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1 UNDERSTANDING THE CEREBROPROTECTIVE ACTIONS OF THE ACE2/ANG (1 7)/MAS AXIS DURING ISCHEMIC AND HEMORRHAGIC STROKE By ROBERT W. REGENHARDT A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PA RTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Robert W. Regenhardt
3 ACKNOWLEDGMENTS First, I would like to thank my Ph.D. mentor, Colin Sumners. He has been a great personal and professional role model since I was admitted to medical school in 2006 through an early acceptance program he directs. Since starting my Ph.D. in his lab in 2009, I have grown as a scientist and a person under his mentorship. Dr. Sumners has guided me thr ough graduate school and kept m e directed, while teaching me how to be a great scientist. I have a learned a great deal about designing experiments, writing manuscripts and grants leadership, administration, and mentoring. We have had some great times in a nd out of the lab over the past 3 years, and I am truly grateful for his friendship. I have enjoyed the opportunities to travel with him around the country and present our work at professional meetings. I would also like to thank my committee members, Moh an Raizada, Mike Katovich, Judy Delp, J Mocco, and Mike Waters Each has provided invaluable input toward my dissertation project over these last 3 years. Their guidance has helped me broaden my skillset as a scientist and shape the way I think. Dr. Raiza da, the PI for the adjacent lab and long time collaborator with my PI, has given me much advice over the years. One to approach a lot of problems. Dr. Katovich has also been instrumental in my scientific development and has provided great input in the design of our experiments. His expertise in the renin angiotensin system was a great complement to that of Dr. biology has given us great insight into the pathophysiology of our stroke models. I was also fortunate to learn how to do vascular reactivity experiments in her lab, while none are presented in this dissertation. Drs. Mocco and Waters have been great for not only their expertise as
4 scientists, but also clinicians. They have encouraged me to always think about translation to the bedside and given me great clinical perspectives on stroke. The many people I have worked with in the Sumners lab and other labs have been instrumental to my progress and have become good friends. Many thanks are owed to Adam Mecca, Fiona Desland Pioquinto, Neal Patel, Phillip Ritucci, Jacob Ludin, David Greenstein, Peng Shi, Ho ngwei Li, Nan Jiang, Ying Dong, Annette de Kloet, Cristina Banuelos C ristina Kitchen and Kat Marulanda I would also like to thank Ron Mandel for his guidance with behavioral exams and Jennifer Bizon for her expertise in stereology. In addition, I would like to acknowledge Marda Jorgensen from the Histology Resource lab for her i mmunohistochemistry expertise and Mary Reinhard for her expertise as a licensed veterinary pathologist. Glen Walter and Huadong Zeng were also helpful in teaching us the technical aspects of MRI. Chris Baylis also deserves special recognition as director of the Hypertension Center and PI for the NIH T32 training grant I was awarded. Finally and most importantly, I would like to thank my friends and family for their unwavering suppo rt throughout my life and during graduate school. I would especially like to thank Adam Mecca. He is one of the most outstanding friends I have ever known, and his mentorship in the lab and in life can not be overstated. I would also like to thank my home town friends Kevin Vickers and Jeremiah Ross. They have always been there for me even when we have lived hours apart. Alex Dickert and Matt Bienkowski have been truly amazing friends since I met them just before Alex and I started medical school in 2007. V ermali Rodriguez has b een one of the best people to have a few drinks with during my Ph.D studies. I cannot thank her enough f or many rides home and
5 showing me around Puerto Rico Jason Joseph and I have become great friends since meeting in lab, and I wil l never forget our amazing times driving some of the world most exotic super cars and traveling throughout Asia and Europe. There are many other friends I wish I had room to acknowledge. My grandmother, father, mother, and sister also deserve a very spec ial acknowledgement for putting up with me when I have been burdened with work and unable to spend time with them
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 3 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 STRO KE AND THE ROLE OF THE RENIN ANGIOTENSIN SYSTEM .................. 15 Stroke Epidemiology ................................ ................................ ............................... 15 Pathophysiology of Ischemic Stroke ................................ ................................ ....... 16 Pathophysiology of Hemorrhagic Stroke ................................ ................................ 21 Renin Angiotensin System Components ................................ ................................ 26 Ang (1 7)/Mas Opposes Ang II/AT1R Signaling ................................ ..................... 28 Targeting the Renin Angiotensin System for Stroke Therapy ................................ 30 Summary ................................ ................................ ................................ ................ 35 2 ANTI INFLAMMATORY EFFECTS OF ANGIOTENSIN (1 7) IN ENDOTHELIN 1 INDUCED MIDDLE CEREBRAL ARTERY OCCLUSION: POTENTIAL MECHANISM FOR CEREBROPROTECTION ................................ ....................... 46 Introduction ................................ ................................ ................................ ............. 46 Methods ................................ ................................ ................................ .................. 47 Animals and Ethical Approval ................................ ................................ ........... 47 Implantation of Intracranial Cannulae and Osmotic Pumps .............................. 48 ET 1 Induced Middle Cerebral Artery Occlusion (MCAO) ................................ 48 Pos t Mortem Analyses ................................ ................................ ...................... 49 PCR Array Hypoxia Signaling Pathway ................................ ............................ 50 qRT PCR ................................ ................................ ................................ .......... 50 Western Blotting ................................ ................................ ............................... 51 Mas Immunohistochemistry ................................ ................................ .............. 52 Nitric oxide Production by Primary Glial Cultures ................................ ............. 53 HPLC Analysis of Ang (1 7) ................................ ................................ ............. 54 Chemicals ................................ ................................ ................................ ......... 54 Data analysis ................................ ................................ ................................ .... 54 Results ................................ ................................ ................................ .................... 55 Cerebroprotective Ang (1 7) Reduces Infarct Size 24 h after ET 1 induced MCAO ................................ ................................ ................................ ........... 55 Increased Expression of Hypoxia related Genes in the Ipsilateral Cerebral Cortex following ET 1 induced MCAO; Inhibitory Action of Ang (1 7) ........... 55
7 Effects of Ang (1 7) on the Expression of Ni tric Oxide Synthase (NOS) Isozymes, PICs, and Inflammatory Cell Markers in the Ipsilateral Cerebral Cortex following ET 1 induced MCAO ................................ .......................... 56 Cellular Localization of Mas within Rat Cerebral Corte x ................................ ... 58 Ang (1 7) Blunts LPS Induction of NO in Primary Mixed Glial Cultures ............ 59 Discussion ................................ ................................ ................................ .............. 60 3 ANGIOTENSIN (1 7) HAS CEREBROPROTECTIVE POTENTIAL IN HEMORRHAGIC STROKE AND INCREASES SURVIVAL OF STROKE PRONE SPONTANEOUSLY HYPERTENSIVE RATS ................................ ........... 77 Introduction ................................ ................................ ................................ ............. 77 Methods ................................ ................................ ................................ .................. 79 Ethical Approval and Animals ................................ ................................ ........... 79 Implantation of Intrac ranial ICV Cannulae and Osmotic Pumps ....................... 79 Hemorrhage Number and Severity Assessment ................................ .............. 80 Hermorrhage Volume ................................ ................................ ....................... 81 Brain Water Content ................................ ................................ ......................... 81 Video Tracking and Locomotor Activity ................................ ............................ 82 Sunflower Seed Task ................................ ................................ ....................... 82 Morris Water Maze ................................ ................................ ........................... 83 Mas Co localization Immunohistochemistry ................................ ..................... 83 Stereology and Immunostaining ................................ ................................ ....... 84 qRT PCR ................................ ................................ ................................ .......... 86 Direct Blood Pressure ................................ ................................ ...................... 87 Indirect Blood Pressure ................................ ................................ .................... 87 Body weight ................................ ................................ ................................ ...... 88 Corticosterone ................................ ................................ ................................ .. 88 Creatinine and BUN ................................ ................................ ......................... 88 Kidney and Heart Pathology ................................ ................................ ............. 88 Chemicals ................................ ................................ ................................ ......... 89 Data Analysis ................................ ................................ ................................ ... 90 Results ................................ ................................ ................................ .................... 90 Ang (1 7) Treatment Improves Survival of Stroke prone Spontaneously Hypertensive Rats ................................ ................................ ......................... 90 Ang (1 7) Treatment Decreases the Number and Severity of Hemorrhages in the Subcortex/Striatum, but not in the Cortex at the Time of Death of spSHR ................................ ................................ ................................ ........... 90 Ang (1 7) Treatment has no Effect on Brain Hemorrhage Volume (Hemoglobin Content) or Water Content ................................ ....................... 91 Ang (1 7) Treatment Improves the Neurological Status of spSHR, but has n o Effect on Visuospatial Memory ................................ ................................ 91 Cellular Localization of Immunoreactive Mas in spSHR Striatum ..................... 92 Ang (1 7) Decreases Microgli a and Increases Neuron Survival in the Striatum ................................ ................................ ................................ ......... 93 Ang (1 7) does not Alter the Expression of Genes Related to Inflammation in Whole Brain Homogenate ................................ ................................ ......... 93
