at the three microenvironments (bifurcation, proximal segment, branching limbs) within the bifurca tion flow model was highly significant (n=24 for each group, p<0.0001 by mixed effects linear model)(Figure 3 2E ). CXCL1 expression at the bifurcation was estimated to be 16% higher than at the proximal straight segment (1.16 vs 1.00 RFU, p<0.001), and 7% higher than at the branching limbs (1.16 vs 1.08, p=0.039). CXCL1 expression at the proximal straight segment was estimated to be 8% lower than at the branching limbs (1.00 vs 1.08 RFU, p=0.001). These findings confirmed that the bifurcation micro environ ment was the primary contributor of increased CXCL1 in the circulating medium in the bifurcation flow chamber model. CXCL1/GRO protein expression analyzed by RF ICC in the bifurcation aneurysm flow chamber by location was also highly significant (n=24 for each group,

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133 p<0.0001 by mixed effects li near model) as shown in Figure 3 2F . CXCL1 expression at the aneurysm sac was estimat ed to be 29 % higher than at the proximal straight segment (1.44 vs 1.00 RFU, p<0.001), 16 % higher than at the bifurcation (1.44 vs 1.23 RFU, p<0.001) and 10 % higher than at the branching arms (1.44 vs 1.25 RFU, p=0.002). CXCL1 expression at the proxim al straight segment was estimated to be 14 % lower than at the bifurcation (1.00 vs 1.23 RFU, p<0.001) and 19 % lower than at the branching arms (1.00 vs 1.25 RFU, p<0.001). No differences were found in CXCL1 expression between the bifurcation and the arm s (p=0.224). This confirmed that the aneurysm sac micro environment was the primary contributor of increased CXCL1 in the circulating medium in the bifurcation aneurysm flow chamber model. ELR+ CXC Chemokines IL 8 and CXCL1 Are Expressed in Human Aneurysm s We studied IL 8 and CXCL1 expression in human aneurysm specimens. Of the specimens analyzed, all had intact endothelium lining the dome and were positive for IL 8 (n=5/5). As control, superficial temporal arteries (STAs) were analyzed. Out of 3 STAs anal yzed, none exhibited endothelial staining for IL 8 (n=0/3) (Figure 3 3A). Of the aneurysm specimens analyzed, all (n=5/5) were positive for CXCL1/GRO (5/5), while control superficial temporal arteries (STA) exhibited no endothelial staining for CXCL1/GRO (n=0/3) (Figure 3 3B). CXCL1/GRO is the Primary ELR+ CXC Chemokine Expressed in Murine Cerebral Aneurysms Since mice do not express CXCL8/IL 8, we decided to analyze the expression of ELR+ CXC chemokines, CXCL 1, 2, and 5 6/LIX in mouse 2 week aneur ysms (Fig ure 3 4A ). Consistent antibodies against CXCL3 are not available for use in mice. ELR+ CXC chemokines attract neutrophils and thus act as functional homologues of IL 8 in the

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134 mouse species. 247 Out of 5 tissues analyzed, CXCL1/GRO was expressed in the intima and the media of 2 week (5/5) aneurysms and aneurysmal vessels (Fig ure 3 4B ), whereas CXCL2/GRO and CXCL5 6/LIX did not show any signal (0/5 for both). Furthermore, CXCL1 was expressed in 3 day and 1 week aneurysmal tissues (Fig ure 3 4C ). In all cases the endothelial cells were visualized with MECA 32. In control arteries from sham animals, no CXCL1 expression was observed (0/5, 0/5, 0/5) (Fig ure 3 4C ). Moreover, CXCL1 expression in murine aneurysms was highly associated wi th inflammatory cell infiltration at one week (Figure 3 5) suggesting that CXCL1 expression might have a role in inflammatory cerebral aneurysm formation . In particular there was a high correlation between CXCL1 expression and neutrophil infiltration (p=0. 0001, R 2 =0.79) (Figure 3 5A and B) , and a weak moderate correlation between CXCL1 expression and monocyte and macrophage infiltration ( p=0.0002, R 2 =0.59) (Figure 3 5C and D). CXCL1 Blockade Reduces Aneurysm Formation in Mouse Intracranial Aneurysm Model by Preventing Inflammatory Cell Infiltration Mice treated with anti CX CL1 antibody every 48 hrs (Figure 3 6 A) developed significantly less saccular aneurysms than IgG treated control mice two weeks after aneurysm induction (13.3% vs 66.7%, p=0.0078 by Fisher (Fig ure 3 6 B and C). CXCL1 neutralizing antibody over two weeks also resulted in significantly less severe aneurysms ( average grade of 1.3 vs 2.4, p=0.0046 by Mann Whitney Test , n=15 each ) than in IgG2A treated mice (Figure 3 7A a nd B) . Mice treated with 100 g/mL CXCL 1 neutralizing antibody also appeared to be less likely to suffer from rupture and SAH associated death (15.8 vs 48.0%) than IgG2A

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135 treated mice during the experimental time period , however the sample size was not large enough to use Kaplan Meier s urvival curve analysis (Figure 3 8) . Neutrophil infiltration in the anti CXCL1 treated mice was significantly decreased at 2 weeks when compared with IgG control treated mice (435 vs 2410 cells/mm 2 , n=15 for both, p=0.0 43 by Mann Whit ney test) (Figure 3 9 A and B). We quantified macrophage infiltration since neutrophils have been shown to attract these inflammatory cells during remodeling. 127 Macrophage infi ltration in the anti CXCL1 treated mice was not found to be significantly different at 2 weeks when compared with IgG control treated mice (7163 vs 9496 cells/mm 2 , n= 15 for both, p=0.056 by Mann Whitney test) (Fig ure 3 9 C and D). In addition to endothelia l and mural cells, infiltrating neutrophils were found to be positive for CXCR2, the receptor for CXCL1. All human aneurysm specimens analyzed were found to be positive for neutrophils and ma crophages (n=5/5, Figure 3 9 E), while control STAs were negative (n=0/3). Infiltrating macrophages in anti CXCL1 and IgG treated aneurysms were not found to have a significantly different balance between M1 and M2 phenotype s (n=6 for all, c p=1.0, r p=1.0, r x c p=0.37 by Two way ANOVA) (Figure 3 10A and B) . Hemodynamic Shear Stress at the Aneurysm Region Promotes Increased Protein Expression of VCAM 1 VCAM 1 staining in the endothelial cells in the anti CXCL1 treated mice was significantly decreased (Figure 3 11 A) at 2 weeks when compared with IgG control treated mice (33.4% vs 76.4% positive cells/section, n=15 for both, p<0 .00 1 by Mann Whitney test, Figure 3 11 B). VCAM 1 protein expression in the bifurcation aneurysm flow chamber by location was highly significant (n=24 for each group, p<0.001 by mixed effects linear model)

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136 (Figure 3 11 C). VCAM 1 expression at the aneurysm sac was estimated to be 51% higher than at the proximal straight segment (1.53 vs 1.00 RFU, p<0.001), 10 % higher than at the bifur cation (1.53 vs 1.38 , p<0.001) and 25% higher than at the branching VCAM 1 expression at the proximal straight segment was estimated to be 41% lower than at the bifurcation (1.00 vs 1.38 RFU, p<0.001) and 25% lower than at the branching arms (1.00 vs 1.27 RFU, p<0.001). VCAM 1 expression at the bifurcation was estimated to be 16% higher than at the arms (1.38 vs 1.27 RFU, p<0.001). Similarly, human aneurysm specimens were positive for VCAM 1. Of the human aneurysm specimens analzyed, each w as strongly positive for VCAM 1 (5/5), while control superficial temporal arteries (STA) exhibited only weak or no endothelial stain ing for VCAM 1 (n=0/3) (Figure 3 11 D). Discussion Role of shear stress in aneurysm formation and growth Low or oscillatory s hear stress such as the one found in arterial bifurcations, stenosed vessels, or atherosclerotic vessels results in pro inflammatory phenotype 75 through upregulation of NF and IL 8 via MAPK 229 . Low wall shear stress increases levels of pro inflammatory mediators such as MCP 1 leading to vascular atheros clerosis. 145 , 163 Once an aneurysm forms, a change occurs in the local blood flow and shear stress pattern re sulting in a low shear stress micro environment within the dome. 77 , 80 , 135 Further changes to this WSS patter n occur during aneurysm growth causin g pathological remodeling. 77 , 135 Recently, aneurysm growth has been shown to occur in regions of low WSS of the aneurysm dome. 77 , 135

