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Targeting the ACE2/Ang-(1-7)/Mas Axis for Cerebroprotection during Ischemic Stroke

Permanent Link: http://ufdc.ufl.edu/UFE0042182/00001

Material Information

Title: Targeting the ACE2/Ang-(1-7)/Mas Axis for Cerebroprotection during Ischemic Stroke
Physical Description: 1 online resource (137 p.)
Language: english
Creator: Mecca, Adam
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: ace2, ang, angioensin, arb, at1r, at2r, candesartan, cerebroprotection, endothelin, excitotoxicity, ischemic, mas, neuron, stroke
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Recent progress in cardiovascular therapy suggests that stimulation of Angiotensin Converting Enzyme 2 (ACE2), production of Angiotensin-(1-7) Ang-(1-7), and activation of the Ang-(1-7) receptor, Mas, are viable targets for disease prevention and treatment. The ACE2/Ang-(1-7)/Mas axis has been shown to counteract many of the physiological effects of the Angiotensin II (Ang II) Type 1 Receptor (AT1R), including vasoconstrictor and proliferative actions. In addition, activation of the ACE2/Ang-(1-7)/Mas axis also attenuates many of the pathophysiological states that involve increased production of Ang II by Angiotensin Converting Enzyme (ACE), and subsequent activation of the AT1R (ACE/Ang II/AT1R axis). For example, many studies targeting the ACE2/Ang-(1-7)/Mas axis have revealed its broad therapeutic potential for the treatment of hypertension, hypertension-related pathology, myocardial infarction, and heart failure. Furthermore, ACE2 can form endogenous Ang-(1-7) from Ang II and has recently been a target for cardiovascular disease therapy. In fact, several small molecule ACE2 activators have been identified that selectively increase ACE2 activity without having an effect on ACE activity. One of these molecules, Diminazene aceturate (DIZE) has been shown to decrease blood pressure dramatically and dose dependently when administered acutely. In addition, a modest decrease in blood pressure and associated reductions in end organ damage are observed with chronic administration of DIZE to spontaneously hypertensive rats. Therefore, small molecule ACE2 activators are promising compounds for ACE2/Ang-(1-7)/Mas axis activation and treatment of cardiovascular disease. Based on previous studies indicating the presence of ACE2 and Mas in the brain as well as a protective role for ACE2/Ang-(1-7)/Mas axis during cardiovascular disease, we have developed the general hypothesis that activation of the brain ACE2/Ang-(1-7)/Mas axis will have cerebroprotective action during ischemic stroke. Specific Aim 1 was designed to evaluate the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for investigations of the rennin angiotensin system (RAS) during stroke. Following ET-1 induced MCAO, rats had significant neurological impairment that correlated with the size of infarction. Systemic pre-treatment with AT1R blocker (ARB), candesartan, for 7 days attenuated both the infarct size and neurological deficits caused by ET-1 induced MCAO without altering blood pressure. The effect of candesartan pre-treatment on ET-1 induced vasoconstriction of the MCA was also evaluated by visualization of the middle cerebral artery (MCA) through a cranial window. It was determined that candesartan pre-treatment did not alter ET-1 induced constriction of the MCA, which validates the use of this stroke model during ARB pharmacotherapy. This study solidifies the cerebroprotective properties of ARBs during ischemic stroke and validates the ET-1 induced MCAO model for examination of the brain RAS s role in ischemic stroke. Specific Aim 2 was designed to test whether central administration of Ang-(1-7) via lateral ventricular cannula would provide cerebroprotection during ET-1 induced MCAO. Sprague Dawley rats were treated via the intracerebroventricular route with Ang-(1-7) or artificial cerebrospinal fluid (aCSF) prior to ET-1 induced MCAO. Ang-(1-7) treatment reduced the cerebral infarct size, neuronal damage and neurological deficits measured 72 h after MCAO induction. Ang-(1-7) treatment also reduced the neurological deficits produced by ET-1-induced ischemic stroke, as indicated by a battery of neurological tests including the Bederson Exam, Garcia Exam, and the sunflower seed eating task. These protective actions of Ang-(1-7) were reversed by blockade of the Ang-(1-7) receptor, Mas, with A-779. In addition, the effect of Ang-(1-7) pre-treatment on ET-1 induced vasoconstriction of the MCA was also evaluated by visualization of the MCA through a cranial window. It was determined that central Ang-(1-7) pre-treatment did not alter ET-1 induced constriction of the MCA, which validates the use of this stroke model during Ang-(1-7) pharmacotherapy. In order to investigate alterations in cerebral blood flow (CBF) as a mechanism of Ang-(1-7) induced cerebroprotection, we measured CBF in the penumbra during ET-1 induced MCAO. Ang-(1-7) did not affect the reduction of CBF in the penumbra which ruled out the possibility of a protective mechanism of Ang-(1-7) mediated through improved CBF during MCAO. This is the first demonstration of cerebroprotective properties of Ang-(1-7) during ischemic stroke. Specific Aim 3 was designed to test whether central pre-treatment with DIZE will provide cerebroprotection in a rat model of ET-1 notinduced MCAO. Adult male Sprague Dawley Rats were pre-treated with intracerebroventricular DIZE or H2O for 7 days prior to ET-1 induced MCAO. DIZE treatment reduced neurological deficits and infarct size measured 72 h after MCAO induction. Additionally, neurological deficits were reduced in DIZE treated rats as determined by the Bederson Exam, Garcia Exam, and the sunflower seed eating task. Furthermore, the histological and neurological benefits of pre-treatment with DIZE were attenuated when DIZE was co-administered with the Ang-(1-7) receptor antagonist, A-779. In order to investigate alterations in CBF as a mechanism of DIZE induced cerebroprotection, we measured CBF in the penumbra during ET-1 induced MCAO. DIZE did not affect the reduction of CBF in the penumbra which ruled out the possibility of a protective mechanism of DIZE mediated through improved CBF during MCAO. This data indicates that central administration of DIZE prior to stroke is cerebroprotective and extends the known cardiovascular protective effects elicited by stimulation of the ACE2/Ang-(1-7/)Mas axis.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Adam Mecca.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Sumners, Colin.
Local: Co-adviser: Katovich, Michael J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042182:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042182/00001

Material Information

Title: Targeting the ACE2/Ang-(1-7)/Mas Axis for Cerebroprotection during Ischemic Stroke
Physical Description: 1 online resource (137 p.)
Language: english
Creator: Mecca, Adam
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: ace2, ang, angioensin, arb, at1r, at2r, candesartan, cerebroprotection, endothelin, excitotoxicity, ischemic, mas, neuron, stroke
Physiology and Pharmacology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Recent progress in cardiovascular therapy suggests that stimulation of Angiotensin Converting Enzyme 2 (ACE2), production of Angiotensin-(1-7) Ang-(1-7), and activation of the Ang-(1-7) receptor, Mas, are viable targets for disease prevention and treatment. The ACE2/Ang-(1-7)/Mas axis has been shown to counteract many of the physiological effects of the Angiotensin II (Ang II) Type 1 Receptor (AT1R), including vasoconstrictor and proliferative actions. In addition, activation of the ACE2/Ang-(1-7)/Mas axis also attenuates many of the pathophysiological states that involve increased production of Ang II by Angiotensin Converting Enzyme (ACE), and subsequent activation of the AT1R (ACE/Ang II/AT1R axis). For example, many studies targeting the ACE2/Ang-(1-7)/Mas axis have revealed its broad therapeutic potential for the treatment of hypertension, hypertension-related pathology, myocardial infarction, and heart failure. Furthermore, ACE2 can form endogenous Ang-(1-7) from Ang II and has recently been a target for cardiovascular disease therapy. In fact, several small molecule ACE2 activators have been identified that selectively increase ACE2 activity without having an effect on ACE activity. One of these molecules, Diminazene aceturate (DIZE) has been shown to decrease blood pressure dramatically and dose dependently when administered acutely. In addition, a modest decrease in blood pressure and associated reductions in end organ damage are observed with chronic administration of DIZE to spontaneously hypertensive rats. Therefore, small molecule ACE2 activators are promising compounds for ACE2/Ang-(1-7)/Mas axis activation and treatment of cardiovascular disease. Based on previous studies indicating the presence of ACE2 and Mas in the brain as well as a protective role for ACE2/Ang-(1-7)/Mas axis during cardiovascular disease, we have developed the general hypothesis that activation of the brain ACE2/Ang-(1-7)/Mas axis will have cerebroprotective action during ischemic stroke. Specific Aim 1 was designed to evaluate the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for investigations of the rennin angiotensin system (RAS) during stroke. Following ET-1 induced MCAO, rats had significant neurological impairment that correlated with the size of infarction. Systemic pre-treatment with AT1R blocker (ARB), candesartan, for 7 days attenuated both the infarct size and neurological deficits caused by ET-1 induced MCAO without altering blood pressure. The effect of candesartan pre-treatment on ET-1 induced vasoconstriction of the MCA was also evaluated by visualization of the middle cerebral artery (MCA) through a cranial window. It was determined that candesartan pre-treatment did not alter ET-1 induced constriction of the MCA, which validates the use of this stroke model during ARB pharmacotherapy. This study solidifies the cerebroprotective properties of ARBs during ischemic stroke and validates the ET-1 induced MCAO model for examination of the brain RAS s role in ischemic stroke. Specific Aim 2 was designed to test whether central administration of Ang-(1-7) via lateral ventricular cannula would provide cerebroprotection during ET-1 induced MCAO. Sprague Dawley rats were treated via the intracerebroventricular route with Ang-(1-7) or artificial cerebrospinal fluid (aCSF) prior to ET-1 induced MCAO. Ang-(1-7) treatment reduced the cerebral infarct size, neuronal damage and neurological deficits measured 72 h after MCAO induction. Ang-(1-7) treatment also reduced the neurological deficits produced by ET-1-induced ischemic stroke, as indicated by a battery of neurological tests including the Bederson Exam, Garcia Exam, and the sunflower seed eating task. These protective actions of Ang-(1-7) were reversed by blockade of the Ang-(1-7) receptor, Mas, with A-779. In addition, the effect of Ang-(1-7) pre-treatment on ET-1 induced vasoconstriction of the MCA was also evaluated by visualization of the MCA through a cranial window. It was determined that central Ang-(1-7) pre-treatment did not alter ET-1 induced constriction of the MCA, which validates the use of this stroke model during Ang-(1-7) pharmacotherapy. In order to investigate alterations in cerebral blood flow (CBF) as a mechanism of Ang-(1-7) induced cerebroprotection, we measured CBF in the penumbra during ET-1 induced MCAO. Ang-(1-7) did not affect the reduction of CBF in the penumbra which ruled out the possibility of a protective mechanism of Ang-(1-7) mediated through improved CBF during MCAO. This is the first demonstration of cerebroprotective properties of Ang-(1-7) during ischemic stroke. Specific Aim 3 was designed to test whether central pre-treatment with DIZE will provide cerebroprotection in a rat model of ET-1 notinduced MCAO. Adult male Sprague Dawley Rats were pre-treated with intracerebroventricular DIZE or H2O for 7 days prior to ET-1 induced MCAO. DIZE treatment reduced neurological deficits and infarct size measured 72 h after MCAO induction. Additionally, neurological deficits were reduced in DIZE treated rats as determined by the Bederson Exam, Garcia Exam, and the sunflower seed eating task. Furthermore, the histological and neurological benefits of pre-treatment with DIZE were attenuated when DIZE was co-administered with the Ang-(1-7) receptor antagonist, A-779. In order to investigate alterations in CBF as a mechanism of DIZE induced cerebroprotection, we measured CBF in the penumbra during ET-1 induced MCAO. DIZE did not affect the reduction of CBF in the penumbra which ruled out the possibility of a protective mechanism of DIZE mediated through improved CBF during MCAO. This data indicates that central administration of DIZE prior to stroke is cerebroprotective and extends the known cardiovascular protective effects elicited by stimulation of the ACE2/Ang-(1-7/)Mas axis.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Adam Mecca.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Sumners, Colin.
Local: Co-adviser: Katovich, Michael J.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042182:00001


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1 TARGETING THE ACE2/ANG (1 7)/MAS AXIS FOR CEREBROPROTECTION DURING ISCHEMIC STROKE By ADAM P. MECCA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS F OR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Adam P. Mecca

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3 To Marcia

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4 ACKNOWLEDGMENTS First, I would like to thank my mentors, Colin Sumners and Michael Katovich. Working for Mike Katovich as an undergraduate s tudent was a great experience for someone with little career direction, but a lot of scientific curiosity. My time in the Katovich lab taught me how truly great it can be to work with people who care about you. In addition to Dr. Katovich, I owe a great deal to Justin Grobe, Tony Cometa, and Caren Beck, all members of Dr. Katovichs Lab who provided guidance and support early in my training. It was hard to imagine having another mentor and I am extremely lucky to have met and worked with Colin Sumners. Dr. Sumners has guided me through graduate school and kept me directed, while teaching me a great deal about designing experiments, writing, leadership, and mentoring. Together Colin and Mike have helped me to become a competent scientist and a better per son. Ill always be grateful for their guidance and friendship. I would also like to thank my committee members, Mohan Raizada, Jeff Kleim, Debbie Scheuer, Anatoly Martynyuk, and Michael Waters. Ive collaborated with Dr. Raizada since I was an undergraduate research student in 2002. His advice and inspiration have been invaluable. Dr. Kleim and the members of his lab were instrumental in helping us establish our stroke model and neurological testing. Dr. Scheuer has provided a lot of insight to my work as well as life in general. Dr. Martynyuk and Dr. Waters have also given much support, insight, and collaboration for which I am thankful. The many people I have worked with in the Sumners, Katovich, and Raizada Labs during graduate school have been instrumental to my progress and have become good friends. Many thanks are owed to Robbie Regenhardt, Tim OConnor, Jason Joseph,

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5 Peng Shi, Hongwei Li, Nan Jiang, Ying Dong, Yanfei Qi, Vinayak Shenoy, and Jenny Shan. In addition, I would like to acknowledge Marda Jorgensen from the Histology Resource lab for her immunohistochemistry expertise. Finally and most importantly, I would like to thank my friends and family for their unwavering support throughout my life and during graduate school. I would especially like to thank my amazing wife Marcia Mecca, and my friends Logan Schneider, and Yukari Takada, for their constant love and valuable advice.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 ABSTRACT ................................................................................................................... 12 1 THE RENIN ANGIOTENSYSTEM AND ITS ROLE IN ISCHEMIC STROKE .......... 16 Stroke Epidemiology ............................................................................................... 16 Pathophysiology of Isc hemic Stroke ....................................................................... 17 Acute Ischemic Cell Death ............................................................................... 18 Programmed Cell Death ................................................................................... 19 Reperfusion Injury ............................................................................................ 20 Inflammation ..................................................................................................... 21 Renin Angiotensin System Components ................................................................. 21 Angiotensin(1 7) Opposes AT1R Signaling ........................................................... 24 AT1R Signaling ................................................................................................ 25 G protein coupled signaling pathways ....................................................... 25 Non G protein coupled signaling pathways ................................................ 25 Mas Signaling ................................................................................................... 26 Targeting the Renin Angiotensin System Axes for Stroke Therapy ........................ 27 ACE/Ang II/AT1R Axis ...................................................................................... 28 ACE2/Ang (1 7)/Mas Axis ................................................................................ 31 Summary ................................................................................................................ 32 2 CANDESARTAN PRE TREATMENT IS CEREBROPROTECTIVE IN A RAT MODEL OF ENDOTHELIN1 INDUCED MIDDLE CEREBRAL ARTERY OCCLUSION .......................................................................................................... 38 Introduction ............................................................................................................. 38 Methods .................................................................................................................. 40 Animals ............................................................................................................. 40 Chemicals ......................................................................................................... 40 Endothelin 1 Induced Middle Cerebral Artery Occlusion ............................... 40 Visualization of MCA Branches vi a a Cranial Window ...................................... 41 Implantation of Telemetry Transducers ............................................................ 42 Neurological Deficits and Infarct Size ............................................................... 42 Implantation of Osmotic Mini pumps ................................................................ 42 Cardiovascular Measurements ......................................................................... 43 Data Analysis ................................................................................................... 43 Results .................................................................................................................... 4 3 Discussion .............................................................................................................. 47

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7 3 ANGIOTENSIN (1 7) IS CEREBROPROTECTIVE IN A RAT MODEL OF ISCHEMIC STROKE ............................................................................................... 58 Introduction ............................................................................................................. 58 Methods .................................................................................................................. 60 Animals ............................................................................................................. 60 Chemicals ......................................................................................................... 60 Placement of Intracerebroventricular and Guide Cannulae .............................. 60 Endothelin 1 Induced Middle Cerebral Artery Occlusion ................................. 61 Visualization of MCA Branches via a Cranial Window ...................................... 62 Cerebral Blood Flow Monitoring ....................................................................... 63 Indirect Blood Pressure Monitoring .................................................................. 64 Neurological Deficits and Infarct Size ............................................................... 64 Immunohistochemistry ...................................................................................... 65 Real time Reverse Transcriptase PCR (qRT PCR) .................................... 65 Data Analysis ................................................................................................... 66 Results .................................................................................................................... 66 Cerebral Blood Flow in the Ischemic Core and Penumbra during ET 1 Induced MCAO .............................................................................................. 66 Localization of Mas and ACE2 in the Cerebrum ............................................... 66 Mas mRNA Levels are increased in the Lesioned Cortex 24 h after Stroke ..... 67 Cerebroprotective Action of Ang (1 7) .............................................................. 67 Ang (1 7) does not Alter ET 1 Induced MCA Constriction ................................ 70 Ang (1 7) does not Alter ET 1 Induced Cerebral Blood Flow in the Cortex Distant from the Primary Branch of the MCA ................................................ 71 ICV Infusion of Ang (1 7) does not Alter Systemic Blood Press ure .................. 72 Discussion .............................................................................................................. 72 4 ACE2 ACTIVATION AS A TARGET FOR CEREBROPROTECTION DURING ISCHEMIC STROKE ............................................................................................... 85 Introduction ............................................................................................................. 85 Methods .................................................................................................................. 87 Animals ............................................................................................................. 87 Chemicals ......................................................................................................... 87 Placement of Intracerebroventricular and Guide Cannulae .............................. 88 Endothelin 1 Induced Middle Cerebral Artery Occ lusion ................................. 88 Cerebral Blood Flow Monitoring ....................................................................... 89 Indirect Blood Pressure Monitoring .................................................................. 90 Neurological Deficits and Infarct Size ............................................................... 90 Data Analysis ................................................................................................... 91 Results .................................................................................................................... 91 Cerebroprotective Action of DIZE ..................................................................... 91 DIZE does not Alter ET 1 Induced Cerebral Blood Flow in the Cortex Distant from the Primary Branch of the MCA ................................................ 93 ICV infusion of DIZE Decreases Blood Pressure ............................................. 94 Discussion .............................................................................................................. 94

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8 5 SUMMARY AND CONCLUSIONS ........................................................................ 103 Summary .............................................................................................................. 103 Specific Aim 1 ................................................................................................. 103 Specific Aim 2 ................................................................................................. 104 Specific Aim 3 ................................................................................................. 105 Discussion ............................................................................................................ 107 Mechanism of ACE2/Ang (1 7)/Mas Ce rebroprotection ................................. 107 Common Mechanisms for AT2R and Mas Mediated Cerebroprotection ........ 110 CNS Pharmacotherapy ................................................................................... 112 Modeling Middle Cerebral Artery Occlusion ................................................... 113 Strategies for Targeting the Brain ACE2/Ang (1 7)/Mas Axis in Humans ....... 116 Additional Considerations about Timing and Dose of Therapy ....................... 117 Conclusion ...................................................................................................... 117 LIST OF REFER ENCES ............................................................................................. 120 BIOGRAPHICAL SKETCH .......................................................................................... 136

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9 LIST OF TABLES Table page 2 1 Neurological exam results before and after ET 1 induced MCAO ...................... 51 2 2 Cardiovascular variables before, during, and after ET 1 induced MCAO ........... 52

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10 LIST OF FIGURES Figure page 1 1 Prevalence of silent cerebral ischemia wit h respect to age.2 .............................. 34 1 2 Pathophysiology of ischemic stroke ................................................................... 34 1 3 RAS components ............................................................................................... 35 1 4 Ang (1 7) opposes AT1R signaling .................................................................... 36 1 5 Renin Angiotensin System axes are involved in the pathophysiology of stroke .................................................................................................................. 37 2 1 Visualization of MCA branches during ET 1 induced vasoconstricti on ............... 53 2 2 Candesartan pretreatment decreases cerebral infarct size after ET 1 induced MCAO ................................................................................................... 55 2 3 Pre treatment with Candesartan leads to improvement of functional outcomes after ET 1 induced MCAO. ................................................................. 56 2 4 Neurological exam scores correlate with % infarcted gray matter ..................... 57 3 1 Cerebral blood flow in the ischemic core and penumbra during ET 1 induced MCAO ................................................................................................................. 76 3 2 Localization of Mas and ACE2 in the cerebrum ................................................. 77 3 3 Mas mRNA levels are increased in the lesioned cortex 24 h after stroke .......... 77 3 4 Intracerebral pretreatment with Ang (1 7) reduces CNS infarct size 72 h after ET 1 induced MCAO .................................................................................. 78 3 5 Intracerebral pretreatment with Ang (1 7) reduces neuronal damage 72 h after ET 1 induced MCAO .................................................................................. 79 3 6 Intracerebral pretreatment with Ang (1 7) reduces neurological deficits 72 h after ET 1 induced MCAO .................................................................................. 80 3 7 Central Ang (1 7) does not alter ET 1 induced MCA constricti on ....................... 81 3 8 Central Ang (1 7) does not alter ET 1 induced cerebral blood flow in the cortex distant from the primary branch of the MCA ............................................ 83 3 9 Central Ang (1 7) pre treatment does not alter systolic blood pressure. ............ 84