8 Ang (1 7) has no Effect on Mean Blood Pressure during Infusion in spSHR, but Rats Treated with Ang (1 7) have Lower Mean Blood Pressure later in Life after Treatment Ends ................................ ................................ .............. 94 Ang (1 7) has no Effect on Body Weight, Corticosterone, Creatinine, BUN, or Kidney/Heart Pathology ................................ ................................ ............ 94 Discussion ................................ ................................ ................................ .............. 95 4 SUM MARY AND CONCLUSIONS ................................ ................................ ........ 111 Summary ................................ ................................ ................................ .............. 111 Specific Aim 1: Investigate the Mechanism of the Cerebroprotective Actions of Ang (1 7) in Ischemic Stroke ................................ ................................ ... 111 Specific Aim 2: Determine whether Ang (1 7) also Exerts Cerebroprotective Actions in Hemorrhagic Stroke ................................ ................................ .... 112 Discussion ................................ ................................ ................................ ............ 114 LIST OF REFERENCES ................................ ................................ ............................. 123 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 145
9 LIST OF TABLES Table page 2 1 PCR array data showing fold changes in gene expression in ipsilateral (right) cerebrocortical tissue 6 h after endothelin 1 (ET 1) induced middle cerebral artery occlusion (MCAO) strok e compared to sham controls (0.9% saline instead of ET 1). ................................ ................................ ................................ 67 2 2 PCR array data showing fold changes in gene expression in ipsilateral (right) cerebrocortical tissue 24 h after ET 1 induced M CAO stroke compared to sham controls. ................................ ................................ ................................ .... 67 2 3 Degradation of Ang (1 7) in media of primary mixed glial cultures ..................... 68 3 1 Parameters used for stereology. ................................ ................................ ......... 99
10 LIST OF FIGURES Figure page 1 1 Prevalence of silent cerebral i schemia for age ranges ................................ ....... 38 1 2 Two types of stroke ................................ ................................ ............................ 39 1 3 Time course of mediat ors in ischemic stroke damage ................................ ........ 40 1 4 The two sides of NO. ................................ ................................ .......................... 41 1 5 RAS components ................................ ................................ ................................ 42 1 6 Ang (1 7) opposes AT1R signaling ................................ ................................ ..... 43 1 7 Renin Angiotensin System axes are involved i n the pathophysiology of stroke 44 1 8 Summary of our focus on the therapeutic potential of the RAS .......................... 45 2 1 Intracerebral pretreatment with Ang (1 7) reduces infarct size 24 h after ET 1 induced MCAO ................................ ................................ ................................ 69 2 2 Gene expression in ipsilateral (right) cerebrocortical tissu e 6 h after ET 1 induced MCAO ................................ ................................ ................................ ... 70 2 3 Gene expression of NOS isozymes and pro inflammatory cytokines in ipsilateral (right) cerebrocortical tiss ue 24 h after ET 1 induced MCAO ............. 71 2 4 Gene expression of cell markers and migratory factors in ipsilateral (right) cerebrocortical tissue 24 h after ET 1 induced MCAO ................................ ....... 72 2 5 Levels of iNOS protein in ipsilateral (right) cerebrocortical tissue 24 h after ET 1 induce d MCAO stro ke or sham procedure ................................ ................. 73 2 6 Cellular localization of immunoreactive Mas in normal rat cerebral cortex a nd 24 h after ET 1 induced MCAO ................................ ................................ .......... 74 2 7 Ang (1 7) blunts lipopolysaccharide (LPS) induced increases in nitrite in the media of primary mixed gli al cultures ................................ ................................ 76 3 1 Ang (1 7) treatment improves su rvival of stroke prone Spontane ously Hypertensive Rats (spSHR) ................................ ................................ .............. 100 3 2 Ang (1 7) treatment decreases the number of hemorrhages in the subcortex/striatum, but not in the cortex at the t ime of death of spSHR ........... 101 3 3 Ang (1 7) has no effect on brain hemor rhage volume or water content ............ 102
11 3 4 Ang (1 7) treatment improves neurological status of spSHR ............................ 103 3 5 Ang (1 7) does not improve performance visuospatial memory as assessed by the Morris Water Maze Task ................................ ................................ ........ 104 3 6 Cellular localization of immun oreactive Mas in spSHR striatum ....................... 105 3 7 Ang (1 7) decreases activated microglia and increases neu ron survival in the stri atum just before treatment ends ................................ ................................ .. 106 3 8 Ang (1 7) does not alter the expression of genes related to inflammation in whole brain homogenate after treatment ends ................................ ................. 107 3 9 Ang (1 7) administered intracerebroventricularly (ICV) has no effect on mean blood pressure during infusion in spSHR, but rats treated with Ang (1 7) have lower mean blood pres sure later after treatment ends ................................ ..... 108 3 10 Ang (1 7) has no effect on body weight, cor ticosterone, creatinine, or BUN ... 109 3 11 Ang (1 7) has no effect on kidney or heart pathology just before tr eatment ends ................................ ................................ ................................ ................. 110 4 1 Schematic of proposed mechanism ................................ ................................ .. 122
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 UNDERSTANDING THE CEREBROPROTECTIVE ACTIONS OF THE ACE2/ANG (1 7)/MAS AXIS DU RING ISCHEMIC AND HEMORRHAGIC STROKE By Robert W. Regenhardt August 201 2 Chair: Colin Sumners Major: Mecial Sciences Physiology and Pharmacology A recently characterized axis of the renin angiotensin system (RAS) has been the focus of much research i n cardiovascular therapeutics. This axis is composed of angiotensin converting enzyme 2 (ACE2), which generates angiotensin (1 7) (Ang (1 7)) from angiotensin II (Ang II). Ang (1 7) the n exerts its effects through its receptor, Mas. Activating this axis in any number of ways is proving to have therapeutic potential in several disease states, including stroke. We previously showed that exogenous central Ang (1 7) is cerebroprotective in an endothelin 1 (ET 1) induced model of middle cerebral artery occlusion performance on neurological exams. These effects were observed without any changes to cerebral blood flow, percent vessel constriction, or blood pressure. Because of this, we began thinking of alternative mechanisms for this cerebroprotection. Specific Aim 1 was desgined to screen for changes in the expression of certain genes that may be involved in decreasing the damage due to ischemic stroke using the ET 1 induced MCAO model In this study a PCR array was used to identify several markers of inflammation that were increased in stroke and blunted by ICV Ang (1 7) therapy. It has previously been shown that damage due to stroke may be amplified by
13 excessive inflammation. The findings from the ar ray and changes in the expression of A western blot was used to show that the changes in iNOS mRNA also existed at the protein level. To uncover which cell types were involve d, we performed a co localization study with the Ang (1 7) receptor Mas and different cell markers. Microglia, endothelial cells, and neurons express Mas. Furthermore, we showed that Ang (1 7) has a Mas dependent in vitro anti inflammatory effect to blunt nitric oxide production in a mixed glia culture system after LPS insult These findings indicate for the first time an anti inflammatory property of Ang (1 7) treatment in the CNS and provide a potential mechanism of cerebroprotection. As we demonstrated A ng (1 7) had an in vivo anti inflammatory effect during MCAO in rats and an in vitro anti inflammatory effect in a mixed glia culture system in Specific Aim 1, Specific Aim 2 was designed to examine the therapeutic potential of this peptide in hemorrhagic stroke. There is much overlap in the pathophysiology of ischemic and hemorrhagic stroke, especially regarding the role of excessive inflammation We fed stroke prone Spontaneously Hypertensive Rats (spSHR) a high salt diet from the time of wea ning and admi nistered Ang (1 7) centrally for 6 weeks starting at 49 days of age. Ang (1 7) increased the survival of spSHR and decreased the number of hemorrhages in the striatum. spSHR treated with Ang (1 7) also showed less lethargy and improved neurological status. We confirmed the presence of the Ang (1 7) receptor Mas on neurons, microglia, and endothelial cells and showed that Ang (1 7) decreases the number of microglia and increases the number of neurons in the striatum. There was no effect of Ang (1 7) on marke rs of peripheral disease, such as
14 ki d n ey pathology, heart pathology, body weight, corticosterone levels, or extent of hypertension. This supports that the actions of this peptide are indeed primarily central in our model These findings indicate for the fi rst time ICV Ang (1 7) has cerebroprotective actions in hemorrhagic stroke and can increase the survival of spSHR.
15 CHAPTER 1 STROKE AND THE ROLE OF THE RENIN ANGIOTENSIN SYSTEM Stroke Epidemiology In the United States, stroke is the fourth leading cause of death and a major cause of severe, long term disability. A stroke occurs every 40 seconds and results in a death every 4 minutes. Each year 795,000 people experience a stroke, with primary strokes accounting for 610,000 and secondary strokes accounting for 185,000. Stroke prevalence in adults is 2.6% for men, 2.6% for women, 2.4% for Caucasians, 4.0% for blacks, 1.4% for Asians, 2.5% for Hispanics, and 5.8% for Native Americans 1 The estimated cost attributed to tre ating stroke in 2009 was $68.9 billion. Risk factors for stroke include: atrial fibrillation, hypertension, diabetes, smoking, advancing age, low levels of physical activity, dyslipidemias, hormone replacement therapy, and pregnancy/postpartum period 2 Patients who have a primary stroke or transient ischemic attack are also at an increase risk of having a recurrent stroke 3 Interestingly, there are many subclinical infarcts th at are discovered incidentally during imaging studies. The prevalence of these silent cerebral infarctions increases dramatically with age (Figure 1 1) 4 A multitude of evidence supports the importance of studies for preventing and treating stroke. Strokes can be classified into two types: ischemic and hemorrhagic (Figure 1 2). Ischemic stroke occurs when blood supply to the brain is decreased by a blocked vessel, depriving the brain of oxygen and nutrients. Hemorr hagic stroke, in contrast, occurs when a blood vessel within the brain ruptures and releases blood into the brain 2 Of all reported strokes, 80 90% are ischemic strokes and 10 20% are an intracerebral hemorrhagic strokes and subarachnoid hemorrhages. The treatment of ischemic