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137 Low level shear stress can induce expression of ELR+ chemokines , primarily IL 8 and CXCL1/GRO . 413 Such conditions lead to higher propensity for endothelial binding of platelets and circulating leukocytes through upregulation of intercellular adhesion prot eins such as ICAM and VCAM 1 and selectins, allowing for inflammatory cell infiltration. 160 , 402 In our ex periments, the bifurcation and aneurysm sac regions of the flow chambers exposed to low a verage hemodynamic shear stress and shear stress gradients were characterized by a much higher expression of ELR+ chemokines, especially IL 8, when compared to the pro ximal straight segment and the distal branching arms micro environments. IL 8 is a chemokine with a powerful ability to attract neutrophils to sites o f inflammation. 260 IL n has been described in stroke 414 and acute vascular injury. 415 IL 8 has been shown to cause endothelial retraction allowing for increased vascular permeability and penetration by proinflammatory cells and cytokines. 416 , 417 Upregulation of IL 8 has been shown in vascular smooth muscle cells of sp ontaneously hypertensive rats. 418 It has also been described in vascular smooth muscle cell (VSMC) proliferation and secretion of MMPs capabl e of causing local remodeling. 419 We also observed increased expression of CXCL1/GRO in the bifurcation and aneurysm sac region of the bifurcation and circulating medium was not as robust as IL 8 for sheared human endothelial cells. Role of ELR+ CXC Chemokines in Inflammation and Cerebral Aneurysm Formation Although IL 8 is not present in the mouse species, there are several murine functional homologues that also attract neutrophils. Within the C XC family, it is the

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138 chemokines with ELR+ (Glu Leu Arg) motif that are capable of attracting neutrophils. 127 , 420 We stained 2 week murine cerebral aneurysms for ELR+ CXC chemokines CXCL1, CXCL2 and CXCL5 6/LIX and determined that CXCL1 was the primary CXC chemokine expressed in murine aneurysms at this early time point. CXCL1 was expressed at 3 day and 1 week time points following aneurysm induction. CXCL1 has been described as a functional murine homologue of human CXCL8/IL 8. 247 CXCL1 shares more than 60% of amino acid sequence with IL 8 and activate s the same receptor CXCR2. 421 , 422 In addition, both IL 8 and CXCL1 are sec reted upon Pro staglandin E2 stimulation. 423 , 424 Murine chemokine CXCL1 has been described to have function in infla mmation, neutrophil chemotaxis, and angiogenesis, similar to human IL 8. 247 , 260 , 413 , 414 , 416 , 419 , 423 , 424 CXCL1 and its receptor CXCR2 have been described as centra l in advanced atherosclerosis. 425 Recently, CXCL1 was descr ibed to have a critical role in rode nt models of encephalomyelitis 426 and ischemic stroke. 427 CXCL1 has the capability to arrest monocytes on atherosclerotic endothelium by inc reasing VCAM 1 but not ICAM 1. 248 Decreased VCAM 1 expression in anti CXCL1 treated mice could account for the marginal decrease in macrophage infiltration in our experiments. Blockade of ICAM 1/CD18 interactions accounts only for 60 to 9 0% of neutrophil infiltration. 125 VCAM 1 has a significant role in neutrophil binding to the activated endoth elium in CD18 null mice 125 and in rat model of arthritic joint and dermal inflammation. 126 In our model, in the IgG treated control group, infiltrating neutrophils were CXCR2 positive confirming th at they were recruited by CXCL1. Neutrophils are cells of the innate immune system that have a role in multiple acute and chronic inflammatory diseases. 426 428 Once activated

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139 by cytokines such as CCL1 and/or CXCL1, neutrophils release pro inflammatory cytokines, matrix degrading MMPs and reactive oxygen species (ROS), and attract monocytes via azurocidin, cathepsin G, and LL37 contribut ing to the local inflammation. 127 Neutrophil depletion has been shown to decrease plaque formation in atherosclerosis. 302 Although neutrophils have been observed in human cerebral aneurysms specimens before by immunohistochemistry, thei r role has not been elucidated. 12 In this work, we show that neutrophil recruitment via shear stress mediated endothelial CXCL1 promotes early cerebral aneurysm formation and progression (Figure 3 12) .

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140 Figure 3 1 . Cytokine array from shear stress experiments. ELR+ chemok ines are the most abundantly expressed inflammatory cytokines in the bifurcation aneurysm flow chamber system. HUVECs were exposed to pulsatile flow at 10 dyne/cm 2 for 24 hrs in straight, bifurcation, and bifurcation aneurysm flow chambers. Out of 40 infla mmatory cytokines analyzed in the perfusing media fo r each of the flow chambers, IL 8 was the most abundant differentially expressed cytoki ne in the aneurysm flow chamber. Data are presented as mean ± s.e.m.

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141 Figure 3 2. ELR+ chemokines IL 8 and CXCL1 are increased in the bifurcation aneurysm flow chamber. A) Cytokine array results were confirmed by IL 8 ELISA (p=0.0238). B) IL 8 expression in the bifurcation flow chamber is not significantly different across different environments (p=0.065). C) HUVECs exp ress more IL 8 in the aneurysm and bifurcation regions of the bifurcation aneurysm flow chamber than other regions. (p<0.0001, representative images for each environment are shown, scalebar=10 m). D) HUVECs express more CXCL1, an ELR+ cytokine closely rel ated to IL 8, in the bifurcation and bifurcation aneurysm flow chamber models than in the straight flow chamber model (p=0.0076). E) HUVECs express more CXCL1 in the bifurcation region of the bifurcation flow chamber (p<0.0001). F) HUVECs express more CXC L1 in the aneurysm sac of the bifurcation aneurysm flow chamber than other regions (p<0.0001, representative images for each environment are shown, scalebar =10 m). (* p<0.05,** p<0.01,*** p<0.001,**** p<0.0001). Data are presented as mean±s.e.m.

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142 Figure 3 3 . ELR+ chemokin e expression in human aneurysm specimens . A) Endothelial cells (CD31+) in human aneurysm tissues are positive for IL 8 (arrows) whereas endothelial cells in control superficial temporal arteries (STA) are negative ( Blue : DAPI, Green : CD31, Red : IL 8, scalebar = 10 m). B) Endothelial cells (CD31+) in human aneurysms are positive for CXCL1 (arrows) whereas endothelial cells in contr ol STAs are negative. ( Blue : DAPI, Green : CD31, Red : CXCL1, scalebar = 10 m).

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143 Figure 3 4 . ELR+ chemokin e expression in murine aneurysm specimens . A ) Two week mouse aneurysm specimens are (scale bar = 2 mm) B ) positive for ELR+ chemokine CXCL1 b ut not CXCL2 or CXCL5 6. ( Blue : DAPI, Green : CD31, Red : CXCL1, CXCL2 or CXCL5 6, scalebar = 100 m) E) CXCL1 expression in mouse aneurysmal vessels, developing aneurysms and aneurysm specimens is present in the media at 3 days, 1 and 2 weeks, while arteri es from sham mice are negative. ( Blue : DAPI, Green : CD31, Red : CXCL1, scalebar = 10 m).

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14 4 Figure 3 5 . CXCL1 expression is associated with i nflammatory cell infiltration. 1 week mouse aneurysms that have higher CXCL1 expression are infiltrated by more A) and B) neutrophils and C) and D) macrophages. ( Blue : DAPI, Green : ANA or CD11b, Red : CXCL1, scalebar = 10 m).

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145 Figure 3 6 . CXCL1 blockade prevents mouse intracranial aneurysm formation . A) Experimental scheme used to study the role of CXCL1 in an eurysm formation. B) Representative pictures of Circle of Willis from IgG and anti CXCL1 treated mice (scalebar = 2 mm). C) Mice treated with 100 g/mL CXCL1 neutralizing antibody over two weeks develop significantly less aneurysms (13.3% vs 66.7%, p=0.00 78, n=15 for both) than IgG2A treated mice. means. * p<0.05, ** p <0.01.

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146 Figure 3 7 . CXCL1 blockade prevents mouse intracranial aneurysm progression . A ) Mice treated with 100 g/mL CXCL1 neutralizing antibody over two weeks develop significantly less severe aneurysms ( average grade of 1.3 vs 2.4, p=0.0046 by Mann Whitney Test , n=15 for both) than IgG2A treated mice. B) Aneurysm Grade: 0 = normal, 1 = aneurysmal, 2 = aneurysm, 3 = two or more aneurysms, 4 = rupture d aneurysm. 0 1 2 3 4

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147 Figure 3 8 . Aneurysm rupture in anti CXCL1 and IgG2A treated mice. Mice treated with 100 g/mL CXCL 1 neutralizing antibody appear to be less likely to suffer from rupture and SAH associated death (15.8 vs 48.0%) than IgG2A treated mice during the experimental time period. Sample size: anti CXCL1 n=19, IgG2A n=25.

