Myxomaviral Immune Modulating Proteins Alter Gene Expression of Monocyte Apoptotic Pathways

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Myxomaviral Immune Modulating Proteins Alter Gene Expression of Monocyte Apoptotic Pathways
Davids, Jennifer A
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[Gainesville, Fla.]
University of Florida
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1 online resource (134 p.)

Thesis/Dissertation Information

Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences
Immunology and Microbiology (IDP)
Committee Chair:
Lucas, Alexandra Rose
Committee Members:
Baker, Henry V
Mccormack, Wayne T
Oh, Suk
Moldawer, Lyle Leonard
Graduation Date:


Subjects / Keywords:
Animal models ( jstor )
Apoptosis ( jstor )
Cells ( jstor )
Chemokines ( jstor )
Homologous transplantation ( jstor )
Inflammation ( jstor )
Macrophages ( jstor )
Mice ( jstor )
Physical trauma ( jstor )
Serpins ( jstor )
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
atherosclerosis -- davids -- dissertation -- inflammation -- serp-1 -- serp-2
Electronic Thesis or Dissertation
born-digital ( sobekcm )
Medical Sciences thesis, Ph.D.


Atherosclerosis ischaracterized by chronic inflammation and cell death (apoptosis) whichinitiates plaque growth and thrombotic occlusion. Serine protease inhibitors, or serpins, regulate apoptotic, inflammatory, and coagulation (thromboticand thrombolytic) pathways driving atherosclerosis and vasculitis. Large doublestranded DNA viruses, such as the Myxoma virus, encode anti-inflammatoryproteins to regulate the host’s immune response. Some anti-inflammatoryproteins, such as serine protease inhibitors, are capable of modulating avariety of host regulatory pathways. Serp-1significantly reduces inflammatory cell activation, invasion and plaque growthin animal models and has successfully completed a Phase 2A clinical trial inacute unstable coronary syndromes (ACS) with stent implant. Serp-2 inhibits caspases 1, 8 andserine protease Granzyme B, with anti-inflammatory and anti-apoptotic effectsin rodent balloon angioplasty and transplant models. M-T7, also markedly reduces plaque growth after angioplasty andtransplant in animal models. The following studies aim to understand how these highlydistinct viral proteins, with particular emphasis on Serp-2, cause reductionsin inflammatory cell invasion and apoptosis in a series of studies of vascularinjury in mouse models. From the studies contained herein, we have examined theroles of Serp-2 and CrmA both in vivo inmouse and in vitro in human celllines treated with apoptosis-inducing chemicals. Common effects of each of thethree proteins, Serp-1, Serp-2, and M-T7 on gene expression in human vascular endothelial,monocyte and T cells were assessed. Camptothecin treatment with concurrentSerp-1, Serp-2 or M-T7 treatment for 30 min resulted in 48 consensus genes inhuman monocytes. Serp-2 and CrmAtarget caspase 1 and granzyme B. To determine whether the effects of Serp-2 andCrmA on altered monocyte activation and gene expression were mediated througheither the caspase 1 or the granzyme B pathways, knockout mouse models wereexamined.  Caspase 1 and Granzyme Bknockout mouse models provide insights confirming that these viral proteinsmodulate gene expression through interactions with both caspase 1 and granzymeB. This work establishes a potential molecular mechanism. All three viralanti-inflammatory treatments significantly altered expression of BAG3, whichhas a wide array of roles in cell migration and apoptosis. The consistentregulation of BAG3 between these disparate mouse and human models makes it anideal candidate for further investigation as part of a consensus pathway fordisparate viral anti-inflammatory proteins. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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Thesis (Ph.D.)--University of Florida, 2013.
Adviser: Lucas, Alexandra Rose.
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by Jennifer A Davids.

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2 201 3 Jennifer Ann Davids


3 To all the mice that gave their all to the cause of Science


4 ACKNOWLEDGMENTS I would like to thank my family for their love, support, and encouragement. Without them, none of this would have happened. I am unbelievably lucky and forever grateful. I am forever indebted to Knox College for my fantastic undergraduate experience, the f inancial aid, and the innumerable amazing experiences! I would like to thank Professor Janet Kirkley at Knox College for being honest enough to tell me graduate school was a terrible idea. I also want to thank Cameron Lilly for also ignoring ce and coming down to UF with me. All of our adventures and growth will forever be burned into my memory. Thank you, my squidge. I thank my roller derby teammates and roommates for tolerating my grumpiness as I tried to finish my research and write this most pleasant experience but your support has meant the world to me. I would also like to thank my mentor, Dr. Lucas, for allowing me to join her lab and giving me so many opportunities to represent the lab at conf erences. Without her, I would never had learned about the fascinating world of serpins, and I am grateful for all of the time she took out of her day to talk me out of some panic about mouse colony problems. I would like to thank my committee members Dr. B aker, Dr. Moldawer Dr. McCormack and Dr. Oh for their various insights and inquiries throughout my doctoral program. I appreciate your contributions to my academic rigor and scientific progress. Lastly, I want to thank my coworkers and students from over the years. Ganesh, Li, Erbin, Jen, Mee, Hao, Donghang, Nichole and Sami each of you have a special place in my heart. I appreciate your calming words, your different interpretations on protocols, your scientific wizardry and sometimes, your brute force a pproach. I hope that distance does not keep us apart for too long.


5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 IN TRODUCTION: INNATE IMMUNITY IN ATHEROSCLEROSIS ......................... 15 Hypothesis ................................ ................................ ................................ .............. 15 Plaque Development ................................ ................................ ............................... 16 Apoptosis in Atherosclerosis ................................ ................................ ................... 18 CTL Mediated Apoptosis of Host Cells ................................ ................................ ... 18 Serine Protease Inhibitors ................................ ................................ ....................... 19 Vi ral Evasion of Host Immune Responses ................................ .............................. 20 Serpins in the Myxoma Pox Virus ................................ ................................ ........... 21 The Innate Immune / Inflammatory Response ................................ ........................ 22 Viral Anti Inflammatory Proteins that Target the Chemokine Axis .......................... 25 Chemokine Modulating Proteins ................................ ................................ ............. 26 Serine Proteases and Serpins in the Innate Immune Response S ystem ................ 28 Serpin Mediated Regulation of the Thrombolytic Cascade ................................ ..... 30 Serpins that Regulate Connective Tissue Degradation and Apoptosis ................... 32 Viral Proteins that Target the Serine Protease / Serpin Axis ................................ ... 33 Thrombolytic Protease Inhibi tors ................................ ................................ ...... 33 Apoptotic Protease Inhibitors ................................ ................................ ............ 35 2 METHODS AND MATERIALS ................................ ................................ ................ 38 Anim al Models ................................ ................................ ................................ ........ 38 Rodents ................................ ................................ ................................ ............ 38 Angioplasty, Aortic Allograft and Carotid Cuff Compression Models ................ 38 Histological, Morphometric, and Fluorescence Analysis ................................ .. 40 Enzyme Activity Analyses ................................ ................................ ................. 42 Histological, Immunohistochemical, and Morphometric Analysis ..................... 44 Mouse Peritoneal Exudate Model ................................ ................................ ..... 45 RNA Isolation, cDNA Synthesis and qRT PCR for Mouse Peritoneal Cells ..... 46 In Vitro Models ................................ ................................ ................................ ........ 47


6 Cell Culture ................................ ................................ ................................ ....... 47 Source and Purification of Proteins ................................ ................................ .. 47 qRT PCR for Cultured Cell Lines ................................ ................................ ..... 48 Adhesion assays ................................ ................................ .............................. 49 Membrane fluidity assays ................................ ................................ ................. 49 Stat istical Analysis ................................ ................................ ............................ 49 3 VIRAL CROSS CLASS SERPIN INHIBITS VASCULAR INFLAMMATION AND T LYMPHOCYTE FRATRICIDE; A STUDY IN RODENT MODEL S IN VIVO AND HUMAN CELL LINES IN VITRO ................................ ................................ .... 50 Introduction ................................ ................................ ................................ ............. 50 Results ................................ ................................ ................................ .................... 53 Serp 2 Reduces Plaqu e Growth in Arterial Surgical Injury Models, In Vivo ...... 53 Serp 2 Reduces Plaque Growth in Apolipoprotein E Deficient (ApoE / ) Mice .. 56 Cross Class Serpin Treatments Modify Apoptotic Responses in Vitro ............. 56 Serp 2 Reduces T Cell Apoptotic Responses to Cytotoxic T Lymphocyte (CTL) Granzyme B ................................ ................................ ........................ 59 Serp 2 Binding to T Cells is Reduced with Granzyme B Inhibition ................... 60 Serp 2 Inhibition of Aortic Plaque is Reduced in Granzyme B Knockout Aortic Allografts ................................ ................................ ............................. 61 Serp 2 Reduces Early Apoptosis in Invading Inflammatory Cells after Aortic Allograft Transplant ................................ ................................ ....................... 62 Discussion ................................ ................................ ................................ .............. 63 4 VIRAL PROTEINS TARGET DIVERGING IMMUNE PATHWAYS WITH CONVERGING EFFECTS ON ARTERIAL DILATATION, PLAQUE, AND APOPTOSIS ................................ ................................ ................................ ........... 80 Introduction ................................ ................................ ................................ ............ 80 Results ................................ ................................ ................................ .................... 84 Increased Plaque and Arterial Dilatation in ApoE / Mice after Angioplasty Injury ................................ ................................ ................................ ............. 84 Anti Inflammatory Protein Treatment Reduces Plaque Growth and Arterial Dilatation ................................ ................................ ................................ ....... 84 Inflammatory Cell Invasion is Reduced with Viral Immunomodulatory Protein Treatment ................................ ................................ ......................... 86 Viral Anti inflammatory Protein Treatment Alters Gene Expression ................. 87 Anti inflammatory Protein Treatment Reduced Early Caspase 3 Activity ......... 88 Discussion ................................ ................................ ................................ .............. 89 5 VIRAL ANTI APOPT OTIC PROTEINS SERP 2 AND CRMA EFFECTS IN CASPASE 1 AND GRANZYME B DEFICIENT MICE ................................ ........... 101 Introduction ................................ ................................ ................................ ........... 101 Results ................................ ................................ ................................ .................. 102


7 CD4 and CD11b are Unaffected by Treatment with Serp 2 or CrmA when Compared to PMA Treatment Alone ................................ ........................... 102 Serp 1 and Serp 2 Treatment Increase CD11b+ Ly6Ghi Populations in Nod Mice Compared To PMA Treatment ................................ ............................ 102 PMA and Serp 2 Treatment Increase CD11b+ Ly6Chi Populations in GzmB / Mice Compared to Equivalent B6 Treatments ................................ .......... 103 Serp 1, Serp 2 and CrmA Treatment Increases CD11b+ Ly6Chi Populations in Casp1 / Mice Compared to Equivalent Nod Treatments ...... 104 BAG3 is Significantly Regulated by Serp 2 and CrmA ................................ ... 104 Discussion ................................ ................................ ................................ ............ 106 6 CONCLUSIONS ................................ ................................ ................................ ... 115 LIST OF REFERENCES ................................ ................................ ............................. 119 BIOGRAPH ICAL SKETCH ................................ ................................ .......................... 134


8 LIST OF TABLES Table page 4 1 Mouse strains, protein treatments and post surgical complications. ................... 99 4 2 Gene expression changes in THP 1 cells. ................................ ........................ 100 5 1 Apoptosis Gene Array ................................ ................................ ...................... 114


9 LIST OF FIGURES Figure page 1 1 Thrombolytic and apoptotic serine / cysteine protease pathways as well as endogenous serpin regulatory proteins.. ................................ ............................ 37 3 1 Serp 2 reduces plaque gr owth in arterial surgery models ................................ 68 3 2 Serp 2 reduces plaque growth in Apolipoprotein E deficient ( ApoE / ) mice. ...... 70 3 3 Viral cross class serpins alter apoptotic responses in T cells and monocytes, in vitro ................................ ................................ ................................ ............... 72 3 4 Blockade of granzyme B reduces viral cross class serpin inhibition of T cell induced T cell apoptosis. ................................ ................................ .................... 73 3 5 Serp 2 binds T cells in vitro with greater affinity than CrmA. ............................. 75 3 6 Granzyme B deficiency ( GzmB / ) in donor aorta interferes with Serp 2 inhibition of plaque growth after aortic allograft transplant. ................................ 76 3 7 CD3 and active Caspase 3 populations 72 h rs after mouse aortic allograft ..... 77 3 8 Viral cross class serpins alter Staurosporine induced apoptotic responses in T cells and monocytes, in vitro ................................ ................................ ........ 78 3 9 Animal Models ................................ ................................ ................................ .... 79 4 1 Mouse aortic histology. ................................ ................................ ....................... 94 4 2 Viral protein t reatment.. ................................ ................................ ...................... 95 4 3 CD3+ T cells or CD11b+ monocytic cells.. ................................ ......................... 96 4 4 Overlapping gene expression changes. ................................ ............................. 97 4 5 Caspase 3 staining.. ................................ ................................ ........................... 98 5 1 Flow cytometry of CD11b+ monocytic cells or CD4+ T cell populations. .......... 109 5 2 CD11b+ Ly6G staining m ouse peritoneal cells. ................................ ................ 110 5 3 CD11b+ Ly6C staining m ouse peritoneal cells.. ................................ ............... 111 5 4 Apoptotic genes altered by CrmA or Serp 2 treatment in mouse peritoneal cells, normalized to PMA treatment.. ................................ ................................ 113


10 LIST OF ABBREVIATIONS AT Alpha1 antitrypsin AAA Abdominal aortic aneurysms ANOVA analysis of variance ApoE Apolipoprotein E BCL B cell lymphoma bFGF Basic fibroblast growth factor CD Cluster of differentiation CTL Cytotoxic T cell CPT Camptothecin CMP Chemokine modulating proteins DISC Death induced signaling cascade DMSO Dimethyl sulfoxide DKO Double knock out EEL External elastic lamina EGFR E pidermal growth factor receptor ERK Extracellular signal related kinases FACS Fluorescence activated cell sorting FASL Fas Ligand FITC Fluorescein isothiocyanate GAGs Glycosaminoglycans GPCRs G coupled chemokine receptors HIV Human immunodefi ciency virus HUVECs Human umbilical vascular endothelial cells


11 iCAD Inhibitor of caspase activated DNAse IEL Internal elastic lamina IFN Interferon gamma LRPR Low density lipoprotein receptor related protein receptor MAPK Mitogen activated p rotein kinase MC Monocyte MCP 1 Monocyte chemoattractant protein 1 MMPs Matrix metalloproteases NSP Neuroserpin PARP Poly ( ADP ribose) polymerase PAI 1 Plasminogen activator inhibitor 1 PI3K Phosphotidylinositol 3 kinase PMA Phorbol myristic acid PMN Polymorphonuclear neutrophils PN Protease nexin qRT PCR Quantitative real time polymerase chain reaction RCL Reactive center loop STS Staurosporine Transforming growth factor beta TL T lymphoc ytes tPA Tissue type plasminogen activator uPA Urokinase type plasminogen activator


12 VEGF Vascular endothelial growth factor VSMC Vascular smooth muscle cells


13 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 MYXOMAVIRAL IMMUNE MODULATING PROTEINS ALTER GENE EXPRESSION OF APOPTOTIC PATHWAYS IN MONOCYTES By Jennifer Ann Davids May 2013 Chair: Alexandra R. Lucas Major: Medical Sciences Atherosclerosis is characterized by chronic inflammation and cell death ( apoptosis) which initiates plaque growth and thrombotic occlusion. Ser ine p rotease in hibitors, or serpins regulate apoptotic, inflammatory, and coagulation ( t hrombotic and thrombolytic) pathways driving atherosclerosis and vasculitis. Large double stranded DNA viruses, such as the Myxoma virus, encode anti inflammatory proteins to regulate inflammatory proteins, such as ser ine protease in hibitors, are capable of modulat ing a variety of host regulatory pathways. Serp 1 significantly reduces inflammatory cell activation, invasion and plaque growth in animal models and has successfully completed a Phase 2A clinical trial in acu te unstable coronary disease ( ACS) with stent implants. Serp 2 inhibits caspases 1, 8 and serine protease Granzyme B, with anti inflammatory and anti apoptotic effects in rodent balloon angioplasty and transplant models. M T7 also markedly reduces plaque growth after angioplasty and transplant in animal models. The following studies aim to understand how these highly distinct viral proteins with particular emphasis on Serp 2,


14 cause reductions in inflammatory cell invasion and apoptosis in a diverse set of models From the studies contained herein, we have examined the roles of Serp 2 and CrmA both i n vivo in mouse and i n vitro in human cell lines treated with different apoptosis inducing chemicals. Common effects of each of the three proteins, Serp 1, Serp 2, and M T7 on gene expression in human vascular endothelial, monocyte and T cells were assessed. Camptothecin treatment with concurrent Serp 1, Serp 2 or M T7 treatment for 30 min resulted in 48 consensus genes in human monocytes. Serp 2 and CrmA target caspase 1 and granzyme B To determine whether the effects of Serp 2 and CrmA on altered monocyte activation and gene expression were mediated through either the caspase 1 or the granzyme B pathway s knockout mouse models were examined. Caspase 1 and Gran zyme B knockout mouse models provide insights confirm ing that these viral proteins modulate gene expression through interactions with both caspase 1 and gra n zyme B. This work establishes a potential molecular mechanism These viral anti inflammatory treatm ents significantly alter ed expression of BAG3, which has a wide array of roles in cell migration and apoptosis. The consistent regulation of BAG3 between these disparate mouse and human models makes it an ideal candidate for further investigation as part o f a consensus pathway for disparate viral anti inflammatory proteins.


15 CHAPTER 1 INTRODUCTION: INNATE IMMUNITY IN ATHEROSC LEROSIS Hypothesis 1 Based on prior research demonstrating the anti inflammatory abilities of the divergent poxviral serpins Serp 1, Serp 2, CrmA and viral chemokine binding protein M T7, we set out to determine how these unique proteins effected gene expression changes in hum an and mouse models. We hypothesized that: : 1. Serp 2 and CrmA alter inflammatory cell responses in mouse models through modification of apoptotic responses in inflammatory cells 2. the anti inflammatory actions of the three viral proteins with proven anti infla mmatory activities will alter expression of a family of shared apoptotic pathway genes indicating a final response pathway common to all three protein treatments 3. Serp 2 and CrmA modify inflammatory mononuclear cell responses and apoptotic gene expression v ia interactions with caspase 1 and / or granzyme B We studied the effects Serp 1 as a positive control for a proven effective anti inflammatory serpin therapeutic and M T7 as negative control for serpin specific effects. Here, we will outline some of the relevant background for atherosclerosis and inflammatory models as well as the known effects of each protein, which sets the stage for the later experiments. 1 Parts of this chapter can be found in Davids J.A.; Munuswamy Ramunujam, G.; Liu, L.Y.; Dai, E.; Lucas, A. Anti Inflammatory & Anti Allergy Agents in Medicinal Chemistry Volume 7, Number 2, June 2008, pp. 130 149 ( 20) *Contributed sections and edited document


16 Plaque Development Although genetics contribute to atherosclerosis, environmental factors such a s smoking, high fat or cholesterol diets, lack of exercise or diabetes can cause initial injury to the arteries ( 1 3) Early lesions form in childhood and are typically asymptomatic b ecause the arteries can extensively remodel to preserve blood flow ( 4,5) However, advanced lesions may rupture, which can clinically present as strokes and myocardial infarctions due to thrombus formation ( 6) Total arterial occlusi on and resulting death may occur if the thrombogenic plaque contents are exposed and initiate blood coagulation and thrombus formation ( 7) The branching points of arterie s are especially susceptible to plaque formation as they have low shear stress and unpredictable blood flow, which leads to up regulation of adhesion molecules and subsequent immune cell invasion ( 8 10) On e typical treatment for atheroscle rosis is surgical intervention; either angioplasty with stent implantation to widen the luminal space, or bypass surgery to re route blood flow using a healthy blood vessel to skip over a damaged section. These treatments have suboptimal success rates due to re stenosis or reoccurrence of lesions ( 11,12) because t hey do not address the underlying cause of plaque formation and subsequent enlargement. In a cholesterol rich environment, endothelial cells at sites prone to lesion formation up regulate vascular cell adhesion molecule 1 ( VCAM 1) before deposition of leu kocytes occurs in both rabbit and mouse models of cholesterol induced lesion formation ( 13,14) VCAM 1 binds to cells expressing the integrin very late antigen 4 ( VLA 4), such as monocytes, T and B lymphocytes, all of which are prominent in plaques, inducing them to diapedese into the arterial intima ( 15) Monocytes secrete cytokines such as interleukin 2 ( IL ( IFN ( 16) to alert other