16 strokes is focused on restoring cerebral blood flow to the affected vascular territory, currently most commonly through the use of systemic administration of recombinant tissue Plasminogen Act ivator (rtPA). Despite the existence of rtPA, only 4 8% of patients with stroke are candidates for thrombolytic therapy because of the high risk of hemorrhagic transformation and a limited window of time for treatment after stroke onset 4 On the other hand, hemorrhagic strokes, while being less prevalent, can be even more difficult to treat with virtually no medical therapies. Furthermore, hemorrhagic stroke has a disproportionate affinity for Asians and African Am ericans 5 Patients with hemorrhagic strokes have very poor prognoses, often much worse than ischemic strokes of similar size. In fact, up to 30% of patients with ischemic stroke undergo hemorrhagic transformation 6 and many patient s with ischemic strokes develop hemorrhages and symptomatic hematomas following thrombolysis 7 Regardless of type, the outcome of stroke is essentially the same. Neuro nal death and consequent behavioral and motor symptoms result from the absence of oxygen and nutrients and/or sudden bleeding into the brain. It is of critical importance to advance our understanding of the mechanisms underlying stroke and to investigate n ew strategies for therapeutics as stroke has an extremely high prevalence and there are very few therapeutic options. Pathophysiology of Ischemic Stroke The greatest risk factors for ischemic stroke are hypertension and other cardiovascular dysregulation/ disease. The etiology of ischemic stroke can be classified into a few different categories that aid in understanding the pathophysiology and treatment 8 : cardioembolic stroke, large artery atherosclerosis, small arte ry occlusion, stroke of other determined causes, and stroke of undetermined causes 9 A majority of
17 ischemic strokes are thromboembolic, with the middle cerebral artery being most commonly affected 10 Most patients who suffer from ischemic stroke have some degree of reperfusion either spontaneously or as a result of thrombolytic therapy 10, 11 A cascade of pathologic al events is associated with this sequence of ischemia followed by reperfusion, progressing through cerebral hypoperfusion, excitotoxicity, oxidative stress, blood brain barrier dysfunction, microvascular injury, hemostatic activation, post ischemic inflam mation, and cell death 12 Within the ischemic core this cascade progresses quickly, resulting in a loss of neuronal function demonstrated by EEG flattening and irreversible cell death due to severe hypoperfusion. In co ntrast, within the surrounding tissue, or the ischemic penumbra, there is less severe hypoperfusion and the tissue remains viable. The penumbra is believed to be the primary target for treatment since it can be saved. In the penumbra there is a loss of neu ronal function demonstrated by EEG flattening, but there is no cell death 13 16 Tissue in this area can, however, progress to irreversible injury and expand the ischemic core depending on the severity and duration of ischemia 12 Necrosis, the type of cell death involving energy depletion and loss of ionic gradients resulting in cellular swelling and membrane dis ruption, occurs during stroke. It occurs primarily in the ischemic core, where a lack of oxygen and glucose results in diminished ATP levels 17, 18 This results in inhibition of the Na+ K+ ATPas e causing cell depolarization and release of neurotransmitters, including glutamate, from neurons and astrocytes 19 21 Excess glutamate activates ionotropic glutamate recepto rs on neurons and astrocytes that causes release of more glutamate in positive feedback fashion to increase intracellular calcium and cause excitotoxicty 22 Furthermore, with decreased
18 oxygen levels there is electron leakage from the electron transport chain generation of reactive oxygen species 23 Protons and lactate also accumulate due to glycolysis in the absence of oxidative phosphorylation 18 These cells in the core undergo terminal depolarization as a result of the dramatic decrease in blood flow. Cells in the penumbra undergo transient ischemic depolarization as the reduction in blood flow is not as great. Still, transient depolar ization can evolve into terminal depolarization and expand the core 24 Apoptosis, parthanatos, and autophagy, types of programmed cell death, also occur during stroke. In contrast to necrosis, programmed cell death o ccurs primarily in the ischemic penumbra, but can also occur in the core to some extent 25 The process of apoptosis differs from necrosis in that cell death occurs with minimal inflammation and energy must be expend ed during the process 12 When mitochondria release cytochrome c, apoptotic cascades are activated that activate caspase 3, a common effector molecule in apoptosis. Parthanatos is programmed cell death that occurs indep endent of caspase activity. In parthanatos, ischemic reperfusion injury involves an increase in p oly(ADP ribose) (PAR) polymer from poly(ADP ribose) polymerase 1 (PARP1) 26 PAR polymer translocates from the nucleu s to the cytoplasm and mitochondria, where it causes the release of apoptosis inducing factor (AIF) from mitochondria 27 Parthanatos is believed to be very involved in ischemic penumbra injury 28 AIF can also be released independent of parthanatos. The calcium dependent protease calpain I can cleave an N terminal fragment of AIF that results in its release from mitochondria. AIF and calpain I have been implicated as being importa nt in cell death during stroke as inhibiting them is neuroprotective 28 Autophagy is another
19 programmed cell death that can be triggered by endoplasmic reticulum stress and oxidative stress 29 It is normally involved in the organelle and protein turnover, but can be involved in cell death during ischemia. Autophagy promoting beclin 1 and microtubule associated protein 1 are upregulated in the penumbra, and inhibition of these h as been shown to decrease cell death during ischemia 30 32 Reperfusion injury also plays a big role in the pathophysiology of ischemic stroke. Reperfusion can occur after ischemia either spontaneously or due to thrombolytic therapy. It has the potential to create a paradoxical injury to brain tissue despite the improved cerebral blood flow that can occur. This is due primarily to the production of reactive oxygen species by NADPH o xidase 23 Reperfusion also leads to the release of proinflammatory cytokines which activate inflammatory cascades. These can lead to breakdown of the blood brain barrier by matrix matalloproteinases and microvascul ar injury resulting in brain swelling and hemorrhagic transformation 12, 33 Despite the risks of reperfusion injury, much evidence suggests that early reperfusion is associated with improved fi nal outcomes compared to delayed reperfusion 10 Excessive inflammation plays a significant role in the damage due to stroke, as well. During stroke, proinflammatory cytokines are released by astrocytes, microglia, s mooth muscle cells, and endothelial cells 12 These cytokines, including TNF IL 1, and IL 6, are associated with an increase in inducible nitric oxide synthase (iNOS), activation of microglia, early invasion of neut rophils, and transmigration of adhesion molecules. Inflammation that occurs at the blood endothelium interface, involving cytokines, adhesion molecules, chemokines, and leukocytes is a major cause of the tissue damage in stroke 34 After middle cerebral artery occlusion, TNF IL 1, IL 6,
20 iNOS, and phosphorylated ERK1/2 are increased in smooth muscle cells of the middle cerebral artery and in associated intracerebral microvessels. Inhibiting the phosphorylation of ERK decreases the tissue damage and the cytokine and iNOS pro duction after cerebral ischemia 35 Inflammatory effects take place over a time course following the initial ischemic event (Figure 1 3). Early immediate genes are activated within a few hours. These include c fos, c jun, and zif 268. Soon after, the heat shock proteins, HSP70 and HSP72, are activated. Beginning after a few hours and peaking after a day, the cytokines and adhesion molecules are upregulated. Lastly, other late inflammatory mediators and apoptosis genes, such as iNOS, COX 2, and p53, are upregulated beginning around 12 hours and peaking at 2 days 36, 37 The expression of iNOS has been shown to be dramatically upregulated in the brain following cerebral ischemia 38 Furthermore, iNOS specific inhibition and gene knockout has been shown to decrease infarct size and improve performance of neurological exams in a rat model of MCAO 39, 40 The expression of iNOS has been confirmed in human cerebral infarcts 41 It is important to point out that nitric oxide (NO), the product of NOS isozymes, plays an important phy siologic al role in lower concentrations in the pM nM range. At these levels, it is usually produced by endothelial NOS (eNOS) and is important for vasodilation and neurotransmission. However, at mM range, it is usually produced by iNO S and leads to nitrosylation of proteins and cell damage (Figure 1 4) 36, 37 It should be stated that inflammation in general is not a bad process. There is much evidence about inflammatory injur y after stroke, but inflammation can also be a protective process that attempts to limit damage and restore tissue architecture by eliminating damaged cells
21 and repairing extracellular matrix 42 The damage occurs wh en inflammatory responses are uncontrolled. Pathophysiology of Hemorrhagic Stroke Hypertension is the main risk factor for spontaneous hemorrhagic stroke. Increased blood pressure can result in the formation of microaneurysms at the bifurcation of arterio les. Persistent hypertension has been shown to damage the walls of small vessels 43, 44 Hemorrhagic strokes most commonly occur in the putamen, caudate, and thalamus. They can also occur in the h emispheres ( lobar ) cerebellum, or pons. The key factor that determines outcome in hemorrhagic stroke is hemorrhage volume; bleeds that are greater 150 mL cause cerebral perfusion pressure to drop to zero, resulting in death. Most patients with bleeds smal ler than 140 mL, however, will survive the initial ictus. Unfortunately, the hematoma itself can lead to secondary brain injury in these patients resulting in severe neurological deficits and possibly delayed death 45 Early pathological changes and injuries of hemorrhagic stroke include hematoma expansion, midline shift, apoptosis, and necrosis. The physical disruption of adjacent tissue and mass effect of the resulting hematoma accounts for part of the injury during hemorrhagic stroke. More than two thirds of patients stop bleeding shortly after the ictus 46 and about one third experience hematoma enlargement 47 52 This enlargement contributes to midline shift and hastens neurological deterioration 47, 53 Most re hemorrhage occurs wit hin the first 24 hours, however the precise mechanism for this is unknown 50, 52 Various models show that apoptosis takes place in the brain adjacent to a hemorrhage 45 50, 52 59 Apoptosis has also been detected in the perihematomal zone in
22 humans 60 Necrosis, likely caused by mechanical forces and chemical toxicity, is also found adjacent to th e hematoma 61 Edema also develops almost immediately after hemorrhagic stroke and peaks a few days later 61 64 Unfortunatel y, this increasing edema raises intracranial pressure and can result in herniation 65 In animal models, perihematomal edema has been shown to be present primarily in white matter 15, 66 Edema occurs in three phases. First, in the initial few hours, hydrostatic pressure increases and clot retraction occurs where serum moves from the clot into the surrounding tissue 62 Second, in the first two days, the coagulation cascade is activated and thrombin production is increased. Third, erythrocytes lyse and hemoglobin toxicity occurs. The main form of edema in hemorrhagic stroke is vasogenic. This is primarily due to disrupti on of the blood brain barrier following stroke. Although the blood brain barrier remains intact to large molecules for the first several hours following hemorrhage 62 the permeability is dramatically increased by 8 12 hours 67 A prolonged mass effect resulting in physical trauma and reduced cerebral blood flow also contributes to injury mechanisms that may account for damage in hemorrhagic stroke. A comparison of autologous whol e blood to an oil wax mixture showed that although edema was present following both injections, it was more severe around the clots formed from the blood 61 This suggests that the edema observed in hemorrhagic stroke is not generated by a mass effect alone. In a rat model, microballoon inflation in the basal ganglia increased intracranial pressure, transiently reduced cerebral blood flow, and caused brain damage 68 In patients, h ematoma mass causes neurological deficits, but the mass alone induces little edema immediately after hemorrhage.