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148 Figure 3 9. Effects of CXCL1 blockade on inflammatory cell infiltration. CXCL1 blockade with 100 g/mL of anti CXCL1 antibody over two we eks results in A and B) significantly less neutrophil infiltration (435 vs 2410 cells/mm 2 ,arrows, n=15 for both, p=0.043) ( Blue : DAPI, Green : NIMP R14, Red : CXCR2, scalebar = 10 m). C and D) No significant differences in macrophage infiltration were foun d (7163 vs 9496 cells/mm 2 , arrows, n= 15 for both, p=0.056) ( Blue : DAPI, Green : F4/80, Red : CXCR2, scalebar = 10 m). E) Human aneurysm specimens are positive for infiltrating neutrophils (open arrows) and macrophages (full arrows) while control STAs are n egative ( Blue : DAPI, Green : neutrophil elastase, Red : CD68, scalebar = 10 m).

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149

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150 Figure 3 10. Macrophage phenotype in CXCL1 blockade in mouse 2 week intracranial aneurysms. A) Representative immunohistochemistry image s of macrophage phenotype in mouse aneurysms. Scalebar = 20 m. B) There is no difference in the ratio of M1 to M2 phenotype of macrophages in anti CXCL1 and IgG treated mice (r p=1.0, c p=1.0, r x c p=0.37) . M1 marker = iNOS, M2 marker = Arg I. n=24 total , n= 6 for each . Data presented as simple arithmetic means. Two way ANOVA with Tukey HSD. *p<0.05.

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151 Figure 3 11 . CXCL1 blockade decreases endothelial VCAM 1 expression. A and B) VCAM 1 expression in mouse aneurysms is significantly decreas ed in anti CXCL1 treated mice at two weeks (33.4% vs 76.4% positive cells/section, arrows, n=15 for both, p<0.001) ( Blue : DAPI, Green : MECA32, Red : VCAM 1, scalebar = 10 m). C) HUVECs express more VCAM 1 in the aneurysm sac of the bifurcation aneurysm flow chamber than other regions (* p<0.05,** p<0.01,*** p<0.001,**** p<0.0001, representative images for each environment are shown). D) Endothelial cells (CD31+) in human a neurysm tissues (arrows) are positive for VCAM 1 (arrows) whereas endothelial cells in control STAs are negative. ( Blue : DAPI, Green : CD31, Red : VCAM 1, scalebar = 10 m).

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152 Figure 3 12 . Proposed mechanism by which hemodynamic changes lead to aneu rysm formation, progression and rupture. Hemodynamic shear stress and shear stress gradients cause initial endothelial dysfunction by increasing proliferation and apoptosis, which leads to degeneration of the IEL. Once an aneurysm develops, the hemodynamic s change and the endothelial cells within the dome are then exposed to low shear stress leading to increased secretion of inflammatory mediators such as IL 8 (human) or CXCL1 (human and mouse). This leads to upregulation of VCAM 1 and causes inflammatory c ell infiltration, primarily neutrophils, in the low shear stress region of the dome, further degeneration of the vascular wall and eventual rupture.

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153 CHAPTER 4 PROTECTIVE EFFECTS OF PPAR AGONISTS IN CEREBRAL ANEURYSM FORMATION Introduction Under Specific Aim 2 , w e have shown strong data that suggests early shear stress mediated inflammation in cerebral aneurysms is facilitated by IL 8 and / or CXCL1. Higher CXCL1 expression in mouse intracranial aneurysms is associated with higher i nfiltration by neutrophil s and macrophages. IL 8 is a chemokine that has originally been described in its ability to attract neutrophil s to sites of inflammation. 260 , 416 , 417 , 419 , 421 , 4 29 , 430 Vascular smooth muscle cells (VSMCs) prolife rate and migrate towards IL 8, 430 while se creting pro inflammatory PGE2 419 and mat rix metalloproteinases (MMPs) 431 capable of causing local rem odeling. Early VSMC proliferation and MMP secretion have been previously describe d in human aneurysm studies. 12 , 29 , 73 , 82 , 98 Although IL 8 is not expressed by mouse cells, its murine homologue CXCL1 (GRO 1, KC, or NAP 3) shares more than 60% of amino acid sequence, activates the same receptor CXCR2 and the two cytokines are functional homologues. 248 , 416 419 , 423 , 426 , 430 , 431 Fina lly, we showed that blockade of CXCL1 with a neutralizing antibody in the mouse elastase based intracranial aneurysm model leads to significantly less a neurysm development and rupture than in IgG treated control mice . Clearly, early inhibition of the IL 8/ CXCL1 signaling is beneficial in preventing aneurysm formation and rupture. However, a therapy that prevents the influx of new inflammatory cells and allows for modulation of their phenotype to induce tissue healing would be superior to a single cytokine b lockade. PPARs are a family of nuclear receptors ( , , and ) that regulate transcription of genes and play a role in cell differentiation, metabolism, indirect response to shear

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154 stress and inflammation . 52 , 322 , 354 , 367 , 372 , 375 , 383 , 391 , 400 The PPARs are localized to cell cytoplasm , and upon activation, translocate to the nucleus where they can exert their effects on gene expression. 322 Recently, the three peroxisomal proliferator activating receptor (PPAR) pathwa ys ( , and ) have been implicated in having a role in cerebrovascular developmental abnormalities, 354 and shown to have profound effects on the inflammatory sta te of macrophages, 326 , 355 , 432 which are the primary immune cells found in aneurysms. 12 , 72 , 83 In fact, PPAR activation has been shown to drive macrophages into the M2 anti inflammatory phenotype. 326 Next, the PPAR pathway has been shown to regulate the inflammatory s tate of smooth muscle cells via the ligands secreted by endothelial cells due to shear stress. 52 In addition, several studies have shown that PPAR dysfunction plays a role in progression of atherosclerosis and development of abdominal aortic aneurysms. 326 , 355 , 379 , 432 Specific Aim 3 was designed to study the potential of treatment with PPAR agonists , which could be used to prevent cerebral aneurysm formation and rupt ure by decreasing 1) IL8/CXCL1 secretion and adhesio n/influx of inflammatory cells , 2) smooth muscle cell proliferation, and 3) by mitigating the effects of M1 pro inflammatory macrophages. In addition, we hypothesized that PPAR agonist treatment would 4) imp rove endothelial cell function by decreasing the inflammatory profile and 5) indirectly (PPAR ) or directly (PPAR ) cause macrophages to switch to M2 anti inflammatory, pro wound healing phenotype 326 within the aneurysm lesion.

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155 Methods PPAR Agonist Screen Using Cytokine Array and ELISAs Levels of 40 different human inflammatory factors in the perfusing medium from the bifurcation aneurysm flow chambers were m easured using the Human Inflammation Array C3 (AAH INF 3, RayBiotech, Norcross, GA). Shear stress experiments were performed as described earlier. HUVECs were exposed to pulsatile flow in the bifurcation aneurysm flow chamber with either 5 M rosiglitazone ( 71742 , Cayman Chemical) , 25 M Wy 14 , 643 ( 70730 , Cayman Chemical) , 30 nM GW501516 (Cayman Europe, Tallinn, Estonia) , 5 M rosiglitazone and 30 nM GW501516, or 0.1% 1:3 DMSO:PBS ( carrier vehicle control ) over 24 hrs. Quantification of in tensity (integrated density) values was performed using the MicroArray Profile plugin for ImageJ. In addition, IL 8 levels in the perfusing medium were quantified using the Human CXCL8/IL 8 Quantikine ELISA Kit (D800C, R&D Systems, Minneapolis, MN). Immun ohistochemistry of PPAR Expression on CXCL1 Challenge Based on the promising results obtained cytokine arrays following PPAR agonist treatment, we decided to study the relationship between CXCL1 and PPAR . HUVECs were grown in Nunc Lab Tek II Tissue Culture slides (154534, T hermo Fisher Scientific ) and the cells were exposed to CXCL1 at 0, 10, and 100 ng/mL CXCL1 ( 453 KC 050 , RnD Systems, Minneapolis, MN ) with or without 25 uM Wy14643 (PPAR agonist) for 24 hrs. Cells were then stained for PPAR using anti PPAR antibody ( ab8934 , Abcam) . Visualization of immunohistochemistry was performed on an Olympus IX71 inverted fluorescent scope with QImaging Retiga 2000R CCD camera using Image Pro Plus software .