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11 4 1 Intracerebral pretreatment with DIZE reduces CNS infarct size 72 h after ET 1 induced MCAO .......................................................................................... 99 4 2 Intracerebral pretreatment with DIZE reduces neurological deficits 72 h after ET 1 induced MCAO ........................................................................................ 100 4 3 Central DIZE does not alter ET 1 induced cerebral blood flow in the cortex distant from the primary branch of the MCA ..................................................... 101 4 4 Central DIZE pretreatment decreases systolic blood pressure ....................... 102 5 1 ACE2/Ang (1 7)/Mas axis is cerebroprotective during stroke ........................... 119

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12 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 TARGETING THE ACE2/ANG (1 7)/MAS AXIS FOR CEREBROPROTECTION DURING ISCHEMIC STROKE By Adam P. Mecca August 2010 Chair: Colin Sumners Cochair: Mich ael J. Katovich Major: Mecial Sciences Physiology and Pharmacology Recent progress in cardiovascular therapy suggests that stimulation of Angiotensin Converting Enzyme 2 (ACE2), production of Angiotensin(1 7) [Ang (1 7)], and activation of the Ang (1 7 ) receptor, Mas, are viable targets for disease prevention and treatment. The ACE2/Ang (1 7)/Mas axis has been shown to counteract many of the physiological effects of the Angiotensin II (Ang II) Type 1 Receptor (AT1R), including vasoconstrictor and proli ferative actions. In addition, activation of the ACE2/Ang (1 7)/Mas axis also attenuates many of the pathophysiological states that involve increased production of Ang II by Angiotensin Converting Enzyme (ACE), and subsequent activation of the AT1R (ACE/A ng II/AT1R axis). For example, many studies targeting the ACE2/Ang (1 7)/Mas axis have revealed its broad therapeutic potential for the treatment of hypertension, hypertensionrelated pathology, myocardial infarction, and heart failure. Furthermore, ACE2 can form endogenous Ang (1 7) from Ang II and has recently been a target for cardiovascular disease therapy. In fact, several small molecule ACE2 activators have been identified that selectively increase ACE2 activity without having an effect on ACE acti vity. One of these molecules, Diminazene

PAGE 13

13 aceturate (DIZE) has been shown to decrease blood pressure dramatically and dose dependently when administered acutely. In addition, a modest decrease in blood pressure and associated reductions in end organ damag e are observed with chronic administration of DIZE to spontaneously hypertensive rats. Therefore, small molecule ACE2 activators are promising compounds for ACE2/Ang (1 7)/Mas axis activation and treatment of cardiovascular disease. Specific Aim 1 was designed to evaluate the endothelin1 (ET 1) induced middle cerebral artery occlusion (MCAO) model for investigations of the rennin angiotensin system (RAS) during stroke. Following ET 1 induced MCAO, rats had significant neurological impairment that correlated with the size of infarction. Systemic pretreatment with AT1R blocker (ARB), candesartan, for 7 days attenuated both the infarct size and neurological deficits caused by ET 1 induced M CAO without altering blood pressure. The effect of candesartan pretreatment on ET 1 induced vasoconstriction of the MCA was also evaluated by visualization of the middle cerebral artery (MCA) through a cranial window. It was determined that candesartan pre treatment did not alter ET 1 induced constriction of the MCA, which validates the use of this stroke model during ARB pharmacotherapy. This study solidifies the cerebroprotective properties of ARBs during ischemic stroke and validates the ET 1 induced MCAO model for examination of the brain RASs role in ischemic stroke. Based on previous studies indicating the presence of ACE2 and Mas in the brain as well as a protective role for ACE2/Ang (1 7)/Mas axis during cardiovascular disease, we have developed the general hypothesis that activation of the brain ACE2/Ang (1 7)/Mas axis will have cerebr oprotective action during ischemic stroke.

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14 Specific Aim 2 was designed to test whether central administration of Ang (1 7) via lateral ventricular cannula would provide cerebroprotection during ET 1 induced MCAO. Sprague Daw ley rats were treated via the intracerebroventricular route with Ang (1 7) or artificial cerebrospinal fluid (aCSF) prior to ET 1 induced MCAO. Ang (1 7) treatment reduced the cerebral infarct size, neuronal damage and neurological deficits measured 72 h after MCAO induction. Ang (1 7) treatment also reduced the neurological deficits produced by ET 1 induced ischemic stroke, as indicated by a battery of neurological tests including the Bederson Exam, Garcia Exam, and the sunflower seed eating task. These protective actions of Ang (1 7) were reversed by blockade of the Ang (1 7) receptor, Mas, with A 779. In addition, the effect of Ang(1 7) pre treatment on ET 1 induced vasoconstriction of the MCA was also evaluated by visualization of the MCA through a cranial window. It was determined that central Ang (1 7) pre treatment did not alter ET 1 induced constriction of the MCA, which validates the use of this stroke model during Ang (1 7) pharmacotherapy. In order to investigate alterations in cerebral blood flow (CBF) as a mechanism of Ang (1 7) induced cerebroprotection, we measured CBF in the penumbra during ET 1 induced MCAO. Ang (1 7) did not affect the reduction of CBF in the penumbra which ruled out the possibility of a protective mechanism of Ang (1 7) mediated through improved CBF during MCAO. This is the first demonstration of cerebroprotective properties of Ang (1 7) during ischemic stroke. Specific Aim 3 was designed to test whether central pretreatment with DIZE will provide cerebroprotection in a rat model of ET 1 induced MCAO. Adult male Sprague Dawley Rats were pretreated with intracerebroventricular DIZE or H2O for 7 days prior

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15 to ET 1 induced MCAO. DIZE treatment reduced neurological deficits and infarct size measured 72 h after MCAO i nduction. Additionally, neurological deficits were reduced in DIZE treated rats as determined by the Bederson Exam, Garcia Exam, and the sunflower seed eating task. Furthermore, the histological and neurological benefits of pre treatment with DIZE were at tenuated when DIZE was co administered with the Ang (1 7) receptor antagonist, A 779. In order to investigate alterations in CBF as a mechanism of DIZE induced cerebroprotection, we measured CBF in the penumbra during ET 1 induced MCAO. DIZE did not affe ct the reduction of CBF in the penumbra which ruled out the possibility of a protective mechanism of DIZE mediated through improved CBF during MCAO. This data indicates that central administration of DIZE prior to stroke is cerebroprotective and extends t he known cardiovascular protective effects elicited by stimulation of the ACE2/Ang (1 7/)Mas axis.

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16 CHAPTER 1 THE RENIN ANGIOTENSY STEM AND ITS ROLE IN ISCHEMIC STROKE Stroke Epidemiology There are two forms of stroke: ischemic stroke, where blood supply to the brain is restricted by a blocked vessel, and hemorrhagic stroke, which occurs when a blood vessel within the brain ruptures.1 During stroke the lack of oxygen and nutrients, and/or sudden bleeding into the brain elicits neuronal death and a host of consequent behavioral and motor symptoms. Stroke is the third leading cause of death in the United States and is a major cause of serious, long term disability.2 Each year 795,000 people experience a primary or secondary stroke accounting for 1 of every 17 deaths. 610,000 are primary strokes and 185,000 are secondary strokes. Stroke prevalence in adults is 2.7% for men and 2.5% for women. Someone suffers a stroke every 45 seconds and someone dies from a stroke every 3 minutes.2 The estimated cost attributed to treating stroke in 2009 was $68.9 billion. Risk factors for stroke include hypertension, atrial fibrillation, diabetes, smoking, advancing age, low levels of physical activity, dyslipidemias, and hormone replacement therapy. Women also have an increased risk of stroke during pregnancy and the postpartum period.1 Patients who have a primary stroke or transient ischemic attack are also at an increase risk of having a recurrent stroke.3 In addition to the patients who present with stroke symptoms, there are also a number of strokes dis covered incidentally by brain imaging studies temporally dissociated from the ischemic event. In fact, the prevalence of silent cerebral infarction increases dramatically with age (Figure 11).2 When applied to the 1998 United States population estimates, these statistics indicate that about 13 million people had a

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17 prevalent silent stroke. This is clear evidence supporting investigations into both preventative and acute treatment strategies for stroke. Of all reported strokes, 87% are ischemic, 10% are an intracerebral hemorrhage, and 3% are a subarachnoid hemorrhage. Thus, ischemic strokes are the most prevalent and are treated with attempts to restore cerebral blood flow to the effected vascular territory. This is most commonly attempted through the use of systemic administration of recombinant tissue Plasminogen Activator (rtPA). Despite the existence of rtPA treatment protocols, only between 4 and 8 percent of patients with a stroke are eligible for throm bolytic therapy.2Pathophysiology of Ischemic Stroke This is due to the high risk of ischemic transformation and a limited window of time for rtPA treatment after stroke onset. In summary, stroke is a highly prevalent disease and there is a pauci ty of effective stroke prevention and acute treatment strategies. Thus, understanding the mechanisms that underlie stroke and the development of new therapeutic strategies are crucial. Stroke etiology can be classifie d into several relatively homogenous categories and this has helped in studies evaluating stroke pathophysiology and treatment.4 These classifications include cardioembolic stroke, largeartery atherosclerosis, smallartery occlusion, stroke of other determined causes, and stroke of undetermined causes.5 Many of these strokes are thromboembolic and most affect the middle cerebral artery.6 The majority of patients with an ischemic stroke have some degree of reperfusion either spontaneously or as a result of thrombolytic therapy.6, 7 This sequence of occlusion and ischemia followed by reperfusion is associated with a cascade of pathological events. These events include cerebral hypoperfusion, excitotoxicity, oxidative stress, blood brain barrier dysfunction, microvascular injury, hemostatic activation, post ischemic

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18 infla mmation, and cell death (Figure 12).8 In areas of severe cerebral hypoperfusion, the cascade of ischemic pathology occurs quickly resulting in irreversible cell death of the ischemic core. In contrast an area of tis sue on the periphery of the ischemic core undergoes a less dramatic reduction of blood flow and remains viable despite functional impairment. This region is called the ischemic penumbra and can be thought of as the front line in a battle for tissue surviv al. Flattening of EEG occurs in animals and humans associated with a specific decrease in cerebral blood flow indicating a loss of neuronal function. This loss of function in the absence of cell death correlates with the ischemic penumbra, whereas a more dramatic reduction in cerebral blood flow occurs in association with both EEG flattening and cell death in the ischemic core.9 12 Therefore, depending on the duration and degree of ischemia, tissue in the ischemic penumbra can progress to irreversible injury, which leads to expansion of the ischemic core.8Acute Ischemic Cell Death Stroke leads to an energetic depletion and collapse of ionic gradients that causes swelling and disruption of cellular and organelle membranes. This process of cell death is known as necrosis and takes place primarily in the ischemic core. During stroke, the lack of oxygen and gluco se delivery to the brain results in a rapid decrease in ATP concentration due to disruption of oxidative phosphorylation and continued consumption of ATP.13, 14 In addition to this reduction in ATP, oxygen depletion results in electron leakage from the electron transport chain and production of reactive oxygen species.15 Finally, glycolysis in the absence of oxidative phosphorylation leads to the accumulation of protons and lactate.14 Together, these events lead to a cellular environment that consists of ATP depletion, acidification, and free radical formation. Depletion of ATP inhibits the Na+ K+ ATPase and leads to cell depolarization, as well as the release of

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19 neurotransmitters, including glutamate, from neurons and astrocytes.1618 Released glutamate activates ionotropic glutamat e receptors on neurons and astrocytes that results in further release of glutamate.19 This positive feedback loop ultimately causes dramatic increases in intracellular calcium and ends in excitotoxic cell death. Ter minal depolarization describes this deleterious cascade of events that is triggered in ischemic core after dramatic reductions in blood flow. Cells outside of the ischemic core in the penumbra have less severe reductions in blood flow and undergo transien t ischemic depolarization. The transient depolarization of cells in the ischemic penumbra can ultimately evolve into terminal depolarization and expansion of the infarct.20Programmed Cell Death Alternatively, released neurotransmitters and oxidative stress can activate pathways of programmed cell death. Although, programmed cell death can occur in the ischemic core, it is the predominant type of cell death that occurs in the penumbra. Several programmed cell death pathways are activated in the ischemic penumbra during stroke.21 Apoptosis is one of these pathways which consist of activation of cellular processes that require metabolic expenditure.8 Activation of apoptotic cascades causes cell death with minimal inflammatory response. Cytochrome c release from the mitochondria and activation of apoptotic cascades ultimately activates caspase 3, which is a common effector molecule in apopt otic pathways. Parthanatos is another type of programmed cell death that is independent of caspase activity. During ischemic reperfusion injury, poly(ADP ribose) polymerase 1 (PARP1) causes an increase in Poly(ADP ribose) (PAR) polymer.22 PAR polymer translocation from the nucleus to the cytoplasm and mitochondria leads to the release of apoptosis inducing factor (AIF) from mitochondria.23 Poly(ADP ribose) glycohydrolase (PARG ) is involved in degrading PAR

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20 polymer and therefore inhibits this system. In a mouse model of focal cerebral ischemia reperfusion injury, reduction of PARG caused an increase in infarct volume. In addition, over expression of PARG decreased infarct volume in the same model.24 Together, these studies provide evidence for the involvement Parthanatos during ischemic penumbral injury. In addition, there are several pathways that lead to AIF release from the mitochon dria that may be independent of parthanatos. For example, calpain I, a calcium dependent protease, can cleave an N terminal fragment of AIF that causes its dissociation from HSP10 in the mitochondria. The involvement of calpain I and AIF in programmed cell death have been substantiated by experiments showing that both inhibition of calpain activity and knockdown of AIF are neuroprotective during cerebral ischemia.24 Furthermore, cyclophilin A can interact with AIF after ischemic injury and facilitate AIF translocation to the nucleus.25 Another process of programmed cell death is autophagy. Autophagy is a process that can be triggered by endoplasmic reticulum stress and oxidative stress. Normally, autophagy is involved in the turnover of organelles and proteins through a lysosomal pathway.26 Autophagy promoting proteins such as Beclin 1 and microtubuleassociated protein 1 light chain 3 are upregulated in the ischemic penumbra following stroke and inhibition of these pathways can decrease cell death due to ischemia.2729Reperfusion Injury Ultimately, reperfusion ca n occur after several hours of ischemia either spontaneously or due to thrombolytic therapy. Reperfusion injury refers to a paradoxical injury of tissue despite improved cerebral blood flow that can occur. Production of reactive oxygen species by the NAD PH oxidase (NOX) may contribute to this injury.15 In addition, reperfusion leads to proinflammatory cytokine release and

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21 activation of inflammatory cascades. Breakdown of the blood brain barrier by matrix matallopr oteinases, as well of microvascular injury can lead to brain swelling and hemorrhagic transformation.8, 30 Despite these mechanisms of reperfusion injury, early reperfusion is associated with improved final outcomes compared to delayed reperfusion.6Inflammation Stroke is associated with proinflammatory cytokine release by astrocytes, microglia, smooth muscle cells, and endothelial cells.8 These cytokines, including TNF IL 1 and IL6, are associated with an increase in inducible nitric oxide synthase (iNOS), as well as early invasion of neutrophils and transmigration of adhesion molecules. Inflammatory inte ractions that occur at the blood endothelium interface, involving cytokines, adhesion molecules, chemokines and leukocytes are critical to the pathogenesis of tissue damage in cerebral infarction .31 For example, after middle cerebral artery occlusion TNF IL 1, IL 6 and iNOS, as well as phosphorylated ERK1/2 are increased in smooth muscle cells of the middle cerebral artery and in associated intracerebral microvessels. An inhibition of ERK phosphorlyation decreases the tissue damage, as well as the cyt okine and iNOS production after cerebral ischemia.32 Despite this evidence for inflammatory injury after stroke, there is also evidence that inflammation is a protective process that attempts to limit damage and rest ore tissue architecture by eliminating damaged cells and repairing extracellular matrix.33Renin Angiotensin System Components The Renin Angiotensin System (RAS) was initially discovered as a circulating endocrine sy stem with the components produced in various organs and secreted into the blood.

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22 For example, Renin was discovered when the injection of kidney homogenates were shown to increase blood pressure.34 Later, it was discovered that Renin is an enzyme that converts Angiotensinogen (ATG) to Angiotensin I (Ang I) and that Ang I is converted to Angiotensin II (Ang II) by the enzyme Angiotensin Converting Enzyme (ACE) in the pulmonary endothelium.35 It is now known that Ang II is one of the primary mediators of RAS activity and is involved in blood pressure (BP), fluid, and electrolyte homeostasis.34, 36, 37 Later, it was discovered that several Ang II receptor types existed.34, 3840 The Ang II Type 1 Receptor (AT1R) mediates most of the classical physiological effects attributed to Ang II and the Ang II Type 2 Receptor primarily opposes AT1R action.34, 41 The obvious physiological role of the RAS in BP regulation led to drugs which targeted this system for the treatment of hypertension. Currently, ACE inhibitors (ACEi) and AT1R blockers (ARBs) are among the first line drugs to treat hypertension.42 More rece ntly, the concept of the RAS has evolved from that of a circulating endocrine system to a more ubiquitous system consisting of both circulating endocrine and tissue specific paracrine signaling systems. Tissue specific RASs have been identified in the brain, heart, kidneys, pancreas, skin, intestines, and many other organs. 43In addition to the important physiological actions of Ang II in regulating fluid homeostasis and blood pressure, it is clear that this peptide i s of primary importance in the promotion of cardiovascular diseases and stroke. In both the circulation and in specific organs, the disease promoting arm of the RAS is mediated by increased Ang II stimulation of AT1R to increase BP and promote organ damag e. Interestingly, this AT1R arm is opposed by Ang II stimulation of AT2R which counteracts many of the

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23 deleterious actions of AT1R stimulation.44 The cardiovascular protective arm of the RAS has expanded with the discovery of Angiotensin Converting Enzyme 2 (ACE2) in 2000 (Figure 13).45, 46 ACE2 converts Ang II to Ang (1 7) with high efficiency. Ang (1 7) effects counteract many of the physiological and pathological processes of AT1R activation.47 In 2003, the Ang (1 7) receptor was discovered to be a G proteincoupled receptor, Mas, which was initially identified as an orphan protooncogene.48Several pathways for Ang (1 7) production exist. The pathways leading to Ang (1 7) can be either ACE dependent or ACE independent. ACE dependent pathways include conversion of Ang I to Ang II by ACE and subsequent conversion of Ang II to Ang (1 7) by ACE2. 45 In addition, ACE2 is capable of converting Ang I to Ang (1 9) which can then be converted to Ang (1 7) by ACE.46 This second ACE dependent rout e involving Ang (1 9) production as an intermediate step is likely less relevant because ACE2 is 400 times more efficient at catalyzing the conversion of Ang II to Ang (1 7) than Ang I to Ang(1 9).49 In addition to the ACE dependent pathways for Ang (1 7) production, various enzymes contribute to the production of Ang (1 7) via ACE independent mechanisms. These enzymes include Neprilysin, proyl endopeptidase, proyl carboxypeptidase, Chymase, and Cathepsin A.5053 The contributions of ACE independent pathways likely become much more relevant during ACE inhibition. However, these enzymes lack specificity for ang iotensin peptide metabolism and they are unlikely to be major contributors to Ang (1 7) synthesis under physiological conditions. Although, multiple pathways leading to Ang (1 7) production exist, it is clear that ACE2 is a key regulator of this angiotens in peptide.

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24 The complexity of the RAS is increasing with the discovery of new components and pathways. For example, studies show that circulating Ang II levels do not decrease after chronic treatment with an ACEi. This phenomenon has been termed angiotensin escape and the enzyme Chymase is thought to contribute via is high efficiency for converting Ang I to Ang II.54 Additionally, the pro(renin) receptor ((P)RR)was discovered in 2002 and is a receptor for both reni n and its precursor prorenin. (P)RR binding to its ligand can increase prorenin activity and also directly activate an independent intracellular signaling cascade.55 Finally, it is established that various other RAS components including Ang III and Ang IV, as well as the enzymes that produce them, aminopeptidase A and aminopeptidase N elicit physiological and pathological actions.56Angiotensin(1 7) Opposes AT1R Signaling Clearly, the numerous components of the RAS collectively provide a system of checks and balances during cardiovascular health and disease. These components and their pathways are, therefore, good targets for the treatment of cardiovascular disease. Activati on of AT1R is known to contribute to the pathophysiology of many cardiovascular diseases including hypertension, cardiac hypertrophy, cardiac fibrosis, atherosclerosis, myocardial infarction, and many others.47 Ev ents mediated by Ang II stimulation of the AT1R include contraction growth, migration, endothelial dysfunction, expression of proinflammatory cytokines, and modification of extracellular matrix.57 Mas activation counteracts many of the pathophysiological signaling events of AT1R stimulation (Figure 14).58 Therefore, the details of signaling events after AT1R and Mas activation are important because of the interplay between respective signaling cascades.