17 lymphocytes to the injury and stimulate them to mature into macrophages and effector T cells. A fter having crossed the vascular endothelial cell lining, macrophages ingest significant amounts of low density lipoproteins and transform into foam cells ( 16) Newly activated macrophages and T cells in the plaque a rea produce pro inflammatory cytokines such as Tumor Necrosis Factor ( TNF) 1, INF ( 17 19) TNF 1 expression, and IFN expression of chemoattractant pro teins ( chemokines), which recruit more leukocytes to the area ( 20) Activated T cells express CD154 which binds to CD40 ligand present on macrophages and allows for cross talk an d cross activation of the innate and humoral immune systems. Natural Killer T ( NKT) cells are a special class of cytotoxic T lymphocytes that do not require activation in order to kill cells that lack MHC class I recognize glycolipid antigens via CD1d and may exacerbate atherosclerosis ( 21,22) Foam cells undergo apoptosis due to the cytotoxic effects of and antibody formation against oxysterols, the oxidized form of low density lipoprotein ( LDL) and ATP depletion, subsequently enlarging the necrotic lipidic core of the plaque ( 23 25) This necrotic core is typically enclosed by a large fibrotic cap composed of connective tissue, smooth muscle cells, collagen, elastin and some foam cells, which prevents the thrombogenic contents of the core from contacting the circulating blood. Over repeated cycles of plaque fissure, or incomplete rupture, the cap is converted from a primarily fatty lesion to a more fibrotic lesion. IFN inflammatory cytokines can inhibit the co llagen production that keeps the fibrotic cap


18 intact, leading to the activation of matrix metalloproteases 1, 2, 8, 9 and 13 and eventual plaque rupture ( 26,27) One way to prevent maturation of lesions and eventual plaque rupture is to shut down this pro i nflammatory free for all and associated apoptosis of macrophages and vSMCs. Apoptosis in Atherosclerosis As briefly mentioned in the preceding section, a poptosis of endothelial cells and monocytes/ macrophages in the plaque leads to an increased r elease of cytokines and activation of thrombolytic serine proteases tissue and urokinase type plasminogen activators ( tPA and uPA, respectively) and the matrix metalloproteases ( MMPs), which breakdown the collagen and elastin of nearby connective tissue, weaken ing the ( 28 30) Apoptosis of vascular smooth muscle cells ( vSMCs) in the shoulder region of the plaque also contributes to the thinning of the fibrotic cap ( 31,32) In addition to these activated and apoptotic cells, the newly exposed necrotic core and eroded cap structure also activate leukocytes and may initiate plaque rupture and subsequent thrombus formation, leading to heart attacks and strokes. Thus the serine proteases in the coagulation and fibrinolytic pathways interact on many levels with the inflammatory and apoptotic responses. CTL Mediated Apoptosis of Host Cells Cytotoxic T Lympho cytes ( CTLs) induce apoptosis in somatic cells that are infected by intracellular bacteria or viruses by releasing granzyme B into the infected host cells via perforin pores. Granzyme B, a serine protease, enters the target cell and induces apoptosis via t he extrinsic pathway ( 33 35) The extrinsic apoptotic pathway is typically initiated by extracellular ligands binding membrane bound receptors, such as Tumor Necrosis Factor ( TNF) Receptor/TNF or Fas/Fas Ligand ( 34,35) Each triggers


19 proximal initiator caspase 8 and/ or 10 are activated by receptor ligand interactions and in turn activate downstream effector caspases 3, 6, and 7 and thereby initiate apoptosis ( 33) Granzyme B, however, d irectly activates caspase 3 and also initiates the intrinsic apoptotic pathway by cleaving the anti apoptotic Bcl 2 family protein Bid ( 36,37) The intrinsic, or mitochondria mediated, pathway requires disruption o f the mitochondrial membrane, causing subsequent release of cytochrome c. Cytochrome c upon release forms a complex with adenosine triphosphate ( ATP), apoptotic protease activating factor 1 ( Apaf 1) and procaspase 9 called the apoptosome. The apoptosome in turn induces activation of caspase 9 and leads to activation of the caspase cascade. Regardless of the method of initiation, apoptosis is characterized by DNA fragmentation, membrane blebbing and cell shrinkage ( 34,35,3 8,39) and, in the case of a virally infected cell, does not release infectious virus particles into the environment. In this manner, CTLs work to suppress the spread of a virus throughout the host ( 36) so viruses have evolved ways to shut down the CTL mediated apoptosis ( 37,40) Viruses can evade this process by inhibiting Granzyme B, a variety of ca spases and a host of intrinsic pathway proteins. Serine Protease Inhibitors Serine proteases, like Granzyme B, are a class of proteins that utilize serine residues in the catalytic site to cleave a target polypeptide. These proteins are involved in divers e cascades, controlling aspects of digestion, blood clotting, the immune system and inflammation. Serine proteases thus must be tightly regulated, and have a variety of inhibitors, including a family called ser ine p rotein ase in hibitors or serpins As a class, serpins share up to 30% sequence identity and have significant structural similarities, sheets ( helices


20 ( 41) When the reactive center loop (RCL) is intact, the serpins exist in a structurally strained form ( 42) The RCL of the serpin st icks out above the globular domain and offers the reactive P1 ( 43,44) Upon cleavage of the scissile bond at the P1 covalently linked to the active site. Cleavage of the RCL results in a dramatic conformational change where the c ovalently linked RCL strand is dragged acr oss the molecule and inserted sheet A ( 41,42) This virtually irreversible covalent bond suicide inhibition ( 42) Despite this 1:1 inhibition ratio, serpins are remarkably effective even when present in low concentrations. Viral Evasion of Host Immune Responses A variety of viruses ha inflammatory response for efficient self propagation ( 45 48) Viral immunomodulatory proteins have a wide range of function s, which fall into two strategies; modulating or blocking extracellular activities or altering intracellular anti viral mechanisms such as apoptosis. The virus may produce cytokine mimics, cytokine receptor mimics, cytokine or complement inhibitors or infl ammatory cell inhibitors ( 37,40,47 49) Some microbes, tumors and viruses, including the pox viruses ( 50) down regulate host extracellular MHC I in an attempt to prevent the humoral immune system from detecting non self antigens ( 45,49,51,52) Natural Killer T cells ( NKTs) protect the host against this tactic. In response, viruses have escalated this evolut ionary arms race by encoding proteins that inhibit NKT cells. Mouse Cytomegalovirus ( MCMV) protein m157 binds to an inhibitory NK cell surface receptor to inhibit NK cell induced lysis ( 45) sarcoma associated herpesvirus protein K5 down regulate s MHC I, ICAM 1


21 and B7 2 molecules to inhibit lysis by NK cells ( 46) TNF apoptosis, inflammation and viral replication, so it is no surprise that secreted TNF Receptors have been described for a variety of pox and herpes viruses ( 50 ) The presence of IL 18 and IFN encoded in the pox virus family emphasizes the importance of suppressing these cytokines in vivo ( 53 55) Serpins in the Myxoma Pox V irus Among its arsenal of immunomodulatory proteins, the leporipoxvirus, myxoma, encodes three serpins, Serp 1, Serp 2 and Serp 3 ( 56) Interestingly, poxviruses are the only viruses known to encode functional serpins ( 22,44,57) Serp 1 has established roles in regulation of inflammation and targets thrombolytic plasmin, tPA and uPA, respectively and thrombotic factor Xa ( 57 59) Serp 3 has significant coding and structural deletions, but has a potential role in virulence in vivo as animals infected with myxoma viruses lacking Serp 3 do not form secondary lesions ( 60) Of particular interest is Serp 2, a 34kD cross class intracellular serpin which inhibits the serine protease Granzyme B as well as caspases 1, 8 and 10 i n vitro and has been shown to prevent apoptosis of immune cells in vivo ( 58,61 64) Serp 2 also has a role in viru lence, as demonstrated by the reduced myxomatosis, mortality and immune cell invasion into lesions in rabbits infected with a Serp 2 null myxoma virus ( named ( 65) In addition, Messud not seen in wild type viral infections ( 63) This was, however, contradicted by a 2006 pape r with a slightly different experimental design ( 61) Preliminary research on Serp blocking abilities shows that it is successfully able to bl ock apoptosis and


22 terminal caspase activity when replacing CrmA, the native anti apoptotic serpin, in the CrmA deleted vaccinia virus ( 64) The specificity of a serpin is based on the residues at the P1 1 has an arginine that t argets serine proteases ( 66) while Serp 2 has a glutamate resid u e, which confers cross class targeting to serine and cysteine proteases ( 65) In prior work, mutation of the Serp 2 P1 site to alanine ( D294A) ablated all protease inhibitor functions, but mutation to Glutamate ( D294E) altered the specificity to only inhibit caspase 1 ( 61) Preliminary research suggests that Serp inflammation rather than apoptosis ( 61) The Innate Immune / Inflammatory Response The innate immune system, which triggers inflammation, is the first line of defense against invading infectious organisms. This non specific, antigen independent, inflammatory response system also acts as a first responder to cell and tissue injury, settin g up wound healing activity wherever tissue trauma occurs. While the antigen dependent, secondary or antibody mediated immune system has been extensively studied for many years, this very powerful innate immune system was previously ignored as a more simpl e and primitive system than secondary, antigen dependent, antibody generating response. Recent work has now demonstrated that the inflammatory response system is, in fact, an extraordinarily powerful and effective response to infection and injury, often cl earing infectious organisms, establishing scar formation and beginning tissue reconstruction in areas of cellular damage before the adaptive immune response is activated. The inner layer of the arterial wall is named the intimal layer and is made up of en dothelial cells. The endothelium forms a single sheet of interconnected cells


23 throughout the vascular tree that is in constant contact and communication with inflammatory and immune defenses as well as inflammatory mediators in the circulating blood. It is the endothelial cell layer together with circulating polymorphonuclear neutrophils ( PMN), monocytes ( MC) and T lymphocytes ( TL) that form this front line Activated endotheli al cells display selectins on their surface. Passing leukocytes recognize these selectins, pause in their travel along the currents of the blood stream, and begin to roll along the endothelial cell surface. These cells then detect local chemoattractants th at, together with newly expressed integrins, induce these cells to that are threatened. Chemokines are small 8 14kDa chemoattractant peptides that attach to glycosaminogl ycans ( GAGs) on the endothelial cell surface or in connective tissue layers and form gradients that direct inflammatory cellular migration and invasion ( 67 69) Chemokines recruit circulating inflammatory response cells by attaching to G coupled chemokine receptors ( GPCRs) on the cell surface, then activating and directing the cells into the tissue ( 68) Larger, more complex viruses have evolved to manipulate the chemokine system such that the virus can hide from or literally block inflammatory and immune responses Another system that viruses target in order to evade the inflammatory and immune systems are the serine protease and serpin cascades. Cell invasion and migration through the arterial wall and into the surrounding tissues and organs often requires that a path be cut through collagen and elastin connective layers so that cells can migrate


24 into or invade the affected spaces. With injury and or infection there is often simultaneous activation and adhesion of platelets and activation of the thrombotic and thr ombolytic cascades on the platelet and connective tissue surfaces. The thrombotic and thrombolytic serine proteases also have a long established interaction with the inflammatory mediators. The thrombotic and thrombolytic cascades, through continuous cross talk, can activate elements in the innate immune response. Platelets, the small fragmented cells that lack nuclei, often function as more than simple clotting platforms by carrying storage organelles full of inflammatory mediators and growth factors that can similarly drive the innate immune response to the site at risk. The thrombotic and thrombolytic cascades are composed of a series of sequentially activated serine proteases which, in turn, are regulated by ser ine p rotease in hibitors, termed serpins. T he serine protease / serpin pathways also regulate many other activities such as complement activation, connective tissue breakdown, hormone expression, placental growth during pregnancy, and some intracellular signaling pathways. Serpins have been reporte d to represent up to two percent or more of circulating plasma proteins in mammalian blood. The urokinase type plasminogen activator ( uPA) that binds to the uPA receptor ( uPAR) is up regulated with increased expression in cells after injury or activation ( 70) The uPA/ uPAR complex in turn activates plas min, which activates the matrix degrading matrix metalloproteinase ( MMP) pro enzymes as well as acting directly on collagen and elastin and activating growth factors stored in the connective tissue layers ( Fig. 1 1A) ( 71,72) T cell induced apoptosis is also mediated th rough release of granzyme B, a serine protease that is part of the extrinsic apoptotic pathway ( 73,74) As for the chemokine / chemokine receptors,


25 the uPA/ uPAR complex as well as serine and cysteine proteases that initiate cellular apoptosis are actively blocked and / or mimicked by viruses, providing another mechanism for evasion of inflammatory and immune responses. The innate immune response developed prior to the evolution of the secondary adaptive immune responses system and still represents a highly effective and very powerful, primitive, non antigen dependent, front line of defense. The chemokine / chemokine receptor and the serine protease / protease receptor / serpin pathways have central roles in this innate immune response system. Viruses have evolved to modulate these two pathways for their own needs in ways that enhance proliferation and spread to other tissues and organs in the mammalian body. Viral Anti Inflammatory Proteins that Target the Chemokine Axis Viruses express both chemokine antagonists and chemokine mimics in order to alter the immune balance either to enhance inflammatory cell migration and activity or to block chemokine mediated inflammation. Many of these chemokine modulators have evolved over millions of years to become highly efficient agents that are s electively evolved to work for the virus. As a result, these proteins often have very low antigenic profiles, allowing them to function beneath host immune surveillance detection and are not subjected to the same regulatory mechanisms that generally act to counterbalance the native host pathways ( Fig. 1 1B). Viral chemokine modulators can then function by directing inflammatory cells to sites of viral infection. Chemokine mimics such as vMIP II, can attract cells to a site where they are then recruited for infection and viral proliferation, or as carriers that are intended to bring the viruses to fresh sites for infection. Others, termed chemokine modulating proteins ( CMPs), such as M T1, M T7


26 and M3, inhibit chemokines and their receptors, blocking host imm une responses so that the virus is protected from host defenses ( 67,75 78) Chemokine Modulating Proteins M T1, a chemokine modulating protein expressed by myxoma virus and vCCI expressed by vaccinia virus share 40% homology. Both of these CMPs bind to the N terminus of CC chemokines inhibiting the chemokine: receptor interaction. Interference with the chemokine N terminus and GPCR interaction prevents or disrupts cell signaling as well as chemokine enhanced chemotaxis and cell activation ( 79) M T1 has been tested in mouse and rat models of balloon angioplasty injury ( 80) and aortic allograft transplants where a reduction in monocyte and early T cell invasion was detected ( 81,82) Together with the early reduction in mononuclear cell invasion, a mark ed reduction in atherosclerotic plaque and transplant vasculopathy was also detected in animal models, indicating potential as an anti inflammatory therapeutic agent for inflammation based diseases such as atherosclerosis, transplant rejection and potentia lly various stages of cancer progression, invasion, angiogenesis or metastasis. vCCI has been reported to bind mammalian MCP 1 with high affinity, suggesting a potential for anti inflammatory activity ( 83) M T7 is a CMP secreted by myxoma virus that binds interferon gamma ( GAG binding domain of a wide range of mouse, rat, rabbit and human C, CC, and CXC chemokin es. This C terminal chemokine binding activity has low affinity and is without species restriction. Purified M T7 protein has been tested in rat and rabbit models of angioplasty injury and stent implant ( 80) as well as in aortic ( 81) models in mouse, and rat models of aortic allograft ( 81) and renal allograft transplant ( 84) When M T7 was


27 administrated subcutaneously as plasmid DNA in AO rats with degradable cross linked dermal sheep collagen discs, a model for angiogenesis, M T7 trea tment reduced new vessel formation, MCP 1 and VEGF levels ( 85) M T7 was also found to reduce IFN in mouse cells. M T7 thus has the ability to target and block important chemokine pathways such as the MCP 1 and vascular endothelial gr owth factor ( VEGF) pathways that are involved in inflammatory diseases such as atherosclerosis, vasculitis, arthritis and potentially invasive or metastatic cancers ( 86) Recently drugs have been designed to deliberately target the chemokine axis through interruption of chemokine: GAG binding and hence disruption of chemokine: GAG gradients ( 87) This design was originally based upon studies with the M T7 chemoki ne modulating protein is believed to bind to th e C terminal GAG binding domain of chemokines ( 76) Several studies of interest have assessed the capacity of unfractionated and low molecular weight heparin infusions or subcutaneous injections to block tumor invasion and metastasis, again presumably through interference with chemokine: GAG gradient formation ( 67,75,87) ( 50) M3 binds to both the C terminal GAG binding domain of chemokines and the N terminal receptor binding domain of all classes of C, CC, CXC, and CX3C chemokines. Intravenou s injection of M3, similar to M T1 and M T7 infusions, reduced early and late monocyte and T cell invasion and plaque growth in mouse and rat models of balloon angioplasty injury as well as aortic transplant in mouse and rat models ( 81) MC148 is a selective human CC chemokine receptor ( CCR8) antagonist encoded by the poxvirus molluscum contagiosum ( 88) MC148 has been tested in


28 murine models where allograft survival was prolonged after vector delivery ( 89) The purified protein has not yet been tested as a therapeutic agent. Serine Proteases and Serpins in the Innate Immune Response System Serine proteases regulate the clotting ( thrombotic) and clot dissolving ( thrombolytic) pathways as well as some stages of connective tissue breakdown, complement activation, and apoptosis ( cell suicide) granzyme mediated pathways and other pathways that will not be discussed here. In the thr ombo tic pathway, tissue factor, factor VII factor Xa and thrombin up regulate inflammatory responses. While less well defined, the role of the thrombolytic cascade in activation of inflammation has now also been discovered to have a very central role in infla mmatory cell responses ( 89 94) In the thrombolytic cascade, tissue type plasminogen activator ( tPA) is the main circulating activator for plasmin, acting at sites of fibrin d eposition. Urokinase type plasminogen activator ( uPA) acts primarily at the tissue interface initiating cellular adhesion and invasion ( 90) uPA, tPA, the uPA receptor ( uPAR), all hav e increased expression at sites of tissue injury throughout the mammalian body ( 44,90,91,93,95) Increased uPAR expression is associated with monocyte / macrophage activation in acute myocardial infa rction, traumatic brain injury, placental cell invasion, renal allograft rejection and most relevant to the topics discussed herein, tumor cell invasion ( 91) The uPA/uPAR pathway is thus implicated in a wide range of disorders where ther e is inflammation. Increased tPA expression is also detected after injury and has been reported to be up regulated in the central nervous system after seizures or stroke. A substantial body of literature now attests to the wide range of influence of the th rombolytic process on innate immune responses and cancer progression ( 92,93,95) ( Fig. 1 1). The uPA/uPAR complex migrates to the leading edge of invading leukocyte


29 membranes during inflammatory responses, forming part of a large grouping of membrane associated, lipid raft proteins ( 96) This uPAR super complex is formed in a lipid raft and includes integrin subunits ( ( CD18), epidermal growth factor receptor ( otein receptor related protein receptor ( LRPR), L selectin, and CXCR4, G protein coupled receptor FPLR1, and caveolin ( 90,93,97,98) uPA, PAI 1, vitronectin and high molecular weight kininogen ( HKa) all associate with uPAR as it sits on the cell surface membrane ( 90,93,98) uPA binds to uPAR to activate plasmin which, in turn, activates matrix degrading enzymes, the matrix metalloproteinases ( MMPs) to break down collagen and elastin ( Fig. 1 1A) ( 72,90,92,99) The plasminogen activators and plasmin can also directly activate MMPs and even degrade connective tissue proteins independently ( 72) Matrix destruction allows cells to invade and liberates tissue stores of growth factors such as basic fibroblast growth factor ( bFGF) and transforming gro wth factor beta ( bound tPA and uPA independently activate MMPs and growth factors. uPAR is glycophosphatidylinositol ( GPI) integrins an d LRPR, altering Ras/ERK, JAK/STAT, MAPK and calcium signaling ( 44,72,90 93,95,96) Ser ine p rotease in hibitors or serpins represent an extensive grouping of proteins that regulate coagulation and the thromboly sis, connective tissue remodeling, inflammation, and complement activation ( Fig. 1 1A). In mammals, serpins represent two percent, or more, of circulating proteins in the blood and mediate a wide range of


30 homeostatic functions. Well known serpins with defi ned functions include anti thrombin III ( AT III), heparin cofactor II ( HC II), alpha 1 ( 94) anti chymotrypsin, alpha 2 anti plasmin ( AP), plasminogen activator inhibitor 1 ( PAI 1), protease nexin ( PN), neuroserpin ( NSP), and heat shock protein 47 ( HSP 47) ( 44,90 93,95) The majority of serpins act as pseudosubstrates for serine proteases, forming a 1:1 complex. Protein size is determined by variable C or N terminal domains. Diseases with serpin dysfunction include clotting disorders, emphysema, and cirrhosis. Some serpins, such as ovalbumin, also have non inhibitory roles. One such serpin, maspin, is widely expressed in a wide range of mammalian cells and combines c haracteristics structurally for both active and inactive, ovalbumin like serpins ( 100 102) The inactive serpins have structures with a relatively shorter RCL and do not undergo the classical conformational changes seen with the active serpin s such as PAI 1. Maspin is both an intracellularly and extracellularly active serpin associated with tumor suppression ( 100 103) Serpins thus have vital roles in regulating normal human physiology. Serpin Mediated Regulation of the Thrombolytic Cascade The roles of serpins that target plasminogen activators, tPA and uPA, in inflammatory cell responses and their potential interactio ns with uPAR remain only partially understood. There are five known mammalian vascular inhibitors of tPA and uPA; neuroserpin ( NSP), PAI 1, PAI 2, protease nexin ( PN) and PAI 3 ( Fig. 1 1A) ( 90,91) PAI 1 is considered the most important inhibitor of tPA and uPA in the vasculature. PAI 1 also binds to thrombin and activated protein C ( APC), but is inactivated by the latter. Increased circulati ng PAI 1 is found in obese, hypertensive, insulin resistant patients who are at increased risk for myocardial infarction ( 104)