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156 Relative Fluorescence Immunocyto chemistry of PPAR Expression on CXCL1 and IL 8 Challenge Based on the promising results obtained from simple immunohistochemistry, we proceed to study dose dependent relationship between CXCL1 or IL 8 and PPAR a. HUVECs were grown in Nunc Lab Tek II Tissue Culture slides (154534, Thermo Fishe r Scientific) and the cells were exposed to CXCL1 (453 KC 050, RnD Systems) or IL 8 ( 618 IL 050, RnD Systems) at 0, 10, and 100 ng/mL with or without 25 uM Wy 14 , 643 (PPAR agonist) for 24 hrs. Cells were then stained for PPAR using anti PPAR antibody (ab8934, Abcam). Relative protein expression was quantified using a DSU Spinning Disk Confocal Scanner mounted on an Olympus IX81 inverted fluorescent microscope (Olympus) with a 40x dry objective and C4742 80 12AG Monochrome CCD camera (Hamamatsu Photonics) as described earlier. Immunohistochemistry of PPAR Expression in Bifurcation Aneurysm Flow Chamber Model Shear stress experiments using bifurcation aneurysm flow ch ambers were performed as described earlier. HUVECs were exposed to pulsatile flow in the bifurcation aneurysm flow chamber over 24 hrs. Cells were fixed in ice cold methanol (Sigma Aldrich) for 5 minutes, washed twice in PBS and then stored in PBS at 4 o C for immunocytochemistry. Cells were then stained within 3 days for PPAR using anti PPAR antibody (ab8934, Abcam). In Vitro CXCR1 /CXCR2 and CXCR2 Blockade and PPAR Expression HUVECs grown in Nunc Lab Tek II Tissue Culture slides (154534, Thermo Fisher Scientific ) were exposed to 100 ng/mL CXCL1 or IL 8 wi th or without CX CR2 antagonist SB225002 ( 13336 , Cayman Chemical) or CXCR1/2 antagonist reparixin ( HY 15251 ,

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157 MedChem Express, Monmouth Junction, NJ ) in 1:3 DMSO:PBS for 24 hrs. At the end of the experiment, cells were stained for PPAR using anti PPAR antibody ( ab8934 , Abcam). Expression was quantified using confocal microscopy as described for the similar experiments performed for Specific Aim 1 and 2 . Animals All animal experimentation was performed in accordance with a protocol approved by our institution's Institutio nal Animal Care and Use Committee. At all times, the principles governing the care and treatment of animals, as outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication no. 85 323, rev ised 1996) and adopted by the American Physiological Society, were strictly adhered to during the course of this study. Mice had ad libitum access to water and standard mouse chow (or special diet as indicated) and were housed in a well ventilated, specifi c pathogen free, temperatu re controlled environment (24± 1 °C; 12 h 12 h light dark cycle). Immunohistochemistry of Mouse Aneurysm Specimens Murine aneurysm specimens obtained from prior experiments (from the non treated control group) in Specific Aim 2 wer e first fixed in 4% PFA for 24 hours, and then dehydrated in 18% sucrose solution. Tissues were mounted in Tissue Tek OCT antigen retrieval in Dako Target Retrieval Solution (Dako, Carpinteria, CA) was performed for all murine immunohistochemistry studies. Following a block in 2% normal horse serum (S 2000, Vector Labs) for 1 hour, th e specimens were incubated with rabbit anti PPAR antibody (ab8934, Abcam) overnight at 4 o C, and then washed. The

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158 secondary antibody used was Alexa Fluor 568 donkey anti rabbit antibody (A 21206, Life Technologies ). For this step, samples were incubated for 1 hour at room temperature. Finally, nuclei were counterstained with DAPI (H 1200, Vecto r L abs ). Murine Intracranial Aneurysm Model Murine intracranial aneurysms were created in female 8 12 week old C57BL/6 mice (Charles River Laboratories, Wilmington, MA) using a method described previously. 114 Anesthesia was induced usi ng ketamine/xylazine. Briefly, the left common carotid artery and the right renal artery are ligated to induce hypertension. One week later, an Alzet micro osmotic pump model 1004 (DURECT Corp, Cupertino, CA) is implanted subdermally to deliver Angiotensi n II (Bachem AG, Switzerland) at 1000ng/kg/min; and 10 uL of 0.8% porcine elastase (Worthington Biochemical Corp, Lakewood, NJ ) in normal saline is injected into the right basal cistern using stereotactic coordinates: 1.2 mm rostral of bregma, 0.7 mm late ral of midline and 5.3 mm ventral of the dorsal aspect of the skull. The animals are fed a hypertensive diet with 8% NaCl and 0.12% BAPN (Harlan Laboratories, Indianapolis, IN). For the PPAR treatment , 120 L of 1:3 DMSO:PBS (control), 25 mg/kg/day Wy 14, 643 (PPAR agonist) or 10 mg/kg/day rosiglitazone (PPAR agonist) was injected i.p. daily for 2 weeks. 1:3 DMSO:PBS was used as vehicle for all agonists. Mice were weighed daily throughout the experiment to ensure proper body mass and IACUC protocol comp liance since PPAR agonists can induce weight loss. Mice were euthanized two weeks after aneurysm induction and aneurysm formation was quantified .

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159 Quantification of Aneurysm Formation At the end of the experimental period mice were euthanized by 3 mL 4% PFA in PBS cardiac perfusion into the left ventricle followed by injection of 1 mL brilliant blue in 20% gelatin in PBS. The brilliant blue in gelatin injection was used for visualization of blood vessels to allow a blinded observer to determine saccular aneu rysm formation in Circle of Willis (COW) from each mouse. The blinded observer did not perform any surgeries or treatments for this experiment. Representative images were taken using Leica dissection microscope with Volocity 3D analysis software . Results P PAR Agonists Decrease Endothelial Expression of Inflammatory Mediators We evaluated the anti inflammatory potential of three different PPAR agonists within bifurcation aneurysm flow chamber ( n= 2 3 each) by using cytokine arrays and quantifying the levels o f 40 different cytokines . HUVECs were exposed to pulsatile flow in the bifurcation aneur ysm flow chamber with 0.1% 1:3 DMSO:PBS (carrier vehicle control), 25 µM Wy 14 , 643 (PPAR agonist) or 5 µM rosiglitazone (PPAR gonist) and the inflammatory cytokine profile was analyz ed. Mul tiple markers were found to be down regulated in the Wy 14 , 643 (PPAR agonist) treated and rosiglitazone (PPAR treated flow chambers. Both PPAR and PPAR showed robust anti inflam matory effects in lowering IL 8 expression (Fig u re 4 1 ). PPAR Agonists Decrease Endothelial Expression of IL 8 Analysis of perfusing medium from PPAR agonist treated bi furcation aneurysm flow chambers by ELISA revealed that IL 8 secretion was significantl y different among the five treatment regimens studied (p<0.0001 by One way ANOVA with Tukey HSD)

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160 (Figure 4 2) . Significantly less IL 8 was secreted in the bifurcation aneurysm flow chamber treated with rosiglitazone ( 136.4 pg/mL, n=3 ) than the 1 :3 DMSO:PBS treated ( 280.5 pg/mL, n=2, p<0.01 ) flow chamber system . Wy 14,643 treated HUVECs secreted significantly less IL 8 (3.1 pg/mL, n=3) than the 1:30 DMSO control group (280.5 pg/mL, n=2, p<0.01), rosiglitazone treated group ( 136.4 pg/mL, n=3, p<0.01 ) , and GW 501516 treated group (92.1 pg/mL, n=3, p<0.01). IL 8 concentration was found to be significantly lower in the GW501516 treated flow chambers (92.1 pg/mL, n=3) than in 1:3 DMSO:PBS control chambers (280.5 pg/mL, n=2, p<0.01). Finally, the combination rosig litazone GW501516 treated group had significantly lower IL 8 levels (4.0 pg/mL, n=3) than 1:3 DMSO:PBS control (280.5 pg/mL, n=2, p<0.01), rosiglitazone only treated group (136.4 pg/mL, n=3, p<0.01), and GW501516 only treated group (92.1 pg/mL, n=3, p<0.0 1). The differences in IL 8 levels between other pairs were not found to be statistically significant. Dose Dependent Relationship Between PPAR Expression and CXCL1 Levels Based on the promising results obtained cytokine arrays following PPAR agonist treatment, we decided to study the relationship between CXCL1 and PPAR . Immunohistochemistry of HUVECs exposed to CXCL1 at 0, 10, and 100 ng/mL CXCL1 ( 453 KC 050 , RnD Systems ) with or without 25 uM Wy14643 (PPAR agonist) for 24 hrs suggests that there is a dose dependent relationship between CXCL1 concentration and PPAR expression (Figure 4 3). Relative Fluorescence Immunocytochemistry of PPA R Expression on CXCL1 and IL 8 Challenge Human umbilical vein endothelial cells (HUVECs) exposed to CXCL1 or IL 8 at 0, 10, and 100 ng/mL with or without 25 uM Wy 14,643 (PPAR agonist) for 24 hrs