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25 AT1R Signaling The AT1R is a seven transmembrane G protein coupled receptor that mediates most of the known physiological and pathological actions of Ang II.59 Many signaling pathw ays are activated by AT1R stimulation. Broadly, these pathways include G protein coupled pathways and nonG protein coupled pathways. The G protein coupled signaling events are mainly mediated by phospholipase C (PLC), phospholipase A2 (PLA2), and phospholipase D (PLD).60, 61 Among st the non G protein coupled pathways are signaling through NOX, MitogenActivated Protein Kinases (MAPK), Src, JAK/STAT, FAK and Pyk2, as well as Receptor Tyrosi ne Kinases.62G protein coupled signaling pathways AT1R activation of PLC converts ( Phosphatidylinositol 4,5bisphosphate) PIP2 into inositol 1,4,5 triphosphate (IP3) and diacylglyerol (DAG). IP3 can bind to a receptor on the sarcoplasmic reticulum to increase intracellular Ca2+ which binds to calmodulin and activates myosin light chain kinase (MLCK). MLCK phosphorylates the myosin light chain to cause smooth muscle cell contraction.63 DAG activates Protein Kinase C (PKC) which stimulates the Ras/Raf/MEK/ERK pathway leading to vasoconstriction and growth promoting effects.64 Activation of PLD can cause hydrolysis of phosphatidylcholine ( PC) to choline and phosphatidic acid (PA).57 PA is converted to DAG that contributes to PKC activation. Finally, activation of PLA2 cleaves arachidonic acid (AA) from PC. Lipoxygenase (LO) and cyclooxygenase (COX) convert AA to leukotrienes (LT) and prostaglandins (PG) respectively.62Non G protein coupled signaling pathways The above cascade of G protein mediated events is complex. In fact, as the pathways of AT1R signaling expand, it is becoming clear that the G protein dependent

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26 and independent mechanisms of singling are closely integrated. For example, Ang II stimulation of AT1R activates NOX via phosphorylation of Src/EGFR/PI3K/Rac 1 and PLD/PKC/p47phox. Generation of superoxide by NOX leads to the production of hydrogen peroxide and other reactive oxygen species. This activity is required for phosphorylation of p38MAPK, Akt/PKB, Src, EGFR and many other components of the AT1R signaling cascade.65 In addition, superoxide generated by AT1R activation can inactivate NO.66 These signaling events are known to be involved in a cell proliferation, endothelial dysfunction, inflammation, a nd atherosclerosis. MAPK signaling pathways are also involved in AT1R signaling. In particular ERK1/2, JNK, and p38MAPK contribute to cell differentiation, proliferation, migration, and fibrosis in vessel walls. In addition, nonreceptor tyrosine kinases are activated by AT1R stimulation. For example, cSrc is a tyrosine kinase that activates many downstream components such as Ras, JAK/STAT, PLC, and AP1. JAK and STAT proteins dimerize after AT1R stimulation and translocate to the nucleus where they ha ve transcriptional effects. Finally, Ang II is able to transactivate several receptor tyrosine kinases, EGFR and PDGFR, as well as inhibit insulin receptor signaling via AT1R activation.6 2Mas Sign aling Clearly the pathways involved in AT1R signal transduction are numerous and redundant. Therefore, it should not be surprising that the RAS signaling can regulate some of these pathways via alternate angiotensin receptors such as AT2R and Mas. Like the AT1R and AT2R, the Ang (1 7) receptor, Mas, is a 7 transmembrane domain G protein coupled receptor. Mas may antagonize the AT1R through direct dimerization and inhibition.67, 68 Ang (1 7) stimulation of Mas can inhibit AT1R mediated phosphorylation of p38MAPK, ERK1/2, and JNK.69, 70 SHP 2 is activated by Ang (1 7)

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27 signaling and this molecule is involved in the disruption o f c Src, ERK1/2, and NOX activation by Ang II.58 In addition, Mas activation causes endothelial nitric oxide synthase (eNOS) stimulation via the phosphatidylinositol 3kinase (PI3K) /Akt pathway.71 Mas stimulation also causes AA release and PGI2 production, as well as potentiation of bradykinin signaling.7275Targeting the Re nin Angiotensin System Axes for Stroke Therapy Among the well documented and modifiable risk factors for stroke, hypertension and the RAS have long been key targets for pharmacotherapy and lifestyle modifications. Most investigations into the involvement of the RAS in stroke pathophysiology have focused on classical RAS components. Numerous studies have shown that blockade of either ACE with ACE inhibitors (ACEi) or AT1Rs with AT1R blockers (ARBs) appears to decrease cardiovascular risk and improve strok e prevention in human trials, as well as decrease infarct size and ensuing neurological deficits in animal models of stroke.7678 The cerebroprotective effect of reducing activat ion of the ACE/Ang II/AT1R axis has been hypothesized to occur via either decreased activation of the AT1R or an unopposed activation of the AT2R.7984While investigations of the role of the RAS in stroke have focused on Ang II and its AT1R and AT2R mediated actions, other components of this system may have potential beneficial effects in stroke. Recent progress in cardiovascular therapy suggests that stimulation of ACE2, production of Ang (1 7), and activation of the Ang (1 7) receptor, Mas, are viable targets for disease prevention and treatment. 85 Activation of the ACE2/Ang (1 7 )/Mas axis has revealed broad therapeutic potential for the treatment of hypertension, hypertensionrelated pathology, myocardial infarction, and heart failure.8692 It is apparent that Mas is present in the cerebrum and that activation of this receptor

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28 with chronic and acute administration of Ang (1 7) increases cerebral blood fl ow, bradykinin, bradykinin receptors, eNOS, and NO.9395ACE/Ang II/AT1R Axis In summary, there is evidence for the involvement of the both ACE/Ang II/AT1R axis and ACE2/Ang (1 7)/Mas axis components in the pathophysiology of stroke (Figure 15). Thus, both of these axes contain many viable targets for stroke prevention and acute therapy. While multiple factors influence stroke development and progression, the peptide Ang II appears to be a key player, especially in ischemic stroke. For example, there are reports that Ang II levels are increased bilaterally in the cortex and hypothalamus following stroke.96 Additionally, systemic treatment of stroke prone spontaneously hypertensive rats (spSHR) with ARBs reduces the occurrence of stroke.79, 84. Furthermore, in normal rats or spontaneously hypertensive rats (SHRs) made ischemic by middle cerebral artery occlusion (MCAO), ARBs provide a 4050% reduction of neuronal damage (infarct size), reduce the neurological deficits, and improve recovery. 78, 82, 97100. These actions of ARBs are, at least in part, independent of their blood pressure lowering actions. 78, 82 More evidence for a role of AT1R i n ischemic stroke comes from a report where a smaller lesion area was observed after MCAO in AT1R deficient mice.101 Interestingly, neither adrenergic receptor blockade nor calcium channel inhibition protects against ischemia, so it appears that inhibition of AT1R is essential to alleviate neural damage following stroke.98, 102A few key facts have been revealed by previous studies utilizing candesartan and other ARBs, given both centrally and peripherally, for their cerebroprotective properties 78, 79, 81, 82, 84, 97101, 103. First, it is clear that ARB administration is cerebroprotective in intraluminal occlusion models of MCAO. In addition, it should be noted that certain

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29 hydrophobic ARBs su ch as candesartan and valsartan cross the blood brain barrier readily and that systemic administration of these drugs for cerebroprotection has been utilized in non blood pressure lowering doses in several cases. Therefore, the cerebroprotective effect of ARBs is likely independent of blood pressure changes associated with administration. In fact, several theories exist for the mechanism of ARB cerebroprotection. The site of action of ARBs can be grossly identified as either cerebrovascular or parenchymal. For example, there is some evidence that ARB pretreatment in a rat model of ischemic stroke increases capillary density 103, improves cerebrovascular reserve 104, and improves c erebral endothelial function 105. These cerebrovascular effects may be mediated by unopposed agonism of the AT2 and Ang II type 4 receptors 82, 106 or AT1R alone 107. In addition, AT2R stimulation has been shown to stimulate post stroke neurite outgrowth and improve neuron survival under hypoxic conditions 82. Similarly, inhibition of AT1R may prot ect neurons during hypoxia by decreasing oxidative stress 108These animal studies have bee n substantiated clinically by the Losartan Intervention For Endpoint (LIFE) clinical trial, which demonstrated that ARBs are more effective as anti stroke agents than the traditionally used adrenergic receptor ( AR) blockers in patients with hypertension and left ventricular hypertrophy Therefore, ARB administration can either increase the cerebrovascular reserve allowing improved perfusion of tissues within the affected vascular territ ory, or act directly on neurons to improve their ability to survive an ischemic insult. In reality, the plethora of evidence indicates a role for both vascular and parenchymal targets in the cerebroprotection offered by ARBs. 77. Other clinical trials such as SCOPE (Study on Cognition and Prognosis in the Elderly) have

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30 substantiated the importance of AT1R blockade for stroke prevention 109. Results from the Captopril Prevention Project (CAPPP) study demonstrated that angiotensin converting enzyme (ACE) inhibitors, which reduce the levels of circulating Ang II, are inferior to conventional therapy for stroke prevention 110. This observation led to the suggestion that increased levels of Ang II in response to ARB treatment may have a role in cerebroprotection 82, 110Despit e the focus on AT1R involvement in stroke, evidence suggests that Ang II type 2 receptors (AT2R) have a role in this disease. AT2Rs often mediate effects of Ang II that are exactly opposite to those mediated by AT1R 111, and in fact the tissue levels of AT2R are dramatically increased following injury, such as in the heart following myocardial infarction, in atherosclerotic blood vessels, in wounded skin, and in the peri infarct region in the brain following ischemia 82, 96, 112114. Considering this, and the evidence that Ang II acts via AT2R in neurons to elicit differentiation, regeneration and neurotrophic actions 115117, Unger and colleagues hypothesized that the increased expression of AT2R within the peri infarct region can (in the presence of ARBs to block AT1R) be act ivated by the raised endogenous levels of Ang II and serve a neuroprotective role 82. These investigators have supported this theory by demonstrating that the beneficial action of ARBs after MCAO induced cerebral ischemia is prevented by specific AT2 receptor blockers 82. Additional support is provided by the following experimental findings: i) ARBs are more cerebroprotective than ACE inhibitors in a rat model of ischemia and reperfusion 81; ii) MCAO produces greater ischemic brain damage in AT2R knockout mice compared with wildtype controls 80; and, iii) CNS delivery of an AT2R agonist provides cerebroprotection during ischemic stroke83.

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31 ACE2/Ang (1 7)/Mas Axis The expression of the Ang (1 7) receptor Mas has been reported in both cardiovascular and noncardiovascular control regions of the brain suggesting that the regulat ory effects of Ang (1 7)/Mas extend beyond the regulation of cardiovascular function. In fact, Mas was first characterized as a protooncogene that was found in high levels in the brain 118 and thought to be exclusively located in neurons 119. Original reports of Mas localization revealed Mas mRNA transcripts were present broadly in areas such as hippocampus, cortex, olfactory bulbs and thalamus of rats or mice 118121. In addition, it has been shown that Mas is present in rat cerebral endothelial cells, but is absent from endothelial cells in the periphery 122. Recently, a global presence for Mas in both cardiovascular and noncardiovascular control areas of the brain was verified by immunofluorescence 93In accordance with these findings, ACE2 has also been identified in the brain. Two studies utilizing human tissue have detected low levels of ACE2 mRNA in the human brain This study also indicates a largely neuronal localization for Mas in cardiovascular control regions, but makes no report on its cellular localization in noncardiovascular control regions of the brain such as the motor cortex. 123 and ACE2 immunostaining in human brain vascular smooth muscle as well as endothelial cells 124. More recently, ACE2 has been detected in glia during primary cell culture experiments 125. However, in vivo analyses of mouse brains have localized both ACE2 mRNA and protein to predominately neurons, but in broad areas of cardiovascular and noncardiovascular control including brainstem nuclei, motor cortex, and raphe 126. Thus, it seems that a local ACE2/Ang (1 7)/Mas axis is found in both cardiovascu lar and non cardiovascular control areas of the brain.

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32 It is also of note that activation of Mas with chronic or acute administration of Ang (1 7) increases cerebral blood flow.9395 This increase in blood flow might possibly be explained by data which indicate that central administration of Ang (1 7) shortly following stroke onset has been shown to increase bradykinin levels in the brain, as well as upregulate br adykinin receptors.127 In addition, NO release and eNOS expression are increased with Ang (1 7) treatment in this model.128Summary Together, these results point toward a potential mechanism of cerebroprotection mediated by Ang (1 7) stimulation of Mas and subsequent release of bradykinin, stimulation of bradykinin receptors, and release of NO which could increase the cerebrovascular reserve during an ischemic insult. Further studies are indicated to determine the mechanism of activation of the ACE2/Ang (1 7)/Mas Axis stroke. Despite current knowledge of risk factors for stroke, as well as preventative and acute stroke therapy, this disease remains a leading cause of mortality and mor bidity. Based on the current literature, ACE2, Ang (1 7), and Mas are all present in the brain in various cerebrovascular and parenchymal structures. In addition, acute stimulation of Mas by peripherally administered Ang (1 7), and chronic peripheral over expression of ACE2 increases cerebral blood flow. Furthermore, direct central administration of Ang (1 7) causes increases in bradykinin, NOS activity, and NO production after stroke. In summary, there is ample evidence supporting a protective role for the ACE2/Ang (1 7)/Mas axis in the cardiovascular system, as well as a physiological role for the presence of Mas and its activation by Ang (1 7) in the cerebrovasculature. Therefore, we have developed the general hypothesis that activation of the brain A CE2/Ang (1 7)/Mas axis prior to

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33 endothelin1 (ET 1) induced middle cerebral artery occlusion (MCAO) will provide cerebroprotection during ischemic stroke. Three Specific Aims were designed using a combination of molecular and in vivo approaches. Specifi c Aim 1: Establish and characterize an appropriate model for investigating the brain angiotensin system during ischemic stroke. Hypotheses: The ET 1 induced MCAO model will result in rapid occlusion of the middle cerebral artery and a gradual reperfusio n. Systemic pre treatment of rats with the ARB Candesartan will elicit a decrease in infarct size and in neurological deficits following ET 1 induced MCAO without altering ET 1 induced constriction of the MCA. Lastly, neurological exams will correlate with the amount of tissue damage resulting from ET 1 induced MCAO. Specific Aim 2: Determine whether activation of the ACE2/Ang (1 7)/Mas axis by direct central administration of Ang (1 7) elicits cerebroprotection during ischemic stroke. Hypothesis: Central Ang (1 7) pre treatment will provide cerebroprotection during ET 1 induced MCAO. This effect will be mediated via the Ang (1 7) receptor, Mas. Specific Aim 3: Determine whether pharmacological activation of ACE2 can provide cerebroprotection dur ing ischemic stroke via a Mas mediated action. Hypothesis: Activation of ACE2 via central pretreatment with DIZE will provide cerebroprotection during ET 1 induced MCAO. This effect will be mediated by Ang (1 7) activation of Mas.

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34 Figure 11. Pre valence of silent cerebral ischemia with respect to age. The prevalence of silent cerebral ischemia increases dramatically with respect to age. Data for this figure was adapted from the American Heart Association, Heart Disease and Stroke Statistics 2009 Update.2 Figure 12 Pathophysiology of ischemic stroke. Reduction of blood flow during ischemic stroke results in tissue hypoxia and then gradual reperfusion. These events are followed by post stroke inflammation. The molecular events involved i n stroke pathophysiology include ATP depletion, cell depolarization, Ca+ influx, glutamate release, excitotoxicity, ROS production, and PIC release. The acute cell death that occurs during stroke consists primarily of necrosis, which progresses to modes o f programmed cell death at later stages of pathology. PIC = proinflammatory cytokines, ROS = reactive oxygen species

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35 Figure 13 RAS components. ACE= Angiotensin Converting Enzyme, ACE2 = Angiotensin Converting Enzyme 2, ATG = Angiotensinogen, Ang = Angiotensin, AT1R = Ang II Type 1 Receptor, AT2R = Ang II Type 2 Receptor, BK = Bradykinin, NEP = Neprilysin, PEP = proyl endopeptidase, PCP = proyl carboxypeptidase, (P)RR = (Pro)Renin Receptor.

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36 Figure 14. Ang (1 7) opposes AT1R signaling. = G protein subunits, Ang = Angiotensin, AA = Arachidonic Acid, AT1R = Ang II Type 1 Receptor, B1/B2 = Bradykinin Receptors, BK = Bradykinin, DAG = diacylglycerol, IP3 = inositol 1,4,5triphosphate LT = Leukotrienes, MAPK = MitogenActivated Protein Kinase, NOS = Nitric Oxide Synthase, NO = Nictric Oxide, NOX = NAD(P)H Oxidase, NRTK = Nonreceptor Tyrosine Kinase, PG, Prostaglandins, PKC = Protein Kinase C, PGI2 = Prostacyclin, PLC = phospholipase C, PLA2 = phospholipase A2, PLD = phospholipase D ROS = Reactive Oxygen Species, RTK = Receptor Tyrosine Kinase

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37 Figure 15. Renin Angiotensin System axes are involved in the pathophysiology of stroke. ACE= Angiotensin Converting Enzyme, ACE2 = Angiotensin Converting Enzyme 2, Ang = Angiotensin, AT1R = Ang II Type 1 Receptor, AT2R = Ang II Type 2 Receptor

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38 CHAPTER 2 CANDESARTAN PRE TREATMENT IS CEREBRO PROTECTIVE IN A RAT MODEL OF ENDOTHELIN1 INDUCED MIDDLE CEREBRAL ARTERY OCCLUSI ON Introduction While multiple factors influence stroke development and progression, the peptide angiotensin II (Ang II) appears to be a key player. For example, there are reports that Ang II levels are increased bilaterally in the cortex following stroke and systemic treatment of spontaneously hypertensive rats (SHR) with Ang II type 1 receptor (AT1R) blockers (ARBs) reduces the occurrence of stroke.79, 84, 129 Additionally, ARBs provide a 4050% reduction of infarct volume and reduce the neurological deficits in normotensive rats and SHRs.78, 82, 97100 These actions of ARBs are, at least in part, independent of their blo od pressure lowering actions.78, 82These animal studies have been substantiated clinically by the Losartan Intervention For Endpoint (LIFE) clinical trial, which demonstrated that ARBs are more effectiv e as anti stroke agents than the traditionally used adrenergic receptor ( AR) blockers in patients with hypertension and left ventricular hypertrophy. 77 Other clinical trials such as SCOPE (Study on Cognition and Prognosis in the Elderly) have substantiated the import ance of AT1R blockade for stroke prevention.109 Results from the Captopril Prevention Project (CAPPP) study demonstrated that angiotensin converting enzyme (ACE) inhibitors, which reduce the levels of circulating Ang II, are inferior to conventional therapy for stroke prevention 110. This observation led to the suggestion that increased levels of Ang II in response to ARB treatment may have a role in cerebroprotection.82, 110 Despite the focus on AT1R involvement in stroke, evidence suggests that Ang II type 2 receptors (AT2R) have a role in this disease. AT2Rs often mediate effects of

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39 Ang II that are exactly opposite to those mediated by AT1R111, and in fact the tissue levels of AT2R are dramatically increased following injury, such as in the heart following myocardial infarction, in atherosclerotic blood vessels, in wounded skin, and in the peri infarct region in the brain following ischemia.82, 96, 112114 Considering this, and the evidence that Ang II acts via AT 2R in neurons to elicit differentiation, regenerationand neurotrophic actions115117, Unger and colleagues hypothesized that the increased expression of AT2R within the peri infa rct region can (in the presence of ARBs to block AT1R) be activated by the raised endogenous levels of Ang II and serve a neuroprotective role.82 These investigators have supported this theory by demonstrating that the ben eficial action of ARBs after MCAO induced cerebral ischemia is prevented by specific AT2 receptor blockers.82 Additional support is provided by the following experimental findings: i) ARBs are more cerebroprotective than A CE inhibitors in a rat model of ischemia and reperfusion81; ii) MCAO produces greater ischemic brain damage in AT2R knockout mice compared with wildtype controls80; and, iii)CNS del ivery of an AT2R agonist provides cerebroprotection during ischemic stroke.83Considering that endogenous levels of Ang II are increased in the cortex and hypothalamus following stroke and ARBs have been shown to attenuate the deleterious effects of animal stroke models utilizing intraluminal occlusion, we have developed the general hypothesis that systemic administration of an ARB prior to ET 1 induced MCAO will provide cerebroprotection during ischemic stroke. The ET 1 induced MCAO model of cerebral ischemia is thought to more closely mimic the temporal events of an embolic stroke. This model provides rapid occlusion of the middle cerebral artery and a gradual reperfusion that lasts for 1622 h. 130 Specifically, we determined whether systemic pre-

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40 treatment of rats with the CNS permeant ARB candesartan for 7 days would elicit a decrease in infarct size and in neurological deficits following ET 1 induced MCAO. This study aims to both solidify the cerebroprotective properties of ARBs during ischemic stroke and to validate the ET 1 induced MCAO model for examination of the role of the brain renin angiotensin system in ischemic stroke. Methods Animals For the experiments des cribed here, we used a total of 329adult male Sprague Dawley rats purchased from Charles River Farms (Wilmington, MA). Two rats from the candesartan pretreatment group and 2 rats from the 0.9% saline pretreatment group died following the ET 1 induced MC AO. This is consistent with the approximately 80% mortality rate we have observed with this procedure. There was no significant difference in mortality rate between groups. All experimental procedures were approved by the University of Florida Instituti onal Animal Care and Use Committee. Chemicals Candesartan was a gift from AstraZeneca (Alderley Park, Cheshire, UK). Endothelin1 was purchased from American Peptide Company, Inc (Sunnyvale, CA, USA). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA). Endothelin 1 Induced Middle Cerebral Artery Occlusion The ET 1 induced MCAO procedure used here has been modified slightly from that published previously.130, 131 Eight week old male Sprague Dawley rats were anesthetized with a mixture of O2 (1 L/min) and 4% isoflurane, placed in a Kopf stereotaxic frame, and anesthesia was maintained for the duration of the surgery using an O2/isoflurane (2%) mixture delivered through a nose cone attached to the frame.