31 Less well known PAIs include PAI 2, PAI 3, PN and NSP ( 44,92,95,105) PAI 2 reacts only with uPA and plasmin, but not tPA and is secreted by most cells whereas PAI 3 is secreted by monocytes, polymor phonuclear leukocytes and epithelial cells ( 70) PAI 3 inhibits tPA, uPA and plasmin weakly, as well as protein C ( 106) PN has a wide range of targets including two chain tPA, uPA, thrombin, factor Xa, trypsin and plasmin, although anti plasmin is the primary plasmin inhibitor ( 107) Neu roserpin ( NSP) is a relatively new serpin that has a slightly increased affinity for tPA, co localizing with tPA at sites of nerve damage ( 105) Additionally, NSP binds to, but is cleaved by plasmin. NSP is found predominantly in nervous system tissue and is secreted by axons, with increased expression at sites of damag e, sites corresponding to increased tPA expression ( 108) NSP has not generally been thought to have a role in vascular responses. However, NSP reduced inflammation and the volume of brain tissue damaged ( stroke size) after cerebral arterial occlusio n in animal models ( 108) In summary, the mammalian serpins that regulate thrombolysis are up regulated at sites of tissue injury and in some cases can act to reduce inflammation and damage. In animal models, thrombolytic proteases ( uPA and tPA) and regulatory serpins alter inflammatory cell responses. uPA deficient mice have prolonged thrombolysis, but also have reduced intimal hyperplasia ( 109) Increased uPA expression increased plaque growth in rabbit arteries ( 110) and increased macrophage invasion in ApoE null mice, again consistent with pro inflammatory activity for uPA ( 98) tPA deficiency however had less effect. Conve rsely, PAI 1 deficient mice had increased plaque formation, as reported by Carmeliet ( 109) which was reduced by adenovirus expression of PAI 1 indicating anti atherogenic activity ( Fig. 1 1A). However, in other mouse models, PAI 1


32 appeared to increase inflammation and plaque growth ( 111) PAI 1 can thus promote thrombosis and plaque growth and also reduce plaque, responses that vary with the animal model. Binding of PAI 1 which directly targets the tPA, uPA and thrombin serine proteases internalizes u PA/uPAR which also moderates cell signaling. Vitronectin activates uPA/uPAR complexes and, conversely, increases PAI 1 inhibitory activity ( 109,111) As a result, the uPA/uPAR complex and serpins that regulate thrombolysis can a lso regulate inflammatory responses. Serpins that Regulate Connective Tissue Degradation and Apoptosis A genetic deficiency in which there is a lack of alpha1 antitrypsin ( AT) predisposes patients to early emphysema and cirrhosis ( 94) AT binds and inhibits neutrophil elastase and chymotrypsin while alpha1 anti chymotrypsin ( ACT) targets chymotrypsin ( 112) These proteins, in addition to the MMPs, regulate breakdown of elastin in connective tissue ( 99,113,114) The pancreatic trypsin and chymotrypsin enzymes are also classified as serine proteases. In chronic pancreatitis elevated levels of activated trypsin contribute to continuous tissue dama ge and pain ( 115) Apoptotic cell death induces a pro thrombotic, inflammatory state in endothelial cells ( 116 121) while in monocytes and smooth muscle cells apoptosis has been implicated in plaque rupture ( 116,117,120) Increased numbers of cytotoxic, perforin positive T lymphocytes are present in unstable coronary syndromes and in the accelerated vasculopathy of transp lanted hearts ( 117,122 126) potentially driving apoptosis in endothelium, smooth muscle, monocytes and also inducing apoptotic responses in T cells as well. Interference with T cell apoptosis in rats ( 118) leads to a tolerant state and granzyme B deficiency in mice reduces transplant vasculopathy ( 123) Fas ligand has been reported to either block ( 116) or accelerate atheroma


33 development in ApoE deficient mice. Cellular apoptosis in the vasculature can thus contribute toward accelerated plaque development and progression to rupture ( 118,120 124,127 129) The precise role of apoptosis, the specific cell death pathways and the principal cell types mediating atherosclerotic plaque growth and rupture, however, remain undefined. Viral Proteins that Target the Serine Protease / Serpin Axis Thrombolytic Protease Inhibitors In particular, viruses have emulated normal cell serpin functions to shut down both the adaptive and innate immune response by a variety of mechanisms. The thrombotic ( clot forming) and thrombolytic ( clot dissolving) pathways are compose of a series of sequentially activated serine proteases that act in counterbalance. These protease pathways are regulated by serine proteinase inhibitors, termed serpins. The myxoma virus expresses Ser p 1, a secreted serpin which inhibits thrombolytic pathway proteases and demonstrates a profound inhibition of inflammatory cell activation and plaque growth in a variety of animal models (57,59,66,84,91,130 133) Se rp 1 inhibits uPA, tPA, plasmin and factor Xa with low K a values in vitro, and, depending on tissue concentrations, is either cleaved by or inhibits thrombin. Similarly, Spi3 is a cowpox viral serpin that similarly targets plasminogen activators ( thromboly tic proteases). Serp 1 treatment induces a profound inhibition of inflammatory cell activation and plaque growth in a wide range of animal models (57,59,66,91,130 133) .Based upon our recent studies with a series of Serp 1 reactive center loop ( RCL) chimeras, we know that an intact RCL and uPAR expression are both required for Serp 1 anti inflammatory actions ( 59,66) Treatment of human endothelial, m onocyte and T cells in culture with Serp 1 has demonstrated reduced cell activation ( 57) in response to selected activators.


34 Reductions in cell activation produced by Serp 1 were associated with specific changes in gene expression and these changes were blocked by antibody to uPA R ( 57) Serp 1 inhibits t he uptake of the uPA/uPAR complex involved in triggering inflammatory cell invasion which is often disregulated in cancers. Macrophages in particular often account for a large portion of a tumor, and are thought to be subverted by the tumor to serve its ow n needs. Macrophages have been shown to assist in angiogenesis and ECM breakdown and remodeling in a variety of tumor lineages by providing access to growth factors and facilitating metastasis (134) If Serp 1 treatment reduces macrophage invasion into the tumor via suppressing uPA/uPAR uptake, it could disrupt this proliferative support and reduce tumor invasiveness. Additionally, Serp s suggests it may be able to reduce angiogenesis in cancer. One small study with Serp 1 has demonstrated reduced angiogenesis in a chicken allantoic membrane model ( 91) providing proof of principle for Serp 1 based cancer treatments tha t remains to be expanded. This unique viral serpin has passed Phase 1 safety trials and has completed a Phase 1b/2a clinical trial in patients with acute coronary syndromes in Canada ( TPD) and the US ( FDA) (1 35) Given the markedly effective anti inflammatory activity of Serp 1, an analysis of Serp mediated anti inflammatory activity and direct discovery of new therapeutic approaches for unstable plaque and other inflammation based disorders (135) Serp 1 has been extensively studied to date in over 40 animal models of atherosclerosis, restenosis after stent implant, acute and chronic transplant re jection and collagen induced arthritis in animal models in labs in North America and Europe,


35 with demonstrated marked inhibition of inflammatory cell responses (57,59,6 6,91,130 133) Serp 1 inhibited early mononuclear cell invasion and late plaque formation in mouse, rat, rabbit, rooster and swine models of angioplasty injury, rabbit and sw ine models of stent implant and mouse and rat models of chronic rejection after aortic, renal and cardiac allograft transplant ( 66, 84) Vasculopathy and scarring were both inhibited in the rat renal transplant model at 5 months follow up, indicating long term efficacy ( 84) A study of carotid cuff injury in the ApoE mouse demonstrated 67% reduction in plaque growth with decreased macrophage invasion suggesting plaque stabilization (132) In general, Serp 1 reduced macrophage and T cel l invasion in the arterial wall, activities that are dependent upon an intact Serp 1 reactive site loop and expression of uPAR in vascular cells. This anti inflammatory protein immunotherapeutic represents the forefront of protein drug development based up on viral designs of immunomodulators. Apoptotic Protease Inhibitors Serp 2 is a normally intracellular myxoma viral protein, a cross class serpin that inhibits both serine proteases, granzyme B ( 62) and cysteine proteases ( human interleukin 1 converting enzyme, or caspase 1), thus preventing the conversion of pro IL and activation of caspas e 8 ( 63) Serp 2 has been tested for anti atherogenic effects in rat angioplasty and aortic transplant models, along with carotid cuff injury (136) Serp 2 markedly reduced plaque growth and inflammatory cell invasion in these models as well as in ApoEnull ( hyperlipidemic) mice (136) CrmA ( cytokine response modifier A) is another immunomodulatory, intracellular serpin encoded by poxviruses ( SPI 2) that binds and inhibits caspases 1 and 8 and granzyme B (63,137,138) CrmA inhibition of caspase 1 ( cysteine protease), preve nts


36 (63,139) The same serpin also inhibits IL 18 as shown by recombinant adenovirus vecto r CrmA gene expression in mice (140) CrmA is a more potent inhibitor, in vitro yet with greater inhibition of inflammation in chicken chorioallantoic membranes (136) On the other hand, Serp 2 binds Caspase 1 and Granzyme B with lower affinity in vitro, but has greater effects on viral virulence in vivo in infected rabbits ( 61,63) CrmA, however, had no effect on cell invasion or plaque growth in any of the animal models tested to date (136) Serp 2, conversely, has not been tested in any of the cancer or angiogenesis models. Serp 2 has a more specific GzmB and casp 1 inhibition and thus may provide a more potent anti inflammatory activity which is evidenced in the animal models tested to date (63,136) With the more potent anti inflammatory action of Serp 2 in animal models of vascular injury and transplant, we would propose that Serp 2 may alter either inflammatory cell responses through altered apo ptotic responses ( granzyme B pathway) ( 62) or regulation caspase 1 ( 63) and thus reduce inflammatory cell invas ion. From this, we hypothesized that Serp 2 and CrmA wou ld induce different changes in apoptotic gene expression for cells involved in the innate immune response We further postulated that other viral ant inflammatory proteins with known anti inflammatory functions, namely Serp 1 and M T7, would potentially target apoptotic pathways and potentially share a final common pathway for efficacy. We further hypothesize d that the effects of Serp 2 and CrmA would be modified in mice deficient for their tar get proteases ( e.g. GzmB / and Casp1 / mice) when compared to genotype background mice (C57Bl/6 and Nod, respectively) potentially reveal ing insights into the mechanis m of action via detection of differences in gene expression.


37 Figure 1 1 Thrombolytic and apoptotic serine / cysteine protease pathways as well as endogenous serpin regulatory proteins. T hrombotic and thrombolytic serine protease and apoptotic pathways ar e closely associated with cancer cell invasion, metastasis and angiogenesis. B) Viral serpins are capable of regulating inflammation driven cancer cell invasion, metastasis and angiogenesis.


38 CHAPTER 2 METHODS AND MATERIAL S Animal Models Rodents All resea Research Institute, University of Western Ontario, London, Canada, laboratory animal ethics committee and the University of Florida, Gainesville, USA, Institutional Animal Care Committee, ( IACUC, Protocol number 102004234) and conformed to national guidelines. The rat strains studied were Sprague Dawley and Lewis rats from Charles Rive r Laboratories, (Wilmington, MA, USA ) and ACI and AO rats from Harlan Labs ( Indianapolis, IN, USA ) The mouse strains studied were ApoE / ( B6.129P2 Apoe/J stock number: 002052), Balb/C ( stock number: 000651), C57BL/6J ( Stock number: 000664), Caspase 1 / ( NOD.129S2 ( B6) Casp1tm1Sesh/LtJ, stock number: 004947), Non Obese Diabetic ( NOD/ShiLt, stock nu mber: 001289 ), Granzyme B / ( B6.129S2 Gzmbtm1Ley/J, stock number: 002248) and PAI 1 / ( B6.129S2 Serpine1tm1Mlg, stock number: 002507). GzmB / /ApoE / double knockout mice ( C57Bl/6 background) were generated at the Breeding Facility at the University of Florida from ApoE and GzmB single knockout mice purchased from Jackson Laboratories and tested for homozygous double knockout status before being used in experiments. Rodents were housed at the University of Florida Animal Services facility in accordance w ith University of Florida Animal Care guidelines. Angioplasty, Aortic Allograft and Carotid Cuff Compression Models Effects of each serpin on plaque growth were assessed in three animal models; 1] angioplasty injury in 250 300g male Sprague Dawley rats ( S D, N = 126) 2] aortic


39 allograft transplant from inbred 250 300g ACI to Lewis rats ( N = 60) as well as C57Bl/6 to Balb/C mice ( N = 96) and 3] carotid cuff compression injury in 12 14 week old ApoE / mice ( N = 33) (132) ( TNPO PG, Leiden, the Netherlands) with all surgeries performed as previously described ( Table 4 1). In a second study, ApoE / ( N = 15), GzmB / ( N = 15), PAI 1 / ( N= 14) as well as GzmB / /ApoE / DKO mice ( C57Bl/6 background, N = 15) were used in aortic allograft transplant studies. All research protocols and general animal care were approved by University laboratory animal ethics and conformed to national guidelines. All surgeries were performed under general anesthetic, 6.5mg per 100g body weight Somno trol ( MTC Pharmaceuticals, Cambridge, Canada) in tra muscular injection for rats and subcutaneous 60mg/kg ketamine ( Eurovet Animal Health), 1.26mg/kg Fentanyl, and 2.0mg/kg fluanisone ( Janssen Animal Health) for carotid cuff placement in ApoE / mice. Vira l serpins were infused intravenously ( i.v.) immediately after surgery in rats at doses of 0.3ng 3000ng/rat ( 0.001 10ng/g), wi th follow up at 4 weeks ( Table 4 1). Daily subcutaneous injections of saline, CrmA ( 240ng/mouse/day, 12ng/g/day) and Serp 2 ( 1800 ng/mouse/day, 90ng/g/day) were started two weeks after collar placement in ApoE / mice and continued for 4 weeks until sacrifice. 125 I labeled CrmA and Serp 2 were injected on the first day and the last two days in two ApoE / mice detecting serum concen trations of 0.16nM for CrmA and 1.72nM for Serp 2. For the mouse aortic transplant studies a single i.v. injection ( 15g/ mouse; 500ng/g) of Serp 2, CrmA, or D294A or D294E mutants was administered immediately after allograft surgery. A separate group of 1 00 rats had angioplasty injury with 300ng of each serpin by i.v. injection for early follow up at 0, 12, or 72 hours to assess early apoptotic pathway


40 enzyme activity ( 6 animals/treatment group; Table 4 1). There were no deaths after angioplasty or aortic transplant in the rats, 14 mice died after aortic transplant, and one mouse died during placement of the carotid cuff. In the mouse aortic transplant model two mice died after treatment with Serp 2, 5 after CrmA, 2 after D294A, 2 after D294E, and 2 after s aline treatment with overall survival of 85.4% ( P= NS). Body weight was measured weekly and mice were checked daily for signs of distress and necessity for analgesic. Histological, Morphometric, and Fluorescence Analysis At the designated study end ( 4 weeks for plaque analysis and 0 72 hours for protease activity or apoptosis) rats and mice were sacrificed with Euthanyl ( Bimenda MTC Animal Health Company, Cambridge, Ontario, Canada). For mouse and rat angioplasty and allograft transplant models, arter ial sections were fixed, processed, paraffin embedded, and cut into 5 m sections ( 2 3 sections per site) for histological analysis, as previously described (59,66,81,131,132) For the ApoE / mice with carotid cuff compression, the aortic valve area ( 10m sections throughout the valve area) and the carotid artery from the bifurcation through the site of cuff compression ( 5m sections at 25m intervals) were assessed. Sections were stained with Haematoxylin/ Eosin, Trichrome and Oil Red O for analysis of plaque area, thickness, and invading mononuclear cells, as previously described (59,66,81,131,132) Plaque area as well as intimal thickness and medial thickness were measured by morphometric analysis via the Empix Northern Eclipse trace application progra m ( Mississauga, ON, Canada) on images captured by a video camera ( Olympus, Orangeburg, NY, USA) attached to and calibrated to the Olympus microscope objective. The mean total cross sectional area of the intima as well as diameter of the intima and media we re calculated for each arterial


41 section. For immunohistochemistry, T cells were labeled with rabbit anti mouse CD3 antibody, both then labeled with secondary goat anti rabbit antibody ( CD3; Cat # AB5690, Secondary anti rabbit; Cat# AB80437. Abcam, Cambridg e, MA, USA). For Caspase 3 staining, sections were incubated with anti caspase 3 polyclonal antibody ( Cat# AB3623 1:20) as primary antibody with secondary rabbit specific HRP conjugated antibody ( Cat # AB80437). Either numbers of positively stained cells i n three high power fields in the intimal, medial, and adventitial layers were counted and the mean calculated for each specimen or, when fewer cells were detected, ( displayed in earlier follow up times) cell counts for positive staining in all three layers were averaged. For spectroscopic analysis of Serp 2 and CrmA binding, 1x10 6 cells/ mL were treated with 1ug/ mL of FITC labeled protein for two hours, lysed with cell lysis buffer, and fluorescence emission at 525nm quantified during excitation at 485nm. F or fluorescence microscopy, cells treated with Serp 2 FITC for 2hrs at 4 C, then fixed with 2% formaldehyde, mounted with 10% glycerol mounting solution and viewed with a Zeiss fluorescent microscope as previously described (136) For FACS analysis, 1x10 6 cells/ mL were treated with 1g/ mL of FITC labeled Serp 2 or CrmA for two hours and run on FACS ( FACS Calibur, BD Falcon) acquiring data for 20,000 events with three re plicates ( Cell Quest data analysis program). To measure uptake of Serp 2, Jurkat T cells were treated with PMA and calcium ionophore to stimulate granzyme B production, (141) then FITC labeled Serp 2 was added to the activated cells and incubated. Cells were lysed and the membrane and ins oluble fractions were separated by centrifugation. FITC Serp 2 presence was quantitated by absorbance at 525nm ( Fluorscan) measurements for both fractions.


42 Enzyme Activity Analyses For whole arterial lysates Serp 2, CrmA, 294A, or 294E treated rat femoral arteries ( 2 3 cm length) were excised at 0, 12, and 72 hours after angioplasty injury. The tissues were homogenized, lysed and extracted in 1mM EDTA buffer, centrifuged at 10,000 rpm, 8C for 10 min to remove un dissolved solids and supernatant stored at 8 0C. For cell lysates, 1 X 10 6 / mL volume ( HUVEC, THP 1, or T cells) were treated with saline or apoptosis actuators ( CPT, 2 M for THP 1 and 10M for T cells; STS 0.5M from Sigma, Oakville, ON, Canada; or FasL, 3ng/ mL from Upstate solutions, Charlottesville, VA, U.S.A.), and each actuator given in combination with either Serp 2, CrmA, 294A, or 294E ( 500 ng/ mL ). Cells were collected at 6 hours, washed with cold saline and treated with 60l lysis buffer ( 150 mM NaCl, 20 mM Tris base, 1% ( v/v) Triton X 100 at pH 7.2, for 10 min, 4 C) followed by centrifugation at 10,000 rpm for 10 min, 8C. Supernatant was collected and stored at 80C until use. Protein concentration was measured ( Bio Rad Protein assay, Bio Rad Laboratori es, Hercules, CA, U.S.A.). A subset of T cell cultures were treated with phorbol myristic acid ( PMA, 1ug/ mL ) and Ionophore A23187 ( 1g/ mL ) to induce a CTL like ( cytotoxic T lymphocyte) state. Medium from treated T cell cultures containing GzmB and perfor in was removed after 2 hours incubation and applied to fresh, untreated T cell cultures together with Serp 2, CrmA, or S erp 2 mutants D294A or E, with and without antibody to GzmB or perforin ( Sigma) or the cell permeable small molecule inhibitor, ZAAD CMK ( ZAAD chloromethylketone, Calbiochem, CedarLane, Hornby, ON), incubation for 12 or 24 hours (136) To analyze cell death ELISA assay, fragmented nucleosomes were det ected using quantitative sandwich


43 ( Cell Death ELISA kit, Roche Diagnostics, Germany) with conjugated peroxidase measured photometrically at 405 nm with ABTS ( 2,2 azino di[3] ethylbenzthiazoline sulfo nate) as substrate using a Multiscan Ascent Spectrophotometer ( Thermo LabSystems Inc., Beverly, MA, US). For the DEVDase ( Casp 3 and 7), 10 l of the cell/tissue lysate was incubated at 37 C for one hour in 90l of reaction buffer ( 100 mM Acetyl DEVD ( As p Glu Val Asp) AFC ( 7 amino 4 trifluoromethylcoumarin) ( Bachem, Torrance, CA, U.S.A.), 100 mM HEPES, 0.5 mM EDTA, 20% ( v/v) Glycerol and 5mM DTT, pH 7.5). For IEPDase, 10l of the cell /tissue lysate was incubated at 37 C for one hour in 90l of reaction buffer ( 100 mM Acetyl IEPD ( Ile Glu Pro Asp) AFC ( 7 amino 4 trifluoromethylcoumarin) ( Kamiya Biomedical Company, Seattle, WA), 50 mM HEPES, 0.05% ( w/v) CHAPS, 10% ( w/v) Sucrose and 5mM DTT, pH 7.5). For the Cathepsin K, S, L, and V assays, 10l of the cell /tissue lysate was incubated in 90 l of reaction buffer ( 5 mM Rhodamine 110, bisCBZ L Phenylalanyl L arginine amide, 50mM Sodium acetate, 1 mM EDTA and 4 mM DTT, pH5.5) ( Bachem, Torrance, CA, U.S.A.). For DEVDase and IEPDase hydrolyzed fluorochrome, 7 am ino 4 trifluoromethyl coumarin was measured using a Spectrofluorometer ( Fluoroskan; with excitation 405 nm, emission 527 nm) ( Thermo LabSystems Inc., Beverly, MA, US). For the Cathepsin assay hydrolyzed fluorochrome, Rhodamine110 was measured using excitat ion 485 nm, emission at 527 nm. Final values are corrected for protein concentration. All these experiments were performed in triplicate, with three separate experiments performed for each condition, and the arbitrary fluorescent units from each reading us ed to derive the mean standard error