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161 displayed an apparent dose dependent relationship with r espect to PPAR expression (Figure 4 4) as quantified using relative fluorescence immunocytochemistry. Exposure to CXCL1 (Figure 4 4A) or IL 8 (Figure 4 4B) increased PPAR expression in a dose dependent fashion. Immunohistochemistry of PPAR Expressio n in Bifurcation Aneurysm Flow Chamber Model H uman umbilical vein endothelial cells (HUVECs) exposed to pulsatile flow in the bifurcation aneurysm flow chamber for 24 hrs displayed a differential expression of PPAR by qualitative immunohistochemistry (n= 3 each) (Figure 4 5) . In the aneurysm region, PPAR was highly expressed in the cytoplasm (non active) . In the proximal straight segment, PPAR was expressed at low levels in both cytoplasm (n on active) and nucleus (active). In the bifurcation and branch ing arms regions, PPAR expression appeared to be increased from the proximal straight segment, but less than in the aneurysm sac. Activation of CXCR1 and CXCR2 Has Opposing Effects on PPAR Expression HUVECs exposed to 100 ng/mL CXCL1 or IL 8 wi th or wi thout CXCR2 antagonist SB225002 ( 13336 , Cayman Chemical) or CXCR1/2 antagonist reparixin ( HY 15251 , MedChem Express, Monmouth Junction, NJ ) in 1:3 DMSO:PBS for 24 hrs displayed differential and opposing effects with respect to PPAR expression (Figure 4 6 ) . Upon CXCL1 challenge, HUVECs exposed to CXCR2 and CXCR1/2 antagonists displayed a significantly different response in PPAR expression (p<0.0001 by One way ANOVA with Tukey HSD) (Figure 4 6A) . CXCR1/ CXCR2 antagonist reparixin -

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162 treated HUVECs displayed a significantly higher expression of PPAR than 1:3 DMSO:PBS treated controls (115.8 vs 100% basal expression, n=12 each, p<0.01) and CXCR2 antagonist SB225002 treated cells (115.8 vs 90.6%, n=12 each, p<0.01). HUVECs exposed to CXCR2 antagonist SB225002 did not have a significantly different expression of PPAR than 1:3 DMSO:PBS controls (90.6% vs 100% basal expression, n=12 each, p>0.05). Upon IL 8 challenge, HUVECs exposed to CXCR2 and CXCR1/2 antagonists displayed a significantly different response in PPAR expression (p=0.014 by One way ANOVA with Tukey HS D) (Figure 4 6B) . CXCR1/CXCR2 antagonist reparixin treated HUVECs displayed a significantly higher expression of PPAR a than CXCR2 antagonist SB225002 treated cells (108.1% vs 89.99%, n=12 each, p<0.05 ). HUVECs exposed to CXCR2 antagonist SB225002 did not have a significantly different expression of PPAR than 1:3 DMSO:PBS controls ( 89.99%, vs 100% basal expression, n=12 each, p>0.05). Similarly, HUVECs exposed to CXCR1/CXCR2 antagonist reparixin did not have a significantly different expression of PPAR than 1:3 DMSO:PBS controls (108.1%, vs 100% basal expression, n=12 each, p>0.05). PPAR Shows Robust Expression in Murine Cerebral Aneurysms Out of 5 tissues analyzed, PPAR was highly expressed in the intima and the media of 2 week (5/5) aneurysms and aneurysmal vessels (Fig ure 4 7). The staining was primarily cytoplasmic suggesting that PPAR agonists could be used as a therapeutic agent to prevent cerebral aneurysm formation.

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163 PPAR Treatment Does Not Reduce Aneurysm Formation in Mouse Intracranial Aneurysm Model Mice treated with 120 L of Wy 14,6 43 at 25 mg/kg/day (PPAR agonist) in 1:3 DMSO:PBS, i.p. daily for 2 weeks (Figure 4 8) did not experience severe body mass loss and, in fact, appeared to recover weight lost post surgery faster than 1:3 DMSO:PBS control mice (Figure 4 9). Treatment with P PAR agonist did not significantly reduce cerebral aneurysm formation when compared to 1:3 DMSO:PBS treated control mice ( 28.6% vs 46.2%, respectively, n=13 14 each, p=0.44 b Exact Tes t) (Figure 4 10) . The developed aneurysms were also not less severe ( avera ge grade of 1.3 vs 1.6, p=0. 16 by Mann Whitney Test , n=13 14 each) (Figure 4 10). PPAR Treatment Does Not Reduce Aneurysm Formation in Mouse Intracranial Aneurysm Model Mice treated with 120 L of rosiglitazone at 10 mg/kg/day (PPAR agon ist) in 1:3 DMSO:PBS, i.p. daily for 2 weeks (Figure 4 8) did not experience severe body mass loss and, in fact, appeared to recover weight lost post surgery faster than 1:3 DMSO:PBS control mice (Figure 4 9). Treatment with PPAR agonist did not signific antly reduce cerebral aneurysm formation when compared to 1:3 DMSO:PBS treated control mice (40.0% vs 46.2%, respectively, n= 10 and 13 each, p=1.0 b y 4 11). The developed aneurysms were also not less severe ( avera ge grade of 1 .6 vs 1.6, p=1.0 by Mann Whitney Test , n=10 and 13 each) (Figure 4 11).

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164 Discussion The objective of Specific Aim 3 was to determine whether modulating the peroxisome proliferator activated receptor (PPAR) signaling pathways can be used to attenuate cell p henotype change and early vessel wall pathology that leads to the development of cerebral aneurysms. PPARs are a family of nuclear receptors ( , and ) that regulate transcription of genes and play a role in cell differentiation, metabolism, indirect response to shear stress and inflammation. 52 , 337 , 354 , 355 , 379 , 391 , 400 , 432 Several of the readily available and low cost drugs for diabetes and dyslipidemias that target PPARs have been also found to have potent anti inflammatory functions. Role of PPAR Agonists in Lowering the Endothelial She ar Stress Mediated Cytokine Inflammatory Profile In Specific Aim 2 we used an array panel to screen for 40 different inflammatory cytokines in our in vitro model and found several potential cytokine targets for immune modulation, the most promising of whic h was IL 8. Then, using our bifurcation aneurysm flow chamber model, we screened different PPAR agonists with cytokine arrays and determined that two of them, Wy 14 , 643 (PPAR agonist) and rosiglitazone ( agonist), could prevent aneurysm progression bas ed on the changes seen in the shear stress mediated inflammatory profile, especially by lowering IL 8 (Figures 4 1 and 4 2) . This pan anti inflammatory effect of PPAR agonists can be attributed to transrepression of NF activity. 379 , 381 , 382 PPAR activation with fenofibrate has been reported to decreas e CXCL chemokine ENA 78 expression in a robust manner in static endothelial cells. 433 Paradoxically, and in contrast to endothelial cells in our shear stress expe riments, Wy 14,643 has been reported to increase IL 8 and MCP 1 secretion in static endothelial cells. 434 Since Wy 14 , 643 (PPAR agonist) was so efficacious in

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165 decreasing the inflammatory profile in endothelial cells under shear stress conditions (F igures 4 1 and 4 2) , we decided to focus our in vitro and molecular studies on the relationship between ELR+ chemokines and PPAR . We reserved the use of rosiglitazone for animal experiments only since we expected its primary mode of action to be through regulation of macrophage phenotype. 326 , 435 GW501516, PPAR agonist, was not studied further due to a failed clinical trial and reports of severe side effects, primarily hepatic inflammation and hepatocarcinoma formation. 436 438 Feedback Loop Between PPAR and ELR+ Chemokines CXCL1 and IL 8 Our results indicate that PPAR expression in endothelial cells is increased on ELR+ chemokine CXCL1 and IL8 exposure in a dose dependent f ashion in vitro (Figures 4 3 and 4 4) . PPAR expression can increase in settings of acute inflammation. 379 However, despite increased expression on CXCL1 exposure, PPAR is not active and is l oca lized to the cytoplasm (Figure 4 5 ) in the aneurysm region in the bifurcation aneurysm flow chamber model . In contrast, in the proximal straight segment PPAR has low expression with some nuclear lo calization (active form) in the bifurcation aneurysm flow chamber. However, this overexpression, despite of lack of activation, can be exploited by treating with PPAR agonists (Figures 4 1 and 4 2 ) leading to pan anti inflammatory effects. We then proceeded to study the role of each of the receptors utilized by ELR+ CXCL chemokines, CXCR1 and CXCR2. In our CXCR1/2 and CXCR2 antagonist studies on CXCL1 and IL 8 challenge, PPAR increased expression appeared to be mediated by positive feedback by CXCR2 and nega tive feedback by CXCR1 (Figure 4 6). When signaling through CXCR2 was blocked, PPAR expression was decreased.