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41 The skull was exposed and a small hole was drilled in the cranium dorsal to the right hemisphere using the following stereotaxic coordinates (1.6 mm anterior and 5.2 mm lateral to the bregma). A 26 gauge needle att ached to a 5 L Hamilton microsyringe was lowered 8.7 mm ventral to bregma, after which 3 L of 80 M ET 1 was infused adjacent to the MCA at a rate of 1 L/min. The needle was withdrawn 3 min after the injection was complete. Following this, the wound w as closed and the rat was administered an analgesic agent (buprenorphine; 0.05 mg/kg Visualization of MCA Branches via a Cranial Window sc) before waking. Rats were anesthetized as described above, after which a temporal craniectomy was performed to visu alize the primary and secondary branches of the MCA. An approximately 34 mm square piece of bone was removed from the left squamous portion of the temporal bone just caudal to the orbit. The dura was left in place and debris was cleared away using steri le 0.9% saline. Next, ET 1 induced MCAO was performed as described above except that the needle was left in place until all images were captured so that the focal plane would not be disturbed. The cerebral cortex and associated vessels visible through this cranial window were imaged using a Moticam 1000 digital camera (Motic; Richmond, BC, Canada) coupled to a Revelation surgical microscope (Seiler Instrument and Manufacturing; St. Louis, MO, USA). Vessel diameter was determined for 2 or 3 arterial branc hes per cranial window using ImageJ software (NIH). A baseline image was captured prior to ET 1 injection and subsequent images were captured each 1 min interval for 40 min. Vessel diameter at each time point was normalized to the baseline vessel diameter so that comparisons could be made using multiple MCA branches of several rats.

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42 Implantation of Telemetry Transducers Rats were anesthetized as described above, after which telemetry transducers (DSI, St. Paul, MN, USA) were implanted into the abdominal aorta, as detailed by us previously.132 Rats were administered the Neurological Deficits and Infarct Size analgesic (buprenorphine, 0.05 mg/kg s.c.) following surgery. Neurological evaluations were performed using two s eparate scoring scales reported by Bederson et al .133 and Garcia et al .134, which cumulatively evaluate spontaneous activity, symmetry in limb movement, forepaw outstretching, climbing, body proprioception, response to vibrissae touch, resistance to lateral push, and circling behavior. Additionally, animals were evaluated for neurological deficits using a sunflower seed eating test.135 Animals with a subarachnoid hemorrhage at postmortem examination were excluded from analysis. Infarct volume was assessed by staining brain sections with 0.05% 2,3,5triphenyltetrazolium chloride (TTC) for 30 minutes at 37C. Tissue ipsilateral to the oc clusion, which was not stained, was assumed to be infarcted. After fixation with 10% formalin, brain sections were scanned on a flatbed scanner (Canon) and analyzed using ImageJ software (NIH). To compensate for the effect of brain edema, the corrected inf arct volume was calculated using an indirect method.129Implantation of Osmotic Mini pumps Rats were implanted subcutaneously between the shoulder blades with an osmotic pump (model 2001, ALZET, Cupertino, CA) a s described previously86, which infused candesartan (0.2 mg/kg/day) subcutaneously for 1 week.

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43 Cardiovascular Measurements Measurements of mean arterial pressure (MAP), systolic blood pressure (SBP), diastolic bloo d pressure (DBP), pulse pressure (PP), and heart rate (HR) were made via DSI telemetry transducers.132Data Analysis Measurements were made prior to candesartan or 0.9% saline pretreatment while awake, after 7 days of candesartan or 0. 9% saline pretreatment while anesthetized, during the ET 1 induced MCAO procedure while anesthetized, and 8 hours after ET 1 induced MCAO when awake. Data are expressed as means SEM. Statistical significance was evaluated, as specified in the figure legends, with the use of a oneway ANOVA, Mann Whitney Test, Wilcoxon signedrank test, or an Unpaired t test, as well as Tukey Kramer Multiple Comparisons Test for posthoc analysis when appropriate. A Spearman nonparametric correlation was used to compare the relationship between infarct size and neurological testing data. Differences were considered significant at p < 0.05. Individual p values are noted in the results and figure legends. Results Intracranial injection of 3 L of ET 1 (80 M) into the brain parenchyma adjacent to the MCA resulted in abrupt constriction of the proximal MCA branches to 0% baseline vessel diameter within minutes followed by recanalizaton of the vessel (Figure 21, D F and J). Rats undergoing a sham MCAO were injected with 3 L of 0.9% saline in place of ET 1. Vessel diameter remained relatively stable at baseline values following this saline injection (Figure 21, A C and J). Additionally, rats were pretreated for 7 days with candesartan (0.2 mg/kg/day, s .c.) prior to ET 1 injection. As with the group receiving no pretreatment, vessel diameter decreased to 0% baseline diameter in the

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44 candesartan pretreated group (Figure 21, GI and J). There was no significant difference in vessel diameter at any time point between candesartan pretreated and nonpretreated rats that received an injection of ET 1 adjacent to the MCA. Both groups receiving an ET 1 brain injection displayed a decrease in vessel diameter 3 min after the start of injection that was signif icantly greater than the group undergoing sham MCAO (p < 0.01). These results indicate that ET 1 injection adjacent to the MCA causes constriction of the vessel followed by recanalization. In addition, pretreatment with candesartan does not alter the am plitude or time course of the ET 1 induced MCAO. The effect of candesartan pretreatment on ET 1 induced cerebral damage was assessed by TTC staining, whereby noninfarcted gray matter is stained red after incubation in TTC, delineating the infarct in white (Figure 22A). Pre treatment of rats for 7 days with candesartan (0.2 mg/kg/day, s.c.) significantly reduced the infarct size to 14.35 5.38% of the gray matter compared to 28.98 4.38% in the 0.9% saline pretreated control group (p < 0.05, Figur e 22B). Neurological testing performed 48 h subsequent to the ET 1 induced MCAO indicated improved performance in rats pretreated with candesartan compared to those with 0.9% saline pretreatment (Figure 23, Table 21). Prior to ET 1 induced MCAO all rats scored the maximum of 18 on the Garcia neurological exam indicating that no deficit existed. After the ET 1 induced MCAO, the Garcia neurological exam score decreased significantly from prestroke values to 12.1 1.1 in the 0.9% saline pretreated group (p < 0.001) and 16.7 0.7 in the candesartan pretreatment group (p < 0.05). Two days following ET 1 induced MCAO, the Garcia exam score was significantly higher in the candesartan pretreated

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45 group compared to the 0.9% saline pretreated group (p < 0.05). A similar pattern was observed with the Bederson neurological exam on which all rats received the minimum score of 0 prior to ET 1 induced MCAO, indicating an absence of neurological deficit. After the ET 1 induced MCAO, the Bederson neurological exam score increased from pre stroke values to 2.1 0.2 in the 0.9% saline pretreated group (p < 0.001) and 0.7 0.4 in the candesartan pretreatment group. The post stroke Bederson exam score was significantly higher in the candesartan pretreated gr oup compared to 0.9% saline pretreated group (p < 0.01). Rats also participated in a sunflower seedeating test where they were timed while opening 5 sunflower seeds. Prior to ET 1 induced MCAO rats performed this task in 103.7 14.4 s and broke the sh ell into 13.6 0.7 pieces. Two days following ET 1 induced MCAO, the time to eat 5 sunflower seeds increased from pre stroke values to 215.6 28.3 s in the 0.9% saline pretreated group (p < 0.01) and 114.7 16.5 s in the candesartan pretreatment group (not significant, p = 0.06). In addition, the number of shell pieces increased from prestroke values to 23.8 2.2 pieces in the 0.9% saline pretreatment group (p < 0.01) and 14.8 0.8 pieces in the candesartan pretreatment group (not significant, p = 0.18). The post stroke number of shell pieces was significantly lower for the candesartan pretreated group compared to the 0.9% saline pretreated group (p < 0.05) and a similar although nonsignificant pattern was observed for the time to eat 5 sunfl ower seeds (p = 0.08). Taken together, these results indicate that candesartan pretreatment prior to ET 1 induced MCAO can reduce the size of the cerebral infarct and the extent of neurological deficits produced by this model of focal cerebral ischemia.

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46 In order to evaluate how closely our neurological exam scores predicted the extent of brain tissue damage we grouped rats receiving both candesartan and 0.9% pretreatment together and compared prestroke and post stroke neurological test scores, as well as post stroke scores with % infarcted gray matter. Prior to ET 1 induced MCAO both candesartan pretreated and 0.9% saline pretreated rats showed no neurological deficits as indicated by a minimum score of 0 on the Bederson exam, and a maximum score of 18 on the Garcia exam. As expected, the rats displayed neurological deficits 48 h subsequent to ET 1 induced MCAO with a Bederson exam score that increased to 1.6 0.2 (p < 0.0001) and a Garcia exam score that decreased to 13.6 0.9 (p < 0.0001) for both groups combined. Prior to ET 1 induced MCAO rats from both treatment groups combined required an average of 103.7 14.4 s to eat 5 seeds and broke the shells into an average of 13.6 0.7 pieces. Rats displayed significant deficit in performing this task 48 h subsequent to ET 1 induced MCAO. The time to eat 5 seeds increased to 181.9 22.5 s (p < 0.01) and the number of shell pieces increased to 20.8 1.8 (p < 0.01) for both groups combined compared to prestroke outcomes. It is clear from these data that our model of ET 1 induced MCAO causes significant neurological deficits that can be measured 48 h after stroke induction (Table 21 and Figure 23). For correlation between neurological exam results and infarct size, data showing the Garcia exam score, Bederson exam score, time to eat 5 sunflower seeds, and number of shell pieces were plotted against the associated % infarcted gray matter for each rat (Figure 24, A D). A strong and significant negative correlation was seen for % infarcted gray m atter vs. Garcia exam score (Spearman r = 0.9151, p < 0.0001). Additionally, strong and significant positive correlations were seen for % infarcted gray

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47 matter vs. Bederson exam score (Spearman r = 0.8632, p < 0.0001), % infarcted gray matter vs. time to eat 5 sunflower seeds (Spearman r = 0.6729, p < 0.005), and % infarcted gray matter vs. number of shell pieces (Spearman r = 0.7241, p < 0.005). Lastly, a subgroup of 5 rats was implanted with telemetry blood pressure transducers in the abdominal aorta in order to record arterial blood pressure and heart rate (Table 22). Cardiovascular variables consisting of mean arterial pressure (MAP), systolic blood pressure (SBP), diastolic blood pressure (DBP), pulse pressure (PP), and heart rate (HR) were measured. No differences in cardiovascular parameters were seen between treatment groups prior to candesartan or 0.9% saline pretreatment or after pretreatment for 7 days. A slight increase in all cardiovascular variables was observed within 5 min of ET 1 induced MCAO, but no difference was observed between groups. Similarly, there were no differences in cardiovascular variables between groups recorded at 8 h subsequent to ET 1 induced MCAO. Discussion The significant findings of this study are that pretreatm ent with the CNS permeant ARB, candesartan, attenuates the neurological deficits and CNS tissue damage produced in an ET 1 induced MCAO model of ischemic stroke without altering blood pressure. In addition, we show that this model of stroke provides a rapid constriction, sustained occlusion, and then gradual reperfusion of the proximal MCA. This constriction is not altered by pretreatment with candesartan which is a concern when using the ET 1 induced MCAO as a disease model for investigating manipula tions of the brain angiotensin system during focal cerebral ischemia. Lastly, we have shown that results of the neurological testing methods used here are strongly correlated with the amount of tissue damage produced by ET 1 induced MCAO. Taken together, our

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48 results confirm the deleterious action of Ang II in the presence of uninhibited AT1Rs in the pathophysiology of ischemic stroke and validate the ET 1 induced MCAO model for examination of the brain renin angiotensin systems role in ischemic stroke. The ET 1 induced MCAO model has been used and characterized extensively in rodent models as a method for producing transient focal cerebral ischemia.130, 131, 136 It has ofte n been presented as a model that closely mimics the initial vessel occlusion in other stroke models, such as the intraluminal suture method, in that rapid and complete occlusion is initially achieved. A unique aspect of the ET 1 induced MCAO model is th e reversibility of the chemically induced occlusion over time. Perfusion MRI studies in the ET 1 induced MCAO model have shown that CBF in the ipsilaterial hemisphere decreases to 30 to 50% of normal at 1 h and gradually returns to normal between 16 h and 22 h. This is followed by a period of hyperperfusion that seems to peak between 24 h and 48 h at which point the study was ended.130 This time course for CBF has been verified by doppler flow studies of the cerebral cortex in the MCA territory.137 However, neither vasoconstriction nor regional blood flow of the proximal MCA have been examined in this stroke model. Our data indicate that ET 1 injection adjacent to the MCA does indeed provide an initial rapid occlusion of the proximal MCA. However, vessel diameter seems to return to baseline after approximately 30 min. This profile does not match that of CBF measured previously even though infarct sizes and neurologica l deficits are comparable. It is possible that a cerebrovascular event distal to the proximal MCA mediates ischemia after the initial occlusion, but this fact is yet to be determined.

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49 We have used the ET 1 induced MCAO model to verify the cerebroprotect ive actions of candesartan during ischemic stroke. A few key facts have been revealed by previous studies utilizing candesartan and other ARBs, given both centrally and peripherally, for their cerebroprotective properties.78, 79, 81, 82, 84, 97101, 103 First, it is clear that ARB adminis tration is cerebroprotective in intraluminal occlusion models of MCAO. In addition, it should be noted that certain hydrophobic ARBs such as candesartan and valsartan cross the blood brain barrier readily and that systemic administration of these drugs for cerebroprotection has been utilized in non blood pressure lowering doses in several cases. Therefore, the cerebroprotective effect of ARBs is likely independent of blood pressure changes associated with administration. In fact, several theories exist f or the mechanism of ARB cerebroprotection. The site of action of ARBs can be grossly identified as either cerebrovascular or parenchymal. For example, there is some evidence that ARB pre treatment in a rat model of ischemic stroke increases capillary density103, improves cerebrovascular reserve104, and improves cerebral endothelial function.105 These cerebrovascular effects may be mediated by unopposed agonism of the AT2 and Ang II type 4 receptors82, 106 or AT1R alone.107 In addition, AT2R stimulation has been shown to stimulate post stroke neurite outgrowt h and improve neuron survival under hypoxic conditions.82 Similarly, inhibition of AT1R may protect neurons during hypoxia by decreasing oxidative stress.108 Therefore, ARB administ ration can either increase the cerebrovascular reserve allowing improved perfusion of tissues within the affected vascular territory, or act directly on neurons to improve their ability to survive an ischemic insult. In reality, the plethora of evidence i ndicates a role for both vascular and parenchymal targets in the cerebroprotection

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50 offered by ARBs. Our observations provide support for a role of angiotensin peptides in the pathophysiology of ischemic stroke, and verify the need for further study of the RAS to identify potential targets for stroke therapy.

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51 Table 21 Neurological exam results before and after ET 1 induced MCAO Bederson Exam Score Garcia Exam Score Sunflower Seed Test Time to Eat 5 Seeds Sunflower Seed Test Number of Shell Pieces Treatment 24 h Pre Stroke 48 h Post Stroke 24 h Pre Stroke 48 h Post Stroke 24 h PreStroke 48 h Post Stroke 24 h Pre Stroke 48 h Post Stroke 0.9 % Saline (n = 12) 0 0 2.1 0.2 *** 18 18 12.1 1.1 *** 120.8 19.5 215.6 28.3 ** 14.1 .9 23.8 2.2 ** Candesartan (n = 6) 0 0 0.7 0.4 18 18 16.7 0.7 69.5 10.5 114.7 16.5 12.7 1.1 14.8 0.8 Combined Groups (n = 18) 0 0 1.6 0.2 *** 18 18 13.6 0.9 *** 103.7 14.4 181.9 22.5 ** 13.6 0. 7 20.8 1.8 ** All values are reported as mean SEM. p < 0.05 or p < 0.05, ** p < 0.01 or *** p < 0.001 vs. pre stroke value (Wilcoxon signedrank test) p < 0.05 vs. Candesartan pretreatment (MannWhitney test);

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52 Table 22 Cardiovascular variables before, during, and after ET 1 induced MCAO Treatment MAP SBP DBP PP HR Prior to Candesartan Pretreatment for 7 days; Awake Candesartan 87.19 3.74 103.00 4.91 74.82 3.55 28.13 3.54 333.66 10.01 0.9% Saline 92.36 2.36 107.54 1.82 8 0.35 4.08 27.17 4.22 328.08 14.28 After Candesartan Pre treatment for 7 days; Anesthetized prior to ET 1 induced MCAO Candesartan 80.19 3.25 94.08 2.70 69.35 3.89 24.65 1.80 336.23 9.18 0.9% Saline 83.98 10.01 101.08 8.78 66.51 15. 67 35.15 13.65 311.43 10.75 Change from baseline during 5 min period after ET 1 induced MCAO; Anesthetized Candesartan 7.89 3.69 8.27 3.69 8.15 3.79 0.47 0.27 19.56 6.99 0.9% Saline 10.57 3.28 9.32 2.42 19.51 11.83 13.17 10.75 27.09 15.66 8 hours post ET 1 induced MCAO; Awake Candesartan 90.36 6.45 107.06 6.69 77.76 6.60 29.28 2.35 407.21 19.20 0.9% Saline 105.80 21.41 117.77 21.77 96.94 21.20 20.82 3.23 358.96 16.17 No significant differences between gr oups (unpaired t test)

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53 Figure 21. Visualization of MCA branches during ET 1 induced vasoconstriction. A I) Visualization of MCA branches (arrows) during ET 1 induced vasoconstriction. Primary and secondary branches of the MCA were visualized with a surgical microscope after temporal craniotomy to create a cranial window. Images were captured at a rate of 1 per min starting immediately prior to ET 1 injection (0 min), throughout the ET 1 injection (3 min), and for at least 40 min after completion of ET 1 injection. Shown here are representative images captured from rats that underwent a sham (0.9% saline) injection (n = 2, A C), an MCAO induced by 80 mM ET 1 injection (n = 3, C E), and an ET 1 induced MCAO plus 7 days pretreatment with candesa rtan (0.2 mg/kg/day, s.c .) (n = 2, G I). J) Vasoconstriction of primary and secondary MCA branches was quantified as the % of baseline vessel diameter. Data are means SEM. Baseline vessel diameter was determined prior to ET 1 or 0.9% saline injection ( Time = 0 min). p < 0.01 for both candesartan pretreated and non pretreated groups compared to sham ET 1 injection (oneway ANOVA followed by Tukey Kramer Multiple Comparisons Test).

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54 Figure 21. Continued

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55 Figure 22. Candesartan pretreatme nt decreases cerebral infarct size after ET 1 induced MCAO. Rats were pretreated with candesartan or 0.9% saline for 7 days prior to ET 1 induced MCAO. Brains were removed and sectioned 48 h later. A) Representative brain sections from both a control (0. 9% saline) pretreated and candesartan pretreated rat showing infarcted (white) and noninfarcted (red) gray matter. B) Bar graphs show the % infarcted gray matter in each treatment group. Data are means SEM from 6 (candesartan) and 11 (0.9% saline) pre treated rats. p < 0.05 compared to 0.9% saline pretreated group (MannWhitney Test)

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56 Figure 23. Pre treatment with Candesartan leads to improvement of functional outcomes after ET 1 induced MCAO. Rats were pretreated with candesartan or 0.9% s aline for 7 days prior to ET 1 induced MCAO. Forty eight hours later, neurological deficits were assessed via the Garcia Neurological Exam ( Panel A), the Benderson Neurological Exam ( Panel B), and the Sunflower Seed Eating Test ( Panels C and D). Bar graphs are means SEM from 6 (candesartan) and 12 (0.9% saline) pretreated rats The dotted horizontal lines on each panel indicate the scores obtained on each test prior to ET 1 induced MCAO. p < 0.05, ** p < 0.01, ns not significant (MannWhitney Test) compared to 0.9% saline pretreatment.

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57 Figure 24. Neurological exam scores correlate with % infarcted gray matter. Neurological exams were performed 48 h after ET 1 induced MCAO. Brains were removed, sectioned and TTC stained. Correlation of Garc ia Exam Scores ( Panel A), Benderson Exam Scores ( Panel B), and the Sunflower Seed Eating Test Scores ( Panels C and D) with % infarcted gray matter revealed strong correlations.