44 for individual treatments and presented in the figures as bar graph with error bars indicating standard error. Histological, Immunohistochemical, and Morphometric Analysis Arterial specimens, 0.5 0.6 cm in length, we re harvested from mice at follow up and cut into three equal length sections, fixed, and paraffin embedded. Three 5 m sections were cut per specimen ( total of 6 9 sections per artery) and strained with ological analysis, as previously described (59,66,81,131,132) Plaque, internal elastic lamina ( IEL), and external elastic lamina ( EEL) areas and intimal and medial diameters were measured using an Olympus microscopy system standardized to the microscopic objective as previously described ( Image Pro Plus V6.0, Georgia, MD) (59,66,81) To assess aneurysmal dilatation, both short and long cross sections IEL and EEL diameters were measured. The number of elastic lamina breaks were counted for each specimen within the high power field ( 100x object ive), with triplicate measurements for each animal studied. Mononuclear cell invasion was quantified by randomly outlining 3 areas with increased cell invasion in the intimal, medial and adventitial layers per section and counting the stained nuclei or cel ls contained within, resulting in a cell density measurement. Immunohistochemical staining for CD3 + T cells and CD11b + monocytes was performed as previously described using the ABC technique (59,66,81) T cells were labeled with rabbit anti mouse CD3 antibody and rabbit anti mouse CD11b, with secondary goat anti rabbit antibody ( CD3; Cat # AB5690, CD1 1b; Cat # AB75476, Secondary anti rabbit; Cat# AB80437. Abcam, Cambridge, MA, USA). For Caspase 3 staining, anti caspase 3 polyclonal antibody ( Cat# AB3623) was used with anti rabbit HRP conjugated antibody ( Cat # AB80437). Positively stained cells in thr ee high power


45 fields in the intimal, medial, and adventitial layers were counted and the mean calculated for each specimen. Mouse Peritoneal Exudate Model Mice were injected intra peritoneally with phorbol 12 myristate 13 acetate ( PMA ) ( 50 ng/mouse), foll owed by injection of saline, or 500ng S erp 1, Serp 2 or CrmA. After 18 h, the mice are anesthetized, then the blood was withdrawn via cardiac puncture ( using heparin flushed insulin needle). Next, the abdominal skin was gently dissected away from the perito neal membrane. 8mL sterile saline was injected into peritoneum, the mouse abdomen gently massaged, then cut open to collect the fluid, which was stored on ice. The peritoneal membrane was bisected and the spleen was removed into a waiting labeled tube with 500uL RNAlater ( Ambion). The abdominal aorta was seized just below the renal bifurcation and dissected down to the femoral bifurcation. This section was removed to a labeled tube containing 500uL RNA later. Spleen and Aorta were stored at 20C overnight then moved to 80C. Peritoneal exudates were centrifuged 8min x 1200 rpm, supernatant removed, then the pelleted cells are treated with RBC lysis buffer ( Ammonium chloride 0.15 M, potassium bicarbonate 10 mM, EDTA 0.1 mM, ph 7.4) for 5 min at room temperature. 3mL sterile PBS was added to stop the reaction, then .5mL FBS was layered over the cells at the bottom of the tube. Centrifuge 8min x 1200rpm. The supernatant was discarded, the cells resuspended in 2mL of 2% Fetal Bovi ne Serum / Phosphate Buffered Saline. The sample was them passed over a 70 micron filter into a round bottom 5mm tube, effectively removing contaminating mouse hair. Each sample was split then into two identical containers and centrifuged 8min x 1200rpm. O ne replicate of each sample was then fixed with 10% paraformaldehyde for 30 minutes, centrifuged


46 8min x 1200rpm, and stored in 1mL 2% FBS/PBS. The other sample was stored in 500uL RNA later for later analysis. Total cell migration count into the mouse asc ites lavage was measured by hemocytometer, then 1x 10 6 fixed cells were used for antibody staining and analysis using flow cytometry ( FACS Calibur, Becton Dickinson Canada Inc., Mississauga, ON) as previously described (59,66,81) The fluors used were: FITC CD11b ( EBioscience #11 0112 82, isotype control #11 4031 82), PE Ly6G ( EBioscience #12 5931 82, isotype control #12 4031 81), PE Cy5 CD4 ( EBioscience #35 0042 8 2, isotype control #35 4321 82) and APC Ly6C ( EBioscience #17 5932 82, isotype control Biolegend #400713) RNA Isolation, cDNA Synthesis and qRT PCR for Mouse Peritoneal Cells Samples stored in RNAlater were thawed to room temperature, centrifuged 8min x 1 200rpm, the RNAlater was aspirated and discarded. The RNA was then isolated from the cells using PureLink RNA Mini Kit ( Ambion instructions.RNA content was measured using a NanoDrop spectroflurometer ( Nanodrop, Wilmington, DE), and equal quantities of RNA were synthesized into cDNA using Superscript III ( Invitrogen, Grand Island, NY) cDNA was mixed with KAPA SYBR FAST qPCR Kits ( Kapa BioSystems, Woburn, MA) and applied to an mouse apoptosis array ( Table 5 1 ). Plates were r un on an ABI 7300 Real time PCR Machine ( Applied Biosystems, Foster City, CA) with the following protocol: initial hot start stage 1 ( 2:30 at 95C), then 40 cycles of Stage 2, Step 1 ( 0:03 at 95C), Step 2 ( 0:30 at 60C). Plates were read at Stage 2, Step 2. Data within mouse strains was normalized to GAPDH, then to the average PMA Relative Quantity ( RQ) for each gene in the other treatment groups.


47 In Vitro Models Cell C ulture Human umbilical vein endothelial cells ( HUVEC, CC 2519 Clonetics, Walkersville, MD, passages 2 5), THP 1 cells ( American Type Culture Collection, Rockville, MD, USA, ATCC TIB 202), or Jurkat cells ( E6.1 clone, ATCC TIB 152) ( 0.5 1.0 X106 cells/ mL ) were incubated with saline control, one of the apoptosis inducing agents ( 3ng/ mL memb rane bound FasL, 0.5M STS, or 2 10 M CPT) together with individual serpins ( 500ng/106 cells/ mL ). Media was supplemented with 10% Fetal Bovine Serum ( Invitrogen Canada Inc., Burlington, ON), Penicillin ( 100 units/ mL ), and Streptomycin ( 100 g/ mL Gibco B RL). Source and Purification of Proteins Serp 1 was expressed in Chinese Hamster Ovary cells and M T7 was expressed by a vaccinia vector system in baby green monkey kidney cells as previously described (142) and each protein was purified by two step fast performance liquid chromatography. Serp 1 and M T7 were kindly provided by Viron Therapeutics Inc. ( London, ON). Serp 2, Crm A, D294A, and D294E were His tagged at the amino terminus, expressed in vaccinia/T7 vector in HeLa cells ( Dr Richard Moyer, University of Florida, Gainesville, USA) ( 6), and purified by immobilized metal affinity using His Bind resin ( Novagen) ( 2 8,31,33) The D294A protein is a site directed mutant of Serp 2 with P1 Aspartate 294 changed to Alanine to inactivate the serpin, while the D294E protein has P1 Aspartate 294 replaced by Glutamic acid to alter the inhibition spectrum. Eluted


48 proteins were judged > 90% pure by SDS 12% PAGE, silver staining and immunoblotting. Serp 2 and CrmA were tested for Casp 1 and GzmB inhibitory activity. Serp 2 and CrmA were labeled with Fluorescein Isothionate ( Fluorotag FITC conjugation kit, Sigma Aldrich Canada Ltd., Miss issauga, Ontario) and passed through G 25M gel filtration column to separate unbound FITC. The F/P ( FITC/protein) ratio was 2.2 and 2.1, for Serp 2 and CrmA respectively. The caspase 1 inhibitory activity of FITC labeled proteins was assayed, displaying no rmal activity. BSA was labeled in parallel with FITC and used as control in entry assays. qRT PCR for Cultured Cell Lines For RNA isolation, 5x10 6 of THP 1 cells ( American Type Culture Collection, Rockville, MD, USA, ATCC TIB 202) were treated with saline, 10M Camptothecin ( CPT) in DMSO ( Acros Organics, Geel, Belgium), or CPT and 500ng Serp 1, Serp 2 or M T7 per million cells for either 30 minutes or 4.5 hrs. Cells were lysed and RNA purified using the Qiagen RNeasy kit ( Valencia, CA). RNA content w as measured using a NanoDrop spectroflurometer ( Nanodrop, Wilmington, DE), and equal quantities of ( Frederick, MD). and PCR mix, and applied to a pre manufactured apoptosis array ( Cat. # PAHS 012). Plates were run on an ABI 7300 Real time PCR Machine ( Applied Biosystems, Foster City, CA) according to the recommended protocol. Human umbilical vein endothelial cells ( HUV EC) and Jurkat T cells were similarly examined after Camptothecin treatment with or without control of anti inflammatory protein treatment.


49 Adhesion assays 1 x10 6 THP ester ( Calcein AM) ( M olecular Probes, Inc., Eugene, OR) for 1hr and activated with phorbol 12 myristate 13 acetate ( PMA) for 1hr. Cells were treated with saline or 500ng Serp 1, Serp 2, or albumin per million cells of and allowed to adhere for 2hrs. Non adherent cells were rem oved with two cold PBS washes and adherent cells quantified by calcein fluorescence ( Excitation 485 nm / Emission 527 nm) using an EnVision multilabel plate reader ( PerkinElmer, Fremont, CA). Membrane fluidity assays 1 x10 6 THP 1 were labeled with 2,2 bis ( 4 hydroxy 3 methylphenyl) propane ( BPP) ( diphenylhexatriene ( TMA DPH) ( Cells were activated with PMA ( mL /1 hr), w ashed, resuspended in complete growth medium and treated with Serp 1, Serp 2 or albumin ( 500 ng/million cells). After one hour, cells were washed to remove excess fluorescent probe and monomer, then excimer fluorescence emission intensities were measured a t 390 nm and 485 nm plate reader.The ratio of excimer fluorescence to monomer fluorescence gives the measure of membrane fluidity. Statistical Analysis Significance was assessed by analysis of variance ( ANOVA) with secondary Fishers least significant. Statview software ( SAS, Inc. Cary, North Carolina) was used.


50 CHAPTER 3 1 VIRAL CROSS CLASS SERPIN INHIBITS VASCULAR INFLAMMATION AND T LYMPHOCYTE FRATRICIDE; A STUDY IN RODENT MODELS IN VIVO AND HUMAN CELL LINES IN VITRO The intention of this chapter is to explore the first hypothesis: Serp 2 and CrmA alter inflammatory cell responses in mouse models through modification of apoptotic responses in inflammatory cells. It also explores induction of apoptosis in human inflammatory cell lines to partially address hypothesis 2: the anti inflammatory actions of the three viral proteins with proven anti inflammatory activities will alter expression of a family of shared apoptotic pathway gene s indicating a final response pathway common to all three protein treatments. Introduction Ser ine p rotease in hibitors or serpins have extensive regulatory actions, moderating thrombotic and immune responses ( 1, 2). Poxviruses encode highly active serpins including a secreted serpin Serp 1, which inhibits extracellular thrombolytic and thrombotic proteases and markedly reduces arterial inflammation and plaque growth in animal models ( 2 4). This serpin, when injected as a purified protein, also significant ly reduces markers of myocardial damage after stent implant in patients with unstable coronary syndromes ( 5). These studies suggest that other viral serpins may have therapeutic potential. With the studies reported herein we explore a second class of viral cross class serpins with different protease targets that block apoptosis and inflammation. 1 This chapter is as published in : Viswanathan K, Bot I, Liu L, Dai E, Turner PC, Togonu Bickersteth B, et al. Viral Cross Class Serpin Inhibits Vascular Inflammation and T Lymphocyte Fratricide; A Study in Rodent Models In Vivo and Human Cell Lines In Vitro. PLoS ONE. 2012;7 (9):e44694 *Contributed written sections, generation of figures and generation of data for Fig 3 6 and 3 7. I also edited and submitted document for publication


51 Serp 2, encoded by myxoma ( 6,7), and CrmA ( cytokine response modifier A) encoded by cowpox ( 8,9) are poxviral cross class serpins that inhibit the serine protease, granzyme B, and cysteine proteases, caspases 1 and 8. These serpins are purportedly intracellular defense proteins; however, both serpins inhibit pathways with potential extracellular activity. CrmA is a more potent inhibitor in vitro binding caspase 1, caspase 8, and granzyme B ( GzmB), with greater inhibition in chicken chorioallantoic membranes ( 8 11). In contrast, Serp 2 binds caspases 1 and GzmB with lower affinity in vitro ( 6,7,10,11), but has greater effects on viral virulence in vivo in rabbits inf ected with Serp 2 deficient myxomavirus ( 6 8,11). These discrepancies contribute to our hypothesis that Serp 2 and CrmA would have different expression changes key apoptotic genes in innate immunity cell types. Key pathways to cellular apoptosis, also term ed programmed cell death, are mediated by serine and cysteine proteases ( 10,12 18). Caspases are cysteine proteases, some of which drive intracellular apoptotic pathways, whereas the serine protease GzmB is released by activated T cells into the surroundin g medium and inserted into target cells. GzmB initiates apoptosis, either via interaction with perforin or through less defined pathways ( 13,16 22). Granzyme B thus has both intracellular and extracellular activities, initiating two tiered caspase activati on in which caspases 3, 7, 8, and Bid ( BH3 interacting domain death agonist) play central roles ( 10,12,14,15). Granzyme B also cleaves proteases and inhibitors that protect against DNA degradation, specifically topoisomerase, poly ( ADP ribose) polymerase ( PARP), and inhibitor of caspase activated deoxyribonuclease ( iCAD) ( 15). Topoisomerase is part of


52 the DNA repair machinery, PARP releases topoisomerase stalled in the repair process, and iCAD blocks caspase activation of deoxyribonuclease. Apoptosis of endothelial cells, monocytes, and T cells leads to release of pro inflammatory mediators, creating a cycle of inflammation and cell death. Caspase 1 directly activates interleukin 1beta ( IL macrophage cell death pa thway called pyroptosis ( 23, 24). In atherosclerotic plaques, increased numbers of apoptotic cells, including T cells, are found at sites of plaque rupture. Monocyte and T lymphocyte invasion, together with endothelial cell dysfunction, are closely linked to atherosclerotic plaque growth and vessel occlusion ( 16, 25). Apoptosis induces a pro thrombotic and pro inflammatory state in endothelium ( 13,16 18,25), while in macrophages and smooth muscle cells ( 13,16 18) apoptosis is implicated in plaque rupture, t he underlying cause for sudden arterial thrombotic occlusion in heart attacks and strokes ( 16,17). While environmental factors such as smoking, high fat or high cholesterol diets, lack of exercise or diabetes can cause initial injury to the arteries, plaq ues can be found in otherwise healthy individuals at the branching points of arteries, as they have low shear stress and unpredictable blood flow and thus recruit additional inflammatory cells ( 5, 16 18). Increased numbers of cytotoxic, perforin positive T lymphocytes are present in inflammatory vascular disease, unstable coronary syndromes, and accelerated transplant vasculopathy ( 19 21), potentially driving cell death. Additionally, activated T cells express CD154 which binds to CD40L present on macrophag es and allows for cross talk and cross activation of the innate and humoral immune systems. Monocytes secrete cytokines such as interleukin 2 ( IL (


53 lymphocytes to the injury and stimulate them to mature into macroph ages and effector T cells ( 22). Interference with T cell apoptosis in rats ( 13) leads to a transplant tolerant state whereas GzmB deficiency in mice reduces transplant vasculopathy in some models ( 20). Fas Ligand ( FasL) has been reported to either block ( 2 6) or to accelerate ( 27) atheroma development in ApoE deficient mice. FasL and GzmB are also associated with T cell death induced by other cytotoxic T cells ( CTL); changing the balance of T cell subsets [e.g. CD8 T cells, CD4 T helper cells ( TH1, TH2, TH17 ), and CTL] and altering immune responses ( 28 30). T cell apoptosis may thus contribute toward plaque progression ( 12,13,16,17,19 22,26,27), but the precise role and effects on the balance of different T cell subsets remains only partially defined. We pr esent here a series of studies examining potential extracellular effects of intracellular cross class serpins, Serp 2 and CrmA on inflammatory vascular disease in animal models ( 2 4, 31 33), with selective analysis of GzmB mediated cellular apoptosis and T cell fratricide. Results Serp 2 Reduces Plaque Growth in Arterial Surgical Injury Models, In Vivo To assess potential extracellular effects of Serp 2 and CrmA on arterial plaque growth, we infused a single dose of individual purified proteins immediately after arterial surgery ( Fig 3 9 ) Two animal models were initially examined; 1] a balloon angioplasty injury model ( 2,31,33) and 2] an aortic transplant model ( 3,4). Saline, Serp 2, CrmA and two Serp 2 mutants were individually assessed after balloon angio plasty ( N = 126, Fig 3 9 Fig 3 1, A C). Serp 2 treatment ( 6 rats/dose; total N=24) significantly reduced plaque growth at doses of 30ng ( 0.10ng/g) or higher ( Fig 3 1A: 3ng/ p=0.400; 30ng/p < 0.006; 300ng/p < 0.006; 3000ng/p < 0.004) after mechanical angioplasty injury


54 when compared to saline ( N=6). Treatment with CrmA ( 12 rats/dose; total N=60; Fig 3 9 Fig 3 1B: 0.03ng/p=0.466; 0.3ng/p =0.121; 3ng/p=0.148; 30ng/0.094; 300ng/p=0.279 and 3000ng/p=0.612) or either of the two active site mutations of Serp 2, D294E ( 6 rats/dose; N = 18; Fig 3 1C: 0.3ng/p=0.377; 30ng/p=0.138 and 3000ng/p=0.567) or D294A ( 6 rats/dose; N = 18; 0.3ng/p=0.821; 30ng/p=0.076 and 3000ng/p=0.623, not shown) showed a trend toward reduced plaque at the 30ng dose, but failed to inhibit plaque gro wth at higher concentrations. These results demonstrate that Serp 2 consistently reduced plaque growth in a rat iliofemoral angioplasty model when compared to CrmA, and Serp 2 RCL mutants. Balloon angioplasty injury predominately induces endothelial denuda tion with smooth muscle cell proliferation and connective tissue scarring, but with less pronounced inflammation. Therefore, treatment with each serpin was assessed after aortic allograft transplant where greater inflammatory cells responses are detected ( 2,3,4,31,33). Plasminogen activator inhibitor 1 ( PAI 1) is a mammalian serpin that regulates thrombolytic proteases and PAI 1 / aortic allografts have increased inflammatory cell invasion and plaque growth ( 2,3,4,34). We therefore examined plaque growth i n PAI 1 / donor to Balb/C recipient mouse aortic allografts ( N=33 total transplants). Serp 2 ( N=6) again reduced plaque growth in transplanted aortic segments [p<0.05 compared to saline ( N=6); p<0.026 compared to CrmA ( N=9)], while CrmA and the two Serp 2 mutants, D294A ( N=5) and D294E ( N=7), did not reduce plaque ( Fig 3 1D). Mutant D294E was predicted to increase inhibitory activity due to preserved negative charge, however, no significant anti plaque activity was detected with D294E


55 or D294A ; conversely D294A increased plaque ( Fig 3 1D) when compared to CrmA ( p < 0.047), D294E ( p <0 .025), or Serp 2 ( P p < 0.0005) treatments. Histological cross sections from aortic allograft transplant ( B6.129S2 Serpine1 tm1Mlg PAI 1 / to Balb/C recipient) with saline treatm ent ( Fig 3 1D, E) or CrmA treatment ( Fig 3 1D, F) displayed rapid plaque growth at 4 weeks with mononuclear cell invasion ( Fig 3 1F, indicated by arrow heads). Serp 2 significantly reduced plaque ( 50ng/g), with reduced inflammatory cell invasion ( Fig 3 1D, G) plaque thickness demarcated by arrows; p < 0.044), whereas Serp 2 mutants D294A and E ( Fig 3 1D, H) did not reduce plaque growth. Serp 2 reduced inflammatory cell invasion in the adventitia ( N=10, 39.53+ 4.51 cell counts per high power field) when compared to saline ( N=10, 71.43+ 6.48 cells, p<0.019) and CrmA ( N=10, 77.27+ 9.59 cells, p<0.007). Conversely, D294A increased inflammatory cell invasion in the intima ( N=6, mean: 25.18 +2.21, p<0.034, saline N=10, 23.7 +1.90) and de creased it in the adventitia ( N=6, 46.06 +12.99, p<0.0341, saline N=10, 71.43+ 6.48), whereas D294E had no significant effects compared to saline. Serp 2 treatment also reduced plaque in rat aortic transplant models ( ACI donor to Lewis recipient, p < 0.05, data not shown) and reduced mononuclear cell invasion, when compared to CrmA, in both intimal ( p < 0.0001, data not shown) and adventitial layers. These initial studies demonstrate an arterial anti inflammatory effect for an intracellular viral serpin, Serp 2, in both angioplasty injury as well as aortic allograft transplant models in rodents, when infused into the circulating blood immediately after injury. This effect was specific to Serp 2 and was neither reproduced by another intracellular serpin, Cr mA nor by two active site mutants of Serp 2.