23 Controversy exists in the field about the role of secondary ischemia due to the mass effect. Rat studies have shown that cerebral blood flow ne ar hematomas does decrease, but only partially and transiently 67, 69, 70 A dog model demonstrated that no ischemic zone could be found in the first 5 hours. Furthermore, ne ither thrombin nor hemoglobin, two main causes of brain damage in hemorrhagic stroke, significantly decreases cerebral blood flow in animals 63, 71 In studies of patients, oxygen extraction fraction was reduced in the perihematomal zone compared to the contralateral side due to decreased cerebral metabolic rate of oxygen and cerebral blood flow 72 This suggests that, at least acutely, there is a zone of hypoperfu sion without ischemia. MRI studies confirmed no cerebral ischemia in the area surrounding the hematoma 73 Cerebral blood flow is reduced, however, as shown by single photon CT 74, 75 General atrophy of the brain has been observed in patients with hemorrhagic stroke 76 and in rat models 64, 77 In a rat model ipsilateral striatum volume was reduced by 20% with an increased ipsilateral ventricular size 3 months after the hemorrhage was initiated 77 Thrombin also plays a role in the mechanism of injury in hemorrhagic st roke. A serine protease essential to the coagulation cascade, thrombin is produced immediately in the brain following hemorrhagic stroke. Large dose infusions of thrombin into the brain cause inflammatory cell infiltration, mesenchymal cell proliferation, scar formation, edema formation, and seizures in animal models 71, 71, 78 83 High concen trations of thrombin also induce apoptosis 84 and kill both astrocytes and neurons in vitro 85 87 Further in vitro studies have demo nstrated that thrombin can potentiate glutamate N methyl D aspartate receptor function 88 and activate microglia 89 Two thrombin inhibitors, hirudin and argatroban, have been shown to inhibit edema formation in a rat
24 model of hemorrhagic stroke 82, 90 Argatroban was also shown to reduce edema when given systemically to patients 24 hours after hemorrhage 91 Interestingly, low concentrations of thrombin, however, are believed to be neuroprotective 85, 86, 92 In addition to cleaving fibrinogen to fibrin, th rombin has receptor mediated effects at three protease activated receptors, PAR 1, PAR 3, and PAR 4 93 95 These receptors are expressed in both neurons and astrocytes 96, 97 and have been shown to mediate some of the pathological effects of thrombin in brain pathophysiology 98 100 Hu man brain tissue has also been confirmed to possess thrombin receptor immunoreactivity 100 Hemoglobin toxicity is another mechanism of injury resulting from hemorrhagic stroke. Erythrocyte lysis occurs several days after the initial hemorrhage in rats, dogs, and human patients 78, 101, 102 Evidence exists to suggest that erythrocyte lysis may be related to delayed brain edema and associat ed midline shift 53 In a dog model, hemoglobin concentrations in the cerebrospinal fluid peaked 2 days after blood injection into the subarachnoid space 103 This demonstrated that erythrocyte lysis can occur remarkably early. Furthermore, hemoglobin and heme were present in the perihematomal zone one day after hemorrhagic stroke in a rat model 104 This result is confirmed by other stud ies that erythrocyte lysis can occur within 24 hours 105, 106 Experimental infusion of lysed erythrocytes to show the toxicity of their contents, when compared to whole packed erythrocytes, results i n more edema formation, disruption of the blood brain barrier, strong neuronal expression of heatshock protein 70, and DNA injury within 24 hours 63, 78, 107, 108 In the brain, heme oxygenase degrades heme into iron, carbon monoxide, and biliverdin 109 The infusion of hemoglobin and its degradation products directly has also been shown to cause brain damage 110, 111
25 Furthermore, inhibitors of heme oxygenase and iron chelators reduce this injury and edema 106, 110, 112, 113 Brain non heme iron increases three fold after hemorrhagic stroke in rats, resulting in iron overload 105 Inflammation and complement have a strong role in the mechanism of injury in hemorrhagic stroke, as well. The infl ammatory response begins soon after hemorrhage and peaks several days later in humans and animal models 58, 101, 114, 115 The infiltration of neutrophils occur s within 2 days, and the activation of microglia may continue for 1 month 114, 116 Evidence supports a role of microglia in hemorrhagic stroke pathology because inhibiting their activation reduces d amage 117, 118 Inflammation is also associated with the production of matrix metalloproteinases, which have been shown to increase in collagenase induced models of hemorrhagic stroke. These enzymes hav e been shown to disrupt the blood brain barrier and cause secondary injury, but their precise role remains controversial 119 121 In the healthy brain, complement is unable to cross t he blood brain barrier. In the hemorrhaged brain, however, it can enter the brain parenchyma through the extravasated blood or through the degraded barrier. Evidence supports that the complement cascade is activated in hemorrhagic stroke and that it contri butes to edema; when the complement cascade is inhibited, there is decreased perihematomal edema in rats. One complement component in particular, the membrane attack complex (MAC), is hypothesized to lyse erythrocytes, neurons, glia, and endothelial cells 122 MAC also causes the release of cytokines, oxygen radicals, and matrix proteins. There is believed to be a strong association between inflammation and edema, as evidenced by data that correlates plasma tumor necrosis factor alpha concentrations in patients with hemorrhagic stroke to the degree of brain edema 123
26 Renin Angiotensin System Components The renin angiotensin system (RAS) (Figure 1 5) was first discovered as a circulat ing endocrine system. Its components are produced in various organs and secreted into the blood 124 Renin is an enzyme produced in the kidneys that converts liver derived angiotensinogen (ATG) to angiotensin I (Ang I). Another enzyme, angiotensin converting enzyme (ACE), is made in the pulmonary endothelium and converts Ang I to Angiotensin II (Ang II) 125 Highly involved in blood pressure (BP) regulation, fluid balanc e, and electrolyte homeostasis, Ang II is one of the primary mediators of RAS activity 124, 126, 127 It has been shown that several Ang II receptor types exist 128 130 Most of the classically described physiological effects of Ang II are mediated through the Ang II type 1 receptor (AT1R). Together, these components make up what is r eferred to as the ACE/Ang II/AT1R axis of the RAS. Another receptor for Ang II, the Ang II type 2 receptor (AT2R) primarily opposes AT1R action 124, 131 Several drugs have been developed that target the RAS for the treatment of hypertension, most aiming to block the ACE/Ang II/AT1R axis. At present, ACE inhibitors (ACEi) and AT1R blockers (ARBs) are among the first line drugs to treat hypertension 132 Rec ent advances in the RAS field have demonstrated that this system is more ubiquitous than just a circulating endocrine system. In fact, there are several tissue specific paracrine RAS, which have been identified in the brain, heart, kidneys, pancreas, skin, intestines, and many other organs 133 Ang II acting through AT1R is heavily implicated in the promotion of stroke and cardiovascular disease. Activation of AT1R in both the circulation and specific organs increases BP and promotes organ damage. In contrast, activation of AT2R opposes AT1R and counters the deleterious effects 134 Another axis of the RAS also opposes
27 AT1R, known as the angiotensin converting enzyme 2 (ACE2)/angiot ensin (1 7) (Ang (1 7))/Mas axis. ACE2 was recently discovered in 2000 and has been shown to convert Ang II to Ang (1 7) with high efficiency 135 137 The receptor for Ang (1 7) was discovered to be a G protein coupled receptor, Mas, which was initially identified as an orphan protooncogene in 2003 138 Although the primary way by which Ang (1 7) is generated is through the action of A CE2 on Ang II, several other pathways for Ang (1 7) production exist. These pathways can be either ACE dependent or ACE independent. ACE dependent pathways involve the conversion of either Ang I to Ang II by ACE then Ang II to Ang (1 7) by ACE2 135 or the conversion Ang I to Ang (1 9) by ACE2 then Ang (1 9) to Ang (1 7) by ACE 136 However, ACE2 is 400 times more efficient at catalyzing the conversion of Ang II to Ang (1 7) tha n Ang I to Ang (1 9), so the pathway involving Ang (1 9) as an intermediate is likely not as important 139 ACE independent pathways to generate Ang (1 7) involve other enzymes, including neprilysin, prolyl endopeptid ase, prolyl carboxypeptidase, chymase, and cathepsin A 140 143 Each of these enzymes, however, lacks specificity for angiotensin peptide metabolism a nd is likely only a minor contributor to Ang (1 7) synthesis under physiological conditions. They may become more relevant during pharmacological ACE inhibition. Therefore, the predominant theory in the RAS field is that ACE2 is the key regulator for the g eneration of Ang (1 7). As more is learned about the RAS, more components are being discovered making this system increasingly complex. The (pro)renin receptor ((P)RR), discovered in 2002, has been shown to be a receptor for both renin and its precursor pr orenin. Binding of renin to (P)RR increases the conversion of ATG to Ang I, and ultimately
28 results in activation of ACE/Ang II/AT1R axis 144 In addition, binding of either ligand can have Ang II independent effects th rough an intracellular signaling cascade that can trigger the expression of profibrotic genes 145 Other more recently described RAS components, including Ang III and Ang IV, and the enzymes that produce them, amino peptidase A and aminopeptidase N, have some pressor effects and also their own unique physiological and pathological actions such as memory function 146 The RAS is a complex system, containing checks and balances to reg ulate many physiological actions, including cardiovascular function. Many pathways within this system provide good targets for cardiovascular disease and stroke drug development. Ang (1 7)/Mas Opposes Ang II/AT1R Signaling AT1R activation has been shown t o be involved in the pathophysiology of many cardiovascular diseases, including hypertension, cardiac hypertrophy, cardiac fibrosis, atherosclerosis, myocardial infarction, stroke and many others 137 ment in these disorders is mediated through several functions, such as contraction growth, altering cell migration, endothelial dysfunction, expression of proinflammatory cytokines, and modification of extracellular matrix 147 Interestingly, Mas activation has been shown to counteract much of these pathological functions (Figure 1 6) 148 Targeting this interplay between AT1R and Mas signaling cascades may prove to have novel t herapeutic potential. The AT1R is a seven transmembrane G protein coupled receptor that mediates known effects 149 These effects are mediated through several signaling cascades, some G prot ein coupled and others non G protein coupled. Phospholipase C (PLC), phospholipase D (PLD), and phospholipase A 2 (PLA 2 ) mediate the AT1R induced G protein coupled pathways 150, 151 AT1R indu ced activation of PLC
29 converts phosphatidylinositol 4,5 bisphosphate ( PIP 2 ) into inositol 1,4,5 triphosphate (IP3) and diacylglyerol (DAG). IP 3 can then bind to a receptor on the sarcoplasmic reticulum to increase intracellular Ca 2+ which binds to calmodul in and activates myosin light chain kinase (MLCK). In turn, MLCK phosphorylates the myosin light chain to cause smooth muscle cell contraction 152 At the same time DAG activates Protein Kinase C (PKC) which stimulates th e Ras/Raf/MEK/ERK pathway causing vasoconstriction and promoting cellular growth and proliferation 153 Also, stimulation of AT1R and subsequent activation of PLD hydrolyzes phosphatidylcholine (PC) to choline and p hosphatidic acid (PA) 147 PA is then converted to DAG, which activates PKC to have the effects described above. Lastly, AT1R induced activation of PLA2 cleaves arachidonic acid (AA) from PC. AA, through the actions o f lipoxygenase (LO) and cyclooxygenase (COX), is converted to leukotrienes (LT) and prostaglandins (PG), respectively. LT and PG then influence smooth muscle contraction and inflammation 154 In contrast, NAD(P)H oxid ase (NOX) mitogen activated protein kinases (MAPK), Src, JAK/STAT, FAK, Pyk2, and receptor tyrosine kinases mediate the AT1R induced non G protein coupled pathways 154 Evidence suggests that non G protein and G prot ein pathways may actually be closely integrated. For instance, A T1R stimulation can activate NOX via phosphorylation of Src/EGFR/PI3 K/Rac 1 and PLD/PKC/p47phox. NOX then generates superoxide, hydrogen peroxide, and other reactive oxygen species (ROS). The phosphorylation of p38MAPK, Akt/PKB, Src, EGFR, and other messengers requires this activity 155 These intracellular messengers and signaling molecules are heavily implicated in cell proliferation, endothelial dy sfunction, inflammation, and atherosclerosis. In addition, AT1R stimulation activates MAPK signaling pathways.