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58 CHAPTER 3 ANGIOTENSIN (1 7) IS CEREBROPROTECT IVE IN A RAT MODEL O F ISCHEMIC S TROKE Introduction Angiotensin(1 7) (Ang (1 7) is the latest member of the reninangiotensin system (RAS) that has been shown to exert major biological activity. This heptapeptide is generated predominately from its precursor peptide, angiotensin II (Ang II), by Angiotensin Converting Enzyme 2 (ACE2) and to exert physiological effects mediated by its receptor, Mas. Together, these components make up the ACE2/Ang (1 7)/Mas axis, which has been shown to counteract many of the physiological effects of Ang I I at the angiotensin type 1 receptor (AT1R), including vasoconstrictor and proliferative actions.85 In addition, activation of the ACE2/Ang (1 7)/Mas axis also attenuates many of the pathophysiological states that involve increased production of Ang II by Angiotensin Converting Enzyme (ACE), and subsequent activation of the AT1R (ACE/Ang II/AT1R axis). For example, many studies targeting the ACE2/Ang (1 7)/Mas axis have revealed its broad therapeutic potential for the treatment of hypertension, hypertensionrelated pathology, myocardial infarction, and heart failure.8688, 91, 92Similar to other cardiovascular diseases, it is well known that activation of the ACE/Ang II/AT1R axis contributes to the pathophysiology of stroke. Numerous studies have shown that blockade of either Angiotensin Converting Enzyme (ACE) with ACE inhibitors ( ACEi) or AT1Rs with AT1R blockers (ARBs) appears to decrease cardiovascular risk and improve stroke prevention in human trials, as well as decrease infarct size and ensuing neurological deficits in animal models of stroke. 7678 The cerebroprotective effect of reducing activation of the ACE/Ang II/AT1R axis has been

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59 hypothesized to occur via either decreased activation of the AT1R or an unopposed activation of the angiotensin Type 2 Receptor (AT2R).80, 82, 83Despite efforts to reduce both the incidence of stroke by targeting modifiable risk factors, as well as the morbidity and mortality associated with str oke through the use of techniques to recannulate occluded vessels, stroke remains the third leading cause of death in the United States and a leading cause of serious, long term disability. 1 Among the well documented and modifiable risk factors for stroke, hypertension and the RAS have long been key targets for pharmacotherapy and lifestyle modifications. While most investigations into the involvement of the RAS in stroke pathophysiology have focused on Ang II a nd its AT1R and AT2R mediated actions, it is evident that components of the ACE2/Ang (1 7)/Mas axis may have potential beneficial effects during stroke. It is apparent that Mas is present in the cerebrum and that activation of this receptor with chronic and acute administration of Ang (1 7) increases cerebral blood flow (CBF).9395 Further, central administration of Ang (1 7) shortly following ischemic stroke onse t has been shown to increase levels of the vasodilator bradykinin, upregulate bradykinin receptors, augment nitric oxide release, and increase endothelial nitric oxide synthase expression.127, 128 The c ardiovascular protective effects offered by ARB and ACEi therapy may also be due in part to ACE2/Ang (1 7)/Mas axis activation. In fact, multiple human and animal studies have shown that ARB and ACEi treatment can increase ACE2 expression and production o f Ang (1 7).47Based on the current literature supporting a protective role for the ACE2/Ang (1 7)/Mas axis in the cardiovascular system, as well as a physiological role for the presence of Mas and its activation by Ang (1 7) in the cerebrovasculature, we have

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60 developed the general hypothesis that Ang (1 7) has the ability to exert a Mas mediated cerebroprotective action during ischemic stroke. In the current study we have demonstrated that central administration o f Ang(1 7) prior to ischemic stroke by endothelin1 (ET 1) induced middle cerebral artery occlusion (MCAO) elicits a decrease in infarct size and neurological deficits. This is the first demonstration of a cerebroprotective action of Ang (1 7) during isc hemic stroke. Methods Animals Adult male Sprague Dawley rats were purchased from Charles River Farms (Wilmington, MA). All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee. Chemicals Ang ( 1 7) and A 779 (D Ala7Placement of Intracerebroventricular and Guide Cannulae ) angiotensin (17) were purchased from Bachem Bioscience (Torrance, CA). E T 1 was purchased from American Peptide Company, Inc (Sunnyvale, CA, USA). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA). Ang (1 7) and A 779 were dissolved in artificial cerebral spinal fluid (aCSF). ET 1 was dissolved in 0.9% saline. Eight week old male Sprague Dawley rats were anesthetized with a mixture of O2 (1 L /min) and 4% isoflurane, placed in a Kopf stereotaxic frame, and anesthesia was maintained for the duration of the surgery using an O2/isoflurane (2%) mixture delivered through a nose cone attached to the frame. The skull was exposed and a small hole was drilled for placement of an MCAO guide cannula in the cranium dorsal to the right hemisphere using the following stereotaxic coordinates (1.6 mm anterior and 5.2 mm

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61 lateral to the bregma). A 21 gauge stainless steel guide cannula cut to 4 mm below the ped estal was lowered into the hole and affixed to the skull with 3 mounting screws and dental cement. During the same surgery, a second hole was then drilled in the cranium dorsal to the left hemisphere for placement of an intracerebroventricular cannula ( kit 1, ALZET, Cupertino, CA) coupled to a 2 week osmotic pump (model 2002, ALZET, Cupertino, CA) via vinyl tubing. The following stereotactic coordinates were used (1.3 mm posterior and 1.5 mm lateral to bregma, 4.5 mm below the surface of the cranium). The osmotic pump was implanted subcutaneously between the shoulder blades as described previously.86 Osmotic pumps were used to infuse Ang (1 7) (1 g/h), Ang (1 7) (1 g/h) plus A 779 (5 g/h), A779 (5 g/h) alone, or aCSF into the left lateral cerebral ventricle starting at the time of cannula placement and lasting until the animals were euthanized. These doses were determined based on concentratio ns of Ang (1 7) shown to increase bradykinin and NO levels in the brain following stroke.127, 128 Following this surgery, the wound was closed and the rat was administered an analgesic agent (buprenorphine; 0.05 mg/kg Endothelin 1 Induced Middle Cerebral Artery Occlusion sc) before waking. Seven days after the placement of ICV and guide cannulae, the ET 1 induced MCAO procedure was performed as we have previously reported with a minor modification.138 Eight week old male Sprague Dawley rats were anesthetized as described above, and anesthesia was maintained for the duration of the injection using an O2/isoflurane (2%) mixture delivered through a nose cone attached to the frame. The cannula dummy was removed after which a 26 gauge needle attached to a 5 L Hamilton microsyringe was lowered 8.7 mm ventral to bregma. Once the needle was in

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62 place, 3 L of 80 M ET 1 was infused adjacent to the MCA at a rate of 1 L/min using a Stoelting Quintessential Injector (Stoelting Co., Wood Dale, IL, USA). The needl e was left in place for 3 min after the injection was complete and then removed slowly. The cannula dummy was then replaced and the rat was administered an analgesic agent (buprenorphine; 0.05 mg/kg sc) before waking. We have characterized this model pre viously by showing that injection of ET 1 can cause rapid constriction of the MCA followed by gradual reperfusion. In addition, a strong and significant correlation exists between the size of infarct measured in this model and several test scores used to assess neurological deficits in the work described here.138Visualiz ation of MCA Branches via a Cranial Window In addition, we have used laser doppler flowmetry to investigate the CBF reduction that results in cortical areas both adjacent (ischemic core) and distal ( ischemic penumbra) to the site of ET 1 injection (Figure 31). It is clear from these data that CBF decreases dramatically in tissue adjacent to the proximal MCA and that CBF is reduced to a lesser degree in tissue of more distal MCA territories. An additional group of rats was anesthetized as described above, after which a temporal craniectomy was performed to visualize the primary and secondary branches of the MCA as described previously.138 An approximately 34 mm square piece of bone was removed from the left squamous portion of the temporal bone just caudal to the orbit. The dura was left in place and debris was cleared away using sterile 0.9% saline. Next, E T 1 induced MCAO was performed as described above except that the needle was left in place until all images were captured so that the focal plane would not be disturbed. The cerebral cortex and associated vessels visible through this cranial

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63 window were i maged using a Sony Handycam HDR SR12 (Sony; Minato, Tokyo, Japan) coupled to a Revelation surgical microscope (Seiler Instrument and Manufacturing; St. Louis, MO, USA). Vessel diameter was determined by averaging one primary and two secondary MCA branches per cranial window using ImageJ software (NIH) by an individual who had been blinded to the treatment group. A baseline image was captured prior to ET 1 injection and subsequent images were captured each 1 min interval for 60 min. Vessel diameter at eac h time point was normalized to the baseline vessel diameter so that comparisons could be made using multiple MCA branches of several rats. Cerebral Blood Flow Monitoring An additional group of rats was anesthetized as described above, after which laser dop pler flowmetery was used to measure CBF for a period of time starting 1 min prior to ET 1 injection and lasting for 1 h after ET 1 injection. CBF measurements were performed using a Standard Pencil Probe and Blood FlowMeter coupled to a Powerlab 4/30 with LabChart 7 (ADInstruments, Inc, Colorado Springs, CO, USA). The probe was placed either just posterior to the MCAO guide cannula at the lateral skull ridge, or 1.6 mm anterior to Bregma and on the lateral skull surface. Data was recorded in arbitrary bl ood perfusion units at 1000 Hz. Baseline CBF was calculated by averaging a 1 min interval just prior to ET 1 injection. Changes in CBF were calculated as a percentage of baseline by averaging a 10 s interval every 1 min (Figure 36 ), or a 1 s interval ev ery 1 s (Figure 31). This procedure allowed us to further validate ET 1 induced MCAO as a method for inducing ischemic stroke, as Figure 31 shows that blood flow is decreased by >90% within the area adjacent to the ET 1 injection (ischemic core).

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64 Indire ct Blood Pressure Monitoring After undergoing surgery to implant an intracerebroventricular cannula (kit 1, ALZET, Cupertino, CA) coupled to a 6 week osmotic pump (model 2006, ALZET, Cupertino, CA) via vinyl tubing as described above, animals were allowed to recover for 1 week. Indirect blood pressure was recorded by tail cuff once a week for 2 weeks as previously described.86Neurological Deficits and Infarct Size Animals were warmed by a 200 W heating lamp for 5 min before restraint in a heated Plexiglas cage to which the animals were previously conditioned. A pneumatic pulse sensor was attached to the tail distal to an occluding cuff controlled by a Programmed Electrosphygmomanometer (Narco BioSystems, Austin TX). Voltage outputs from the cuff and pulse sensor were recorded and analyzed by a Powerlab signal transduction unit and associated Chart software (ADInstruments, Colorado Springs, CO). Neurological deficits and infarct size were evaluated as reported pr eviously.138 Neurological evaluations were performed using two separate scoring scales originally described by Bederson et al .133 and Garcia et al.,134 which cumulatively evaluate spontaneous activity, symmetry in limb movement, forepaw outstretching, climbing, body proprioception, response to vibrissae touch, resistance to lateral push, and circling behavior. Additionally, animals were evaluated for neurological deficits using a sunflower seed eating test.135 Infarct volume was assessed by staining brain sections with 0.05% 2,3,5triphenyltetrazolium chloride (TTC) for 30 minutes at 37C. Tissue ipsilateral to the occlusion, which was not stained, was assumed to be infarcted. After fixation with 10% formalin, brain sections were scanned on a flatbed scanner (Canon)

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65 and analyzed using ImageJ software (NIH). To compensate for the effect of brain ede ma, the corrected infarct volume was calculated using an indirect method.129Immunohistochemistry Seventy two hours following ET 1 induced MCAO, rats were anesthetized with isoflurane and decapitated. Brains were removed and rinsed in ice cold PBS. Brains were then sectioned coronally using a brain blocker (David Kopf Instruments, Tujunga, CA, USA). A 4 mm section starting from the frontal pole was frozen fresh in OCT Compound (Sakura Finetek; Torrance, CA, U SA). Twenty m sections were cut using a cryostat, mounted onto glass slides, and dried overnight at room temperature. Sections were then fixed for 30 min with 10 % formalin. NeuN immunostaining procedure was performed as described previously.139 The primary antibody used was mouse monoclonal anti NeuN antibody (1:100) (Chemicon International; Temecula, CA, USA). The secondary antibody was Alexa Fluor 594 goat anti mouse IgG Real time Reverse Transcriptase PCR (qRT PCR) (1:500) (Molecular Probes; Eugene, OR, USA). Sections were mounted and counter stained with Vectashield Mounting Medium with DAPI (Vector Laboratories; Burlingame, CA, USA). For analyses of endogenous Mas and ACE2 mRNA, a 2 mm thick coronal section caudal to that used for TTC staining was isolated and dissected so that left and right cortical and subcortical tissues were separated into individual samples. Total RNA was isolated using an RNeasy kit (Qiagen, Valencia, CA, USA). Isolated RNA underwent DNAse I treatment to remove genomic DNA. Mas and ACE2 mRNA were reverse transcribed with a highcapacity cDNA reverse transcription kit (BioRad Laboratories, CA, USA) and then analyzed via qRT PCR in a PRISM 7000 sequence detection system (Applied Biosy stems, Foster City, CA, USA) as detailed by us previously.139

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66 Oligonucleotide primers and TaqMan probes specific for rat Mas and ACE2 were obtained from Applied Biosystems. Data were normalized Data Analysis to 18S rRNA. Data are expressed as means SEM. Statistical significance was evaluated, as specified in the figure legends, with the use of a Kruskal Wallis test, Two way Row matched ANOVA, One way ANOVA, or unpaired t test, as well as with Dunns Multiple Comparis on Test, the Bonferroni Test, or Turkeys Multiple Comparison Test for posthoc analyses when appropriate. Differences were considered significant at p<0.05. Individual p values are noted in the results and figure legends. Results Cerebral Blood Flow in t he Ischemic Core and Penumbra during ET 1 Induced MCAO Laser doppler flowmetry was used to investigate the CBF reduction that results in cortical areas both adjacent (ischemic core) and distal (ischemic penumbra) to the site of ET 1 injection (Figure 31). Representative tracings show that CBF decreases dramatically in the ischemic core and that CBF is reduced to a lesser degree in the ischemic penumbra. CBF increases gradually toward baseline during the 1 h in which data was captured. Localization of M as and ACE2 in the Cerebrum Prior to determining if Ang (1 7) exerts beneficial actions during ischemic stroke we investigated whether transcripts of ACE2, an enzyme that is of primary importance in synthesizing Ang (1 7), as well as Mas, an Ang (1 7) rece ptor are located in the brain regions that are greatly impacted by ET 1 induced MCAO. mRNA levels of both Mas and ACE2 were measured in both frontal cortex and subcortex (Figure 32). Mas and

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67 ACE2 mRNA were detected in both of these regions with a signif icantly higher level of Mas in the cortex (p< 0.01) and a significantly lower level of ACE2 in the cortex (p < 0.05) compared to subcortical tissue samples. Mas mRNA Levels are increased in the Lesioned Cortex 24 h after Stroke Mas mRNA levels were measur ed in the frontal cortex (Figure 3 3 ) in rats 24 h after either ET 1 induced MCAO or sham stroke surgery (0.9% saline injejcction). Mas mRNA w as significantly higher in the lesioned (right) cortex of rats that underwent ET 1 induced MCAO compared to the r ight cortex of sham stroke rats (p < 0.05). There was no difference in Mas mRNA levels in the unle si oned (left) cortex between the two groups. Cerebroprotective Action of Ang(1 7) The effect of Ang(1 7) pre treatment on ET 1 induced cerebral damage was assessed by TTC staining, whereby noninfarcted gray matter is stained red after incubation in TTC, delineating the infarct region in white. Seventy two hours following ET 1 induced MCAO there was a cerebral infarct can be seen in rats that had been pre treated with aCSF (ICV infusion for 7 days) (Figure 34 ). No such infarcts were observed in rats that underwent sham MCAO (0.9% saline instea d of ET 1 injection) (Figure 3 4 ). Central pretreatment of rats for 7 days with Ang (1 7) (1 g/h, ICV) prior to ET 1 induced MCAO significantly reduced the infarct size compared to an aCSF pretreated control group (p<0.05). The length of treatment was designed to efficiently determine the effectiveness of Ang (1 7) either before, during, or after stroke. The Ang (1 7) induced cerebroprotection was reversed significantly when Ang (1 7) was co infused for 7 days with its receptor antagonist, A 779 (5 g/h, ICV) (p<0.05). Pre -

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68 treatment with an ICV infusion of A 779 alone did not significantly modify the ET 1 induced cerebral damage (Figure 33). The data in Figure 33 were reinforced by qualitative data from immunostaining which revealed that central pre treatment with Ang (1 7) reduced the neuronal damage 72h after ET 1 induced MCAO. Tissue adjacent to that used for TTC staining was sectioned, and stained with antibodies raised against the neuron specific protein, NeuN. The representative fluoresce nc e micrograph shown in Figure 35 ( upper left panel ) is a low power view of NeuN immunoreactivity (red) in the cerebrum of a rat that had been subjected to a sham MCAO. The higher power view in Figure 35 ( upper right panel ) taken from the inset in the top left panel shows co localization (pink cells) of NeuN immunoreactivity (red) with DAPI nuclear stain (blue) in a rat that received a sham MCAO. In rats that underwent ET 1 induced MCAO and ICV aCSF treatment, NeuN immunoreactivity was fragmented and there was much less colocalization with the nuclei (representative micrograph, Figure 35 lower left panel ). Brain tissue from rats treated ICV with Ang (1 7) prior to ET 1 induced MCAO displayed more colocalization of NeuN and DAPI (pink cells, Figure 35 lower left panel ) compared with the rats treated ICV with aCSF. In addition to the gross and histological evidence for cerebroprotection, central pre treatment with Ang (1 7) attenuated the neurological deficits attributable to ET 1 induced MCAO. For example, 72 hr following ET 1 induced MCAO there were significant behavioral deficits in rats that had been pretreated with aCSF (ICV infusion for 7 days), according to the Bederson Exam (score>0) and the Garcia Exam (score<18) (Figures 5A and 5B). No su ch deficits were observed in rats that underwent

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69 sham MCAO (0.9% saline instead of ET 1 injection). Central pretreatment of rats for 7 days with Ang (1 7) (1 g/h, ICV) prior to ET 1 induced MCAO showed a trend toward a reduced Bederson Exam Score compared to the aCSF pret reated control group (Figure 3 6 A). This cerebroprotective trend was reversed when Ang (1 7) was co infused for 7 days with its antagonist, A 779 (5 g/h, ICV). The trend toward cerebroprotection was also seen with an improved Garcia E xam Score compared to the aCSF pret reated control group (Figure 3 6 B). Again, this cerebroprotective trend was diminished when Ang (1 7) was co infused for 7 days with its antagonist, A 779 (5 g/h, ICV). Pre treatment with an ICV infusion of A 779 alone resulted in both Bederson and Garcia Exam Scores which were similar to the score for aCSF pretreatment. Utilization of a sunflower seed eating task allowed a more sensitive evaluation of neurological function that provided strong evidence for the cerebroprotective properties of Ang(1 7) during focal cerebral ischemia. Rats were given 5 unshelled sunflower seeds and then timed while manipulating and opening the shells to eat the seeds. Rats with significant neurological deficits display longer latenc y to remove the shell. In addition, deficits at this task result in rats that are inefficient at removing the shell and therefore break it into many small pieces. In summary, both increasing latency to open the shell and increasing number of shell pieces are indications of more severe neurological deficit. Central pretreatment of rats for 7 days with Ang (1 7) (1 g/h, ICV) prior to ET 1 induced MCAO showed a significant reduction in the time required to eat 5 sunflower seeds compared to an aCSF pretreated c ontrol group (p<0.01, Figure 36 C). This cerebroprotection was reversed when Ang (1 7) was co infused for 7 days with its antagonist, A 779 (5 g/h, ICV). Pre treatment with an ICV infusion of A 779 alone

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70 resulted in a time to eat 5 sunflower seeds which was not significantly different than the aCSF pretreatment group. Neurological evaluation by counting the number of shell pieces produced during this task also supported the conclusion that central pretreatment with Ang (1 7) is cerebroprotective during focal cerebral ischemia. Rats receiving central Ang (1 7) prior to ET 1 induced MCAO produced significant ly fewer shell pieces than the aCSF pretreated control group (p<0.001, Figure 36 D). This cerebroprotection was reversed when Ang (1 7) was co infused for 7 days with its antagonist, A 779. Pre treatment with an ICV infusion of A 779 alone resulted in a number of shell pieces, which was not significantly different than the aCSF pretreatment group. Two rats that underwent sham strokes via injection of 0.9% saline in place of ET 1 demonstrated a rapid ability to eat sunflower seeds while producing few sh ell pieces, indicating a lack of behavioral deficits (Figures 5C and 5D). Ang (1 7) does not Alter ET1 Induced MCA Constriction In order to assess alterations in ET 1 induced MCA constriction that may have occurred due to chronic central Ang (1 7) in fusion, primary and secondary MCA branches (black arrows) were viewed through a cranial window (Figure 37 ). One group of rats was pretreated with central aCSF infusion for 7 days prior to receiving an intracranial injection with 3 L of ET 1 (80 M) int o the brain parenchyma adjacent to the MCA. This ET 1 injection resulted in abrupt maximal constriction of the proximal MCA branches within 5 minutes followed by recanal izaton of the vessel (Figure 37 D F and J). Another group of rats undergoing a sham MCAO was pre treated with central aCSF infusion for 7 days prior to receiving an intracranial injection with 3 L of 0.9% saline in place of ET 1. Vessel diameter remained relatively stable at baseline values