56 Serp 2 Reduces Plaque Growth in Apolipoprotein E Deficient ( ApoE / ) Mice Serpin treatment in hyperlipidemic ApoE / mice after carotid cuff compression injury was examined, both at the site of cuff injury and a t the aortic root where no surgical injury occurs ( Fig 3 2) ( 32). This model provides a means to assess both effects of serpin treatment after arterial surgical injury and also at a site of de novo growth of plaque induced by genetic hyperlipidemia with no arterial surgical injury ( e.g. apolipoprotein E deficiency). Serp 2 significantly reduced plaque area in the aortic root ( N=11, p < 0.001, Fig 3 2B, D) where there was no surgical injury, but with borderline significance at sites of carotid cuff compression injury ( N=11, p=0.06, Fig 3 2E), when compared to saline ( Fig 3 2A, D). Plaque reductions with Serp 2 treatment were comparable at both sites, 42% for aortic root versus 44% for the carotid ( Fig 3 2D, E), while CrmA treatment ( N=11) had no effect ( Fig 3 2 C, D, E). Compared to saline, plaque lipid content was also significantly reduced on Oil red O stained sections with Serp 2 treatment, but not with CrmA ( Fig 3 2F). This reduction in lipid laden cells is similar to the reduction in inflammatory cell invasi on seen with Serp 2 treatment in the aortic angioplasty and allograft models. Cross Class Serpin Treatments Modify Apoptotic Responses in Vitro Effects of serpin treatments on cellular apoptotic responses were assessed both at early times after angioplasty injury in vivo and in vitro in human cell lines. Changes in individual cells in the arterial wall may be masked by analysis of arterial extracts, therefore effects of serpins on apoptosis were examined in individual cell lines in addition to assessing cha nges in the arterial wall. At early times post aortic angioplasty injury ( 12h) in rat arteries, increased fragmented nucleosomes and higher levels of caspase 3, 7, 8, and granzyme B were


57 detected when compared to saline control treatment ( p< 0.0001). Trea tment with individual serpins demonstrated a trend towards reduced caspase and GzmB activity, but no significant reduction was detectable. For instance when DEVDase ( caspase 3 and 7) activity was tested, Serp 2 ( 1.52 0.18; p< 0.017), CrmA ( 1.56 0.18; p < 0.0001), D294A ( 1.8 0.31; p< 0.037), or D294E ( 1.67 0.25; p< 0.008) treatment produced significant differences when compared to saline ( 2.3 0.4), but did not show any difference in activity between Serp 2, Serp 2 mutants, and CrmA treatments. To test for potential effects on individual cell types associated with arterial inflammation and plaque growth, inhibition of apoptotic responses were measured in HUVEC ( human umbilical vein endothelial cells), THP 1 monocytes, and Jurkat T lymphocytes in vit ro with and without serpin treatment. Apoptotic responses were induced through three pathways using staurosporine ( STS; intrinsic pathway), and camptothecin ( CPT; double strand break initiation). In T cells, caspase 3 and 7 activity ( as measured by DEVDa se assay) ( 35) were significantly increased after camptothecin ( Fig 3 3A, P<0.001) and staurosporine ( Fig 3 3B, P<0.0001) treatment ( 48). Granzyme B and caspase 8 ( as measured by IEPDase assay, 35) were significantly increased by STS ( P<0.0001, Supp. Fig. 3 1). Serp 2 reduced caspase 3 and 7 in T cells after camptothecin ( Fig 3 3A, P<0.001) and after staurosporine ( Fig 3 3B, P<0.033) treatment, while CrmA did not alter T cell responses after CPT or STS ( Fig 3 3A). Serp 2 inhibition was more pronounced with CPT treatment ( Fig 3 3A) than after STS ( Fig 3 3B) in T cells. Serp 2 also significantly reduced apoptosis measured by cell death ELISA in T cells after CPT treatment ( Fig 3 3C, p < 0.012), while CrmA did not.


58 In THP 1 monocytes, camptothecin ( Fig 3 3D, p < 0 .001) and staurosporine ( p < 0.0009, not shown), both significantly increased caspase 3 and 7 activity, but had little effect on caspase 8 and GzmB ( Fig 3 8B). In monocytes, caspase 3 and 7 were significantly reduced by both Serp 2 ( p < 0.032) and CrmA ( p < 0.00 1) after CPT treatment ( Fig. 3 3D), with CrmA producing greater inhibition. Conversely, neither Serp 2 nor CrmA significantly altered GzmB and caspase 8 in THP 1 cells treated with STS ( Fig. 3 8). The Serp 2 mutants, D294A and D294E, did not alter caspase activity in T cells after camptothecin treatment ( Fig 3 3A, p=0.11), but D294E ( although not D294A) did reduce caspase 3, 7, 8 and GzmB with staurosporine ( Fig 3 3B, p < 0.016), indicating that the D294E RCL mutant retains some anti apoptotic activity that differs from endogenous Serp 2. D294A and D294E had no inhibitory activity when tested in THP 1 monocytes after camptothecin or staurosporine treatment ( Fig 3 3D, p=0.104). In HUVEC cultures treated with serum deprivation, staurosporine, or camptothecin caspase 3, 7, 8 and GzmB were increased ( p < 0.001, not shown). Serp 2 and CrmA both significantly reduced caspase 8 after CPT treatment ( p < 0.0001) in HUVEC, but had no effect on caspase 3 and 7 ( not shown). STS induced apoptosis was not altered by Serp 2, C rmA, D294A, or D294E in HUVEC ( not shown). In all cell lines tested after FasL treatment Serp 2, CrmA, and D294A had no inhibitory activity ( not shown). The Serp 2 RCL mutant, D294E did, however, reduce caspase 3, 7, 8 and GzmB activity after FasL treatment of T cells ( p < 0.0005, not shown). Cathepsin K,S,L,V activity in T cells was not affected by serpin treatment ( p=0.386, not shown).


59 In summary, Serp 2, but not CrmA, inhibited camptothecin and staurosporine induced caspase activity in T cells i n vitro Both Serp 2 and CrmA inhibited camptothecin induced caspase activity in monocytes with CrmA displaying greater inhibitory activity in monocytes, suggesting T lymphocytes as a primary target for Serp 2. No differential effects were produced by Serp 2 and CrmA in vivo in arterial sections isolated early after angioplasty injury which may be due to the fact that multiple cell types are assessed in arterial sections. Serp 2 Reduces T Cell Apoptotic Responses to Cytotoxic T Lymphocyte ( CTL ) Granzyme B A ctivated T cells ( TH1) and cytotoxic T lymphocytes/ natural killer ( CTL/NK) cells release GzmB into the surrounding medium, initiating death responses in other cells, and also in T lymphocytes ( 12,14,15). The role of GzmB in Serp 2 mediated anti apoptotic activity was assessed using medium from T cells activated to a CTL like state with phorbol myristic acid ( PMA) and ionophore ( 12,14). These activated CTL like cells express and secrete increased GzmB. Naive HUVEC, THP 1, and Jurkat cells were then treated with medium from the CTL like T cells ( CTLm) with and without serpin treatments ( Fig 3 4, Fig. 3 8). Increased secreted extra cellular GzmB was detected in PMA and ionophore treated Jurkat cultures ( Fig 3 4A). Both anti GzmB antibodies and the tetrapeptide ZAAD Chloromethylketone ( ZAAD CMK), a chemical intracellular inhibitor of GzmB, reduced granzyme B and caspase 8 activity ( IEPDase) in these CTL cultures ( Fig 3 4A, P<0.001). IEPDase ( GzmB and caspase 8 p < 0.0001) activity and DEVDase ( caspase 3 and 7 p < 0.0001) activity were increased significantly in T cells treated with CTL medium


60 ( Fig 3 4C, D), but not significantly in CTLm treated HUVECs ( Fig 3 4B, p=0.836) nor monocytes ( not shown) ( 36). Serp 2, reduced caspase 3 and 7 ( Fig 3 4B, p < 0.01) and caspas e 8 ( not shown, p < 0.01) in HUVEC cultures, but not in THP 1 ( not shown). CrmA did not decrease caspase activity in HUVEC cultures treated with CTLm ( p=0.249, Fig 3 4B). In T cells treated with CTLm, Serp 2 produced a significant reduction in CTLm mediated increases in caspase 8/GzmB ( p < 0.01, Fig 3 4C) and in caspase 3/7 ( p < 0.0009, Fig 3 4D). Concomitant treatment with antibody to GzmB or perforin blocked Serp 2 inhibition of caspase 8/GzmB ( Fig 3 4D, p < 0.0004 compared to Serp 2 and CTLm treatment for GzmB a ntibody, p= 0.412 for perforin antibody compared to CTLm alone) and caspase 3/7 activity ( Fig 3 4D, p=0.149 compared to CTLm treatment alone, p < 0.0193 for perforin antibody compared to CTLm tre atment alone). Serp 2 Binding t o T Cells is Reduced w ith Granzyme B Inhibition To determine whether Serp 2 or CrmA binds to T cells, FITC labeled Serp 2 and CrmA binding was measured by flow cytometry ( Fig 3 5A) and fluorescence microscopy ( Fig 3 5B). In these studies Serp 2 displayed a greater binding affi nity than CrmA ( Fig 3 5A, B) for T cells in vitro and mouse peripheral circulating lymphocytes in vivo ( not shown). Serp 2 significantly reduced caspases 3/7 activity in CTLm treated T cells ( Fig 3 5C, p < 0.001) and treatment with the GzmB inhibitor ZAAD CM K, which predominately inhibits intracellular granzyme B, further increased Serp 2 mediated inhibition of caspases, indicating that Serp 2 actions may be predominately extra cellular ( Fig 3 5C, p < 0.005). The uptake of FITC labeled Serp 2 into Jurkat T cell s, as measured by soluble lysate content, was also reduced after treatment with antibody to GzmB ( Fig 3


61 5D, p < 0.003) or perforin ( Fig 3 5D, p < 0.012), further supporting a role for Serp 2 binding to GzmB and inhibiting of T cell responses. Serp 2 Inhibition of Aortic Plaque is Reduced in Granzyme B Knockout Aortic Allografts We postulated that Serp 2 mediates extracellular anti inflammatory and anti atherogenic activity via selective targeting of granzyme B ( GzmB). Plaque growth in GzmB / single and ApoE / GzmB / double knockout ( ApoE / GzmB / DKO) aortic allografts was assessed with and without Serp 2 or CrmA treatment. Donor ApoE / GzmB / DKO aortic allografts ( N = 18) were compared with ApoE / ( N = 16) and GzmB / ( N = 29) allografts ( B6 into C57Bl/6 background). ApoE / hyperlipidemic mice are expected to have greater de novo plaque buildup and thus are predicted to enhance the capacity to detect changes / reductions in plaque growth in GzmB deficient mice. GzmB deficiency has the potential to reduce baseline plaque and thus prevent detection of a further reduction in plaque size after serpin treatment. ApoE / GzmB / DKO and single GzmB / or single ApoE / knockout allografts were therefore assessed for differences in plaque production. In ou r model, 4 weeks post transplantation, saline treated ApoE / GzmB / DKO allografts had a significant trend toward reduced plaque area ( Fig 3 6A) and IMT ( Fig 3 6B) when compared to saline treated ApoE / allografts ( Fig 3 6A), suggesting that GzmB deficie ncy in donors has variable effects on plaque growth. At 4 weeks follow up Serp 2 significantly reduced plaque area ( Fig 3 6A, p < 0.036) and intimal to medial thickness ( IMT) ratios ( Fig 3 6B, p < 0.045) in the ApoE / allografts. Serp 2 no longer significantl y reduced plaque area ( Fig 3 6A) or IMT ratios ( Fig 3 6B) in GzmB / ( p=0.995 for plaque area and p=0.992 for IMT) or in ApoE / GzmB / DKO allografts ( p=0.704 for plaque area, p=0.353 for IMT). Conversely, CrmA significantly


62 increased plaque area in ApoE / donor allografts ( Fig 3 6A, P<0.026). CrmA increased plaque area in GzmB / ( p < 0.041), but not in ApoE / GzmB / DKO ( p=0.973) allografts ( Fig 3 6A) and had no significant effect on IMT ( Fig 3 6B). The increase in plaque area detected with CrmA treatment in ApoE / donor allografts ( Fig 3 6A, p < 0.026) was significantly reduced in CrmA treated ApoE / GzmB / DKO allografts ( Fig 3 6A, B, p < 0.021 for plaque area and p < 0.006 for IMT). Serp 2, but not the mutant D294E protein, was able to reduce the a mount of active Caspase 3 staining in cross sections of mouse aorta 4 weeks after transplant injury ( Fig 3 6C). Reduced staining was visible in mononuclear cells in the adventitia ( p<0.024) for Serp 2 treatment when compared to the less active D294E mutant ( Fig 3 6D). These studies support GzmB as one of the central targets for Serp 2 mediated anti inflammatory and anti atherogenic activity. Serp 2 Reduces Early Apoptosis in Invading Inflammatory Cells after Aortic Allograft Transplant Early changes in T cell and macrophage responses can initiate inflammatory responses that drive plaque development and plaque growth at later times in arterial plaque growth and disease. To assess effects of Serp 2 and CrmA on inflammatory T cell responses in vivo in a mouse model, we examined markers for T cell invasion and apoptosis in aortic allograft transplants in mice at early 72 hour follow up ( Fig 3 7). Serp 2 treatment ( Fig 3 7) induced no significant T lymphocyte responses at 72 hrs follow up, although there is a mi nor trend toward an increase in CD3 positive T cells. On immunohistochemical staining, however, Serp 2 but not CrmA, markedly reduced inflammatory cell apoptosis ( Fig 3 7G, p <0.0002).


63 Discussion Many cell types are associated with atherosclerotic plaque g rowth. Injury to the arterial wall is believed to cause endothelial cell dysfunction and activation of inflammatory cells, specifically monocytes that transform into macrophages, T lymphocytes as well as smooth muscle cells, and other cells types such as m ast cells, neutrophils and even B lymphocytes. Damage to the arterial wall and loss of supporting connective tissue can additionally cause programmed cell death or apoptosis, which can lead to release of increased levels of inflammatory cytokines. Activate d or dysfunctional T cells can also induce transformation of other cells to a suicidal or apoptotic state. These initial changes in inflammatory cell responses are believed to then drive further damage to the arterial wall and cause intimal plaque growth a nd arterial narrowing. It is evident that there are multiple factors that drive plaque growth with some known shared or common pathways. We elected to assess the effect of an anti apoptotic serpin, as the apoptotic pathways are becoming recognized as a dr iving force in arterial injury responses and inflammation. Apoptosis in endothelial and in macrophage cells has been reported, as has apoptosis in SMC and T cells in plaque development. However, apoptosis altering the many T cell sub populations remains po orly defined. It is not known whether interruption of apoptotic responses will alter plaque development and whether this applies to the wide range of arterial injury states that can cause plaque formation. It is for this reason we have elected to assess pl aque growth and responses to the viral anti inflammatory serpin, Serp 2, in a range of models to determine whether the effects of this protein will be evident in different animal models of arterial disease in


64 order to assess whether this will be of more wi despread potential interest. Furthermore, in order to better isolate the contributions of individual cellular subpopulations, individual serpins were tested on human cell lines. Activated or dysfunctional T cells can also induce a poptosis of endothelial ce lls and monocytes/ macrophages, among other cell types; in the plaque, this leads to an increased release of cytokines and activate thrombolytic serine proteases tissue and urokinase type plasminogen activators ( tPA and uPA, respectively) and the matrix m etalloproteases ( MMPs), which breakdown ( 45 47) In addition to these activated and apoptotic cells, the newly exposed necrotic core and eroded cap structure also activate leukocytes and may initiat e plaque rupture, subsequent thrombus formation, leading to heart attacks and strokes. Through a series of complex cross talk and feedback mechanisms, the serine proteases in the coagulation and fibrinolytic pathways interact on many levels with the inflam matory and apoptotic responses and vice versa. Intravenous infusion of Serp 2, a reputed intracellular myxomaviral cross class serpin, effectively inhibited plaque growth in a series of animal models of vascular disease ( Fig s. 3 1, 3 2) irrespective of the model being vascular surgery based or hyperlipidemic mice. These studies demonstrate marked extracellular, GzmB dependent inhibitory actions for Serp 2, previously thought to function in a predominantly intracellular capacity This inhibitory activity was unique to Serp 2; the cowpox viral serpin, CrmA and two Serp 2 active site mutants were inactive in these models.


65 Serp 2 blockade of plaque growth in donor aortic allografts was absent when the transplanted tissue was from Gz mB / donors. Local deficiency of GzmB was not sufficient to reduce plaque growth in donor allografts treated with saline, but did block increased plaque in ApoE / mice treated with CrmA. Thus, granzyme B may have greater effects on vascular disease when active locally rather than systemically during inflammatory cell responses. Although Serp 2 was infused systemically, the loss of activity in donor allografts from knockout mice suggests that Serp 2 acts locally on donor aorta after infusion ( Fig 3 6). In vitro studies suggest that Serp 2 specifically inhibits T cell apoptosis. Further work using isografts, GzmB / transplant recipients, and caspase 1 deficient transplants is needed to assess and contrast the roles of systemic GzmB and caspase 1 in allograf t vasculopathy and as a target for Serp 2. To try to separate out the effects of different cell lineages found in plaques, the effects of the serpins on apoptosis induced cell lines were examined. Serp 2 bound to T cells in vitro in culture and selectivel y inhibited caspase 3/7 in camptothecin ( CPT) treated Jurkat T cells ( Fig 3 3, 3 4) and was dependent upon GzmB and perforin ( Fig 3 5, 3 6). Additionally, Serp 2 but not Serp 2 D294E was able to reduce levels of active caspase 3 in ApoE and ApoE/GzmB knockout mouse aortic cross sections after transplant ( Fig 3 6). This Serp 2 mediated reduction for apoptosis was substantiated when comparing Serp 2 to CrmA and saline 72hrs after C57Bl/6 aortic transplant into Balb/C mice ( Fig 3 7 A C, H). Based upon the se studies we postulate that Serp 2 decreases T cell mediated apoptosis, inducing a generalized reduction in vascular inflammation.


66 The extracellular activity for this viral serpin is predicted to begin with binding to GzmB. GzmB mediates apoptosis upon r elease from T cells and can also induce apoptosis in other T cells. Many viral proteins have multiple functions ( 2,8,31) and expanded actions of these cross class serpins upon release from infected cells is predicted ( 2 9,31 33). This inhibitory activity i s present either with camptothecin treatment of T cells or after treatment with CTL medium from PMA and ionophore treated T cells, containing granzyme B. Serp 2 may thus either bind GzmB outside the cell or may be internalized via perforin pathways. The Gz mB inhibitor ZAAD CMK, which is cell membrane permeable and inhibits GzmB inside the cell, further increased Serp 2 inhibition of caspase activity indicating that Serp 2 actions are extra cellular. Serp 2 inhibition of camptothecin mediated apoptosis is co nsistent with GzmB inhibition. Many viral proteins are also reported to derive functions through mimicking mammalian genes, as well as the converse. Two mammalian serpins, murine serine protease inhibitor 6 ( SPI 6) ( 36) and human protease inhibitor 9 ( PI 9) ( 37) target GzmB and protect cells from CTL induced apoptosis. Serp 2 protein may thus mimic this mammalian serpin pathway, hindering T cell apoptosis and inflammation, e.g. T cell fratricide. We have not yet, however, determined whether selected T cell subsets are targeted by Serp 2 protection. It is undeniable that Serp 2 is protecting both T cells and other lineages from GzmB mediated apoptosis ( Fig. 3 4) in vitro GzmB mediates DNA degradation, interfering with DNA repair responses. Topoisomerase, iC AD, and PARP are involved in DNA damage repair and are GzmB substrates. Camptothecin binds topoisomerase I, an enzyme class that alters DNA topography, interfering with DNA re ligation ( 38) and creating persistent DNA breaks.