30 ERK1/2, JNK, and p38MAPK are some examples that contribute to cell differentiation, proliferation, migration, and fibrosis in vessel walls. AT1R induced activation of nonreceptor tyrosine kinases, such as c Src, activate many downstream components, including Ras, JAK/STAT, PLC, and AP 1. JAK and STAT proteins can then dimerize and translocate to the nucleus, where they influence transcription. Last ly, AT1R stimulation can activate receptor tyrosine kinases, such as EGFR and PDGFR, and inhibit insulin receptor signaling 154 Given the multitude of signaling pathways and redundancies, it is not surprising that ot her RAS receptors, namely AT2R and Mas, might have interacting/counteracting intracellular signaling cascades. Similar to AT1R, Mas is a seven transmembrane G protein coupled receptor. In fact, there is evidence that Mas can directly dimerize and inhibit AT1R 156, 157 In addition, Mas stimulation can have effects to counter signaling, such as inhibiting AT1R mediated phosphorylation of p38MAPK, ERK1/2, and JNK 158, 159 Furthermore, Mas induced activation of SHP 2 can disrupt AT1R mediated acti vation of c Src, ERK1/2, and NOX 148, 160 Where AT1R stimulation causes vasoconstri ction, Mas stimulation causes vaso relaxation by inducing an increase in eNOS activity through the phosphatidylinositol 3 kinase (PI3K)/Akt pathway 148 Stimulation of Mas also causes AA release and PGI 2 production t o potentiate bradykinin signaling 161 164 Targeting the Renin Angiotensin System for Stroke Therapy Many factors are involved in the pathophysiology of stro ke, yet the RAS appears to play a key role (Figure 1 7). As hypertension is one of the most important modifiable risk factors for both types of stroke and manipulating the RAS through the use of ARBs and ACEi is the standard of care for hypertension, there is much interest in the therapeutic potential of RAS manipulation in stroke. The classical ACE/Ang II/AT1R axis
31 has received the most attention for its potential involvement in stroke pathophysiology. Ang II levels have been shown to increase in the corte x and hypothalamus following stroke 165 Blocking the effects of Ang II by ARB treatment has been shown to have therapeutic potential in both hemorrhagic and ischemic stroke. Many studies, utilizing both animal models and human clinical trials, have shown a decrease in cardiovascular risk and improvement in stroke prevention when blocking this axis 166 168 The occurrence of hemorrhagic stroke is reduced in stroke prone Spontaneously Hypertensive Rats (spSHR) when ARBs are administered systemically 169, 170 Ischemic stroke is also a therapeutic target of ARBs as demonstrated by several s tudies in normal rats and spontaneously hypertensive rats (SHR) that undergo ischemia by middle cerebral artery occlusion (MCAO). ARBs were shown to reduce the infarct size by 40 50%, reduce the neurological deficits, and improve recovery 168, 171 175 In addition, our lab has shown a robust cerebroprotective effect of the ARB candesartan in an endothelin (ET 1) induced MCAO model in rats 176 Interestingly, these therapeutic effects are, at least partly, independent of BP lowering effects 168, 175 In addition, AT1R deficient mice have smaller infarcts compared to controls after undergoing MCAO 177 Further support that blocking AT1R has additional effects than simply lowering BP is provided by the fact that other antihypertensive drugs, such as adrenergic receptor blockers and Ca channel blockers, do not protect against ischemia 171, 172 Also the cerebroprotective effects of ARBs are seen when given peripherally and centrally 168 175, 177, 178 That the ARBs candesartan and valsartan cross the blood brain barrier rea dily and that systemic administration of these drugs for cerebroprotection has been utilized
32 in non blood pressure lowering doses provides further evidence that these drugs have effects independent of BP lowering. There are a few theories that have been pr oposed for the mechanism by which ARBs afford cerebroprotection. The two broad sites of action would have to be either the cerebrovasculature or the brain parenchyma. Examples of effects to the cerebral vessels in rat pretreatment models include increasing capillary density 179 improving cerebrovascular reserve 180 and improving cerebral endothelial function 181 There are different proposed theori es to how these cerebrovascular effects may occur, including AT1R blockade alone 182 or unopposed agonism of the AT2 or Ang II type 4 receptors 175, 183 An example of a parenchymal effect is that agonism of the AT2R stimulates post stroke neurite outgrowth and improves neuron survival under hypoxic conditions 175 Another is that AT1R blockade has been shown to protect neurons during hypoxia by decreasing oxidative stress 184 As more evidence mounts, there is support for both theories working in concert. In addition to many animal studies, many clinical trials show the therapeutic effects of ARB s. Interestingly, the Losartan Intervention For Endpoint (LIFE) clinical trial adrenergic receptor blockers, common antihypertensive agents, in patients with hypertension and left ventricular hypertrophy 167 The Study on Cognition and Prognosis in the Elderly (SCOPE) has demonstrated the effectiveness of ARBs in stroke prevention 185 In the Captopril Prevention P roject (CAPPP) study, ACE inhibitors were shown to be inferior to conventional therapy for stroke prevention 186 This lead to the idea that perhaps increased levels of Ang II in response to ARBs, presumably acting at another receptor,
33 may play a role in their cerebroprotective effects 175, 186 This and other studies lead to further investigation of the role of AT2R in stroke. As mentioned above, there is debate abou t whether the cerebroprotective effects of ARBs occur because of decreased activation of AT1R or unopposed activation of AT2R 169, 170, 175, 178, 187, 188 The role of AT2R in stroke has been the topic of many recent studies. The effects of Ang II at this receptor are often exactly opposite those at AT1R 189 Furthermore, tissue levels o f AT2R are increased following many injuries and disease states, including the heart after myocardial infarction, atherosclerotic blood vessels, wounded skin, and the peri infarct region in the brain following ischemia 165, 175, 190 192 There is also evidence that AT2R stimulation in neurons leads to differentiation/regeneration and plays a role in neurotrophic actions 193 195 Because of this, it has been theorized that the increased expression of AT2R in the peri infarct region can be activated by the raised endogenous levels of Ang II during ARB treatment to ex ert neuroprotective effects 175 There is much support for this hypothesis. In an experiment utilizing an MCAO model of stroke where ARBs were given with an AT2R antagonist, the beneficial action was lost 175 Also, ARBs have been shown to be more effective in stroke than ACEi in a rat model of ischemia and reperfusion 178 When comparing AT2R knockout mice to wild type controls using MCAO, the AT2R knock out mice show more brain damage 187 Furthermore, central administration of an AT2R specific agonist has cerebroprotective effects during ischemic stroke 188 We have also shown that peripheral, post stroke administration of the AT2R agonist C21 has robust cerebroprotective effects in MCAO, where infarct size is reduced and rats perform better on neurological exams ( unpublished data ).
34 Similar to activation of the AT2R, activation of the ACE2/Ang (1 7)/Mas axis is just beginning to receive more attention for its therapeutic potential in cardiovascular disease 196 Activating this axis has been demonstrated to be effective for the treatment o f hypertension, hypertension related pathology, myocardial infarction, and heart failure 197 203 There is also evidence that activating this axis may have therapeutic potential in stroke. Peripheral administration of Ang (1 7), both acutely and chronically, has been shown to increase cerebral blood flow 204 206 Another study showed that central administration of Ang (1 7), given shortly after stroke, increased bradykinin levels and upregulated bradykinin receptors in the brain 207 Using this same model, Ang (1 7) caused an increase in NO release and eNOS activity 208 Furthermore, the components of the ACE2/Ang (1 7)/Mas axis are present in the brain providing support tha t this axis may be targeted for stroke therapy. In fact, Mas was first characterized as a protooncogene found highly expressed in the brain 209 It was originally thought to be present only in neurons 210 In rats and mice, Mas mRNA was detected throughout the brain in areas such as the hippocampus, cortex, olfactory bulbs, and thalamus 209 212 Mas has also been detected in rat endothel ial cells in the brain 213 Immunofluorescence studies have confirmed the mRNA studies, showing Mas immunoreactivity in both cardiovascular control areas and ot her areas of the brain 204 Neurons were described as being the main cell type with Mas immunoreactivity in cardiovascular control regions of the brain, but there was no description of other brain areas. ACE2 has also been detected in the brain, including in human tissue where low levels of mRNA were present 214 Furthermore, an immunofluoresence study using human brain showed positive staining in vascular smooth muscle and endothe lial cells
35 215 Cell culture experiments have shown that glia have ACE2 216 In vivo mouse studies have shown ACE2 mRNA and protein to be predominantly located in neurons, how ever. It was shown to be present throughout the brain, in cardiovascular control regions, brainstem, raphe, motor cortex, and many other areas 217 In summary, Mas and ACE2 are present throughout the brain, implying the possibility of a local ACE2/Ang (1 7)/Mas axis. We have recently demonstrated for the first time that ICV Ang (1 7) affords cerebroprotection in an ET 1 induced MCAO model of ischemic stroke 218 Ang (1 7) had no e ffect on % baseline vessel diameter during stroke, nor on % baseline cerebral blood flow during stoke. ET 1 injection very significantly decreased both of these parameters. Treatment of Ang (1 7) did decrease infarct size by more than 50% compared to contr ol stroked animals. The decrease in infarct size was prevented by co administration of the Mas antagonist, A 779, indicating a Mas dependent pathway. Ang (1 7) also Mas dependently improved performance of several neurological exams, including the Bederson exam, Garcia exam, and sunflower seed eating task. We also demonstrated that pharmacological activation of the ACE2/Ang (1 7)/Mas by administration of the putative ACE2 activator diminazine aceturate (DIZE) had the same effects as exogenous Ang (1 7). Furt her studies are indicated to determine the mechanism of this cerebroprotective action of stimulating the ACE2/Ang (1 7)/Mas axis during stroke. Summary Although the risk factors for stroke are well known, few effective preventative or acute therapies exist making it a leading cause of mortality and morbidity. In recent years, the RAS has been given more attention for its therapeutic potential (Figure 1 8).
36 Targeting the ACE2/Ang (1 7)/Mas axis in stroke makes sense as the components are present within the b rain, in both the cerebrovasculature and parenchyma. In addition, peripheral acute stimulation of Mas by Ang (1 7) administration and chronic peripheral over expression of ACE2 both increase cerebral blood flow. Central administration of Ang (1 7) causes i ncreases in bradykinin, eNOS activity, and NO production after stroke. Furthermore, we have shown a cerebroprotective effect of central stimulation of the ACE2/Ang (1 7)/Mas axis in an ET 1 induced MCAO model of ischemic stroke. However, in this model we d id not observe any effect on blood flow. In summary, there is much evidence supporting a protective role for the ACE2/Ang (1 7)/Mas axis in the cardiovascular system, including the ET 1 induced MCAO model of ischemic stroke. Therefore, we aim to uncover t he mechanism of protection in this stroke model and also to examine if activation of this axis has any therapeutic potential in hemorrhagic stroke. Therefore, two Specific Aims were designed using a combination of in vivo, in vitro, and molecular approache s. Specific Aim 1: Investigate the mechanism of the cerebroprotective actions of Ang (1 7) in ischemic stroke. Hypotheses: Ang (1 7) will modify the expression of some genes that will play a role in mediating the observed cerebroprotective effects. Furthe rmore, Mas will be present on certain cell types, indicating which cells are important in mediating the cerebroprotection. Specific Aim 2: Determine whether Ang (1 7) also exerts cerebroprotective actions in hemorrhagic stroke. Hypothesis: Since there is great overlap in the pathophysiology of both ischemic and hemorrhagic stroke, especially with regard to the
37 role of inflammation, Ang (1 7) will also have a cerebroprotective effect in hemorrhagic stroke.
38 Figure 1 1. Prevalence of silent cerebral is chemia for age ranges. 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.
39 Figure 1 2. Two type s of stroke. Hemorrhagic strokes occur when a blood vessel ruptures and blood leaks into the brain. Ischemic strokes occur when a clot prevents the flow of blood to an area of the brain. Figure was inspired by the Heart and Stroke Foundation of Canada.