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71 following t his saline injection (Figure 37 A C and J). Finally, a third group of rats was pre treated with central Ang (1 7) infusion for 7 days prior to receiving an intracranial injection with ET 1. Compared with the group receiving control aCSF pretreatment prior to ET 1 injection, there was a similar, nonsignificantly different, and abrupt maximal vessel diameter decrease within 9 min (Figure 37 GI and J). Additionally, it is of note that there was no significant difference in vessel diameter at any time point between Ang (1 7) pre treated and aCSF pre treated rats that received an injection of ET 1 adjacent to the MCA. Lastly, both the aCSF and Ang(1 7) pre treatment groups receiving an ET 1 brain injection displayed a decrease in vessel diameter 3 min after the start of injection that was significantly greater than the group undergoing sham MCAO (p<0.05) and remained significantly different f or 31min and 23 min respectively. These results indicate that ET 1 injection adjacent to the MCA causes constriction of that vessel followed by recanalization. In addition, pretreatment with Ang (1 7) does not significantly alter the amplitude or time c ourse of the ET 1 induced MCA constriction. Ang (1 7) does not Alter ET1 Induced Cerebral Blood Flow in the Cortex Distant from the Primary Branch of the MCA To assess the effects of chronic central Ang (1 7) infusion on blood flow in microvascular beds during ET 1 induced MCA constriction, CBF was monitored transcortically for 1 h via laser doppler flowmetry during stroke induction. The data presented in Figure 31 demonstrate the reduction in cerebral blood flow at regions of the brain correspondin g to the ischemic core and the ischemic penumbra following injection of 3 L of ET 1 (80 M ) into the brain parenchyma adjacent to the MCA. These data confirm that the ET 1 injection produces a significant ischemic action. In

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72 rats that received an ICV in fusion of Ang (1 7) for 7 days, ET 1 injection as above resulted in abrupt reduction of CBF in the ischemic penumbra region followed by a gradual return to baseline over the period of monitoring. There were no significant differences in CBF at any time between rats infused ICV wi th Ang (1 7) or aCSF (Figure 3 8 ). Therefore, Ang (1 7) had no effect on the reduction in CBF in the cortical areas distant from the primary branch of the MCA. ICV Infusion of Ang(1 7) does not Alter Systemic Blood Pressure In order to assess SBP changes that might be caused by chronic central Ang (1 7) infusion, two groups of 6 rats each were treated with either Ang (1 7) (1 g/h, ICV) or aCSF starting at eight weeks of age and SBP was recorded by the tail cuff method after 1 and 2 weeks of treatment. The data shown in Figure 39 indicate that ICV infusion of Ang (1 7) under the same conditions that attenuated the ET 1 induced cerebral infarcts and behavioral deficits produced no changes in systemic blood pressure. Discussi on The most significant findings of this study are that central pretreatment with Ang (1 7) and activation of the Ang (1 7) receptor, Mas, attenuate the neurological deficits and brain tissue damage produced in an ET 1 induced MCAO model of ischemic strok e independent of any effects on cerebral blood flow or systemic blood pressure. Previously, we have shown that ET 1 induced MCAO is a minimally invasive model of ischemic stroke that provides a rapid constriction, sustained occlusion, and then gradual rep erfusion of the proximal MCA. 138 The current study provides evidence that this constriction is not altered by pretreatment with Ang (1 7), which was a concern when using the ET 1 induced MCAO as a disease model for investigating manipulations of the brain RAS. Our data also suggests that the cerebroprotective effect of central Ang (1 -

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73 7) pre treatment is not due to attenuation of the decrease in CBF in the vascular territory of the MCA. In addition, our results c onfirm the presence of Mas and ACE2 transcripts in relevant brain regions affected by the ET 1 induced MCAO model as well as increased Mas mRNA levels in the lesioned cortex 24 h after stroke This is the first report of cerebroprotection in a model of i schemic stroke elicited via Ang (1 7) stimulation of its receptor, Mas. The presence of both Mas and ACE2 in multiple cell types of the cerebral cortex and subcortical structures has been confirmed previously. Mas is expressed broadly in areas such as hip pocampus, cortex, olfactory bulbs and thalamus of rats or mice.118121 In addition to Mas, ACE2 has been identified as a key component in the vasoprotec tive axis of the RAS and is known to be present in the brain. Several investigations into the CNS localization of ACE2 have detected low levels of ACE2 mRNA in the human brain123 and ACE2 immunostaining in human brai n vascular smooth muscle as well as endothelial cells.124 More recently, ACE2 has been detected in glia during primary cell culture experiments.125 However, in vivo analyse s of mouse brains have localized both ACE2 mRNA and protein to predominately neurons, but in broad areas of cardiovascular and noncardiovascular control.126 Our data support the literature, which indicates the pres ence of key components of the ACE2/Ang (1 7)/Mas axis in brain tissue affected by the ET 1 induced MCAO model of focal cerebral ischemia. Furthermore, we have demonstrated for the first time that Mas mRNA levels increase in the lesioned cortex after strok e Increased expression of Mas implicates the Ang (1 7) receptor as a key player with a potential protective role during stroke pathophysiology.

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74 Based on the diverse localizations of both Mas and ACE2 described in the literature, we cannot preclude the possibility that Ang (1 7) cerebroprotection is mediated by its actions in either the parenchymal cells of the CNS or the cells of the cerebrovasculture. Activation of Mas with chronic or acute peripheral administration of Ang (1 7) increases cerebral blood flow.9395 This increase in blood flow can possibly be explained by data which indicate that central administration of Ang (1 7) shortly following stroke onset has been shown to increase bradykinin levels in the brain, as well as upregulate bradykinin receptors.127 In addition, nitric oxide release and endothelial nitric oxide synthase expression are increased with Ang (1 7) treatment in this model.128Furthermore, we have begun an investigation to determine the role of CNS parenchymal cells (neurons and glia) in Ang (1 7) cerebroprotection. Preliminary data with PCR arrays and qRT PCR show that levels of proinflammatory cytokines such as IL 1 IL 6 and TNF and also of inducible nitric oxide synthase are increased in the ipsilateral cortex at 24 h after ET 1 induced MCAO. Our data also indicate that these effects are attenuated by central Ang (1 7) pretreatment, suggesting an interruption o f pro inflammatory signaling as a mechanism of cerebroprotection. We are currently These results point toward a potential mechanism of cerebroprotection mediated by Ang (1 7) stimulation of Mas and subsequent release of bradykinin, stimulation of bradykinin receptors, and release of NO. Our results indicate that central Ang (1 7) at a dose equal to that which leads to release of NO did not inhibit the decrease in CBF within the MCA territory during ET 1 induced MCAO. Therefore, we have provided evidence against alterations in cerebrovascular dynamics as a mechanism of Ang (1 7) cerebroprotection.

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75 pursuing further investigations into this mechanism as well as studies to discover the cellular localization of Mas in the cerebral cortex. It is also of note that there are reports of both systemic Ang (1 7) administration leading to a decrease in local cardiac Ang II levels,140 as well as Ang (1 7) inhibiting the conversion of Ang I to Ang II by ACE in kinetic studies.141In summary, our findings support a protective role for Ang (1 7) and stimulation of Mas during cerebral ischemia. The high prevalence of stroke and its resulting morbidity and mortality indicate the importance of investigations into novel therapeutic strategies for stroke prevention and treatment. This study demonstrates the therapeutic benefit of Mas activation with Ang (1 7) during stroke. Additional, unpublished data from our laboratory indicate that pharmacological activation of ACE2 provides a similar benefit through the activation of Mas. Finally as the first report of Ang(1 7) elicited cerebroprotection in a model of focal cerebral ischemia our results highlight the ACE2/Ang (1 7)/Mas axis as a promising target for future stroke therapies. Further experiments investigating Ang II levels in the ET 1 model of ischemic stroke could determine if a similar event is occurring in the brain due to Ang (1 7) treatment.

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76 Figure 31. Cerebral blood flow in the isch emic core and penumbra during ET 1 induced MCAO. Laser doppler flowmetry was used to investigate the CBF reduction that results in cortical areas both adjacent (ischemic core) and distal (ischemic penumbra) to the site of ET 1 (80 mM) injection. Represe ntative tracings from one rat are shown for each probe position. The data is presented as a percent of the baseline signal.

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77 Figure 32. Localization of Mas and ACE2 in the cerebrum. mRNA levels of both Mas (A) and ACE2 (B) were measured by qRT PCR in both frontal cortex (n = 4) and subcortex (n = 2) (Panels G and H). Data are presented as means SEM of the levels of ACE2 or Mas mRNA, normalized against 18s rRNA. p < 0.05, ** p < 0.01 (unpaired t test) Figure 33. Mas mRNA levels are increased in the lesioned cortex 24 h after stroke mRNA levels of Mas were measured by qRT P CR in the frontal cortex of rats that underwent ET 1 induced MCAO surgery (stroke, n = 5) or 0.9% saline injection (sham stroke, n = 5). Data are represented as means S EM of the levels of Mas mRNA normalized against 18s rRNA p < 0.05 (One way ANOVA with post hoc Tukeys Multiple Comparison Test)

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78 Figure 34 Intracerebral pretreatment with Ang (1 7) reduces CNS infarct size 72 h after ET 1 induced MCAO. Rats wer e pretreated via the ICV route with either Ang (1 7) (1 g/h; n = 9), aCSF (n = 23), Ang (1 7) + A 779 (5 g/h; n = 9), or A 779 (n = 7) alone for 7 days prior to MCAO induced by intracranial injection of ET 1 (80 mM). In addition, two rats received a sham MCAO with 0.9% saline injection in place of ET 1. Bra ins were removed for TTC staining 72 h after stroke. (A) Bar graphs show the % infarcted gray matter in each treatment group. Data are presented as means SEM. Kruskal Wallis Test (p = 0.025), p < 0.05 vs. Ang (1 7) (Dunns Multiple Comparison Test) (B) Representative brain sections show infarcted (white) and noninfarcted (red) gray matter under the treatment conditions indicated.

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79 Figure 35 Intracerebral pretreatment with Ang (1 7) reduces neuronal damage 72 h after ET 1 induced MCAO. Rats were pretreated via the ICV route with either Ang (1 7) (1 g/h) or aCSF for 7 days prior to ET 1 induced MCAO. In addition, a group was pretreated with aCSF and underwent a sham stroke surgery with injection of 0.9% saline in place of ET 1. Representative fluorescence micrographs (two upper panels) are from rats that underwent sham stroke surgery. The upper left panel shows strong NeuN immunoreactivity (red) in healthy brain tissue. The remaining panels show NeuN immunoreactivity co localized with DA PI nuclear stain (pink color cells) in areas of the frontal cortex within the MCA territory corresponding to the white dotted outline in the upper left panel. In rats that underwent MCAO (aCSF panel), NeuN immunoreactivity was fragmented and there was lit tle co localization (pink color) with the nuclear marker (DAPI). Tissue from rats pre treated with Ang (1 7) displayed neuron morphology and colocalization that was similar to tissue from the sham stroke brain tissue (Sham Stroke panel). NeuN (red), DAP I (blue), co localization (pink). Magnifications: 20X (upper left panel), 100X (all other panels)

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80 Figure 36 Intracerebral pretreatment with Ang (1 7) reduces neurological deficits 72 h after ET 1 induced MCAO. Rats were pretreated via the ICV route with either Ang (1 7) (1 g/h; n = 9), aCSF (n = 23), Ang (1 7) + A 779 (5 g/h; n = 9), or A 779 (n = 7) alone for 7 days prior to ET 1 induced MCAO. Seventy two hours later, neurological deficits were assessed via the Bederson Neurological Exam ( Pa nel A ) and the Garcia Neurological Exam ( Panel B ), as well as the Sunflower Seed Eating Test for the time to eat 5 seeds ( Panel C) and the number of shell pieces (Panel D). Data are represented as means SEM. Bederson Exam p < 0.05 (Kruskal Wallis Test) with no post hoc significance (Dunns Multiple Comparison Test). Garcia Exam p = 0.08 (Kruskal Wallis Test). p < 0.01 or ** p < 0.001 vs. Ang (1 7)

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81 Figure 37 Central Ang(1 7) does not alter ET 1 induced MCA constriction. ( A I) Visualization of MCA branches (arrows) during ET 1 induced vasoconstriction. Primary and secondary branches of the MCA were visualized with a surgical microscope after temporal craniotomy to create a cranial window. Images were captured at a rate of 1 min1, starting imm ediately prior to ET 1 injection (0 min), throughout the ET 1 injection (3 min) and for at least 60 min after initiation of the ET 1 injection. Representative images are shown for rats that underwent 7 days of ICV aCSF pretreatment prior to a sham MCAO ( 3 mL of 0.9% saline injection, n = 4, A C), 7 days of ICV aCSF pretreatment prior to an ET 1 induced MCAO (3 mL of 80 mM ET 1 injection, n=4, D F), and 7 days of ICV Ang (1 7) (1 g/h) pre treatment prior to an ET 1 induced MCAO (n=4, G I). ( J) Vasoconst riction of primary or secondary MCA branches was quantified as the percentage of baseline vessel diameter. Data are means SEM. Baseline vessel diameter was determined prior to ET 1 or 0.9% saline injection (time = 0 min). *p < 0.05 for aCSF vs Sham MCA O and p < 0.05 for Ang (1 7) vs Sham MCAO [two way RM ANOVA (p < 0.001) followed by Bonferroni post test].

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82 Figure 37 Continued

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83 Figure 38 Central Ang(1 7) does not alter ET 1 induced cerebral blood flow in the cortex distant from the primar y branch of the MCA. Lasar Doppler flowmetry was used to monitor CBF in the vascular territory of the MCA distal to the site of ET 1 injection. Data are presented as means SEM of the percent change from baseline CBF. ET 1 injection takes place over a period of 3 minutes starting at 0 min on this graph. No significant differences exist between Ang (1 7) (n = 6) and aCSF (n = 6) treatment groups at any time point [twoway RM ANOVA].

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84 Figure 39 Central Ang(1 7) pre treatment does not alter systol ic blood pressure. Rats were administered either Ang (1 7) (1 g/h; n = 6) or aCSF (n = 6) via the ICV route over a period of 2 weeks. SBP was measured using the tail cuff method at weekly intervals. Data are represented as means SEM of SBP.

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85 CHAPTE R 4 ACE2 ACTIVATION AS A TARGET FOR CEREBROPROTECTION DURING ISCHEMIC STROKE Introduction We have recently shown that central pretreatment with Angiotensin(1 7) [Ang (1 7)] can greatly reduce tissue damage and neurological deficits that result during foc al cerebral ischemic injury.142Thus far, we have focused on c entral administration of exogenous Ang (1 7) to combat the pathophysiology of stroke. However, endogenous sources of Ang (1 7), as well as several pathways for Ang (1 7) production exist. The pathways leading to Ang (1 7) can be either Angiotensin Conver ting Enzyme (ACE) dependent or ACE independent. ACE dependent pathways include conversion of Angiotensin I (Ang I) to Angiotensin II (Ang II) by ACE and subsequent conversion of Ang II to Ang (1 7) by Angiotensin Converting Enzyme 2 (ACE2). These studies were performed using a rat model of endothelin1 (ET 1) induced Middle Cerebral Artery Occlusion (MCAO) and were the first to reveal the cerebroprotective properties of central Ang (1 7) administration. In addition, co administration of the Ang (1 7) receptor antagonist, D Alanine[Ang (1 7)] (A779) reduced the Ang (1 7) cerebroprotective activity. Thus, Ang (1 7) cerebroprotection was mediated via activation of it s endogenous receptor, Mas. This was the first description of Ang (1 7) activation of Mas mediated cerebroprotection during stroke. Therefore, the ACE2/Ang (1 7)/Mas Axis is a promising target for cerebprotective therepy. 45 In addition, ACE2 is capable of converting Ang I to Ang(1 9) which can then be converted to Ang (1 7) by ACE.46 This second ACE dependent route involving Ang (1 9) production as an intermediate step is likely

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86 less relevant because ACE2 is 400 times more efficient at catalyzing the conversion of Ang II to Ang (1 7) than Ang I to Ang (1 9).49 In addition to the ACE dependent pathways for Ang (1 7) pr oduction, various enzymes contribute to the production of Ang (1 7) via ACE independent mechanisms. These enzymes include Neprilysin, proyl endopeptidase, proyl carboxypeptidase, Chymase, and Cathepsin A.5053Thus, ACE2 h as recently been a target for cardiovascular disease therapy. The contributions of ACE independent pathways likely become much more relevant in the presence of ACE inhibitors (ACEi). However, these enzymes lack specificity for angiotensin peptide metabol ism and they are unlikely to be major contributors to Ang (1 7) synthesis under physiological conditions. Although, multiple pathways leading to Ang (1 7) production exist, it is clear that ACE2 is a key regulator of this angiotensin peptide. 47 In fact, several small molecule ACE2 activators have been identified that selectively increase ACE2 activity without having an effect on ACE activity .143, 144 One of these molecules, Diminazene aceturate (DIZE) has been shown to decrease blood pressure dramatically and dose dependently when administered acutely. In addition, a modest decrease in blood pressure and associated reductions in end organ damage are observed with chronic administration of DIZE to spontaneously hypertensive rats. Another of these activators, XNT (1[(2 dimethylamino) ethylamino] 4 (hydroxymethyl) 7 [( 4 methylphenyl) sulfonyl oxy] 9H xanthene9 one), had similar antihypertensive effects and also prevented the pathophysiology right heart failure and pulmonary fibrosis in a rat model of pulmonary hypertension induced by monocrotaline.90

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87 Therefore, these small molecule ACE2 activators are promising compounds for ACE2/Ang (1 7)/Mas axis activation and treatment of cardiovascular disease. Based on our previous studies supporting a protective role for ACE2/Ang (1 7)/Mas axis during stroke, as well as a protective effect of ACE2 activation by DIZE in various models of cardiovascular disease, we have developed the general hypothesis that DIZE can activate ACE2 in the brain, leading to increased production of Ang (1 7), a nd a subsequent Mas mediated cerebroprotective action during ischemic stroke. In the current study we have demonstrated that central administration of DIZE prior to ischemic stroke by endothelin1 (ET 1) induced middle cerebral artery occlusion (MCAO) eli cits a decrease in infarct size and neurological deficits. This is the first demonstration of a cerebroprotective action of the ACE2 activator, DIZE. Methods Animals Adult male Sprague Dawley rats were purchased from Charles River Farms (Wilmington, MA). All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee. Chemicals Diminazene aceturate (DIZE) was purchased from SigmaAldrich (St. Lous, MO, USA). A779 (D Ala7) angiotensin (17) was purchas ed from Bachem Bioscience (Torrance, CA). E T 1 was purchased from American Peptide Company, Inc (Sunnyvale, CA, USA). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA). DIZE and A 779 were dissolved in H2O. ET 1 was dissol ved in 0.9% saline.

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88 Placement of Intracerebroventricular and Guide Cannulae Eight week old male Sprague Dawley rats were anesthetized with a mixture of O2 (1 L/min) and 4% isoflurane, placed in a Kopf stereotaxic frame, and anesthesia was maintained for the duration of the surgery using an O2/isoflurane (2%) mixture delivered through a nose cone attached to the frame. The skull was exposed and a small hole was drilled for placement of an MCAO guide cannula in the cranium dorsal to the right hemisphere using the following stereotaxic coordinates (1.6 mm anterior and 5.2 mm lateral to the bregma). A 21 gauge stainless steel guide cannula cut to 4mm below the pedestal was lowered into the hole and affixed to the skull with 3 mounting screws and dental cement. During the same surgery, a second hole was then drilled in the cranium dorsal to the left hemisphere for placement of an intracerebroventricular cannula (kit 1, ALZET, Cupertino, CA) coupled to a 2 week osmotic pump (model 2002, ALZET, Cupertino, CA) via vinyl tubing. The following stereotactic coordinates were used (1.3 mm posterior and 1.5 mm lateral to bregma, 4.5 mm below the surface of the cranium). The osmotic pump was implanted subcutaneously between the shoulder blades as described previously.86 Osmotic pumps were used to infuse DIZE (5 g/h), DIZE (5 g/h) plus A 779 (1 g/h), A779 (1 g/h) alone, or H2O into the left lateral cerebral ventricle starting at the time of cannula placement and lasting until t he animals were euthanized. Following this surgery, the wound was closed and the rat was administered an analgesic agent (buprenorphine; 0.05 mg/kg Endothelin 1 Induced Middle Cerebral Artery Occlusion sc) before waking. Seven days after the placement of ICV and guide cannulae, the ET 1 induced MCAO procedure was performed as we have previously reported with a minor

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89 modification.138 Eight week old male Sprague Dawley rats were anesthetized as described above, and anesthesia was maintained for the duration of the injection using an O2/isoflurane (2%) mixture delivered through a nose cone attached to the frame. The cannula dummy was removed after which a 26 gauge needle attached to a 5 L Hamilton microsyringe was lo wered 8.7 mm ventral to bregma. Once the needle was in place, 3 L of 80 M ET 1 was infused adjacent to the MCA at a rate of 1 L/min using a Stoelting Quintessential Injector (Stoelting Co., Wood Dale, IL, USA). The needle was left in place for 3 min a fter the injection was complete and then removed slowly. The cannula dummy was then replaced and the rat was administered an analgesic agent (buprenorphine; 0.05 mg/kg sc) before waking. We have characterized this model previously by showing that injecti on of ET 1 can cause rapid constriction of the MCA followed by gradual reperfusion. In addition, a strong and significant correlation exists between the size of infarct measured in this model and several test scores used to assess neurological deficits in the work described here.138Cerebral Blood Flow Monitoring In addition, we have used laser doppler flowmetry to investigate the CBF reduction that results in cortical areas both adjacent (ischemic core) and distal (ischemic penumbra) to the site of ET 1 injection (unpublished). It is clear from these data that CBF decreases dramatically in tissue adjacent to the proximal MCA and that CBF is reduced to a lesser degree in tissue of more distal MCA territories. An addi tional group of rats was anesthetized as described above, after which laser doppler flowmetery was used to measure CBF prior to ET 1 injection and lasting for 1 h after ET 1 injection. CBF measurements were performed using a Standard Pencil