67 Inhibition of topoisomerase al so leads to caspase activation ( 37). Poly ( ADP ribosylation, PAR) is a post translational modification driven by the PAR polymerase 1 ( PARP 1) that reactivates topoisomerase complexes, preventing further damage ( 39) and is also a transcription initiation f ( 40). Once cleaved, iCAD no longer inhibits caspase activated DNAse permitting DNA degradation ( 41). Serp 2 mediated inhibition of GzmB or inhibition of secondary induction of caspase 3 may alter the balance between DNA repair and damage in T cells ( 42 44). We conclude that Serp 2, a viral anti apoptotic cross class serpin, has the potential to inhibit arterial vascular disease progression in animal models through inhibition of GzmB dependent T cell apoptosis. Granzyme B inhibition of T cell fratricide may represent a potential new target for intervention in inflammation based disease. Serp 2 inhibition is generalized, with expanded inhibitory function for plaque growth in hyperlipidemic ApoE / mice and after arterial surgery, indicative of blockade of central regulatory pathways. The experiments described in this chapter help to characterize differences in the functions of Serp 2 and CrmA. Changes to the active Casp3 detected both in vitro (Fig 3 3) and in mouse aortas (Fig 3 3) provide evi dence for the purported anti apoptotic activities of Serp 2 and confirm a previously observed lack of activity of CrmA in vivo (Fig 3 7). To further define the potential anti inflammatory functions of Serp 2 and to examine the capacity of Serp 2 to modify gene expression in human cells known to b e central mediators of the innate immune responses we have proceeded examine the effects of Serp 2 on apoptotic gene expression in endothelial cells, monocytes and T cells in vitro in culture in Chapter 4. This work was further expanded to assess the


68 potential for any common effects or shared final effects on apoptotic pathways in response to Serp 2 with comparison to two other effective myxomaviral anti inflammatory proteins, Serp 1 and M T7. Figure 3 1 Serp 2 reduces plaque growth in arterial surgery models. Treatment of rat models of arterial injury with viral cross class serpins demonstrates reduced vasculopathy. Hematoxylin and eosin stained sections of rat iliofemoral arterial sections harvested at 4 weeks and the mean plaque area were measured and presented as mean SE. The results demonstrated reduced plaque growth with Serp 2 ( 6 rats/dose; 3 3000ng total N=24) treatment at doses > 30ng ( A ). CrmA ( 6 12 rats/dose, 0.3 3000ng; total N=60) ( B ) and th e Serp 2 reactive center loop mutant D294E ( 6 rats/dose, 0.3 3000ng; total N=18) treatments ( C) demonstrated a non significant trend toward reduced plaque at 30ng, with no significant inhibition of plaque growth at higher concentrations. ( D ) In the mouse a ortic allograft transplant model ( B6.129S2 Serpine1tm1Mlg donor to Balb/C recipient) Serp 2 again significantly reduced plaque ( p<0.026), whereas CrmA and the D294A and D294E Serp 2 RCL mutants did not reduce plaque ( total mice 33). ( E H ) Cross sections of mouse aortic allograft transplants demonstrate the marked intimal hyperplasia and associated mononuclear cell invasion in the adventitial layers in saline ( E ) or CrmA ( F ) treated mice. Treatment with Serp 2 ( G ) but not Serp 2 D294E mutants ( H ), displayed reduced plaque and inflammation. Arrows bracket intimal plaque limits. Arrowheads point to areas of mononuclear cell invasion. Magnification 100X.




70 Figure 3 2 Serp 2 reduces plaque growth in Apolipoprotein E deficient ( ApoE / ) mice. Hyperlipidemic ApoE / mice were infused with a bolus of Serp 2, CrmA or control saline after carotid cuff compression injury. Histological sections taken at the aortic root, where no surgical injury occurs, and distally at the site of cuff injury were examined. Cross se ctions taken at the aortic valve level ( Oil red O staining) demonstrate plaque growth in saline treated control mice ( A) ( N=11). A significant reduction in plaque area is detectable with Serp 2 ( B, N=11), but not with CrmA ( C, N=11) treatment compared to s aline treated controls. Morphometric analysis of plaque area at the aortic root in ApoE / mice, where no surgery has been performed, demonstrated that Serp 2 inhibited aortic plaque and macrophage/foam cell invasion ( D, p < 0.001) to a greater extent than a t sites of vascular carotid compression surgery in the same model ( E, P= 0.06). Oil red O staining confirmed a reduction in fatty plaque in the ApoE / aortic root ( F), indicating decreased foam cell/macrophage invasion ( p < 0.001). Thin arrows bracket intim al plaque limits, larger arrows identify an aortic leaflets, large arrow with open base points to area of fatty, foam cell ( macrophage) invasion; Magnification 100X.




72 Figure 3 3 Viral cross class serpins alter apoptotic responses in T cells and monocytes, in vitro Apoptotic responses were induced in T cells and monocytes using camptothecin or staurosporine. Inhibition of granzyme B and caspase 8 activity was measured after treatm ent with Serp 2, CrmA, or the two Serp 2 mutants by analysis of changes in IEPDase activity with comparison to untreated controls. Serp 2, but not CrmA nor D294A and D294E treatment of Jurkat T cells reduced caspase 3 activity ( DEVDase) after camptothecin ( CPT) ( A, p<0.001) or staurosporine ( STS) ( B, p<0.033) apoptosis actuator treatment. Cell death in T cells measured as fragmented DNA by ELISA was also blocked in CPT treated T cells ( C, p<0.012). Serp 2 ( p<0.032) and CrmA ( p<0.001) both significantly redu ced CPT induced elevations in caspase 3 and 7 activity in monocytes ( D). The results shown here represent mean SE from 3 to 5 replicates for each experiment. Significance was assessed by analysis of variance ( ANOVA) with secondary Fishers least significa nt difference and Mann Whitney analysis.


73 Figure 3 4 Blockade of granzyme B reduces viral cross class serpin inhibition of T cell induced T cell apoptosis. Jurkat T cells were treated with PMA and ionophore ( PI) and the level of granzyme B expressed wa s measured ( A). There was an increased level of granzyme B ( GzmB, p<0.001) secreted by these cells and was inhibited by treating the cells with an intracellular inhibitor of granzyme B, ZAAD CMK ( p<0.001) or anti granzyme B antibody ( p<0.001) ( A). The medi um containing granzyme B from PI treated T cells ( CTLm) was applied to naive HUVECs to induce apoptosis ( B). Treatment with Serp 2, but not CrmA, reduced caspase activity in CTLm treated HUVECs ( B). The CTLm was also applied to naive T cells in culture to induce apoptosis and increase levels of caspase 3 and granzyme B were observed as IEP Dase activity ( C, p<0.0001) and DEV Dase activity ( D, p<0.0001) respectively. Treatment with Serp 2 reduced both caspase 3 ( C, p<0.01) and granzyme B ( D, p<0.0009) activities significantly. Antibody to granzyme B ( GzmB) blocked Serp 2 mediated reductions in CTLm induced caspase 3 ( C) when compared to Serp 2 treatment alone ( p<0.0004), but with a still significant decrease ( p< 0.005) when compared to CTLm treatment al one. Antibody to granzyme B also blocked the Serp 2 mediated decrease in granzyme B ( D, p<0.149) when compared to CTLm activation. This Serp 2 mediated inhibition of CTLm induced caspase 3 activity was also blocked by incubation of cells with antibody to p erforin ( C, p= 0.412) but not the granzyme B activity ( D, p<0.0193). The results shown here represent mean SE from 3 to 5 replicates for each experiment.




75 Figure 3 5 Serp 2 binds T cells in vitro with greater affinity than CrmA. Jurkat T cells we re treated with FITC labeled Serp 2 or CrmA and binding/ association of these viral proteins with T cells was analyzed using Flow cytometry ( FACS) analysis ( A)and fluorescence microscopy ( B, Magnification 10X). Extracellular, surface FACS analysis shows bo th Serp 2 and CrmA binds to the T cell surface ( A). FITC labeled protein treated cells were washed lysed and the total fluorescence measured, indicating that significant amount of Serp 2 and CrmA associate with the T cells compared to the control BSA ( B). This observation was also supported by the fluorescent microscopic analysis ( B). Intracellular ZAAD CMK granzyme B inhibitor decreased Serp 2 mediated inhibition of caspase 3 activity in response to treatment with CTLm ( cytotoxic like T cell medium) ( C, p< 0.005). Treatment with antibody to granzyme B ( GzmB Ab) or perforin ( PF Ab) partially blocked Serp 2 binding ( D, p<0.003 and p<0.012 respectively). The results represent mean SE from 3 to 5 replicates for each experiment. Significance was assessed by analysis of variance ( ANOVA) with secondary Fishers least significant difference and Mann Whitney analysis.


76 Figure 3 6 Granzyme B deficiency ( GzmB / ) in donor aorta interferes with Serp 2 inhibition of plaque growth after aortic allograft transplant. ApoE / ( C57Bl/6) donor aortic allograft transplant into Balb/C recipient mice ( N = 16) induced plaque growth at 4 weeks follow up as measured by plaque area ( A) and intimal to medial thickness ( IMT) ratios ( B). Serp 2 treatment significantly reduced plaqu e area ( A, p<0.036) and IMT ratios ( B, p<0.045) when compared to saline treatment. CrmA treatment markedly increased plaque area ( A, p< 0.026) and non significantly increased IMT ratios ( B, p=0.312). Saline treated ApoE / GzmB / DKO allografts ( N= 18) ha d non significant reductions in plaque area and IMT when compared to ApoE / donor allografts ( A, B). CrmA treated ApoE / GzmB / DKO allografts had significantly reduced plaque area ( A, p<0.021) and IMT ( B, p< 0.006) when compared to CrmA treated ApoE / donor allografts. Serp 2 no longer reduced plaque area ( A) or IMT ( B) in either GzmB / allografts ( p= 0.995 for plaque area and p= 0.992 for IMT) or ApoE / GzmB / DKO ( p= 0.704 for plaque area and p=0.353 for IMT). Serp 2 but not Serp 2 mutant D294E si gnificantly reduced caspase 3 staining in the intima ( C, p>0.008), media ( C, p>0.001) and adventitia ( C, p>0.024) in PAI 1 / mice 4 weeks post aortic transplant.


77 Figure 3 7 CD3 and active Caspase 3 populations 72 hrs after mouse aortic allograft. C57B l/6 donor aortic allografts were transplanted into Balb/C recipient mice ( N = 3 per treatment) and followed up at 72hrs. Compared to Saline, Serp 2 but not CrmA treatment resulted in a decrease in caspase 3 activity ( panels A C, G; P = 0.0224). Neither pro tein treatment significantly reduced CD3 positive T cells ( panels D F, H).


78 Figure 3 8 Viral cross class serpins alter Staurosporine induced apoptotic responses in T cells and monocytes, in vitro A poptotic responses were induced in T cells and monocytes using staurosporine. The ability of Serp 2, CrmA, or Serp 2 mutants to counteract this induction was measured by granzyme B and caspase 8 activity by IEPDase activity. Serp 2, but not CrmA nor D294A and D294E treatment of Jurkat T cells reduced c aspase 8 and Granzyme B activity after staurosporine ( STS) ( A, p<0.001) apoptosis actuator treatment. In THP 1 human monocytes, no cross class serpins significantly reduced granzyme B or caspase 8 activity ( B). The results shown here represent mean SE fr om 3 to 5 replicates for each experiment. Significance was assessed by analysis of variance ( ANOVA) with secondary Fishers least significant difference and Mann Whitney analysis.


79 Figure 3 9 Animal Models


80 CHAPTER 4 VIRAL PROTEINS TARGET DIVERGING IMMUNE PATHWAYS WITH CONVERGING EFFECTS ON ARTERIAL DILATATION, PLAQUE, AND APOPTOSIS This chapter further investigates hypothesis 2: the anti inflammatory actions of the three viral proteins with proven anti inflammatory activities will alter expression of a family of shared apoptotic pathway genes indicating a final response pathway common to all three protein treatments. This chapter expands the knowledge of the potential mechanism of action for how these diverse proteins effect similar changes in vivo, exploring analogous alterations in vitro human cell culture Introduction 1 Although the risk associated with atherosclerotic cardiovascular disease is now markedly improved, the incidence of abdominal aortic aneurysms ( AAAs) is increasing (143 148) AAA is a localized dilatation of the aorta with diameter greater than 3cm ( or more than 50% increase) with loss of media l elastin and associated cell death, as well as often marked increase in local atherosclerotic plaque and thrombosis. Undiagnosed AAAs affect 5% of men over age 65, often causing sudden death (143 148) Nearly 2 million Americans are at risk for AAAs, with 4 8.9% prevalence worldwide, which is increasing as the population ages (143) Sudden rupture of the destabilized aneurysmal vessel has 50% to 80% associated mortality (143 148) While control of atherosclerotic risk factors, e.g. smoking, hypertension and hyperlipidemia, is recommended for patients with AAAs, efficacy of preventative treatment varies. Expectant m onitoring of aneurysm progression with eventual surgical repair or urgent surgery for aneurysm rupture remain the standard of care, but have higher associated risk. Additionally, many 1 This chapter as submitted to Journal of Atherosclerosis, Thrombosis and Vascular Biology. *I wrote this paper and generated the graphs and analysis using data generated by Dr. Dai.


81 aneurysms are silent with unheralded rupture requiring surgical repair. Surgical repair can also induce further hazards due to arterial damage, recurrence or leak after repair, thrombotic occlusion, or neurological damage (148) Recurrence is reported to be particularly high in subsets of patients with hig hly inflamed aneurysms. The maturation of aneurysms occurs through chronic inflammation, matrix ( collagen and elastin) degeneration, apoptosis, and vascular remodeling (31,144,145,149) sharing causative factors with atherosclerosis (20,149) Inflammatory cell invasion occurs in all arterial layers and is accompanied by elastin fragmentation, resulting in large, fragile vessel walls at risk for rupture and hemorrhage (144) Inflammatory cells that invade the vessel include macrophages, CD8+ cytotoxic T lymphocytes ( CTL), and CD4+ T helper cells (19,149) as well as neutrophils, fibroblasts, and mast cells (20,149) Enlargement and degradation of the arterial wall is due, in part, to destruction of elastin and collagen by fibrinolyt ic serine proteases known as tissue and urokinase type plasminogen activators ( tPA and uPA, respectively) that activate matrix metalloproteinases ( MMPs) (144,149) Protease mediated degradation of structural proteins weakens the arterial wall and induces apoptosis of endothelial cells, vascular smooth muscle cells ( VSMCs), and even invading cells, causing further inflammation and thinning of the arterial wall (19,144,150) Many of the proteolytic, inflammatory, and apoptotic respon ses driving atherosclerotic plaque development and rupture overlap with those causing aneurysms and may result in simultaneous progression of both atherosclerotic lesions and aneurysms (143 148) Apoptotic cells in the arterial wall release cytokines and chemokines and can enhance inflammatory cell activation (149,150) Chemokines from


82 activated cells attract inflammatory cells into the arterial wall ( 20) and, together with breaks in the elastin connective tissue layers, can indu ce cell invasion and arterial dilatation. Apoptosis of VSMCs reduces secreted elastin and collagen in the arterial wall with reduced tensile strength of the vessel wall (149,150) Apoptosis of endothelial cells, macrophage, or T lymphocytes leads to release of clotting factors and cytokines and produces pro thrombotic and pro inflammatory states (19,144,145,150) Apoptosis in VSMCs is closely associated with aneurysm formation; however, the contribution of apoptotic inflammatory cells to aneurysm progression is less well defined. We have postulated that modulation of inflammatory cell responses through inhibition of a variety of protease or chemokine pathways has the potential to alter apoptotic responses, inflammation, and aneurysm formation. Large DNA viruses, such as the poxviruses, encode a variety of proteins that have evolved to modify innate and acquired immune responses ( 50,56) Myxoma virus is a leporipoxvirus that expresses anti inflammatory serpins and chemokine modulating proteins ( CMPs), among other immune modifiers. Ser ine p roteinase i n hibitors, termed serpins regulate thrombotic and apoptotic cascades along with multiple other pathways, representing up to 2 10% of plasma proteins (41,151) CMPs, inhibit either chemokine binding to receptors or glycosaminoglycans (80,152,153) Myxomaviral proteins suppress host inflammatory responses during v irus infections, acting as protective elements for the virus (59,66,136,151) These proteins suppress disease progressio n in animal models of inflammatory vascular disease, specifically atherosclerosis and transplant rejection (59,66,80,13 6,152,154) Notably, when these proteins are knocked out in the virus, the viral infection becomes more benign (155) Serp 1 is a secreted


83 55kD serpin ( ser ine p rotein ase in hibitor) that binds and inhibits urokinase and tissue type plasminogen activators ( uPA, tPA, respectively) as well as plasmin and factor Xa ( 18,20,23 27) Serp 2 is a 34kD cross class serpin that inhibits caspase 1 and 8 and granzyme B (61 64,136,156) M T7 represents a separate protein class, inhibiting rabbit interferon gamma ( (55,152) and a broad spectrum of C, CC, and CXC chemokines in a non species dependent manner (152) In multiple animal models, Serp 1 reduced plaque growth and transplant v asculopathy (57,66,84,91,131,132,153) and has been successfully tested in a clinical trial in pat ients with unstable coronary syndromes after stent implantation (135) Serp 2 and M T7 also reduced inflammation and plaque growth in models of atherosclerosis and transplant vasculopathy (80,136,154) Inactive mutants of these proteins, however, do not block inflammation nor plaque growth (136) These three immunomodulatory viral proteins have not as yet been directly compared for potential effects on arterial dilatation and aneurysm formation nor have they been analyzed for shared effects on regu latory pathways. With this study we compare three anti inflammatory viral proteins targeting different innate immune pathways after balloon angioplasty injury in Apolipoprotein E deficient ( ApoE / ) mice. ApoE / mice develop accelerated atherosclerotic pl aque growth after balloon angioplasty injury with aneurysm like dilatations, and while it is known that these proteins can alter atherosclerosis, it is unknown if they also alter incidence of aortic dilatation or formation of AAA (116,142,157 159) Selective changes in gene expression in cells involved in innate immune and apoptotic responses were also examined to investigate potential common molecular targ ets.


84 Results Increased Plaque and Arterial Dilatation in ApoE / Mice after Angioplasty Injury To assess atherosclerotic plaque development and aneurysmal dilatation after balloon angioplasty, both wild type and hyperlipidemic mice were evaluated for aortic plaque area and dilatation in wild type mice ( Fig 4 1A) and ApoE / mice ( Fig 4 1B) at 28 days post angioplasty. Aortic dilatation was measured using IEL and EEL diameters and areas together with elastic lamina fragmentation. ApoE / mice ( N=12, Tab le 4 1) treated with saline after angioplasty injury displayed increased plaque area ( Fig. 4 1B, D; P < 0.001), dilatation ( Fig. 4 1B, E; P < 0.043), and IEL fragmentation ( Fig. 4 1C, F, P < 0.05) when compared to wild type mice ( N=10, Table 4 1, Fig 4 1) Increased arterial dilatation in ApoE / mice was time dependent; smaller changes initially at 24 hours ( Fig 4 2I ) reached significance at 28days ( 4 2G, H P < 0.001). Consistent with prior studies, ApoE / mice displayed spontaneous atheroma and aneury sm formation, however, plaque progression and arterial dilatation occurred more rapidly 4 weeks with balloon injury. Anti Inflammatory Protein Treatment Reduces Plaque Growth and Arterial Dilatation Three myxomavirus derived proteins with proven anti infla mmatory activity were assessed for effects on plaque growth or arterial dilatation in ApoE / mice after balloon angioplasty injury. Mice were given a single i.v. bolus ( 15g) of one of the proteins ( Serp 1, Serp 2, or M T7) immediately after angioplasty, or a saline control treatment ( Table 4 1). Treatment with Serp 1 ( N=5, P < 0.010), Serp 2 ( N=5, P < 0.003), and M T7 ( N=5, P < 0.007) significantly reduced plaque formation at 28 days ( Fig 4 2A, C F; P < 0.004), when compared to saline treatment ( N = 12). I n contrast, treatment of C57Bl/6


85 mice with M T7 after angioplasty did not significantly alter plaque growth, or arterial dilatation ( Data not shown). Mononuclear cell invasion was reduced from early follow up at 24 hours and 7 days to 28days in the intimal ( not shown) and adventitial layers ( Fig 4 2B) with anti inflammatory protein treatment. At late follow up, this reduction was significant for Serp 1 ( N=5, P < 0.0013) and M T7 ( N=5, P < 0.0030) treatments ( Fig 4 2 B F). In ApoE / mice at 28 days post angioplasty, there was a significant reduction in EEL ( Fig 4 2G, I L) and IEL ( Fig 4 2H, I L) diameters. When compared to saline ( N=5) after angioplasty, Serp 1 ( N = 5) reduced the long axis diameter of both the IEL ( Fig 4 2H, P < 0.04 7) and the EEL ( Fig 4 2G, P < 0.025) by 22% Similarly M T7 treatment ( N=5) significantly reduced the IEL ( P < 0.003) and EEL diameters ( P < 0.001) by 41% and 44%, respectively. Serp 2 treatment ( N = 5) also significantly reduced the IEL ( P < 0.0331) and EEL ( P < 0.0213) by 25% and 26%, respectively. Changes in IEL and EEL were not significant at earlier follow up times ( Fig 4 2G, H). There was no significant early difference detected in saline or protein treated ApoE / mice for plaque area, IEL or EEL measure ments after angioplasty from 0 hours up to 7 days ( P = NS), as minimal plaque is present at early follow up times ( Fig 4 2A). Changes in inflammatory cell invasion ( Fig 4 2B, C F) as well as elastin breaks ( Fig 4 2I) were assessed with Serp 1 and M T7 trea tment. Serp 1 ( Fig 4 2K) and M T7 ( Fig 4 2L) significantly decreased ( at 7ds Serp 1 N=5, P < 0.0012; M T7 N=5, P < 0.0059; Fig 4 2I) when compared to saline ( Fig 4 2I, J). This reduction was not significant at later times ( Serp 1, N=5, P= 0.1296; Serp 2, N=5, P = 0.1187; M T7, N=5, P = 0.1804).