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166 On the other hand, stimulation of CXCR1 appeared to have a major negative feedback on PPAR expression as combined CXCR1/2 blockade increased PPAR expression. Additional blocking or knockdown experiments with siRNA could be done in the future to confirm these preliminary results. Both, CXCR1 and CXCR2 are 7 dom ain transmembrane G protein receptors that can bind CXCL chemokines, but their signaling pathways differ in ability of the cell to sustain signaling through each receptor, ligand affinity, and receptor internalization half life . 439 Both receptors transduce signaling through PI3K and PLC, which lead to calcium mobilization and mTOR activity. 440 Initial neutrophil rec ruitment gradually slows down due to overstimulation of CXCR2, although it has been shown that some CXCL chemokines, especially IL 8, can overcome this diminishing effect through stimulation of CXCR1. 69 , 239 , 247 249 However, most studies suggests that CXCR2 is the receptor through which ELR+ CXC chemokines exert their chemoattractant properties. 252 , 253 CXCR1 has been shown to have higher affinity for IL 8 than othe r ELR+ CXCL chemokines. 253 The differences in CXCR1 and CXCR2 effects on PPAR expression in our experiments could also be due to the fact that ELR+ chemokines a ctivate a separate set of signaling cascades depending on the process that is studied. Effects such as proliferation, cell survival, inflammation and angiogenesis are mediated via downstream signaling on NF , HIF 1 , and AP 1, while cytoskeletal dynamics and migration are mediated by Cdc42, Rho and Rac GTPases, STAT3, and catenin. 440 Role of PPAR and PPAR Agonists in Preventing Cerebral Aneurysm Formation O ur immunohistochemistry studies of mouse intracranial aneurysms suggested that PPAR modulation could be beneficial due to its robust expression (Figure 4 7).

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167 We also detected a sizeable population of M1 and undeterminate phenotype of macrophages infiltra ting murine aneurysms (Figure 3 10), which suggested an opportunity to mediate macrophage inflammatory processes. Despite beneficial effects of PPAR agonists on the endothelial cell inflammatory profile and in vitro data (Figures 4 1, 4 2, 4 3, 4 4, 4 5, and 4 6) we could not confirm their efficacy in our murine intracranial aneurysm model (Figures 4 10, 4 11 , and 4 12 ). Although PPAR agonist Wy 14,643 appeared to decrease aneurysm formation in our mouse model (28.6% vs 46.2%) , the differen ce was not significant (Figure 4 10). In order to reach significance, we estimated that we would need to add 30 40 mice into each experimental grou p, an expensive undertaking rendering our results about Wy 14,643 inconclusive. It is worth noting that aneurysm formation rate in 1:3 DMSO:PBS control group appeared to be lower (46.2%), but not statistically significant, than in IgG treated control group (66.7%) in experiments in Specific Aim 2 .This suggests that there could be an additional confounding effect from using DMSO as a carrier vehicle for drug delivery. DMSO has been described to decrease inflammation by acting as a ROS scavenger 441 , decrea sing NLRP3 inflammasome activation 442 , and has been shown to have differential effects on inflammation depending on route of administration 443 . A better drug delivery system could be used in future experiments to fully ascertain the role of PPAR activation in cerebral aneurysm formation (Fig ure 4 12) . Such drug delivery routes could include oral gavage, adding the drug to food, o r using liposomes as carrier vehicles, which have been used with great efficacy in rosiglitazone delivery studies 444 . Next, PPAR agonist rosiglitazone offered n o protective effect agai nst aneurysm formation (Figure 4 11). It is possible that its anti inflammatory effects were offset by

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168 cardiovascular volume overload, which has been described. 445 , 446 Rosiglitazone could also be more beneficial in preventing aneurysm rupture rather than aneurysm formation since a preponderance of the M1 pro inflammatory phenotype has been described in human ruptured aneurysms. 84

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169 Figure 4 1. Effects of PPAR , and expression. HUVECs were exposed to pulsatile flow in the bifurcation aneur ysm flow chamber with 0.1% 1:3 DMSO:PBS (carrier vehicle control), 25 µM Wy 14643 (PPAR agonist) or 5 µM rosiglitazone (PPAR was analyz ed. n=2 3 each. Results are presented as means ± s.e.m.

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170 Figure 4 2. IL 8 secretion under different PPAR agonist treatments. HUVECs were exposed to pulsatile flow in the bifurcation aneurysm flow chamber with either 5 M rosiglitazone, 25 M Wy14643, 30 nM GW501516, 5 M rosiglitazone and 30 nM GW501516, or 0.1% 1:3 DMSO:PBS ( carrier vehicle control ) over 24 hrs. IL 8 levels in the perfusing medium were then analyzed by ELISA. p<0.0001, n= 3 each. *p<0.05, **p<0.01 . Results are presented as means ± s.e.m.

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171 Figure 4 3 . PPAR expression increases on CXCL1 challenge. Human umbilical vein endothelial cells (HUVECs) were exposed to CXCL1 at 0, 10, and 100 ng/mL CXCL1 with or without 25 uM Wy14643 (PPAR agonist) for 24 hrs. Cells were then stained for PPAR . Exposure to CXCL1 appears to increase PPAR expression in the cytoplasm (in active form ) in a dose dependent fashion. Treatment with Wy14643 caused some relocalization to the nucleus (active form).

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172 Figure 4 4 . PPAR expre ssion on CXCL1 or IL 8 challenge. Human umbilical vein endothelial cells (HUVECs) were exposed to CXCL1 or IL 8 at 0, 10, and 100 ng/mL with or without 25 uM Wy 14,643 (PPAR agonist) for 24 hrs. Cells were then stained for PPAR . Exposure to CXCL1 or IL 8 appears to increase PPAR e xpression in the cytoplasm (in active form ) in a dose dependent fashion. Treatment with Wy 14,643 caused relocalization to the n ucleus (active form). N=12 each. Results are presented as means ± s.e.m.

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173 Figure 4 5. PPAR expression within the bifurcation flow chamber. HUVECs were exposed to pulsatile flow over 24 hrs. At the end of the experiment, the coverslips were collected and stained for PPAR In the aneurysm region, PPAR is hi ghly and primarily expressed in the cytoplasm (non active) priming the cells for potential PPAR agonist treatment. In the proximal straight segment, PPAR is expressed at low levels in both cytoplasm (non active) and nucleus (active) suggesting that PPAR is exerting its protective anti inflammatory effects.

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174 Figure 4 6 . CXCR1 and CXCR2 h ave opposing effects on PPAR expression. Human umbilical vein endothelial cells (HUVECs) were exposed to A) CXCL1 or B) IL 8 at 100 ng/mL with or without CXCR2 ant agonist (SB225002) or CXCR1/2 antagonist (reparixin) for 24 hrs. Cells were then stained for PPAR . n=12 each. * p<0.05, ** p<0.01 . Results are presented as means ± s.e.m.

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175 Figure 4 7. PPAR staining of mouse aneurysm specimen s and aneurysmal arteries. Mouse aneurysm and aneurysmal artery speci mens are positive for PPAR indicating that treatment with PPAR agonists may have a therapeutic benefit.

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176 Figure 4 8 . Mouse Intracranial Aneurysm Model: Surgical and PPAR agon ist Treatment Scheme .

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177 Figure 4 9 . B ody mass recovery post aneurysm induction surgery during PPAR agonist treatment . C57BL/6 mice were treated with 120 L 1:3 DMSO:PBS (control), 25 mg/kg/day Wy 14,643 (PPAR agonist) or 10 mg/kg/day rosiglitazone ( PPAR agonist) and their weights were monitored. Sample size: 1:3 DMSO:PBS n=13 16 , Wy 14,643 n=14 17, rosiglitazone n=10 13 .

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178 Figure 4 10 . Treatment with PPAR agonist Wy 14,643 does not appear to prevent mouse intracranial aneurysm formation . A) Mice treated with 25 mg/kg/day Wy 14,643 (in 120 L 1:3 DMSO:PBS, i.p.) over two weeks do not develop significantly less aneurysms (28.6% vs 46.2%, p=0.44, n=13 14) than 1:3 test. B) T he developed aneurysms are also not less severe. Results are presented as means. * p<0.05, ** p<0.01.