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90 Probe and Blood FlowMeter coupled to a Powerlab 4/30 with LabChart 7 (ADInstruments, Inc, Colorado Springs, CO, USA). The probe was placed either just posterior to the MCAO guide cannula at the lateral skull ridge. Data were recorded in arbitrary blood perfusion units at 1000 Hz. Baseline CBF was calculated by averaging a 1 min interval just prior to ET 1 injection. Changes in CBF were calculated as a percentage of baseline by averaging a 10 s interval every 1 min. Indirect Blood Pressure Monitoring After undergoing surgery to implant an intracerebroventricular cannula (kit 1, ALZET, Cupertino, CA) coupled to a 2 week osmotic pump (model 2006, ALZET, Cupertino, CA) via vinyl tubing as described above, animals were allowed to recover for 1 week. Indirect blood press ure was recorded by tail cuff once a week for 2 weeks as previously described.86Neurological Deficits and Infarct Size Animals were warmed by a 200 W heating lamp for 5 min before restraint in a heated Plexiglas cage to which the animals were previously conditioned. A pneumatic pulse sensor was attached to the tail distal to an occluding cuff controlled by a Programmed Electrosphygmomanometer (Narco BioSystems, Austin TX). Voltage outputs from the cuff and pulse sensor were recorded and analyzed by a P owerlab signal transduction unit and associated Chart software (ADInstruments, Colorado Springs, CO). Neurological deficits and infarct size were evaluated as reported previously.138 Neurological evaluations were performed using two separate scoring scales originally described by Bederson et al .133 and Garcia et al.,134 which cumulatively evaluate spontaneous activity, symmetry in limb movement, forepaw outstretching, climbing, body proprioception, response to vibrissae touch, resistance to lateral push, and circling

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91 behavior. Additionally, animals were evaluated for neurological deficits using a sunflower seed eating test.135 Infarct volume was assessed by staining brain sections with 0.05% 2,3,5triphenyltetrazolium chloride (TTC) for 30 minutes at 37C. Tissue ipsilateral to the occlusion, which was not stained, was assumed to be infarcted. After fixation with 10% formalin, brain sections were scanned on a flatbed scanner (Canon) and analyzed using ImageJ software (NIH). To compensate for the effect of brain edema, the corrected infarct volume was calcula ted using an indirect method.129Data Analysis Data are expressed as means SEM. Statistical significance was evaluated, as specified in the figure legends, with the use of a Kruskal Wallis test, Two way Row matched ANOVA, One way ANOVA, or unpaired t test, as well as with Dunns Multiple Comparison Test, the Bonferroni Test, or Tukeys Multiple Comparison Test for posthoc analyses when appropriate. Differences were considered significant at p<0.05. Individ ual p values are noted in the results and figure legends. Results Cerebroprotective Action of DIZE The effect of DIZE pre treatment on ET 1 induced cerebral damage was assessed by TTC staining, whereby noninfarcted gray matter is stained red after incubat ion in TTC, delineating the infarct region in white. Seventy two hours following ET 1 induced MCAO a cerebral infarct can be seen in rats that had been pretreated with H2O (ICV infusion for 7 days) (Figure 41). Central pretreatment of rats for 7 days with DIZE (5 g/h, ICV) prior to ET 1 induced MCAO significantly reduced the infarct size compared to an H2O pre treated control group (p<0.001). The length of treatment was designed to efficiently determine the effectiveness of DIZE either before, during, or after st roke.

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92 The DIZE induced cerebroprotection was reversed by co infusion of the Ang (1 7) receptor antagonist, A 779 (1 g/h, ICV). Pre treatment with an ICV infusion of A 779 alone did not significantly modify the ET 1 induced cerebral damage compared to H2In addition to the gross histological evidence for cerebroprotection, central pretreatment with DIZE attenuated the neurological deficits attributed to ET 1 induced MCAO. For example, 72 hr following ET 1 induced MCAO there were significant behavioral deficits in rats that had been pretreated with H O pre treatment (Figure 41). 2O (ICV infusion for 7 days), according to the Bederson Exam (score > 0) and the Garcia Exam (score < 18) (Figures 2A and 2B). Central pretreatment of rats for 7 days with DI ZE (5 g/h, ICV) prior to ET 1 induced MCAO significantly reduced the Bederson Exam Score compared to the H2O pre treated control group (p<0.05, Figure 42A). This cerebroprotection was reversed when DIZE was co infused for 7 days with the Ang (1 7) receptor antagonist, A 779 (1 g/h, ICV). A trend toward cerebroprotection was also seen with an improved Garcia Exam Score compared to the H2O pre treated control group (Figure 42B). Again, this cerebroprotective trend was diminished when DIZE was co infuse d for 7 days with its antagonist, A 779 (1 g/h, ICV). Pre treatment with an ICV infusion of A 779 alone resulted in both Bederson and Garcia Exam Scores which were similar to the score for H2In addition, performance on the sunflower se ed eating task was also used to evaluate neurological function. This task provided further evidence of the cerebroprotective properties of DIZE during focal cerebral ischemia. Rats were given 5 unshelled sunflower seeds and then timed while manipulating and opening the shells to O pre treatment.

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93 eat the seeds. Rats with significant neurological deficits display longer latency to remove the shell. In addition, deficits at this task result in rats that are inefficient at removing the shell and therefore break it into many small pieces. In summary, both increasing latency to open the shell and increasing number of shell pieces are indicators of more severe neurological deficits. Central pretreatment of rats for 7 days with DIZE (5 g/h, ICV) prior to ET 1 induced MCAO sh owed a nonsignificant trend toward reduction in the time required to eat 5 sunflower seeds compared to an H2O pre treated control group (Figure 4 2C). This cerebroprotective trend was reversed when DIZE was co infused for 7 days with the Ang (1 7) recept or antagonist, A 779 (1 g/h, ICV). Pre treatment with an ICV infusion of A 779 alone resulted in a time to eat 5 sunflower seeds which was similar to the H2O pre treatment group. Neurological evaluation by counting the number of shell pieces produced during this task also produced a pattern suggesting that central pretreatment with DIZE is cerebroprotective during focal cerebral ischemia. Rats receiving central DIZE prior to ET 1 induced MCAO resulted in a nonsignificant trend toward reduction in the number of shell pieces c ompared to the H2O pre treated control group (Figure 42D). This cerebroprotective trend was reversed when DIZE was co infused for 7 days with A 779. Pretreatment with an ICV infusion of A 779 alone resulted in a number of shell pieces, which was similar to the H2DIZE does not Alter ET1 Induced Cerebral Blood Flow in the Cortex Distant from the Primary Branch of the MCA O pre treatment group. To assess the effects of chronic central DIZE infusion on blood flow in microvascular beds during ET 1 induced MCA constriction, CBF was monitored transcortically for 1 h via laser doppler flowmetry during stroke induction. We have

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94 previously demonstrated the reduction in cerebral blood flow at regions of the brain corresponding to the ischemic core and the ischemic penumbra following injection of 3 L of ET 1 (80 M ) into the brain parenchyma adjacent to the MCA.138 These data confirm that the ET 1 injection produces a significant ischemic action. In rats that received an ICV infusion of DIZE for 7 days, ET 1 injection as above resulted in abrupt reduction of CBF in the ischemic penumbra region followed by a gradual return to baseline over the period of monitoring. There were no significant differences in CBF at any time between rats infused ICV with DIZE or H2ICV infusion of DIZE Decreases Blood Pressure O (Figure 4 3). Therefore, DIZE had no effect on the reduction in CBF in the cortical areas distant from the primary branch of the MCA. ICV infusion of DIZE (5 g/h, ICV) produces a significant reduction in SBP after 7 days of treatment (Figure 44). These pretreatment conditions are identical to those that attenuated the ET 1 induced cerebral infarcts and behavioral deficits. Discussion The most significant findings of this study are that central pretreatment with DIZE attenuates the neurological deficits and brain tissue damage produced in an ET 1 induced MCAO model of ischemic stroke. Previously, we have shown a similar protective effect after central pretreatment with Ang (1 7).142 The ET 1 induced MCAO is a minimally invasive model of ischemic stroke that provides a rapid constriction, sustained occlusion, and then gradual reperfusion of the proximal MCA which i s not altered by pretreatment with central Ang (1 7).142 Our current data also suggests that the cerebroprotective effect of central DIZE pretreatment is not due to attenuation of

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95 the decrease in CBF in the vascu lar territory of the MCA, which is consistent with previous studies in involving Ang (1 7) pre treatment. In addition, both the current DIZE cerebroprotection and previously described Ang (1 7) cerebroprotection were attenuated in the presence of the Ang (1 7) receptor, Mas. This is the first report of cerebroprotection in a model of ischemic stroke elicited via ACE2 activation and subsequent stimulation of the Ang (1 7) receptor, Mas. It is noteworthy that 7 days of ICV treatment with DIZE decreased BP i n rats to a modest degree. This decrease was approximately 17 mmHg. It is unclear whether this change in BP contributes to the DIZE medicated cerebroprotection. However, a decrease in blood pressure would be expected to decrease the cerebrovascular rese rve since this decrease is certainly well within the autoregulatory range of the cerebrovasculature. A blood pressure decrease is likely mediated by a central mechanism involving either a decrease in Ang II or an increase in Ang (1 7). Such a decrease co uld potentially have an even greater antihypertensive effect in an animal strain with a well known neurogenic hypertension component. For example, central administration of DIZE to spontaneously hypertensive rats (SHRs) would likely lead to a robust decrease in blood pressure that could potentially decrease the cerebrovascular atherosclerosis and dysfunction in these animals. In addition, DIZE would likely have a protective effect separate from these BP related changes. Therefore, we predict a more robus t cerebroprotective effect in this model of chronic cerebrovascular disease. Additionally, the use of a chronic cerebrovascular disease model, as opposed to the young health rats used here, would more closely mimic human stroke because of the

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96 cardiovascul ar pathology present in many patients due to increased age and comorbidities. The magnitude of cerebroprotection elicited by DIZE was similar to that elicited by Ang (1 7) previously. However, attenuation of the DIZE induced cerebroprotection by the M as antagonist, A 779 seems to be less complete. Thus, either A 779 antagonism of Mas is incomplete, or Ang (1 7) stimulation of Mas may only be a partial mechanism for DIZE induced cerebroprotection. Since ACE2 can breakdown Ang II, one possibility is th at ACE2 mediated decreases in Ang II could decrease Ang II type 1 receptor (AT1R) stimulation. AT1R antagonism is known to provide cerebroprotection during ET 1 induced MCAO. Therefore, decreased levels of Ang II could contribute to a similar mechanism.138 In addition, ACE2 activation could actually participate in a feed forward loop of Ang (1 7) production. Ang (1 7) is broken down to Ang (1 5) by ACE.145 However, Ang (1 7 ) is also an inhibitor of ACE at its c terminal domain.146 Therefore, activation of ACE2 can lead to increased production of Ang (1 7) which directly inhibits the enzyme that causes its degradation, further increas ing the levels of Ang (1 7). Finally, although DIZE significantly reduced infarct size after ET 1 induced MCAO, the reduction of neurological deficits seemed to be less dramatic than our previous studies using Ang (1 7) pre treatment. One possible explanation of this could be an inadequate amount of Ang II substrate that would be necessary to produce Ang (1 7) due to ACE2 activation. Alternatively, the relationship to DIZE dose and ACE2 activation in vivo is unknown and higher doses of DIZE might further increase levels of Ang (1 7) and provide greater cerebroprotection. To answer these questions, further investigation into

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97 the tissue levels of Ang II, Ang (1 7), and Ang (1 5) would help to understand the mechanism of DIZE induced cerebroprotection. It should be noted that ACE2 is able to metabolize several other non renin angiotensin system peptides with similar efficiency to that of Ang II to Ang (1 7). These peptides include apelin13, neurotensin, kinetensin, dynorphin, Des Arg9Bradykinin, and Lys Des Arg9Bradykinin.49 Although the physiological significance of these reactions has not been explored, breakdown of these peptides could be involved in protection of tissue during or after cerebral ischemia. For example, ACE2 can hydrolyse the proinflammatory kinin, Des Arg9Bk. This kinin cannot be broken down by ACE, which suggests that an increased ACE2/ACE ratio could contribute to a decrease in post ischemic inflammation and ensuing tissue injury. Also, important is the fact that DIZE is currently used as an antiprotozoal agent in animal and some human cases of trypanosomiasis. DIZE likely mediates this antiprotozoal activity through its ability to inhibit replication of trypanosomal mitochondrial DNA.147In summary, our findings s upport a protective role for ACE2 activation during cerebral ischemia. The high prevalence of stroke and its resulting morbidity and mortality indicate the importance of investigations into novel therapeutic strategies for stroke prevention and treatment. This study demonstrates the therapeutic benefit of ACE2 activation and Mas stimulation during stroke. Finally, as the first report of pharmacological activation of ACE2 for cerebroprotection in a model of focal cerebral Although this activity is reportedly specific protozoal DNA, we cannot exclude a mechanism of DIZE mediated cerebroprotection that is related to this antiprotozoal activity at this time.

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98 ischemia our results highlight the ACE2/Ang (1 7)/Mas axis and ACE2 activating molecules as promising targets for stroke therapy.

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99 Figure 41. Intracerebral pretreatment with DIZE reduces CNS infarct size 72 h after ET 1 induced MCAO. Rats were pretreated via the ICV route with ei ther H2O (n = 15), DIZE (5 g/h; n = 18), DIZE + A 779 (5 g/h 1 g/h; n = 7), or A 779 (n = 10) alone for 7 days prior to MCAO induced by intracranial injection of ET 1 (80 mM). Brains were removed for TTC staining 72 h after stroke. Bar graphs show t he % infarcted gray matter in each treatment group. Data are presented as means SEM. Oneway ANOVA (p < 0.0001), p < 0.001 vs. DIZE, ** p < 0.05 vs. DIZE (Tukeys Multiple Comparison Test). Representative brain sections show infarcted (white) and noninfarcted (red) gray matter under the treatment conditions indicated.

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100 Figure 42. Intracerebral pretreatment with DIZE reduces neurological deficits 72 h after ET 1 induced MCAO. Rats were pretreated via the ICV route with either H2O (n = 11), DI ZE (5 g/h; n = 16), DIZE + A 779 (5 g/h 1 g/h; n = 6), or A 779 (n = 8) alone for 7 days prior to MCAO induced by intracranial injection of ET 1 (80 mM). Seventy two hours later, neurological deficits were assessed via the Bederson Neurological Exam ( Panel A ) and the Garcia Neurological Exam ( Panel B ), as well as the Sunflower Seed Eating Test for the time to eat 5 seeds ( Panel C) and the number of shell pieces (Panel D). Data are represented as means SEM. Bederson Exam p < 0.05 (Kruskal Wallis Te st), *p<0.05 vs. Ang (1 7) (Dunns Multiple Comparison Test)

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101 Figure 43. Central DIZE does not alter ET 1 induced cerebral blood flow in the cortex distant from the primary branch of the MCA. Lasar Doppler flowmetry was used to monitor CBF in the vas cular territory of the MCA distal to the site of ET 1 injection. Data are presented as means SEM of the percent change from baseline CBF. ET 1 injection takes place over a period of 3 minutes starting at 0 min on this graph. No significant differences exist between DIZE (n = 6) and H2O (n = 6) treatment groups at any time point (twoway RM ANOVA).

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102 Figure 44. Central DIZE pretreatment decreases systolic blood pressure. Rats were administered either H2O (n = 6) or DIZE (5 g/h; n = 6) via the ICV route over a period of 2 weeks. SBP was measured using the tail cuff method at weekly intervals. Data are represented as means SEM of SBP. p < 0.01 vs. H2O (un paired t test)

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103 CHAPTER5 SUMMARY AND CONCLUSI ONS Summary Specifi c Aim 1 Endogenous levels of Angiotensin II (Ang II) are increased bilaterally in the cortex and hypothalamus following stroke and systemic treatment of spontaneously hypertensive rats (SHR) with Ang II type 1 receptor (AT1R) blockers (ARBs) reduces the oc currence of stroke.79, 84, 129 Additionally, ARBs provide a 4050% reduction of infarct volume and reduce the neurological deficits in normotensive rats and SHRs that received a middle cerebral artery (MCA) occlusion via intraluminal occlusion.78, 82, 97100 However, the endothelin1 (ET 1) induced middle cerebral artery occlusion (MCAO) model of cerebral ischemia is thought to more closely mimic the temporal events of an embolic stroke. This model provides rapid occlusion of the middle cerebral artery and a gradual reperfusion that lasts for 1622 h.130 Aim 1 was designed to evaluate whether systemic administration of an ARB prior to ET 1 induced MCAO will provide cerebroprotection during this model of ischemic stroke. Injection of 3 L of 80 M ET 1 adjacent to t he MCA resulted in complete occlusion of the vessel that resolved over a period of 30 min to 40 min. Following ET 1 induced MCAO, rats had significant neurological impairment, as well as, an infarct that consisted of approximately 30% of the ipsilateral g ray matter. Systemic pretreatment with 0.2mg/kg/day candesartan for 7 days attenuated both the infarct size and neurological deficits caused by ET 1 induced MCAO without altering blood pressure. The effect of candesartan pretreatment on ET 1 induced v asoconstriction of the MCA was also evaluated by visualization of the MCA through a cranial window. It was determined that candesartan pretreatment did not

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104 alter ET 1 induced constriction of the MCA, which validates the use of this stroke model during AR B pharmacotherapy. In addition, strong correlations were observed between infarct volumes and neurological deficits. This study solidifies the the view that ARBs can exert a cerebroprotective action during ischemic stroke and validates the ET 1 induced MC AO model for examination of the brain renin angiotensin systems (RAS) role in this disease. Specific Aim 2 Recent progress in cardiovascular therapy suggests that stimulation of Angiotensin Converting Enzyme 2 (ACE2), production of Angiotensin(1 7) [An g (1 7)], and activation of the Ang (1 7) receptor, Mas, are viable targets for disease prevention and treatment. The ACE2/Ang (1 7)/Mas axis has been shown to counteract many of the physiological effects of the AT1R, including vasoconstrictor and proliferative actions.85 In addition, activation of the ACE2/Ang (1 7)/Mas axis also attenuates many of the pathophysiological states that involve increased production of Ang II by Angiotensin Converting Enzyme (ACE), and subsequent activation of the AT1R (ACE/Ang II/AT1R axis). For example, many studies targeting the ACE2/Ang (1 7)/Mas axis have revealed its broad therapeutic potential for the treatment of hypertension, hypertensionrelated pathology, myocardial infarcti on, and heart failure.8688, 91, 92 Aim 2 was designed to test whether central administration of Ang (1 7) via lateral vent ricular cannula would provide cerebroprotection during ET 1 induced MCAO, a rat model of ischemic stroke. Sprague Dawley rats were treated via the intracerebroventricular route with Ang (1 7) (1 g/h) or artificial cerebrospinal fluid (aCSF) prior to ET 1 induced MCAO. Ang (1 7) treatment reduced the cerebral infarct size, neuronal damage and

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105 neurological deficits measured 72 h after MCAO induction. Infarct size was reduced to 15.78 5.54% of i psilateral gray matter in the Ang (1 7) treated rats compared with 40.11 5.48% in aCSF treated controls. Ang (1 7) treatment also reduced the neurological deficits produced by ET 1 induced ischemic stroke, as indicated by a battery of neurological tests including the Bederson Exam, Garcia Exam, and the sunflower seed eating task. These protective actions of Ang (1 7) were reversed by blockade of the Ang (1 7) receptor, Mas, with A 779 (1 g/h). In addition, the effect of Ang (1 7) pre treatment on ET 1 induced vasoconstriction of the MCA was also evaluated by visualization of the MCA through a cranial window. It was determined that central Ang (1 7) pre treatment did not alter ET 1 induc ed constriction of the MCA, which validates the use of this stroke model during Ang (1 7) pharmacotherapy. In order to investigate alterations in cerebral blood flow (CBF) as a mechanism of Ang (1 7) induced cerebroprotection, we measured CBF in the penum bra during ET 1 induced MCAO. Ang (1 7) did not affect the reduction of CBF in the penumbra which ruled out the possibility of a protective mechanism of Ang (1 7) mediated through improved CBF during MCAO. This is the first demonstration of cerebroprotec tive properties of Ang (1 7) during ischemic stroke. Specific Aim 3 Investigations of the ACE2/Ang (1 7)/Mas axis has revealed broad therapeutic potential for the treatment of hypertension, and hypertensionrelated pathology such as stroke, myocardial infa rction, and heart failure. For example, we have shown the beneficial actions of central pretreatment with Ang (1 7) in a rat model of ischemic stroke. Furthermore, ACE2 can form endogenous Ang (1 7) from Ang II and has