86 In summary, treatment with three viral anti inflammatory proteins effectively reduced early inflammatory cell invasion and elastin break s with late reduction in plaque growth and dilatation. Inflammatory Cell Invasion is Reduced with Viral Immunomodulatory Protein Treatment To confirm whether protein treatments reduced invasion of specific inflammatory cell types into the abdominal aorta, macrophage and T cell counts were measured by immunohistochemical staining. Cell counts for positively stained cells ( macrophage and T cells) were assessed in each arterial layer. Mononuclear cell invasion was increased in ApoE / mice after angioplasty i njury at 28 days. When compared to saline treated ApoE / mice, saline treated C57Bl/6 wild type mice had significantly fewer CD3 + cells in the intima ( Fig 4 3A, N= 6, P < 0.016), but not in the adventitial layer ( Fig 4 3B, N=6, P =0.297). For ApoE / mi ce at 28 days follow up, Serp 1 ( N=36 high powered fields, P < 0.001), Serp 2 ( N=9 fields, P < 0.020) and M T7 ( N=12 fields, P < 0.007) treatments significantly reduced CD3+ T cells in the intima ( Fig 4 3A), whereas only Serp 2 ( N=9 fields, P < 0.006) and M T7 ( N=12 fields, P < 0.008) significantly reduced detected adventitial CD3+ T cells ( Fig 4 3B). Serp 1 treatment produced an insignificant trend toward reduced CD3+ T cells in the adventitia ( P =0.082). Conversely, for CD11b + macrophages in ApoE / mice, none of the protein treatments significantly reduced macrophage invasion in the intima ( Fig 4 3C) at 28d post treatment, although there was a trend toward a reduction with Serp 1. Serp 1 ( N=15 fields, P < 0.0163) did, however, significantly reduced adventitial macrophage counts ( Fig 4 3D).


87 Viral Anti inflammatory Protein Treatment Alters Gene Expression Cultured human inflammatory system cells were screened for shared gene response pathways targeted by all three proteins. Jurkat T cells, THP 1 monocytes, and Hu man Umbilical Vein Endothelial Cell ( HUVECs) were treated with Camptothecin ( CPT), a topoisomerase inhibitor, to induce apoptosis and co treated with either saline or Serp 1, Serp 2 or M T7 ( 500ng per million cells). As cell death is now known to drive som e aspects of vascular inflammation, plaque growth, and aneurysm formation, expression of a set of 96 genes in apoptosis specific qRT PCR arrays were analyzed. Although greater and more consistent changes were detected in T cells by immunostaining after an gioplasty, monocytes displayed more changes in gene expression in vitro. Although these viral proteins target different molecular pathways in vivo, after 30 minutes of treatment, a cohort of 4 8 shared genes was detected in monocytes ( Fig 4 4A; Table 4 2) but not in T cells ( Fig 4 4B; Table 4 2). A few shared gene changes were observed for HUVEC cells with Serp 1 and Serp 2 treatments at 30 minutes. Minimal shared changes were detected for all three cell lines at 4.5 hours ( data not shown). For Jurkat cell s there were no genes with shared changes in altered expression at 30 minutes for all three treatments, e.g. Serp 1, Serp 2 and M T7 ( Fig 4 4B). Serp 1 and Serp 2 did have altered expression of three shared genes ( FAS, BNIP1, TP53) and Serp 1 and M T7 did share 1 gene with altered expression ( CARD6). FAS and TP53 were also seen in THP 1 cells at 30 min treatment, but they were not affected in the same way; FAS and TP53 are up regulated in Jurkat T cells and down regulated in THP1 monocytes. CARD6 was down r egulated in Jurkat cells at 30 minutes as in THP 1 cells. For HUVECs treated with Serp 1 or Serp 2, eight genes ( BCL2L10, BIRC8,


88 FASLG, HRK, TNFRSF11B, CD27, TNFSF8, TP73) had shared similar changes at 30 minutes with THP1 consensus genes, but none are sha red with Jurkat gene changes. All three viral proteins demonstrated the capacity to regulate expression of a shared cohort of genes in THP 1 monocytes. A closer look at the co regulated genes ( Table 4 2) demonstrates tha t many of these shared genes are in volved in triggering or repressing apoptosis, are down regulated when compared to the CPT only control. In vivo we have seen that the net effect of the viral protein treatments is a decrease in cellular apoptosis ( Fig 4 5). B cell lymphoma ( BCL) 2 family proteins are highly represented, with 13 candidate genes detected in the screen. This family of proteins has highly divergent effects on apoptosis; many induce apoptosis while others are suppressor proteins ( as reviewed in 41 ) and their roles in apoptosis may change based on relative expression levels. Further investigation into the roles of these genes in aneurysmal dilatation is required, but the role of apoptosis in unstable plaques and aneurysm rupture is promising. Having detected both a reduction in inflammation and elastin breakage in vivo as well as associated gene expression changes in apoptotic human monocytes in vitro, we validated apoptotic changes using active caspase 3 as an indicator of ongoing apoptosis in our mouse balloon angioplasty model. Anti infla mmatory Protein Treatment Reduced Early Caspase 3 Activity To assess the anti apoptotic potential for each protein, caspase 3 levels were assessed in aortic cross sections by immunohistochemical analysis for active caspase 3. Caspase 3 expression varied with location and protein treatment ( Fig 4 5 A; Saline, B; Serp 1 C; M T7 ). In the adventitia ( Fig 4 5F) and media ( Fig 4 5E), the greatest levels of caspase 3 positive cells were present at 24 hours in saline treated mice with a


89 reduction by 7 days. In th e intima ( Fig 4 5D), apoptosis increased over time with an apparent further increase in Serp 1 treated mice ( P < 0.0001) at 7 days. Conversely, at 7 days M T7 further reduced apoptosis when compared to saline ( P < 0.0446) in the intima. In the adventitia, ca spase 3 was reduced at 24hrs ( Fig 4 5F) with Serp 1 ( P < 0.0001) and M T7 ( P < 0.0001) treatment and at 7 days ( Serp 1 P < 0.0106; M T7 P < 0.0221), In the media, both M T7 and Serp 1 reduced active caspase 3 at 24hrs ( Fig 4 5E, P < 0.0001) whereas at 7 days ( P < 0.0020), only M T7 achieved a significant reduction ( P < 0.0013). Discussion ApoE / mice, unlike their wild type counterparts, spontaneously develop plaques when fed normal chow and also spontaneously develop aneurysms (158) In this model of balloon angioplasty injury, ApoE / mice develop significantly more plaque ( Fig. 4 1A) and aortic dilatation occurs at an accelerate d rate at earlier times ( Fig. 4 1C) than wild type mice. Our findings indicate that the aggressive inflammatory response and associated plaque growth in ApoE / mice potentially drive the aneurysm like dilatations detected after balloon angioplasty. Treat ment with proven inflammatory cell inhibitors that targeted different molecular pathways reduced both aneurysm formation and plaque growth. These findings suggest altering immune or inflammatory responses via differing excitatory pathways is capable of red ucing both aneurysm formation and plaque growth, irrespective of the pathway targeted, suggesting a common or shared end stage response in the arterial wall causing plaque growth and associated aneurysm formation. We have demonstrated that the viral prote ins Serp 1, Serp 2 and M T7 significantly reduce inflammation early elastic lamina breakage and late plaque growth


90 and aneurysmal dilatation in this mouse model with balloon angioplasty injury. Serp 1 has completed a phase II clinical trial, reducing mark ers for myocardial damage (135) demonstrating that purified viral anti inflammatory proteins can be used safely in both animal models and in man as therapeutic agents. Further, prior work h a s demonstrated that these virus derived agents do target the differing pathways predicted by their inhibitory functions as inactive serpin controls, including myxomavirus Serp 1 SAA ( 59,66) vaccinia virus serpin CrmA (136) and Serp 2 reactive center mutants (136) do not alter inflammatory cell invasion or plaque growth. Apoptosing cells in early plaques and arterial damage can release a powerful mix of chemokines, cytokines and other pro inflammatory molecules that exacerbate in flammatory cell invasion, plaque growth, artery dilatation and apoptosis, begetting a vicious cycle that can lead to aneurysm formation (143 148) Although the proteins reduced CD3 + T cell invasion in the mouse aorta more than for CD11b + macrophage, we have detected a more consistent gene expression response in human monocytes than in JURKAT or HUVEC cells. This differen ce may reflect an earlier response in monocytes, such that early inhibition of monocyte/ macrophage activation may have greater effects downstream at later times after angioplasty injury on arterial invasion, plaque growth and dilatation. These early chang es in macrophage invasion may also represent altered levels of activation rather than altered numbers of invading cells. The gene expression changes in reflect changes in apoptotic pathways underscoring a possible pivotal effect on macrophage apoptosis in the arterial response. Analysis of 96 genes revealed consensus genes for all three anti inflammatory treatments, highlighting 13 BCL family proteins and 9 TNF receptors and ligands which


91 have diverse roles in apoptosis ( Table 2). For instance, BCL family proteins BIK, BAG4 and BCLAF1 were up regulated in response to protein treatments: BIK is a pro apoptotic protein, BCLAF1 is a transcriptional repressor protein with pro apoptotic effects, and BAG4 is purported to silence TNFR regulated gene TNFSRF1A, culminating in anti apoptotic effects (160) In a similar manner, the 9 down regulated BCL family proteins are also functionall y diverse. The TNFR family has 8 genes significantly down regulated, compared the only two TN F family proteins ( DR6/TNFRSF21 and TRAF3) that are up regulated after treatment with three viral anti inflammatory proteins. The TNF family proteins and ligands are present in a wide variety of cell types and are also known to be responsible for diverse f unctions, including induction of apoptosis as well as inflammatory cell recruitment, inhibition of viral replication, and tumorigenesis (161) Of note, down regulated proteins include the death ligand ( FasL) and its transmembrane death rece ptor ( FAS) which are the main components of the Death Induced Signaling Cascade ( DISC), the platform of the major extrinsic apoptosis cascade. Furthermore, c FLIP, an important adapter protein for activation of caspase 8 is up regulated, as is the downstre am molecule caspase 2. Yet caspase 10, often involved in alternate assemblies of the DISC, is down regulated. The complex regulatory interplay of DISC components is overruled by the down regulation of Fas and FasL, as they are the primary initiators of DIS C assembly. Down regulation of CD27 is noteworthy as it is vital to the long term generation and maintenance of T cell immunity, an important player in atherosclerotic disease. The interplay between these apoptotic and immune pathways merits further invest igation.


92 With these studies we examine the potential effects of three viral anti inflammatory proteins targeting divergent inflammation pathways after angioplasty injury. All three proteins have had prior proven effective anti inflammatory and ant atherog enic functions in rodent models. Our findings demonstrate that inhibition of inflammatory responses, with any of these three different proteins, significantly reduces arterial inflammation, elastic lamina breakage, plaque growth, and aneurysm like dilatati on. Further, our findings suggest that these three diverse proteins may cause altered expression and/or activation of a common downstream pathway, resulting in reduced inflammation, plaque growth and aneurysm like dilatation of the abdominal aorta. The pot ential shared effects on apoptotic and inflammatory cell responses of these three proteins is mirrored by the detection of a shared cohort of genes with altered expression after treatment of human mononuclear cells with each protein in vitro in tissue cult ure. Elucidating this pathway has the potential to lead to a greater understanding of vascular injury progression in atheroma and aneurysm formation and to identify new therapeutic targets for future drug development. Based upon the effects of Serp 2 and control anti inflammatory proteins Serp 1 and M T7 on human monocyte gene expression, as well as shared effects on hallmarks of aortic dilatation, such as elastic lamina breaks, plaque area and inflammatory cell invasion we have proceeded to e xamine the roles of caspase 1 and gra n zyme B in Serp 2 inh i bition of inflammatory cell responses and apoptotic pathway gene expression in mice Thus to t ie the observed phenotype in vivo in the mouse models to gene expression changes in vitro we sought to explore the same gene set in mice. As Serp 2 targets Caspase 1 and Granzyme B, mice deficient for these genes and the appropriate


93 background controls were studied to reveal insights into the mechanism of action of Serp 2 or CrmA.


94 Figure 4 1 Mouse aortic histology. A) Sample 20x H&E C57Bl/6 histology; B) Sample 20x trichrome histology from ApoE / mouse; C) 40x Elastin break and intact elastin from ApoE / D) Measured plaque area in ApoE / ( N = 24) and C57Bl/6 ( N =19) after saline treatment ( P <0.0 01) mice at 4 weeks; E) Elastic lamina measurements across mouse strains at 4 weeks ( P < 0.043); F) Number of elastin breaks at 4 weeks ( P < 0.050).


95 Figure 4 2 Viral Protein Treatment.Treatment with viral proteins significantly reduces plaque formation ( A, P <0.004 for all treatments at 28d, N=5) and inflammatory cell invasion ( B) in ApoE / mice both immediately and over 28d ( C; Saline N=12, D; Serp 1, P <0.0013, N=5, E, Serp 2 N=5, F; M T7, P <0.0030, N=5). Serp 1, Serp 2 and M T7 were sufficient to re duce external elastic lamina diameter ( G [Saline N=12 at 28d, P <0.0248 Serp 1 N=5 at 28d, P <0.0213, Serp 2 N=5 at 28d, P <0.001 M T7 N=5 at 28d]) and internal elastic lamina diameter ( H [Saline N=12 at 28d, P <0.0465, Serp 1 N=5 at 28d, P <0.003, Serp 2 N=5 at 28d, P <0.0331, M T7 N=5 at 28d]). Elastin fragmentation was reduced at 7 days ( I [J; Saline N=5, K; Serp 1 N=5, P <0.0012, L; M T7 N=5, P<0.0059]) in ApoE / mice.


96 Figure 4 3 After staining, 3 high power fields per mouse sample were counted for CD3+ T cells or CD11b+ monocytic cells. Asterisks indicate P < 0.05. For ApoE / mice at 4 weeks post injury, Serp 1 ( N=5, P <0.0199), Serp 2 ( N=4, P <0.0199) and M T7 ( N=4, P <0.0071) treatments significantly reduce CD3+ T cells in the intima ( A), where as only Serp 2 ( N=4, P <0.0061) and M T7 ( N=4, P <0.0088) effect significant changes in adventitial T cells ( B). Compared to saline treated ApoE / mice, C57Bl/6 saline treated mice had significantly less CD3+ cells in the intimia ( N= 5, P <0.0163) but no t in the adventitial layer ( N=5, P <0.2966). For ApoE / mice at 4 weeks post injury, no treatments significantly reduce macrophages in the intima ( C), whereas only Serp 1 ( N=9, P <0.0163) effected significant changes in adventitial macrophages ( D).


97 Figure 4 4. Overlapping gene expression changes in THP 1 cells ( A) or Jurkat T cells ( B) after treatment with 3 viral proteins and CPT for 30 minutes. Numbers represent genes which experienced a greater than 2 fold change in expression compared to CPT tre ated cells.


98 Figure 4 5 Caspase 3 staining. Aft er staining for active Caspase 3 positively staining cells were counted in 3 100x magnification fields per mouse aortic sample for Saline ( A ), Serp 1 ( B ) and M T7 ( C) In the Intima (D ), significant chang es were observed at 7 days post angioplasty. Serp 1 caused a 1.5 fold increase ( N=18 fields, P <0.0198) whereas M T7 caused a 2 fold decrease ( N=18 fields, P <0.0046) in positively staining cells. In the media ( E ) at 0hrs, Serp 1 significantly decreased ca spase 3 activity by 2.2 fold ( N=18 fields, P <0.0076). 24 hours post angioplasty, M T7 caused a 4.4 fold decrease ( N=18 fields, p> 0.0001) whereas Serp 1 caused a 1.8 fold decrease ( N=18 fields, P <0.0013). M T7 also caused a 1.2 fold decrease ( N=18 fields P <0.0112) at 7 days post treatment. Lastly, in the adventitia ( F ) of ApoE / mouse aortas, M T7 caused a 5.9 fold decrease ( N=18 fields, P <0.0001) in positively staining cells, whereas Serp 1 treatment results in a 3.6 fold decrease ( N=18 fields, P <0. 0106) at 24h. However, at 7 days post treatment, M T7 resulted in a 2.4 fold decrease ( N=18 fields, P <0.0221) and Serp 1 resulted in a 1.4 fold decrease ( N=18 fields, P <0.0221) in positively stained cells.


99 Table 4 1 Mouse strains, protein treatments and post surgical complications. Mouse strain # of angioplasty mice Duration Protein Treatment Post surgery complications Serp 1 M T7 Serp 2 Saline ApoE / 12 4wks + 1 hematoma, 1 thrombosis ApoE / 3 1wk + ApoE / 3 24hr + ApoE / 3 0h + ApoE / 5 4wk 15g ApoE / 3 1wk 15g ApoE / 3 24hr 15g ApoE / 3 0hr 15g ApoE / 5 4wk 15g 1 hematoma ApoE / 3 1wk 15g ApoE / 3 24hr 15g ApoE / 3 0hr 15g ApoE / 5 4wk 15g 1 hematoma C57Bl/6 10 + 1 hematoma C57Bl/6 5 6g

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100 Table 4 2 Gene expression changes in THP 1 cells after treatment with 3 viral proteins and camptothecin ( CPT) for 30 minutes. Bolded genes are upregulated in response to these treatments. BCL family gene #TNF family gene Gene RQ Gene RQ Gene RQ BCL2L10a 0.053 FASLG 0.217 TNFSF10 # 0.424 TNFRSF11B # 0.077 AKT1 0.245 CASP7 0.447 HRK 0.091 Casp10 0.251 BFAR 0.452 BCL2A1 0.095 TP53 0.262 BCL2L11 0.472 BAK1 0.110 BCL10 0.266 CRADD 0.472 CD40LG 0.110 BAG1 0.270 CASP2 2.402 BRAF 0.122 CD27 0.276 BCLAF1 2.648 BIRC8 0.124 FAS 0.283 PYCARD 2.716 ABL 0.139 Casp1 0.328 CASP3 2.780 CIDEA 0.148 BAX 0.336 BAG4 2.994 IGF1R 0.151 BAD 0.347 CIDEB 3.003 TNFSF8 # 0.171 TNFRSF10A # 0.356 BNIP2 3.295 TP73 0.171 GADD45A 0.373 TNFRSF21 # 3.345 APAF1 0.187 TNFRSF1A # 0.386 CFLAR 4.123 CARD6 0.188 NAIP 0.389 TRAF3 # 4.178 TNFRSF25 # 0.214 BAG3 0.391 BIK 4.220

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101 CHAPTER 5 VIRAL ANTI APOPTOTIC PROTEINS SERP 2 AND CRMA EFFECTS IN CASPASE 1 AND GRANZYME B DEFICIENT MICE This chapter explores hypothesis 3: Serp 2 and CrmA modify inflammatory mononuclear cell responses and apoptotic gene expression via interactions with caspase 1 and / or granzyme B. These experiments were done to unify hypotheses 1 and 2, highlighting comm onalities between how Serp 2 and CrmA alter mouse peritoneal exudate gene expression and their effects in vivo and the effects of Serp 1, Serp 2 and M T7 in vivo and in vitro, as discussed in the prior chapters. Introduction As reviewed in Chapter 1, Serp 2 and CrmA, homologous proteins from the Myxoma and Vaccinia virus families, respectively, both target granzyme B and caspases 1 and 8 (47,61 65,136,139,140,156,162) Although Serp 2 has weaker coefficients of inhibition than Serp 2 for Casp1 ( K I =80 nM v er s us 4pM, respectively) and for GzmB ( Ki = 420 nM versus 100nM, respectively) ( 62) As discussed In Chapter 3, despite thi s inequity in inhibition, Serp 2 has more profound effects than CrmA in vivo in wild type and ApoE / mouse models of vascular damage (136) In Chapter 4, we investigated the role of Serp 1, Serp 2 and chemokine modulating protein M T7 in human cell lines. Based on the significant gene expression cha nges noted in monocytes in response to all 3 protein treatments ( Table 4 1), and in order to better understand the molecular mechanism of action of these proteins in vivo mice were injected intra peritoneally with a cell activator and saline or Serp 2 or CrmA. 18 hours later, the resulting peritoneal exudates from treated caspase 1 and granzyme B deficient mice were compared to their control strain backgrounds by flow cytometry and qRT PCR. If CrmA and Serp 2 are active in altering inflammatory cell invasi on or gene expression in

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102 background strains, but not in the single gene knockout mice, the relative contribution of that knockout gene to the mechan ism of action can be inferred. Results CD4 and CD11b are Unaffected by Treatment with Serp 2 or CrmA when C ompared to PMA Treatment Alone In order to tease apart the anti inflammatory effects of Serp 2 and CrmA noted in prior studies, peritoneal exudate cell populations from 2 knockout mouse strains and their respective backgrounds were analyzed 18 hours after stimulation with PMA and concurrent treatment with Saline, Serp 2 or CrmA. One million cells from each mouse sample were stained with antibodies to CD4, CD11b, Ly6C and Ly6G. Due to some low total cell counts, some mouse samples had to be pooled, reducing the effective number of replicates. Compared to their respective background phenotype, no significant differences were observed in CB11b+ cell infiltrates between knockouts or within each strain for treatments ( Fig 5 1A & B). Likewise, regardless of treat ment there was no detectable CD4+ T cell population in Casp1 / or NOD mice ( data not shown) or in GzmB / mice, though an insignificant increase was noted in Serp 2 treated B6 mice ( Fig 5 1C). Based on these results, PMA stimulation is not inducing CD4+ T cell migration, nor altering relative CD11b+ leukocyte migration between knockout and background strains. Given that Serp 2 has previously demonstrated reductions in mononuclear cell invasion in vivo ( 66) it was surprising that neither Serp 2 nor Crm A significantly altered the CD11b+ migration in any strain. Serp 1 and Serp 2 Treatment Increase CD11b+ Ly6Ghi Populations in Nod Mice Compared To PMA Treatment While the total CD11b+ leukocyte population may not have changed, there are major subgroups wi thin CD11b+ leukocytes: Ly6Ghi neutrophils, Ly6Chi classical