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179 Figure 4 11 . Treatment with PPAR agonist rosiglitazone does not prevent mouse intracranial aneurysm formation . A) Mice treated with 10 mg /kg/day rosiglitazone (in 120 L 1:3 DMSO:PBS, i.p.) over two weeks do not develop significantly less aneurysms (40% vs 46.2%, p=1.0, n=10 for rosiglitazone, n=13 for 1:3 DMSO:PBS) than 1:3 DMSO:PBS treated control mice. Data xact test. B) The developed aneurysms are also not less severe. Results are presented as means. * p<0.05, ** p<0.01.

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180 Figure 4 12. Proposed Role of PPAR Agonists in Prevention of Cerebral Aneurysm Formation. Under Specific Aim 1 and 2 we showed that low shear stress micro environment leads to pro inflammatory conditions within the aneurysm and causes IL 8 and/or CXCL1 dependent neutrophil infiltration. Blockade of CXCL1 decreases aneurysm formation in mice. PPAR agonists could be a promising treatment modality due to their pan anti inflammatory effects on endothelial cell function and IL 8 and CXCL1 release. However, PPAR agonist treatment did not have a significant effect on cerebral aneurysm formation in the mouse model despite promising in vitro data. We believe that the PPAR in vivo data is inconclusive due to confounding effects of 1:3 DMSO:PBS control, and may necessitate optimization of the delivery system. PPAR agonists did not cause any significant reduction in cerebral aneurysm formation, although their role in aneurysm rupture may warrant a separate study.

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181 CHAPTER 5 SUMMARY AND CONCLUSIONS Summary Specific Aim 1 : Model Shear Stress Inflammatory Micro environment of Cerebral Aneurysm In Vitro We d eveloped a novel in vitro model of a bifurcation aneurysm, and compared its endothelial phenotype to similar models of flow in a straight artery and a bifurcation . We characterized the model using computational fluid dynamics (CFD), enzyme immunoassay (EIA ), real time reverse transcriptase PCR (RT PCR) for cyclooxygenase 1 (COX 1) and 2 (COX 2), and monocyte chemotactic protein 1 (MCP 1), and relative fluorescence immunocytochemistry (ICC). The bifurcation aneurysm model was characterized by significantl y higher circulating PGE2 levels by EIA, and t he aneurysm sac micro environment had significantly higher COX 2 and MCP 1 expression by relative fluorescence ICC. These in vitro findings were confirmed by immunohistochemistry in murine and human cerebral a neurysms. Together, these findings confirm that we have been able to model shear stress micro environments of cerebral aneurysm in vitro . In addition, we were able to show that at cerebral aneurysm micro environments exposed to low wall shear stress are ch aracterized by higher expression of pro inflammatory COX 2 and PGE2. Specific Aim 2 : Study the Role of ELR+ CXC Chemokines in Cerebral Aneurysm Formation Cerebral aneurysms are thought to develop by an inflammatory mediated process related to hemodynamic s hear stress. The molecular mechanism by which shear stress triggers inflammation at the site of aneurysm development has not been elucidated. We conducted a screen using a cytokine array and found that t he bifurcation

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182 aneurysm model was characterized by s ignificantly higher IL 8 concentrations when compared to other models. The bifurcation aneurysm model was characterized by significantly higher IL 8 levels by cytokine array, and IL 8 and CXCL1 levels by ELISA when compared to other models. The bifurcatio n and aneurysm sac micro environments had significantly higher IL 8 and CXCL1 expression by relative fluorescence imunocytochemistry (RF ICC) when compared to the other micro environments, within the model itself. IL 8 and CXCL1 expression in human and C XCL1 in murine cerebral aneurysms was demonstrated by immunohistochemistry. Immunohistochemistry revealed that CXCL1 was the primary ELR+ CXC chemokine expressed in murine aneurysms. CXCL1 antibody blockade in mouse intracranial aneurysm model resulted in significantly less aneurysm formation (13.3 vs 66.7%, p=0.0078) than in IgG treated control mice over a 2 week period. Anti CXCL1 treated mice had significantly decreased neutrophil infiltration and VCAM 1 expression. Bifurcations and bifurcation aneurys ms exposed to hemodynamic shear stress in a parallel plate flow chamber model are characterized by increased local endothelial expression of IL 8 and CXCL1, which are also expressed in human cerebral aneurysms. CXCL1 blockade in mouse int racranial aneurysm model resulted in significantly less aneurysm formation. Specific Aim 3: Determine the Role of PPAR Pathway in Prevent ion of Cerebral Aneurysm Formation. Specific Aim 3 was designed to study the relationship between the ELR+ CXC cytokines IL 8 and CXCL1, and PPAR in cerebral aneurysm formation. Based on a cytokine screen, two PPAR agonists, Wy 14 , 643 (PPAR agonist) and rosiglitazone (

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183 agonist), were found to have protective effects in vitro under shear stress conditions. Increasing concentrations of ELR+ chemokines CXCL1 and IL 8 had a dose dependent relationship and led to increase of PPAR expression. This protective mechanism appeared to be primarily mediated via CXCR2, while CXCR1 activation countered these effects. We then studied the role of PPAR and in aneurysm formation in mice using respective agonists, but the results were inconclusive for PPAR , and PPAR appears to have no protective role. Discussion Although the role of shear stress has been well established in aneurysm initiation, its function in aneurysm progression once a lesion has formed is not well understood. It has been repo rted that aneurysms continue to grow in dome regions where shear stress is much lower than in surrounding environment, but the details of signaling pathways involved are not fully known. Using our novel in vitro model of an arterial bifurcation with an ane urysm we were able to show that IL 8 is the most differentially upregulated cytokine and could have a role in cerebral aneurysm formation. The parallel plate flow chamber (PPFC) has been used successfully for more than two decades to study the behavior of cells under defined and reproducible conditions of flow and shear stress. Our modified PPFC has been designed to replicate the geometry and flow conditions of an idealized artery bifurcation with a saccular aneurysm (Fig ures 2 1, 2 2, 2 3, 2 4, and 2 5 ). We performed computational fluid dynamics (CFD) using Ansys FLUENT to ensure the presence of multiple hemodynamic micro environments based on shear stress and flow velocity patterns (Fig ure 2 6 ).

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184 Then, we performed perfusion experiments by subjecting human u mbilical vein endothelial cells (HUVECs) to flow for 24 hrs at ~60 pulses per minute and shear stress of 10 dyne/cm2, which are typical physiological conditions seen in most mouse intracranial and some human intracranial arteries. Using inflammatory marker s COX 2, PGE2, and MCP 1 we verified the inflammatory conditions in the bifurcation aneurysm flow chamber and compared them to mouse and hum an aneurysm specimens (Figures 2 11, 2 12, 2 13, 2 14, and 2 15). We concluded the experimental studies in Specific Aim 1 by showing that low shear stress hemodynamic micro environment within cerebral aneurysms leads to local inflammatory conditio ns and aneurysm growth (Figure 2 16). Then, we used conventional straight channel an d bifurcation flow chambers to compare th e secretion of 40 different inflammatory cytokines and found that IL 8 was the most differentially upregulated cytokine (Fig ure 3 1 ), whic h was confirmed by ELISA (Figure 3 2 ). IL 8 is a chemokine that has originally been described in its ability to attrac t neutrophils to sites of inflammation. Vascular smooth muscle cells (VSMCs) proliferate and migrate towards IL 8 while secreting pro inflammatory PGE2 and matrix metalloproteinases (MMPs) capable of causing local remodeling. Early VSMC proliferation and MMP secretion have been previously described in human aneurysm studies. Although IL 8 is not expressed by mouse cells, its murine homologue CXCL1 (GRO 1, KC, or NAP 3) shares more than 60% of amino acid sequence, activates the same receptor CXCR2 and the t wo cytokines are functional homologues. We have strong data that suggests early shear stress mediated inflammation in cerebral aneurysms is fac ilitated by IL 8/CXCL1 (Figure 3 2). Both IL 8 and GRO /CXCL1 are expressed in human cerebral aneurysms (Figure 3 3) and the mouse CXCL1 is