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106 recently been a target for cardiov ascular disease therapy.47 In fact, several small molecule ACE2 activators have been identified that selectively increase ACE2 activity without having an effect on ACE activity.143, 144 One of these molecules, Diminazene aceturate (DIZE) has been shown to decrease blood pressure dramatically and dose dependently when administered acutely. In addition, a modest decrease in blood pressure and associated reductions in end organ damage are observed with chronic administration of DIZE to spontaneously hypertensive rats. Another of these activators, XNT (1 [(2 dimethylamino) ethylamino] 4 (hydroxymethyl) 7 [(4 methylphenyl) sulfonyl oxy] 9H xant hene9 one), had similar antihypertensive effects and also prevented the pathophysiology right heart failure and pulmonary fibrosis in a rat model of pulmonary hypertension induced by monocrotaline.90 Therefore, t hese small molecule ACE2 activators are promising compounds for ACE2/Ang (1 7)/Mas axis activation and treatment of cardiovascular disease. Based on our previous studies supporting a protective role for ACE2/Ang (1 7)/Mas axis during stroke, as well as a protective effect of ACE2 activation by DIZE in various models of cardiovascular disease, we have developed the general hypothesis that DIZE can activate ACE2 in the brain, leading to increased production of Ang (1 7), and a subsequent Mas mediated cerebroprotective action during ischemic stroke. Aim 3 was designed to test whether central pretreatment with DIZE will provide cerebroprotection in a rat model of ET 1 induced MCAO. Adult male Sprague Dawley Rats were pretreated with intracerebroventricular DIZE (5 g/h) or H2O for 7 days prior to ET 1 induced MCAO. DIZE treatment reduced neurological deficits and infarct size measured 72 h after MCAO induction. Specifically, infarct size was reduced to 28.75 5.05% of ipsilateral gray matter in DIZE treated rats

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107 compared with 62.40 4.08% in H2O controls. Additionally, neurological deficits were reduced in DIZE treated rats as indicated by a lower Bederson Exam Score of 0.9 0.3 compared to 2.1 0.3 in control rats, a higher Garcia Exam Score of 15.9 0.7 com pared with 13.9 1.0 in control rats, and improvement in a sunflower seed eating task to 187 33 s and 20 3 shell pieces compared to 256 5 s and 25 3 pieces in H2Discussion O control rats. Furthermore, the histological and neurological benefits of pretreatm ent with DIZE were attenuated when DIZE was co administered with the Ang (1 7) receptor antagonist, A 779 (1 g/h). In order to investigate alterations in CBF as a mechanism of DIZE induced cerebroprotection, we measured CBF in the penumbra during ET 1 in duced MCAO. DIZE did not affect the reduction of CBF in the penumbra which ruled out the possibility of a protective mechanism of DIZE mediated through improved CBF during MCAO. This data indicates that central administration of DIZE prior to stroke is c erebroprotective and extends the known cardiovascular protective effects elicited by stimulation of the ACE2/Ang (1 7)/Mas axis. Mechanism of ACE2/Ang (1 7)/Mas Cerebroprotection Our results demonstrate that either direct stimulation of Mas with Ang(1 7) or indirect stimulation through ACE2 activation with DIZE improves the neurological outcomes after ET 1 induced MCAO. However, the cell types and molecular mechanisms involved in this cerebroprotection have not been determined. Previous report s have demonstrated that Ang(17) administered centrally after a stroke can increase bradykinin release, bradykinin receptor stimulation, endothelial nitric oxide synthase (eNOS) activity, and NO production.127, 128 This suggests that Ang (1 7) may be able to increase NO availability and cerebrovascular vasodilation. In addition, both

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108 acute and chronic peripheral administration of Ang (1 7) has been shown to increase cerebral blood flow. However, the peripheral route of administration should not deliver effective doses of Ang (1 7) across the blood brain barrier (BBB).148Some insight into the protective mechanism of Ang (1 7) might be gained from examining the cellular localization of Mas. There has not been an organized investigation into the cell types that express M as in the brain. Mas was first characterized as a protooncogene that was found in high levels in the brain Despite these findings, the data presented in Chapter 2 demonstrate that central infusion of Ang(1 7) at a dose which affords cerebroprotection does not alter cerebral blood flow. Therefore, the mechanism for peripheral Ang (1 7) induced increases in cerebral blood flow cannot be synonymous with the mechanism of central Ang (1 7) potentiatio n of the bradykinin/NO pathway activity after stroke. 118 and thought to be exclusively located in neurons 119. In addition, it has been shown that Mas is present in rat cerebral endothelial cells, but is absent from endothelial cells in the periphery 122. Recently, a global presence for Mas in both cardiovascular and noncardiovascular co ntrol areas of the brain was verified by immunofluorescence 93. This study also indicates a largely neuronal localization for Mas in cardiovascular control regions, but makes no report on its cellular localization i n noncardiovascular control regions of the brain such as the motor cortex. Taken together, these results suggest a diverse localization for Mas in the brain. However, none of these studies have reported various Mas expressing cell types simultaneously. This can be addressed with studies to co localize various cell types with Mas using immunohistochemistry. Identification of cell types expressing Mas as endothelial cells, smooth muscle cells, neurons,

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109 astrocytes, oligodendrocytes, microglia, or peripheral leukocytes (post ischemic stroke) will help lead us toward the mechanism of action of Ang (1 7). Although limited in scope, we have made several advancements in the understanding of Ang (1 7) mediated cerebroprotection. First, we have investigated the cerebral blood flow changes of the penumbra that result from MCAO. It is clear that with either Ang (1 7) or DIZE pre treatment, the reductions of blood flow in the penumbra remain unaltered compared with vehicle pre treated control rats. These experiments rule out a protective mechanism of Mas stimulation that involves improved cerebral blood flow to tissue in the vascular territory of the MCA. Although, the participation of smooth muscle and endothelial cells of the cerebrovasculature is not ruled out by these experiments, it is clear that their involvement in vasodilatory events is not a factor. In addition, our preliminary studies indicate Ang (1 7) mediated interruption of the post stroke inflammatory cascade as a potential mechanism of cerebroprotection. Our results indicate that ET 1 induced stroke can increase levels of mRNAs for proinflammatory cytokines such as IL 1 IL 6 and TNF within the infarct region. In addition, inducible nitric oxide synthase (iNOS) expression is increased in the ipsilateral cortex at 24 h after ET 1 induced MCAO, consistent with what is observed in other models of ischemic stroke.149, 150These findings are supported by report s of proinflammatory cytokine release by astrocytes, microglia, smooth muscle cells, and endothelial cells during stroke. Ang (1 7) attenuates these increases in proinflammatory cytokines and iNOS, providing potential loci for the cerebroprotective actions of this peptide. 8 These cytokines, including TNF IL 1 and IL6, are associated with an increase in iNOS, as

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110 well as early invasion of neutrophils and transmigration of adhesion molecules. These proinflammatory mediators are critical to the pathogenesis of tissue damage in cerebral infarction Fo r example, after middle cerebral artery occlusion TNF IL 1, IL 6 and iNOS, as well as phosphorylated ERK1/2 are increased in smooth muscle cells of the middle cerebral artery and in associated intracerebral microvessels. Inhibition of ERK phosphorylat ion decreases the tissue damage, as well as the cytokine and iNOS production after cerebral ischemia. 32Common Mechanisms for AT2R and Mas Mediated Cerebroprotection Therefore, disruption of post stroke inflammatory pathways may be a mechanism of Ang (1 7) cerebroprotection. Further investigations of the time course and protein expression levels of these inflammatory mediators are underway. AT2Rs often mediate effects of Ang II that are exactly opposite to those m ediated by AT1R 111, and in fact the tissue levels of AT2R are dramatically increased following injury, such as in the heart following myocardial infarction, in atherosclerotic blood vessels, in wounded skin, and in the peri infarct region in the brain following ischemia 82, 96, 112114. Considering this, and the evidence that Ang II acts via AT2R in ne urons to elicit differentiation, regeneration and neurotrophic actions 115117, Unger and colleagues hypothesized that the increased expression of AT2R within the peri infarct re gion can (in the presence of ARBs to block AT1R) be activated by the raised endogenous levels of Ang II and serve a neuroprotective role 82. These investigators have supported this theory by demonstrating that the benefici al action of ARBs after MCAO induced cerebral ischemia is prevented by specific AT2 receptor blockers 82. Additional support is provided by the following experimental findings: i) ARBs are more cerebroprotective than ACE in hibitors in a rat model of ischemia and reperfusion 81; ii) MCAO produces

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111 greater ischemic brain damage in AT2R knockout mice compared with wildtype controls 80; and, iii) CNS deliv ery of an AT2R agonist (CGP 42112) provides cerebroprotection during ischemic stroke83. Although most studies have focused on Ang II stimulation of the AT2R during stroke, there is also evidence that conversion of Ang II to Ang IV which activates the AT4R might be involved in ARB mediated cerebroprotection.106 Similarly, it has been shown that Ang II conversion to Ang III is required for AT2R mediated naturetic effects of ARBs.151The AT2R is a 7 transmembrane domain G protein coupled receptor with 34% sequence homology to the AT1R. Overall, these studies demonstrate a variety of RAS components that counteract AT1R stimulation and lead to cerebroprotection during ischemic stroke. Our studies have added the components of the ACE2/Ang (1 7)/Mas axis to these AT1 opposing mechanisms during stroke. 152 Stimulation of the AT2R activates signaling ca scades that counteract many of the AT1R mediated events.153 For example, several phosphatases such as MAP Kinase Phosphatase 1 (MPK 1), SHP 1, and PP2A are activated following AT2R stimulation by Ang II41, 154. These phosphatases serve to deactivate several of the AT1R signaling members such as ERK1/2, STAT, and JAK. The increase in phosphatase activity involved in AT2R signaling can be both G protein dependent and independent. In addition to increasing phosphatase activity, AT2R stimulation increases NO, cyclic GMP formation, and bradykinin release.155, 156 The interplay between these events is s omewhat uncertain, but soluble adenylate cyclase and the bradykinin B2Like the AT2R, the Ang (1 7) receptor, Mas, is a 7 transmembrane domain G protein coupled receptor that antagonizes the AT1R through direct similar mechanisms. receptor are involved.

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112 F or example, Ang (1 7) stimulation of Mas can inhibit AT1R mediated phosphorylation of p38MAPK, ERK1/2, and JNK.69, 70 SHP 2 is activated by Ang (1 7) signaling and this molecule is involved in the disruption of c Src, ERK1/2, and NOX activation by Ang II.58 In addition, Mas activation causes eNOSstimulation via the phosphatidylinositol 3kinase (PI3K) /Akt pathway.71 Mas stimulation also causes AA release and PGI2 production, as well as potentiation of bradykinin signaling.7275CNS Pharmacotherapy Thus, it is clear that both AT2R and Mas s timulation can contribute to phosphatase activation, bradykinin receptor activation, and NO release. Similar mechanisms of AT1R signaling disruption by these receptors may indicate common mechanisms of cerebroprotection. Targeting the CNS with pharmacotherapy has proven difficult because the BBB excludes many molecules and limits their activity centrally.148 In fact, less than 5% of drugs are active in the CNS due to exclusion by the BBB.157 The limitations of many small molecules to cross the BBB are due to the relatively narrow set of characteristics that seem to allow for BBB transport. Most of the small molecules that cross the BBB have a molecular m ass less than 400 to 500 Da and are very lipophilic.148 These concerns were the rationale for designing our studies using transcranial drug delivery directly into the cerebral ventricle. Ang (1 7) is an 899 Da peptide and is hydrophilic. These characteristics are extremely likely to limit its transport across the BBB. In addition, DIZE is approximately 515 Da and is also hydrophilic. In fact, there is direct evidence of the limited ability of DIZE delivered peripherally to cross the BBB.158 Therefore, we have administered Ang (1 7) and DIZE centrally with a preventative treatment strategy in order to bypass the BBB. This route of administration is more

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113 invasive and less sustainable than peripheral delivery of drugs. Therefore, alternative strategies to activate the central ACE2/Ang (1 7)/Mas axis should be explored. Modeling Middle Cerebral Artery Occlusion There are several methods of inducing focal cerebral ischemia i n rodents and each method has numerous benefits and drawbacks. The specific method used to induce experimental ischemia is important to interpreting results of cerebroprotection studies. This difficulty in reproducing the pathophysiology of human stroke has likely led to the failure of many cerebroprotective agents during clinical trials.159, 160The intraluminal thread induced MCAO consists of inserting a filament into the common carotid artery and passing it through the internal carotid up to the junction of the anterior middle cerebral arteries. Some of the most common models of focal cerebral ischemia include the intraluminal thread in duced MCAO, ET 1 induced MCAO, surgically induced MCAO (clipping, electrocauterization, ligation), photothrombosis, embolization (blood clots, microspheres). 161 Benefits of this method include the absence of craniotomy and the ability to control the duration of isch emia and onset of reperfusion if it is desired. Drawbacks to using the intraluminal thread model include a high rate of subarachnoid hemorrhage, external carotid artery hypoperfusion, retinal damage, and involvement of the hypothalamus.159The ET 1 induced MCAO consists of a stereotactical injection of ET 1 into the brain parenchyma adjacent to the proximal portion of the middle cerebral artery. 136 Benefits of this method inc lude the ability to control artery constriction by altering the dose of ET 1 delivered, as well as no manipulation of the extracranial vessels supplying blood to the brain.162 In addition, ET 1 delivery allows for a g radual as opposed to

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114 abrupt reperfusion of the middle cerebral artery.138 Drawbacks to this method are the need for a craniotomy as well as high variability in stroke volume.159 Our experiments show that ET 1 induced vasoconstriction is at a maximum within a few minutes and gradual recanalization occurs over 30 to 40 min. Although, this clearly causes a significant amount of tissue damage and neurological deficit, the duration of occlusion may not closely mimic that of human stroke where most patients have at least partial reperfusion over a period of hours to days following occlusion.6, 7Surgically induced MCAO consists of occluding the middle cerebral artery using clips, electrocauterization, or ligation. These methods require removal of part of the mandible and zygomatic arch, as well as a craniotomy. Complex and invasive s urgical procedures are among the drawbacks of this model that have led many researchers to prefer alternative methods. It should also be note d that the rapid constriction and gradual reperfusion of the ET 1 induced MCAO might closely mimic the vasospasms seen following subarachnoid hemorrhage. Therefore, this model could also be a useful tool for studying the ischemi that occurs during vasospas m events. 163Photothrombisis can be used to induce cortical ischemia through the use of a photosensitive dye and then irradiation of the cortex. 164 Irradiation can be performed either transcortically or after performing a craniotomy. Benefits of this method are that it is a noninvasive, efficient, and reproducible procedure. Drawbacks include doubts that photothrombosis damages tissue through an ischemic mechanism and generates an ischemic penumbra.165167

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115 Embolization can also be used to induce ischemic stroke. Methods of embolization include injection of microspheres or an ex vivo prepared thrombus into either the common carotid or middle cerebral artery.159 The major benefit of this model i s the similarity to human stroke. In fact, most human ischemic strokes are embolic in nature. In addition, use of an ex vivo prepared thrombus is a good model for testing thrombolytic therapies. The drawbacks to embolizaton models are the high variabili ty of infarct and difficulty in placing the embolus directly into the middle cerebral artery in models of MCAO. In addition, e m bolization procedures have a high rate of subarachnoid hemorrhage.168170It is clear that many factors must be taken into consideration when developing animal models of ischemic stroke. For example, anesthesia is a possible source of variability when modeling ischemic stroke. Isoflurane can protect brain tissue from injury during ischemia and thus differences in dose and duration of anesthesia can affect the tissue damage that occurs during stroke. 171 The experiments of each aim were performed sequentially and the brain tissue damage increased during the ET 1 induced MCAO experiments of each aim. One possible explanation for the increase in tissue damage could be decreased times to complete surgical manipulations with experience in the required surgical techniq ues. Normalization of the data using a log of anesthesia duration should be performed in subsequent experiments to reduce variability. Consideration of factors such as anesthesia, as well as the benefits and drawbacks to various models of focal cerebral ischemia, must be taken into consideration when attempting to translate results from animal stroke experiments to human clinical trials.

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116 Strategies for Targeting the Brain ACE2/Ang(1 7)/Mas Axis in Humans Medicinal chemistry can be used to alter a compound in one of two ways to increase BBB permeability. The drug can either be altered to be more lipophilic or altered to utilize active transport mechanisms.148 The latter is more desirable because extremely lip id permeable drugs would cross the BBB, but also be eliminated faster leading to difficulties achieving an effective concentration in the brain. BBB disruption can be used to effectively deliver drugs to the CNS. Strategies for BBB disruption include the use of poorly diffusible osmotic compounds such as mannitol to shrink endothelial cells, and detergents to destabilize membranes. The use of either of these methods is associated with pathological changes due to the BBB disrupting compounds or neurotoxic plasma proteins.148, 172 In experiments using hyperosmolar lithium chloride to disrupt the BBB, concentrations of DIZE in the brain were increased compared to a vehicle control.158 Interestingly, a hyperosmolar treatment with sucrose in the same study was not effective. The discrepancy in effectiveness between sucrose and lithium to increase DIZE delivery to the brain is interesting and coadministration of lithium and DIZE should be investigated further for cerebroprotective properties. More recent strategies to delivery therapy across the BBB have included transcranial ultrasound and liposome assisted delivery of drugs.173, 174 In addition to more conventional strategies of drug delivery to the CNS, gene therapy approaches with ACE2 or secretable Ang (1 7) should be investigated. Viral delivery and endothelial specific expression of ACE2 or Ang (1 7) in the brain could provide a means of by passing the BBB.

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117 Additional Considerations about Timing and Dose of Therapy Many investigations into stroke therapeutics have focused on acute therapy after onset of stroke symptoms. However, a preventative approach is not unrealistic due to the well defined and strong risk factors for stroke, such as age and hypertension. Our experiments were designed so that treatment was administered prior to and throughout cerebral ischemia and subsequent pathology. This approach was used to efficiently determine the cerebroprotective potential of ACE2/Ang (1 7)/Mas activation. Further experiments should be performed to test the effect of acute therapy after stroke using Ang (1 7) and DIZE. If acute therapy is benefic ial, than a peripheral administration strategy may be warranted due to the BBB disruption that occurs during stroke. This would circumvent the need to investigate strategies for traversing the BBB. In addition, it is unclear whether administration of DIZ E at the dose used in these experiments activates ACE2 and generates a comparable dose of Ang (1 7) when compared to that delivered directly. Proof of principle experiments should be performed to determine the ability of DIZE to alter angiotensin peptide metabolism in the brain. Measurement of Ang II, Ang (1 7), and Ang (1 5) via high performance liquid chromatography and mass spectrometry would be an appropriate way to test this hypothesis. Conclusion Our studies have extended the known cardiovascular pr otective roles of the ACE2/Ang (1 7)/Mas axis (Figure 51). Specifically, we have shown that the ET 1 induced MCAO model of ischemic stroke can provide a rapid onset of ischemia caused by constriction of the MCA. This constriction is followed by a gradual period of vessel relaxation and reperfusion of the involved vascular territory. Our studies, have confirmed that the ET 1 induced MCAO model is appropriate for studies investigating

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118 the involvement of RAS components in stroke. For example, we have show n that neither candesartan, nor Ang (1 7) can alter the ET 1 induced vasoconstriction of the MCA. Most importantly, we have determined that central administration of Ang (1 7) prior to ET 1 induced MCAO can reduce the brain damage and neurological deficit s that occur due to ischemic injury. The effects of Ang (1 7) were mediated via its receptor, Mas. A similar Mas mediated cerebroprotective action was elicited when DIZE, an ACE2 activator, was administered prior to stroke. Neither ACE2 activation, nor Ang (1 7) administration was shown to alter CBF reductions resulting from MCAO. Thus, the mechanism of ACE2/Ang (1 7)/Mas mediated cerebroprotection remains undetermined, but preliminary studies suggest interruption of post ischemic inflammatory responses as a possibility. In summary, stimulation of the ACE2/Ang (1 7)/Mas components is a viable target for the prevention and treatment of ischemic stroke.

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119 Figure 51. ACE2/Ang (1 7)/Mas axis is cerebroprotective during stroke. ACE= Angiotensin Convert ing Enzyme, ACE2 = Angiotensin Converting Enzyme 2, Ang = Angiotensin, AT1R = Ang II Type 1 Receptor, AT2R = Ang II Type 2 Receptor, iNOS = inducible nictric oxide synthase, PIC = proinflammatory cytokines

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136 BIOGRAPHICAL SKETCH Adam P. Mecca was born in West Palm Beach, Florida, USA in 1983. He graduated with highest honors from the University of Florida in 2005 with a Bachelor of Science and a double major in chemistry and m icrobiology. He performed his undergraduate honors thesis research with Michael Katovich, Ph.D. and studied the cardioprotective properties of Angiotensin(1 7) in animal models of chronic hypertension. Adam began medical school at the University of Florida, College of Medicine in 2005 and started his graduate studies as an M.D. Ph.D. student working with Colin Sumners, Ph.D., and Michael Katovich, Ph.D. His research interests include the renin angiotensin system, cardiovascular disease, and stroke. His doctoral thesis is titled "Targeting the ACE2/Ang (1 7)/Mas axis for cerebroprotection during isch emic stroke. Adam completed his graduate research in 2010 for this work which focused on activating endogenous biological pathways in the brain to prevent or treat stroke. He is currently completing the clinical portion of his Medical Doctorate training In addition to his research interests, Adam is a CoDirector of the Equal Access Clinic, a student run free medical clinic in downtown Gainesville. The clinic serves the under insured populations of Gainesville and Alachua County. Adam became interest ed in medically under served populations when he began volunteering at the Equal Access Clinic as an undergraduate student in 2002. Since then, Adam has worked with faculty and student volunteers across the University of Florida health professions to establish and expand the patient services offered by the Equal Access Clinic. Adam is interested in utilizing student run free clinics to enhance the educational experience of health professional students and provide high quality care to patients in need. He is also the Co founder and Conference Coordinator for the Society for Student Run Free Clinics,

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137 an organization dedicated to assisting international collaboration between student run free clinics. Adam aspires to be an effective physician scientist, educat or, and healthcare provider.