40 Figure 1 3 Time course of mediators in ischemic stroke damage. The early immedi ate genes peak at 6 h. Heat shock proteins peak at 12 20 h. The adhesion molecules and cytokines peak around 36 h. Lastly, genes involved in inflammation and apoptosis peak a t around 48 h. Data for this figure was adapted from Iadecola, et al. 1997.
41 Figure 1 4. The two sides of NO. At low concentrations NO has physiological effects to cause vasodilation. At high concentrations, however, its effects are pat hological resulting in nitrosylation of proteins and cell death. Figure was inspired from Iadecola, et al. 1997.
42 Figure 1 5. RAS components. ACE= Angiotensin Converting Enzyme, ACE2 = Angiotensin Converting Enzyme 2, ATG = Angiotensinoge n, 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.
43 Figure 1 6. Ang (1 7) opposes AT1R signali ng. a/b/g = G protein subunits, Ang = Angiotensin, AA = Arachidonic Acid, AT1R = Ang II Type 1 Receptor, B1/B2 = Bradykinin Receptors, BK = Bradykinin, DAG = diacylglycerol, IP 3 = inositol 1,4,5 triphosphate LT = Leukotrienes, MAPK = Mitogen Activated Protein Kinase, NOS = Nitric Oxide Synthase, NO = Nictric Oxide, NOX = NAD(P)H Oxidase, NRTK = Nonreceptor Tyrosine Kinase, PG, Prostaglandins, PKC = Protein Kinase C, PGI 2 = Prostacyclin, PLC = phospholipase C, PLA 2 = phospholipase A 2 PLD = phospholipase D, ROS = Reactive Oxygen Species, RTK = Receptor Tyrosine Kinase
44 Figure 1 7. 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
45 Figure 1 8. Summary of our focus on the therapeutic potential of the RAS. The effects of Ang II at AT1R are deleterious for both ischemic and hemorrhagic stroke. In contrast the effects of Ang II at AT2R and Ang (1 7) at Mas can decrease the damage in both types of stroke. To study ischemic stroke we use and endothelin 1 induced model of middle cerebral artery occlusion. To study hemorrhagic stroke we use stroke prone Spontaneously Hypert ensive Rats.
46 CHAPTER 2 ANTI INFLAMMATORY EFFECTS OF ANGIOTENSIN (1 7) IN ENDOTHELIN 1 INDUCED MIDDLE CEREB RAL ARTERY OCCLUSION : POTENTIAL MECHANIS M FOR CEREBROPROTECTIO N Introduction Stroke is the fourth leading cause of death in the United States and a major cause of serious, long term disability 219, 220 While there have been many efforts to develop therapeutic approaches for stroke, very little progress has been made to counteract stroke damage and limit long term disability. Mounting evidence indicates that the renin angiotensin system (RAS) is a potential therapeutic target for ischemic stroke, as it is highly involved in the processes that induce cerebral damage following ischemia 165, 175, 177, 191 Specifically, it is well known that over activation of the angiotensin converting enzyme/angiotensin II/angiotensin Type 1 receptor (ACE/Ang II/AT1R) axis of the RAS contributes to CNS damage following cerebral ischemia 176, 221 In fact, numerous studies have shown that blocking the actions of Ang II at AT1R with AT1R antagonists (ARBs) decreases c ortical/subcortical infarct size and the ensuing neurological deficits, in animal models of stroke 168, 171, 173 Importantly, some human clinical trials have also indicated that AR Bs can reduce cardiovascular risk and prevent stroke 167, 185, 186, 222 A lesser known axis of the RAS, the angiotensin converting enzyme 2/angiot ensin (1 7)/Mas (ACE2/Ang (1 7)/Mas) axis, has recently begun receiving more attention in the literature. There is accumulating evidence that activation of this axis exerts beneficial actions in several cardiovascular diseases 143, 223 137, 223 136, 222 Activating the ACE2/Ang (1 7)/Mas axis appears to have potential for treating hypertension, hypertension related pathology, pulmonary hypertension myocardial infarction, and heart failure based on its ability to counteract the ACE/Ang II/AT1R axis 224 In the brain,
47 Ang (1 7) is primarily generated by the action of ACE2 on Ang II, and its effects are medi ated by its receptor, Mas 138 In recent studies, we demonstrated that the intracerebral damage and neurological deficits elicited by endothelin 1 (ET 1) induced middle cerebral artery occlusion (MCAO), a model of isc hemic stroke, are significantly reduced by intracerebroventricular (ICV) administration of either exogenous Ang (1 7) or an activator of ACE2, prior to and during the stroke period 218 Since we previously demonstrated a cerebroprotective effect of Ang (1 7) treatment during ET 1 induced MCAO, our aim here was to uncover the mechanism of this protection. This will further support the rationale for activating the ACE2/Ang (1 7)/Mas axis for stroke treatment and preventio n. Furthermore, we believe that uncovering this mechanism will lead us to consider other diseases that may benefit from a treatment involving manipulation of the ACE2/Ang (1 7)/Mas axis. We began by examining changes in gene expression during ET 1 induced MCAO that result from Ang (1 7) treatment. For these experiments, we first tested for alterations in hypoxia related genes in vivo using a PCR Array. Changes were verified by quantitative reverse transcriptase polymerase chain resction (qRT PCR) and Wester n blotting. Immunohistochemical analyses were also performed to examine which CNS cell types in vivo express Mas. Lastly, we measured the effects of Ang (1 7) on isolated glia in vitro The data suggest that Ang (1 7) exerts a cerebroprotective action via a direct inhibitory action on microglia, as well as blunting the production of pro inflammatory cytokines (PIC) and inducible nitric oxide synthase (iNOS). Methods Animals and Ethical A pproval For the experiments described here, we used a total of 127 adul t male Sprague
48 Dawley rats (250 275 g) purchased from Charles River Farms (Wilmington, MA, USA). In addition, 20 Sprague Dawley pups were used to generate the cell cultures. All experimental procedures were approved by the University of Florida Institution al Animal Care and Use Committee. In addition, the principles governing the care and treatment of animals, as stated in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication no. 85 323, revised 1996) and adopted by the American Physiological Society, were followed at all times during this study. Rats had ad libitum access to water and standard rat chow and were housed in a well ventilated, specific pathogen free, temperature controlled environmen t (24 1C; 12 h 12 h light dark cycle). Implantation of Intracranial Cannulae and Osmotic Pumps After a 7 day acclimation period, rats were anesthetized with a mixture of O 2 (1.5 L/min) and 4% isoflurane and placed in a Kopf stereotaxic frame. Anesthesia was maintained for the duration of the surgeries using an O 2 /isoflurane (2%) mixture delivered through a nose cone attached to the frame. Animals then underwent intracranial surgeries, where two cannulae were implanted: one guide cannula for ET 1 injectio n and another cannula for ICV Ang (1 7) [1.1 nM; 0.5 L/h] or control artificial cerebrospinal fluid (aCSF; 0.5 L/h) delivery via osmotic pumps, as detailed previously 218 ET 1 Induced Middle Cerebral Artery Occlusio n (MCAO) Seven days after placement of intracranial cannulae, rats underwent MCAO via intracranial ET 1 (3 uL of 80 uM solution; 1 uL/min) injection through the guide cannula (17.2 mm below the top of the cannula), adjacent to the middle cerebral artery. S ome groups of rats underwent sham MCAO via administration of 3 uL 0.9% saline instead of
49 ET 1. In previous studies we validated this procedure for investigating the role of the brain renin angiotensin system in ischemic stroke 218 Post Mortem Analyses Following neurological testing, using forelimb flexion 176 to ensure that stroke had occurred, all rats were euthanized and their brains were removed at 6 h or 24 h post MCAO, depend ing on the experiment Three coronal sections (2 mm each) were cut through the cerebrum ipsilateral to the ET 1 induced MCAO beginning caudally approximately 6 mm rostral to the top of the ventral pons, and ending rostrally just before the end of the prefr ontal cortex and olfactory bulbs. To further confirm stroke, the rostral most brain section from each rat was stained with 0.05% 2,3,5 triphenyltetrazolium chloride (TTC) for 30 minutes at 37 C 176, 21 8 Tissue ipsilateral to the occlusion, which is unstained by TTC, was assumed to be infarcted. The coronal section immediately caudal to the section used for TTC staining was isolated and dissected so that left and right cortical and subcortical tissues were separated into four individual samples for protein analysis. They were immediately flash frozen in liquid nitrogen and stored at 80 C. Total protein was later extracted using a lysis buffer composed of the following: distilled water, 10X RIPA (Cell S ignaling, Danvers, MA), protease inhibitor cocktail (Sigma, St. Louis, MO), two phosphatase inhibitor cocktails (Sigma), and PMSF (Sigma). Tissue was homogenized with a syringe, first using an 18 gauge needle then a 26 gauge until tissue was dissolved. Sam ples were allowed to incubate on ice for 30 min with occasional vortexing. Samples were then centrifuged at 4C for 30 min at 16.1g. Supernatant was saved for Western analysis at 20 C. The coronal section just caudal to this protein section was similarly s eparated into four
50 individual samples and saved for RNA at 80 C. Total RNA was isolated using an RNeasy Plus kit (Qiagen, Valencia, CA, USA). Isolated RNA underwent DNAse I treatment to remove genomic DNA. Different groups of rats were used for the Mas im munohistochemistry, where the same section described above that was saved for protein analysis was frozen in Tissue Compound (Sakura Finetech, Torrance, CA) to use for immunostaining. PCR Array Hypoxia Signaling Pathwa y RNA extracted from the ipsilateral (right) cortex at either 6 or 24 hr post MCAO was pooled (200 ng RNA/sample, N=5 samples per group) to make 1.0 ug total RNA for each pooled group. The pools were: aCSF+sham, aCSF+ET 1, and Ang (1 7)+ET 1. cDNA was made from these pooled samples by using the RT First Strand Kit (SABiosciences, Frederick, MD) as described by the manufacturer. RT SYBR (SABiosciences, Rat Hypoxia Signaling Pathway PARN 032A) were used with the pooled samples. The array examined 84 hypoxia related genes, additional housekeeping genes, and appropriate controls. Data were first analyzed by comparing fold change in the stroked groups compared to sham. Ang (1 7)+ET 1 fold cha nge was then compared to control aCSF+ET 1 fold change. qRT PCR Levels of iNOS, endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), pentraxin 3 (Ptx3), interleukin 11b (CD11b), glial fibrillary acidic protein (GFAP), myeloperoxidase (MPO), monocyte chemotactic protein 1 (MCP 1), chemokine receptor type 4 (CXCR4), and chemokine