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103 monocytes and Ly6Clo resident monocytes (17,163,164) When cells are gated for CD11b+ populations and then examined for Ly6G versus Side Scat ter, it is possible to examine population statistics for high and low staining cells. There is no significant change in CD11b+ Ly6Ghi neutrophils between GzmB / and B6 strains or between treatments ( Fig 5 2A). GzmB / demonstrates a trend toward increased CD11b+ Ly6G low cells, but due to a low mouse sample size, does not reach significance ( Fig 5 2B). Compared to PMA treatment in NOD mice, treatment with Serp 1 ( p=0.0349) and Serp 2 ( p=0.0193) demonstrated significantly increased Ly6Ghi and consequently decreased Ly6Glo CD11b+ cells ( Fig 5 2C & D). CrmA treatment had no significant change in Ly6 G populations. No significant changes were observed in Casp1 / mice for Ly6Ghi or Ly6Glo populations. PMA and Serp 2 Treatment Increase CD11b+ Ly6Chi Populations in GzmB / Mice Compared to Equivalent B6 Treatments In addition to the CD11b+ Ly6G positive populations, we examined the CD11b+ monocytes, believed to mature into the pro i nflammatory M1 macrophage while Ly6Clo anti inflammatory M2 macrophages (17,165) As shown in Figure 3, no significant changes were observed between treatments in CD11b+ Ly6Chi populations in B6 mice, while granzyme B knocko ut mice have a significantly larger population of Ly6Chi monocytes for PMA ( p=0.0051) and Serp 2 treatments ( p=0.0094) than their background control stain ( Fig 5 3A). No significant changes are seen in CD11b+ Ly6C low populations between treatments in GzmB / or B6 mice or between strains ( Fig 5 3B). Thus, PMA stimulation and Serp 2 treatment of GzmB / results in increased Ly6Chi

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104 recruitment from the bone marrow but no changes in the resident monocyte population, when compared to the equivalent treatments in B6 mice. No significant changes in Ly6Chi were observed for proteins treatments compared to PMA in the GzmB / strain. Serp 1, Serp 2 and CrmA Treatment Increases CD11b+ Ly6Chi Populations in Casp1 / Mice Compared to Equivalent Nod Treatments When co mpared to treated NOD mice, Casp1 knockout mice display increased Ly6Chi populations in Serp 1 ( p=0.0258), Serp 2 ( p=0.0152) and CrmA ( p=0.0027) treatments ( Fig 5 3C). Treatment with Serp 1 ( p=0.0349) and Serp 2 ( p=0.0193) demonstrated increased Ly6Chi mon ocytes when compared to PMA stimulation alone in NOD mice ( Fig 5 3D) ; with a corresponding decrease in Ly6C low cell counts ( Fig 5 3E). CrmA treatment did not significantly alter Ly6C populations on Casp1 / mice. Thus, PMA stimulation, Serp 1, Serp 2 and CrmA treatment of Casp1 / results in increased Ly6Chi inflammatory monocytes and Serp 1 and Serp 2 treatment reduce the resident monocyte population, when compared to the equivalent treatments in NOD mice. This increase in Ly6Chi inflammatory monocytes in Casp1 / mice is despite a statistically significant increase with Serp 1 treatment and significant decrease in this population with Serp 2 treatment in NOD mice. BAG3 is Significantly Regulated by Serp 2 and CrmA Primary mouse peritoneal exudate cells were screened for shared gene response pathways targeted by Serp 2 and CrmA after normalization to GAPDH and PMA. Mice were injected with 50ng PMA in 100L DMSO, a potent cell stimulator, to induce migration and concurrently treated with either 100L salin e or Serp 2 or CrmA ( 500ng per mouse). As Serp 2 and CrmA are known inhibitors of granzyme B and

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105 caspase 1 and inhibit cell death, expression of a set of 96 genes in apoptosis specific qRT PCR arrays were analyzed ( Table 5 1). Nod mouse peritoneal exudate s (Fig 5 4A) evidenced 19 genes responding to CrmA treatment more than 2 fold and 18 genes responding to Serp 2 treatment after stimulation with PMA Caspase 1 deficient mice had 38 genes with a greater than 2 fold response to CrmA and 46 genes responsive to Serp 2 treatment ( Fig 5 4 A). Nod and Casp1 / mice treated with CrmA or Serp 2 displayed 14 genes regulated by both serpin treatments when compared to PMA treatment (Fig 5 4 A). B6 had a moderately more responsive phenotype, with 44 genes responding wit h greater than 2 fold changes to CrmA treatment and 14 to Serp 2 treatment. Granzyme B knockout mice had 64 genes regulated more than 2 fold by CrmA treatment and 39 regulated by Serp 2 treatment. When comparing protein treated Granzyme B mice to backgroun d B6 mice, there are only 3 genes re gulated by both Serp 2 and CrmA (Fig 5 4B) As seen in Fig 5 4C in g ranzyme B deficient mice and B6 mice, only one gene, BAG3, was altered more than 2 fold for each of the Serp 2 and CrmA treatments in all strains of mice after normalization to PMA treatment in their respective strains Serp 2 treated GzmB / mice downregulated B AG3 ( RQ = 0.02) whereas B6 mice experienced an upregulation ( RQ = 7.41) causing an expression change of 345 fold between Serp 2 treated B6 a nd GzmB / mice ( p=0.0235). CrmA treated GzmB / mice expressed BAG3 at a level 2.9 times less than CrmA treated B6 mice ( RQ= 5.99 and 17.6, respectively, p=0.0181). Ser p 2 treatment had significantly lower expression levels than CrmA treated mice in GzmB and B6 mice ( p=0.0267 and 0.0280, respectively). This indicates that Serp 2 and CrmA may utilize Granzyme B in their mechanism of BAG3 regulation.

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106 Caspase 1 knockout mice treated with Serp 2 downregulate BAG3 when normalized to PMA treatment and express 9.7 fold less BAG3 when compared to Nod background mice ( p=0.0006). BAG3 expression after treatment with CrmA in Casp1 / or Nod mice did not significantly vary ( 1.16 fold, p=0.8537). Likewise, there was no significant difference in BAG3 expression between CrmA and Serp 2 treated Nod or Casp1 / mice. Discussion While neither Serp 2 nor CrmA altered the CD11b+ infiltrating population in any of the tested mouse strains, there were significant changes in the Ly6Chi sub population when either knockout strain was compared to its background strain. PMA ( p=0.0051) and Serp 2 treatments ( p=0.0094) were increased in GzmB / compared to B6, and in Casp1 / mice, Serp 1 ( p=0.0258), Serp 2 ( p=0.0152) and CrmA ( p=0.0027) were altered significantly compared to NOD mice ( Fig 5 3A & C). Decreases in Ly6Clo ( Fig 5 3E) cells may be due to increased apoptosis of patrolling monocytes in response to PMA stimulation which is no longer inhibited by Serp 2 or CrmA treatment. NOD mice also demonstrated an increased Ly6Ghi populati on with Serp 1 ( p=0.0349) and reduced Ly6Ghi population Serp 2 ( p=0.0193) when compared to its own PMA treatment ( Fig 5 4B). Ly6Chi cells are considered classical or inflammatory monocytes, which are recruited from the bone marrow in a CCL 2 dependent fash ion (164) and enter the site of inflammation via endothelial cell interactions. Monocytes are immune effector cells, capable of detecting and endocytosing pathogens or toxins as well as secreting their own array of cytokines to alter the host immune response. CD11b+ Ly6Chi monocytes are thought to mature into pro inflammatory M1 macrophages.

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107 Interestingly, in qRT PCR analysis of mouse intra peritoneal cells for apoptosis gene expressi on one gene, BAG3, was represented in almost all strains for regulation by CrmA or Serp 2 when compared to PMA treatment ( Fig 5 4C ). Bcl2 associated athanogene 3 ( BAG3) interacts with heat shock protein ( Hsp) 70 through an ATPase: BAG domain connection (166,167) particularly after stressful stimulation with heat or heavy metals. In humans, BAG3 is only constitutively expressed in myocytes and a handful of other normal cell types, but is transiently expressed after cell stress in other normal cell types, such as leukocytes (166) Generally considered anti apoptotic, is not not surprising that aberrant, constitutive BAG3 expression is commonly found in a wide variety of human solid tumors and tumor cell lines (166) In addition, Bag3 is thought to bind and stabiliz e the a nti apoptotic protein Bcl 2, (168 172) and thus further reduci ng stress induced apoptosis. BAG3 also sustains NFkB signaling pathways in cells, which promotes cell survi val (166) BAG 3 knockout mice demonstrate severe myopathy and a post natal lethal phenotype, around 4 weeks after birth (169) In our model, caspase 1 deficiency resulted in an up regulation of BAG3 for CrmA treatment when compared to the same treatments in NOD background control mice. It is known that the anti apoptotic effects of BAG3 are limited by caspases 3,7, 9 and 10 (170) and subsequent polyubiquitination and proteolysis, but prior to this, caspase 1 has not been implicated in BAG3 regulation BAG3 is well known to be involved in proteolysis and autophagy, so this new involvement of Casp1 in BAG3 induction after treatment with PMA and Serp 2 (p=0.0006 for Serp 2 treatments between Casp1 / and background Nod mice) Likewise, granzyme B deficiency resulted in reduced BAG3 expression induction when compared to the same treatment effect s in B6 mice p= 0.0235 for Serp 2 between strains and

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108 p=0.0181 between strains) Interestingly, BAG3 also showed a decrease in human monocytes ( Table 4 2) for Serp 1, Serp 2 and M T7 treatments ( p < 0.0001). Taken together, these data indicates that Serp 2 and CrmA have important regulatory roles in inflammatory cell invasion and gene regulation. Compared to human monocytes, mouse peritoneal exudates were less responsive to serpin regulation after stimulation ( 48 consensus genes vs 1) but also comprise a mixed cellular population. However, the fact that the singular consensus gene for our mouse models, BAG3, is also one of the 48 co regulated gene in our human cell lines is incredibly promising for the involvement of this gene or protein in the mechanism of action for viral anti inflammatory and anti apoptotic protein treatments. Interestingly, GzmB and Casp1 deficient mice demonstrated significant up regulation of Ly6Chi cells for Serp 2 treatment in comparison to their background strains, indicating that Serp 2 may in fact work to suppress this pro inflammatory monocyte subset in GzmB and Casp1 competent, wil d type mouse exudates. In contrast to the marked reduction in mononuclear cell invasion in ApoE / ( B6 background ) noted in Chapter 4 ( Fig 4 2), Serp 2 did not significantly reduce CD11b+, Ly6Chi or low monocyte populations when compared to PMA treatment i n the same genetic background except in Nod mice ( Fig 4 3D) This difference may be due to the highly inflammatory nature of our vascular injury model and the relatively less invasive intra peritoneal injection of PMA or may indicate that Serp 2 is alteri ng another invading cell population not analyzed in this study Further investigation into the role of caspase 1 in BAG3 regulation will help elucidate the mechanism of action for Serp 2 and CrmA and is a promising clue a possible consensus pathway targete d by viral anti inflammatory proteins.

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109 Figure 5 1 After staining, mouse peritoneal cells were sorted by flow cytometry for CD11b+ monocytic cells or CD4+ T cell populations No significant changes were observed for CD11b+ ( A, B) or CD4+ cell populations ( C) in isolates from B6, Nod, Casp1 / or GzmB / mice, regardless of treatment. indicates significant difference for same treatment between strains (p <0.05). # indicates significant difference between treatments in the same strain (p <0.05) The results shown here represent mean SE from 3 to 5 replicates for each treatment within each strain. N=5 for all strains and treatments, except for Granzyme B mice, where N=5 for Serp 2 and PMA and N=3 for CrmA.

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110 Figure 5 2 Mouse peritoneal cell s were gated by flow cytometry for CD11b+ monocytic cells and then Ly6G staining. No significant changes were observed for CD11b+ Ly6Ghi or Ly6Glow populations in isolates from B6, Casp1 / or GzmB / mice, regardless of treatment ( A, B). Nod mice, however showed a statistically significant increase in Serp 1 and Serp 2 treated animals for Ly6Ghi populations ( C), with a concurrent decrease in Ly6Glo populations ( D). indicates significant difference for same treatment between strains (p <0.05). # indicate s significant difference between treatments in the same strain (p <0.05). The results shown here represent mean SE from 3 to 5 replicates for each treatment within each strain N=5 for all strains and treatments, except for Granzyme B mice, where N=5 for Serp 2 and PMA and N=3 for CrmA.

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111 Figure 5 3 Mouse peritoneal cells were gated by flow cytometry for CD11b+ monocytic cells and then for Ly6C staining.GzmB / mice displayed a significant increase in Ly6Chi cells in response to PMA and Serp 2 treatment, when compared to equivalent B6 treatments ( A) with no change in Ly6Clo populations ( B). Additionally, Casp1 / mice treated with Serp 1, Serp 2 or CrmA display ed significantly higher Ly6Chi staining levels than their Nod equivalents ( C). Nod mice treated with Serp 1 had a statistically significant increase in Ly6Chi populations, while Serp 2 resulted in a statistically significant decrease in Ly6Chi cells ( D). A significant decrease happened in Nod mice for both Serp 1 and Serp 2 treatment in Ly6Glo populations ( E). indicates significant difference for same treatment between strains (p <0.05). # indicates significant difference between treatments in the same st rain (p <0.05). The results shown here represent mean SE from 3 to 5 replicates for each treatment within each strain N=5 for all strains and treatments, except for Granzyme B mice, where N=5 for Serp 2 and PMA and N=3 for CrmA.

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113 Figure 5 4 Apop totic gene expression in treated mice. A) 2 fold gene expression changes in Casp1 / and Nod background mice. B) 2 fold gene expression changes in GzmB / and B6 background mice C) BAG 3 is upregulated compared to PMA treatment by CrmA in all mouse strains. Serp 2 treatment reduces BAG3 expression compared to CrmA in all strains and reaches significance in Granzmye B / B6 and Nod strains. indicates significant difference for same treatment between strains (p <0.05). # indicates signific ant difference between treatments in the same strain (p <0.05). The results shown here represent mean RQ SE from 4 to 5 biological replicates for each treatment within each strain.

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115 CHAPTER 6 CONCLUSIONS AND FUTU RE DIRECTIONS The anti inflammatory and anti apoptotic effects of Serp 2 discussed within these chapters are pronounced. However, there are still a large number of questions regarding how Serp 2 and the other viral anti inflammatory proteins contained within are able to effect these changes. In these studies we show that Serp 2 is capable of reducing plaque area in rat balloon angioplasty ( Fig 3 1A), in mouse aort ic allograft ( Fig3 1D & G Fig 4 2A & E ), in mouse carotid cuff models ( Fig 3 4B, D & E), Casp3 activity ( Fig 3 3A and 3 7G) mononuclear cell invasion in vivo ( Fig 4 2 B ) inflammatory cell invasion in vivo ( Fig 5 1, 5 2 and Fig 5 3) and on gene expression in human cultured cells ( Table 4 2) and in mouse peritoneal exudates ( Fig 5 4 A & B). We questioned the role of Serp Granzyme B and Caspase 1, by utilizing m ice deficient for these genes. We found differential regulation of BAG3 by qRT PCR analysis of inflammatory cell exudates and a further potential role for Casp1 in BAG3 regulation, as deficiency resulted in down regulation of BAG3 for Serp 2 treatment when compared NOD background control mice. Likewise, granzyme B deficiency resulted in a significa nt down regulation of BAG3 in Serp 2 and CrmA treated mice (345 fold and 2.9 fold respectively) when compared to similarly treated B6 control mice. This suggests that Serp 2 and CrmA may utilize GzmB regulated pathways in altering expression of BAG3 in vivo That BAG3, is also co regulated in human monocytes is incredibly promising for the involvement of this gene or protein in the mechanism of action for viral anti inflammatory and anti apoptoti c protein treatments. BAG3 has a tremendous variety of intracellular roles; stabilizing BCL 2, interfering with Hsp70 chaperone activities by

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116 promoting client protein or mRNA release to altering cell adhesion and migration (166) BAG3 is constitutively expressed in human myocytes and few other normal cell types but is often constitutively expressed or overexpressed in a wide variety of tumors (166) The role of BAG3 in lymphocyte differentiation, maturation and maintenance is poorly understood. Though our limited flow data and RTPCR analysis leaves many questions about intricate role BAG3 may play in regulating CD11b+Ly6Chi population, it seems that BAG3 expression may be altering the ability of classical monocytes to migrate to the site of inflammation, as silencing BAG3 expression in tumors decreases metastasis (166) It is also plausible that the anti apoptotic / NFkB sustaining effects of BAG3 expression co uld be stabilizing a monocyte population under stress from the broad effects of the injected Protein Kinase C activator, PMA. Completing the GzmB and Casp1 knockout mouse studies, ( Saline, DMSO and Serp 1 treatment in GzmB mice ) would be the next logical next steps. The two background treatments, Saline and DMSO, will help determine how much the observed effects are from PMA stimulation alone ( which should be neutralized due to normalization) and finishing GzmB treatments as knockout mice from the new colony become available will contribute to statistical rigor. This will also allow completion of the qRT PCR analysis, and allow for confirmation a role for Serp 1 in regulation of BAG3 in knockout mic e, to mirror what is seen in human monocytes These new insights into the direct molecular mechanism are key to future studies. Furthermore, GzmB and Casp1 deficient mice demonstrated significant up regulation of Ly6Chi cells for Serp 2 treatment in comparison to their background

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117 strains, indicating that Serp 2 may in fact work to suppress this pro inflammatory monocyte subset in GzmB and Casp1 competent, wild type mouse exudates. A s CD11b+ cells only compromise a small percentage of the total peritoneal exudates and CD4+ T cells are practically non existent ( Fig 5 1), it may be useful to determine if Serp 1, Serp 2 or CrmA treatment are affecting B 1 cells (CD20+CD27+CD43+CD70 ) which comprise a significant part of the PEC populations in other models (173) Additional flow cytometry using CD11c and CD206 as M 1 and M2 macrophage markers would allow us to track if Ly6C+ populations are maturing, or if the maturation of Ly6Chi into M1 macrophages, for instance, is accelerated or terminated. From the studies contained herein, we have established the roles of Serp 2 and CrmA both i n vivo in mouse and i n vitro in human cell lines treated with different apoptosis inducing chemicals. CrmA consistently fails to inhibit apoptsosis in vivo and for all stimulators except the Topoisomerase I inhibitor Camptothecin (CPT). O n these grounds, CPT was selected as the inhibitor for gene expression studies conducted in human monocytes. CPT treatment with concurrent Serp 1, Serp 2 or M T7 treatment resulted in 48 consensus genes which were then examined in knockout mice. Armed with knockout mice data, we have established a potential molecular mechanism for the anti inflammatory properties of Serp 1, Serp 2 and M T7 and the null effect of CrmA, both i n vivo in mouse and i n vitro in human cell lines. The consistent regulation of BAG3 between these disparate models and species underscores its potential as a lynchpin in a key consensus pathway for disparate viral anti inflammatory proteins.

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118 As serum samples were stored at the time of mouse harvesting, it would be logical to examine syste mic cytokine and complement system responses for the PMA injected knockout mice. This could also provide further clues to details of the mechanism of action for Serp 2 and CrmA, particularly if IL 1B and IL 18 ( downstream products of caspase 1) and regulat ed differentially by Serp 2 vs. CrmA or in Casp1 / vs. Nod background mice. The method of entry into cells has not yet been determined for Serp 2 or CrmA, although some data has been provided that implicates granzyme B with the internalization mechanism o f Serp 2 ( Figure 3 5D). Surface and cytosolic binding partners can also be identified by chemical cross linking and submitting the sample to mass spectrometry analysis, which has already been started. T he results presented in this collection of studies pro vide novel and important contribution to the role of BAG3 expression in modulating the immune and apoptotic responses in vivo for proteins Serp 1, Serp 2, M T7 and CrmA It also reveals a potential novel role of Casp1 in regulating BAG3 expression. These i mportant questions beg to be addressed in follow up research.

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134 BIOGRAPHICAL SKETCH Jennifer was born to Paula and Don Davids of Aurora, Illinois. A love of science and music runs throughout the family, including younger brother Brian, who is also a fairly smart dude. Jennifer graduated from the fine institution of Knox College, Galesburg, IL, in 2002, with a Bach elor of Art in Biochemistry. Interdisciplinary Program in BioMedi cal Sciences and joined the lab of Dr. Alexandra Lucas. Graduating with her PhD in May 2013 Jennifer hopes to follow in the fine impregnability, and nurture academic succes s.