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185 expressed in mouse intracranial aneurysms (Figure 3 4) but not in control. Both IL 8 (Figure 3 2C) and CXCL1 (Figure 3 2F) are more highly expressed in the aneurysm pocket region of the bifurcation aneurysm flow chamber than oth er regions. Higher CXCL1 expression in mouse intracranial aneurysms is associated with higher infiltration by neutr ophils and macrophages (Figure 3 5). Finally, blockade of CXCL1 with a neutralizing antibody in the mouse elastase based intracranial aneurys m model leads to significantly less aneurysm development and rupture than in IgG treated control mice (Figures 3 6, 3 7, and 3 8). This process was mediated by decreased neutrophil infiltration and adhesion, possibly via VCAM 1, but not macrophage infiltra tion (Figures 3 9, 3 10 and 3 11). Clearly, early inhibition of the shear stress mediated IL 8/CXCL1 signaling and neutrophil recruitment is beneficial in preventing aneurysm formation and rupture (Figure 3 12). We then screened three different PPAR agonis ts Wy14643 ( ), GW501516 ( ), and rosiglitazone ( ) in 1:3 DMSO:PBS at ~30 50 times the ED50 concentration in the bifurcation aneurysm flow chamber to study their effects on shear stress induced inflammatory profile (compared to 1:3 DMSO:PBS control c hambers). All PPAR agonists were effective at lowering inflamm at ory cytokine secretion (Figure 4 1 ), especially IL 8, which was a lso confirmed by ELISA (Figure 4 2 ) when compared to 1:3 DMSO:PBS treated controls. However, PPAR and were the most effec tive at overall pan anti inflammatory modulation (Fig ure 4 1 ). Our preliminary results indicate that PPAR expression in endothelial cells is increased on CXCL1 and IL 8 exposure in a dose dependent fashion in vitro ( Figures 4 3 and 4 4 ). However, despit e increased expression on CXCL1 exposure, PPAR is

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186 not active and is lo calized to the cytoplasm in the aneurysm region of the bifurcation aneurysm flow chamber (Figure 4 5) . However, this overexpression, despite of lack of activation, can be exploited by treating with PPAR agonists ( Figure 4 1 and 4 2 ) leading to pan anti inflammatory effects. Our data suggests that the protective upregulation of PPAR by ELR+ ch emokines is positively mediated by CXCR2 and negatively m ediated by CXCR1 (Figure 4 6). We pr opose d that treatment with PPAR agonists could be used to prevent aneurysm formation and rupture by decreasing 1) IL8/CXCL1 secretion (Fig ure 4 1 and 4 2 ) and adhesion/influx of inflammatory cells (Fig ures 3 9 and 3 11 ), 2) smooth muscle cell proliferation , and 3) by mitigating the effects of M1 pro inflammatory macrophages (Figure 3 10) . In addition, PPAR agonist treatment would 4) improve endothelial cell function (Fig ure 4 1 and 4 2 ) by decreasing the inflammatory profile and 5) indirectly (PPAR ) or d irectly (PPAR ) cause macrophages to switch to M2 anti inflammatory, pro wound healing phenotype wit hin the aneurysm lesion . However, PPAR and agonist therapy offered no protective benefits in the mo use intracranial model (Figure 4 12). Rosiglitazone could potentially be used to prevent cerebral aneurysm rupture rather than formation. Wy 14,643 could have a therapeutic potential if a better drug delivery system is utilized. In conclusion, our experiments show that the biological link between shear str ess inflammation and aneurysm formation is mediated by endothelial dysfunction, specifically IL 8 and/or CXCL1 mediated neutrophil recruitment. Currently studied CXCR1/2 antagonists such as reparixin could be used in lieu of CXCL1 antibody blockade in the clinic to prevent aneurysm formation and progression. 414 , 447 , 448

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187 LIST OF REFERENCES 1. Brisman JL, Song JK, Newell DW. Cerebral aneurysms. The New England journal of medicine . 2006;355:928 939 2. Wiebers DO, Whisnant JP, Huston J, 3rd, Meissner I, Brown RD, Jr., Piepgras DG, Forbes GS, T hielen K, Nichols D, O'Fallon WM, Peacock J, Jaeger L, Kassell NF, Kongable Beckman GL, Torner JC, International Study of Unruptured Intracranial Aneurysms I. Unruptured intracranial aneurysms: Natural history, clinical outcome, and risks of surgical and e ndovascular treatment. Lancet . 2003;362:103 110 3. Wijdicks EF, Kallmes DF, Manno EM, Fulgham JR, Piepgras DG. Subarachnoid hemorrhage: Neurointensive care and aneurysm repair. Mayo Clinic proceedings. Mayo Clinic . 2005;80:550 559 4. Feigin VL, Lawes CM, Bennett DA, Anderson CS. Stroke epidemiology: A review of population based studies of incidence, prevalence, and case fatality in the late 20th century. Lancet neurology . 2003;2:43 53 5. Connolly ES SR. Management of unruptured aneurysms. In: Le Roux PD WH, Newell DW, ed. Management of cerebral aneurysms . Philadelphia: Saunders; 2004:271 285. 6. Roos YB, Dijkgraaf MG, Albrecht KW, Beenen LF, Groen RJ, de Haan RJ, Vermeulen M. Direct costs of modern treatment of aneurysmal subarachnoid hemorrhage in the f irst year after diagnosis. Stroke; a journal of cerebral circulation . 2002;33:1595 1599 7. Starke RM, Ali MS, Jabbour PM, Tjoumakaris SI, Gonzalez F, Hasan DM, Rosenwasser RH, Owens GK, Koch WJ, Dumont AS. Cigarette smoke modulates vascular smooth muscle phenotype: Implications for carotid and cerebrovascular disease. PloS one . 2013;8:e71954 8. Greenberg MS. Handbook of neurosurgery . New York: Thieme Publishers; 2010. 9. de Rooij NK, Linn FH, van der Plas JA, Algra A, Rinkel GJ. Incidence of subarachnoid haemorrhage: A systematic review with emphasis on region, age, gender and time trends. Journal of neurology, neurosurgery, and psychiatry . 2007;78:1365 1372 10. Johnston SC, Selvin S, Gress DR. The burden, trends, and demographics of mortality from subar achnoid hemorrhage. Neurology . 1998;50:1413 1418 11. Chalouhi N, Hoh BL, Hasan D. Review of cerebral aneurysm formation, growth, and rupture. Stroke; a journal of cerebral circulation . 2013;44:3613 3622

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230 BIOGRAPHICAL SKETCH Kamil W. Nowicki was born in Starachowice, Poland in 1986 . He spent his early childhood in , Poland where he attended the Gimnazjum Nr 3 i.m. Jan III Sobieski junior high school . He immigrated to the United States in 2001. He graduated in the top portion of his class in 2005 from Buchholz High School , Gainesville, Florida. In 2005 , he started his undergraduate studies and was admitted to the Honors Program at the University of Florida . He joined the Quantum Theory Project group in 2006 as part of his undergraduate research experience in computational and quantum chemistry under directi on of Jeffrey Krause, PhD. During that time, his involvement in doing team science taught him the importance of efficient communication in a collaborative interdisciplinary and multidisciplinary research environment. In 2007 he received the Anderson Schola r of High Distinction award. In 2008 he graduated summa cum laude with a degree in chemistry. He was named one of the top graduating seniors in chemistry and was co awarded the Colonel Allen R. and Margaret G. Crow Undergraduate Scholarship for his underg and Förster Energy Transfer in Bifunctional Non Conjugated Benzothiadiazole based He matriculated into the combined MD PhD program at the University of Florida College of Medicine in 2008 following his passion for medicine and research. In 2009 he rotated through Yiider Tseng , PhD 's molecular mechanics and interactomics lab studying cell migration in wound healing. During his time with Dr. Tseng, he learned many skills relevant to research in biologi cal sciences as well as biomedical engineering. research studies in the field of molecular cell biology. His research interests encompass

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231 mechanisms of cerebral aneurysm formation, model ing vascular inflammation in vitro, and development of potential pharmacological treatments to prevent aneurysm progression. mediated inflammation, ELR+ CXC chemokines , and peroxisomal proliferator a ctivated receptor showing that shear stress mediated inflammatory cerebral aneurysm formation and growth i s mediated by ELR+ chemokines IL 8 and CXCL1. During his research years , Kamil remained active in medical school activities such as teaching and volunteering. He acted as the Physical Exam Teaching Assistant (PETA) for Essentials of Patient Care I and II. He served as a teaching assistant for Dr. ce course. Furthermore, he has been involved with the Equal Access Clinic Network since 2008. He became director of the mobile clinic site known as Equal Access Clinic at Tower Road in 2012. He was awarded the Equal Access Clinic Service award for his work with the undeserved population of Gainesville in 2012 and 2013 . Kamil plans on continuing his journey into the world of medical research and wishes to further his training by completing a residency in a surgical field.