51 (C X C motif) ligand 12 (CXC L12) mRNAs within the cerebral cortex were analyzed by quantitative real time reverse transcriptase polym erase chain reaction (qRT PCR). This was used to confirm all differences from the PCR array and examine other genes A group where Ang (1 7) was given to sham animals was also included. Extracted RNA was reverse transcribed with a high capacity cDNA reverse transcription kit (Bio Rad Laboratories, Hercules, CA) and then analyzed via qRT PCR in a PRISM 7000 sequence detection system (Applied Biosystems, F oster City, CA) as detailed by us previously 225 Oligonucleotide primers and TaqMan probes specific for rat iNOS, eNOS, from Applied Biosystems (Carlsbad, Cal ifornia). Data were normalized to 18S rRNA. Western Blotting Rad, mercaptoethanol (Bio Rad). After heating for 5 min at 90 C, samples were loaded into a preca st TGX Criterion gel (Bio Rad). The gel was run at 200V using Tris Glycine SDS running buffer (Bio Rad) and Dual Color MW markers (Bio Rad). Transfer buffer was prepared using Tris base 25mM (Bio Rad), Glycine 192mM (Fisher), and methanol 20% (Sigma). The protein was then transferred from the gel to a nitrocellulose membrane (Bio Rad) at 80V for 1 hour. After transfer, the membrane was rinsed with Tris buffered saline (TBS), then TBS with Tween (TBST), and placed on a shaker in TBST+milk for 1 hour of block ing. The membrane was then incubated with primary antibodies [rabbit anti iNOS (1:333) and rabbit anti cofilin (1:10,000; used as a loading control)] overnight at 4 C with slow agitation on a shaker. The following day the membrane was washed six times with TBST (5 min each) on a
52 shaker with fast agitation. Then, the secondary antibody (goat anti rabbit IgG horse radish peroxidase [HRP]; 1:2,000) was added to the membrane, which was then placed on a shaker for 1 h. The membrane was then washed with TBST six times (5 min each) on a shaker with fast agitation, exposed to ECL, and developed. Densitometry was used to quantify the blots using the integrated density (ImageJ, NIH). Samples were normalized to the housekeeping protein cofilin. A positive control, prot ein extracted from mixed cultured glia cells treated with LPS, was also used. Mas Immunohistochemistry Twenty four hours following ET 1 or sham injections, a 2 mm coronal section of brain was sectioned and fresh frozen in Tissue Tek Optimal Cutting Temp erature (Sakura Finetech, Torrance, CA) to be used for co immunostaining of Mas with NeuN (neuronal specific nuclear protein), GFAP (glial fibrillary acidic protein), RECA 1 (rat endothelial cell antigen), OX 42 (CD11b), and MPO (myeloperoxi dase), which are respective specific markers for neurons, astroglia, microglia, endothelial cells, and neutrophils. Procedures were as follows: Brain sections (at 5um) were cut from the 2 mm coronal section block and air dried at room temperature overnight OCT was removed in a wash of 1X TBS for 5 minutes. Slides were drained and wiped, then blocked for 1 hour in 2% horse serum diluted in 1X TBS. Slides were wiped and blocking buffer was replaced with antibody cocktails (rabbit anti Mas [1:400] combined w ith either mouse anti NeuN [1:100], anti GFAP [1:100], anti OX 42 [1:100], anti RECA [1:100] or anti MPO [1:50] diluted in antibody diluent (Invitrogen, Carlsbad, CA), and incubated overnight at 4C. Following two 5min washes in TBS, Alexafluor donkey anti rabbit 594 and donkey anti mouse 488 were added to the slides, both at 1:500. After a 45 minute room temperature incubation, slides were again double
53 washed in TBS. Sections were then post fixed for 5 minutes in 10% neutral buffered formalin, washed twic e, then wiped dry before mounting in DAPI vectashield (Vector Labs, Burlingame, CA). Nitric oxide Production by Primary Glial Cultures Primary mixed glial cultures were grown from newborn SD rat pups as previously described 226 The cortex and striatum were dissected out and homogenized. Trypsin containing 10% fetal bovine serum (FBS), were filtered though sterile gauze, plated in poly L lysine coated dishes, and grown for 10 14 days at 37 C in 5% CO 2 / 95% air. Cells were then dissociated by mild treatment with trypsin (0.25%; 2 min, 37 C), re plated in 35mm dishes at a density of 1 x 10 5 cells/dish, and cultured for a further 7 days. A t this time, cultures consisted of both astrocytes and microglia, but not neurons, as determined by immunostaining with GFAP, Iba 1, and NeuN antibodies. Cultures were treated as follows: control solution (PBS), lipopolysaccharide (LPS; 10 ng/mL), LPS + An g (1 7) [0.1 nmol/mL], and LPS + Ang (1 7) + A779 (Mas inhibitor; 1.0 nmol/mL). Ang (1 7) and A779 were applied 12 h before, at the time of, and 12 h after LPS treatment. Growth media was replaced with serum free DMEM just before the first treatment. Twent y four hours after the LPS treatment, the media was sampled for analysis of nitrite (NO 2 a stable breakdown product of nitric oxide) by the Greiss reaction, as previously described 226 Media samples (100 ul) were added to 100 ul Greiss reagent consisting of 0.75% sulfanilamide in 0.5M HCl and 0.075% N (1 naphthyl)ethylenediamine dihydrochloride, in water. The mixture was incubated for 10
54 min and the absorbance measured at 543 nm. NO 2 concentration was then calculat ed from a standard curve, using known concentrations of Na NO 2 HPLC Analysis of Ang (1 7) Growth media was sampled at the following timepoints: 0 h, 14 h, 24 h, and 48 h. These samples were sent to a core facility for measurement of Ang (1 7) by standard HPLC. Chemicals Ang (1 7) and A779 were purchased from Bachem Bioscience (Torrance, CA, USA). ET 1 was from American Peptide Company, Inc (Sunnyvale, CA, USA). Rabbit anti Ang (1 7) Mas receptor antibody was from Alomone Labs (Jerusalem, Israel). Mouse ant i rat RECA 1 was from AbD Serotec (Raleigh, NC). Mouse anti MPO, mouse anti GFAP, rabbit anti iNOS (ab15323), rabbit anti Cofilin (ab11062) and goat anti rabbit IgG HRP (ab6721) antibodies were from Abcam (Cambridge, MA). Mouse anti OX 42 antibody was fro m BD Biosciences (San Jose, CA). Mouse anti NeuN was from Millipore (Bedford, MA). Rabbit anti Iba 1 (01919741) was from Wako (Richmond, VA). Alexafluor donkey anti rabbit 594 and anti mouse 488 were from Molecular Probes [Invitrogen] (Carlsbad, CA, USA). Vectashield mounting medium with DAPI ( 4',6 diamidino 2 phenylindole ) was from Vector Labs (Burlingame, CA, USA). All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA). Data analysis Data are expressed as means SEM. Statisti cal significance was evaluated, as specified in the figure legends, with the use of a one way ANOVA, Newman Keuls test, test as appropriate. Differences were considered significant at p<0.05.
55 Results Cerebroprotective Ang (1 7) Reduces I nfarct Size 24 h after ET 1 induced MCAO In previous studies, we demonstrated a cerebroprotective effect of central treatment with Ang (1 7) prior to and during induction of ischemic stroke via ET 1 induced MCAO 218 An g (1 7) treatment decreased the size of the intracerebral infarct and improved performance on several neurological exams 72 h after MCAO. Furthermore, Ang (1 7) did not alter blood pressure, percent change in MCA branch vessel diameter or percent change in cerebral blood flow. Here we show the percent infarcted grey matter at 24 h after MCAO. Ang (1 7) blunts the infarct size by 50% (Figure 2 1) in agreement with our previous results at 72 h 218 Increased Expression o f Hypoxia related Genes in the Ipsilateral Cerebral Cortex following ET 1 induced MCAO; Inhibitory Action of Ang (1 7) It is well known that the expression of many genes, including PICs and some NOS isozymes, is increased in the cerebral cortical infarct z one following ischemic stroke, contributing to neurotoxicity 37, 40 Therefore, we became interested in assessing changes in gene expression within the brain parenchyma to investigate potential me chanisms underlying the protective action of Ang (1 7). We began by using a PCR array to examine the expression of hypoxia related genes in pooled ipsilateral samples of cerebral cortex. Of the 84 hypoxia related genes on the array, five were up regulated by more than 4 fold at 6 hours post MCAO stroke compared to sham (Table 2 1): heme oxygenase (9 fold increase in aCSF controls vs. 5 fold increase in Ang (1 7) treated); fold vs. 3 fold) and interleukin 6 (IL6; 24 fold vs. 4 fold); myosin light polypeptide 2 (6 fold vs. 2 fold); pentraxin related gene (Ptx3; 4 fold for both). At 24 hours post stroke, five genes were up regulated by more than 4 fold (Table
56 2 2): heme oxygenase (34 fold vs. 40 fold); the PIC IL6 (45 fold vs 38 fold); iNOS (60 fold vs. 8 fold); plasminogen activator/urokinase (12 fold for both); and Ptx3 (72 fold vs. 42 fold). Ang (1 stroke. Only those data that were increased in stroke by gr eater than four fold are presented, as we believed these to be the most important and most reliable given the nature of PCR arrays. Effects of Ang (1 7) on the Expression of Nitric Oxide Synthase (NOS) Isozymes PICs, and Inflammatory Cell Markers in the I psilateral Cerebral C ortex following ET 1 induced MCAO The PCR array data presented in Tables 2 1 and 2 2 confirm the view that the expression of several inflammatory cytokines and iNOS is increased in the ipsilateral cerebral cortex following ischemic str oke, and also indicate potential loci of Ang (1 protective action. Thus, in order to confirm the results of the PCR array, we performed qRT PCR on individual ipsilateral cortical samples from rats that had undergone ET 1 induced MCAO or sham strokes i n the absence or presence of ICV infusion of Ang (1 7) as described in the Methods. Specifically, we were interested in determining the relative levels of mRNAs for NOS isozymes, PICs, chemokines and their receptors, and of inflammatory cell markers (MPO f or neutrophils, CD11b for monocytes/microglia, and GFAP for astrocytes) under each treatment condition as a function of time following increased at 6 hours post MCAO, whereas l evels of iNOS, nNOS, CXCL12, CD11b (Figure 2 2) and IL6 (not shown) were unaltered. The data also demonstrate that Ang (1 7) significantly decreased the levels of CXCL12 mRNA 6 h after MCAO, and also produced non significant reductions in the post MCAO ex 2 2), and IL 6 (not shown). At 24 hours post MCAO there were dramatic increases in
57 1, CD11b, and GFAP mRNAs by factors of 300, 7, 100, 10, 10, 80, 10, 40, 1200, 12, and 5 respectively, when compared with sham rats (Figures 2 3 and 2 4), whereas nNOS, CXCL12, and MPO mRNA levels were unaffected. Of the genes that were increased in d by ICV Ang (1 1, and GFAP exhibited non significant reductions in the presence of this peptide. Ang (1 7) alone had no effects on gene expression in sham animals that did not undergo MCAO (Figures 2 2 through 2 4). To control for ET 1 effects independent of stroke, we injected at a site 2 mm medial and 2 mm superior to the stroke coordinates and confirmed the absence of stroke by TTC staining. There was no effect of ET 1 at this site compared to saline on gene Since the inhibitory effects of Ang (1 7) on iNOS mRNA levels were observed to be the most profound, we tested whether these changes were detectible at the protein level via Western blotting (Figure 2 5). Densitometry analysis showed that iNOS protein was increased by ~ 4 fold 24 h after MCAO. This increase was blunted by ICV Ang (1 7) administration. As with the mRNA, Ang (1 7) alone had no effect on the expression of iNOS protein in sham ani mals that did not undergo MCAO These data corroborated our earlier finding where levels of iNOS mRNA in the cerebral cortical infarct zone were reduced by Ang (1 7) at 24 hours post ET 1 induced MCAO 218 and also expa nded our understanding of the potential mechanism of Ang (1 No effect of stroke or Ang (1 7) treatment was measured on Mas mRNA levels (not shown).
58 Cellular Localization of Mas within Rat Cerebral Cortex Having demonstrated that Ang (1 7) reduces the expression of various proinflammatory markers in the ipsilateral cortex during ischemic stroke, we used immunostaining to investigate the cellular location of the Ang (1 7) receptor, Mas in the brains of normal, healthy rats and in those that had undergone ET 1 induced MCAO. The representative fluorescence micrographs presented in Figure 2 6 indicate that Mas immunoreactivity within normal rat cerebral cortex was primarily associated with neurons, as evidenced by co localization w ith the neuron specific marker NeuN. However, it was clear that Mas immunoreactivity is also associated with non NeuN positive cells. Further immunostaining experiments revealed the presence of Mas on macrophages/microglia, as illustrated by co localizatio n with immunoreactive CD11b (OX 42 immunostaining), a specific marker of these cells (Figure 2 6). Mas immunoreactivity was also observed within endothelial cells of small cerebral vessels (Figures 2 6), and was abundant within the endothelium of large ves sels such as the MCA (not shown), as demonstrated by co localization with immunoreactive RECA. In contrast, Mas immunoreactivity was not associated with astroglia or neutrophils (the latter present only within the blood vessel lumen in the normal brain), b ased on a lack of co localization with immunoreactive GFAP and MPO, respectively (Figure 2 6). Similar localization of Mas immunoreactivity was observed in the subcortex of normal rats, i.e. presence of Mas on neurons, macrophages/microglia and endothelial cells, but not on astroglia nor on neutrophils (not shown). In comparison with the normal brains, stroked brains were also examined in the ipsilateral cortex at 24 hours following ET 1 induced MCAO (Figure 2 6). Consistent with the control brains, Mas imm unoreactivity was associated with neurons and macrophages/microglia, but not with MPO positive
59 neutrophils that invade the brain parenchyma (Figure 2 6 ). We have verified the specificity of the Mas antibody using brain sections from a Mas knockout mouse (F igure 2 6). We also confirmed that each immunopositive area was a cell by co localizing with DAPI nuclear staining (not shown). Ang (1 7) Blunts LPS Induction of NO in Primary Mixed Glial Cultures Considering that macrophages/microglia within the cerebral cortex contain immunoreactive Mas, and that ICV infusion of Ang (1 7) decreases the stroke induced expression of PICs, iNOS, and CD11b (indicating reduced activation of microglia), we wanted to test the direct effects of Ang (1 7) on NO production by glia As our hypothesis is that Ang (1 7) is cerebroprotective by blunting inflammation, using an LPS induced model of inflammation is relevant. T his was achieved using primary glial cultures that contain a mixture of astrocytes and microglia, as evidenced by i mmunostaining using anti GFAP and anti Iba 1 antibodies (Figure 2 7). Incubation of cultures with LPS (10ng/mL), which is well known to elicit PIC production and secretion, produced a highly significant (3 fold) increase in NO production after 24 h, as dem onstrated by accumulation of NO 2 (a stable breakdown product of NO) in the growth media (Figure 2 7). This increase in NO 2 was significantly blunted by Ang (1 7) treatment (0.1 nmol/mL), administered 12 h before LPS, just before LPS, and 12 h after LPS (F igure 2 7). This multiple treatment model was designed to account for the breakdown of Ang (1 7) as measured by HPLC (Table 2 3). Furthermore, this blunting was reversed by co treatment with the Mas antagonist, A779 (1.0 nmol/mL), when administered at the same time points.
60 Discussion In previous studies we demonstrated a significant cerebroprotective action of the peptide Ang (1 7) against ischemic stroke when administered centrally prior to and during the insult 218 H ere we have investigated the potential mechanisms that underlie this cerebroprotective action of Ang (1 7). Our data suggest that the cerebroprotective action of Ang (1 7) involves an anti inflammatory effect, as evidenced by the following findings: (i) An g (1 7) blunts MCAO induced increases in the levels of iNOS, PICs and CD11b (a marker of macrophage/microglial activation), that occur in the ipsilateral hemisphere following MCAO; (ii) Ang (1 7) blunts the LPS induced increases in NO production in culture d glia (mixed culture of microglia and astrocytes). Considering our immunostaining results which show that Mas is localized to microglia, our findings are consistent with the idea that the cerebroprotective action of Ang (1 7) is mediated through reduction s in microglial action and the intracerebral pro inflammatory response that occurs following MCAO. Despite the above findings, the present study raises many important questions. One of the major issues concerns the precise locus of action of Ang (1 7) in reducing the damage due to stroke. Over activation of microglia and increased iNOS expression have been shown to be detrimental in stroke 37, 40 Furthermore, inhibiting iNOS and microglia has bee n shown to have a therapeutic effect in experimental models of stroke 40 Based on our data we believe that Ang (1 7) reduces the damage due to stroke through a reduction of an over activated microglia response and an excessive production of iNOS. Evidence for this is as follows. First, Mas is present on microglia so Ang (1 7) could have a direct action on this cell type. Furthermore, Ang (1 7) blunts the increase in CD11b mRNA during stroke, implying that fewer activa ted
61 microglia are present in the ipsilateral cortex 24 h after MCAO. Lastly, downstream effects of microglia are also blunted by Ang (1 7), namely the increases in iNOS and PICs during stroke. While direct actions of Ang (1 7) on microglia are a possibilit y, we cannot conclusively rule out other indirect mechanisms. Other cell types, including neurons and endothelial cells, contain Mas immunoreactivity. Thus, it is possible that Ang (1 7) has an effect on neurons, such as inhibition of monocyte chemotactic protein 1 (MCP 1) production and secretion, which normally recruits monocytes/microglia, astroglia, or other inflammatory cells towards neurons 227, 228 There was a strong trend for Ang (1 7) to blunt the increase in MCP 1 during stroke that almost reached significance. Although it has been reported that Mas stimulation increases cerebral blood flow through increasing bradykinin secretion and eNOS activity 3 hours after stroke 208 our previous measurements of cerebral blood flow with central Ang (1 7) treatment during stroke do not support these alterations as a mechanism for cerebroprotection 218 Further, our in vitro da ta with a cultured glial system support that Ang (1 7) has a direct effect to blunt an LPS induced increase in NO Since the culture system lacked neurons or endothelial cells, it demonstrates that these cells are not required for an anti inflammatory effe ct. Many of the genes that were altered by MCAO on the PCR array have been discussed in the literature. Heme oxygenase, PICs, iNOS, myosin light polypeptide 2, Ptx3, and plasminogen activator/urokinase have been shown to be increased in several pathologic al conditions, including hypoxia, ischemia, and inflammation. Heme oxygenase gene expression is up regulated in response to cerebral ischemia. Furthermore, it has been shown that over expression of heme oxygenase is
62 neuroprotective in an MCAO model in mice 229 In addition, PICs, such as IL and iNOS have been shown to be increased in stroke models and to cause damage 40, 230, 231 Myosin light polypeptide 2 has been reported to be upregulated during hypo xia or inflammation in several tissues, including the aorta in stroke prone spontaneously hypertensive rats 232 Ptx3, a gene believed to modulate the phagocytic activity of microglia 233 has been shown to increase in acute ischemic cardiomyopathy 234 In an MCAO model in rats, mRNA levels of both tissue type plasminogen activator and urokinase type plasminogen activator were increased in the cerebra l cortex and hippocampus 24 h post MCAO 235 Confirming these changes further supported the validity of our stroke model. The changes in gene expression due to Ang (1 7) treatment, however, have not been previously report ed to our knowledge. As stated in the methods, we verified changes with qRT PCR. iNOS induction 24 h after MCAO was significantly blunted by Ang (1 7), affirming our earlier study 218 We focused much of our attention o n iNOS, as there is substantial evidence in the literature to suggest it is highly involved in the pathology of stroke, by increasing NO levels 40 Interestingly, while we did not see an effect of Ang (1 7) on eNOS ex pression at 6 or 24 h post MCAO, it is still possible that Ang (1 7) may have increased eNOS activation, but not gene expression, at least in the early post stroke time period as described previously 208 Further support for the anti inflammatory effect of Ang (1 (1 7) at 24 h, but these changes did not reach statistical significance. Fu rthermore, blunting of these PICs only reached statistical significance at 24 h post stroke and not 6 h post
63 stroke. This suggests that the effects of Ang (1 7) may not be measurable early after stroke since there is not yet sufficient inflammation to reve al an effect. It is also worth mentioning that several of these trends may have reached statistical significance if we examined other time points, but we limited our examination to 6 and 24 h post stroke. In addition to the above, CXCL12 and its receptor C XCR4 were examined. These molecules are involved in the trafficking of stem cells and are known to be upregulated in inflammation 236 While CXCR4 was significantly increased 6 h and 24 h after stroke, CXCL12 showed onl y a trend to increase at these time points. Furthermore at 6 h, the increase in CXCR4 was significantly blunted by Ang (1 7) treatment, as was the increase in CXCL12 at 24 h post stroke. CXCL12 has been shown to be upregulated in stroke in the ischemic pen umbra 237 It is possible that our samples contained less penumbra compared to the total tissue collected. It has been shown that the CXCL12/CXCR4 pathway stimulation can increase IL6 production in microglia, indicating a role for this pathway in activating microglia 237 Interestingly, CD11b was increased by stroke at 24 h and found to be blunted by Ang (1 7), as well. This suggests that fewer macrophages/microglia are becoming activated or fewer are migrating to the cortex in the stroked area after Ang (1 7) treatment 238 This was not observed at 6 h post MCAO, but CD11b levels were not observed to increase due to stroke until 24 h. It appeared that th e early inflammatory response was mediated more by neutrophil invasion at 6 h, as there was a statistically significant increase in MPO at this time point due to stroke. It should also be mentioned that all of the gene expression studies presented here, i ncluding the PCR array and the qRT PCR, were performed using only cortex and
64 only from a precisely measured 2 mm coronal section determined to be in the stroked area (see Methods). It contained core, penumbra, and some healthy tissue. This same section was used for every rat. We believed that this was the most appropriate way to measure the expression of these genes in the tissue, since in a previous study 218 we showed no increases in iNOS ipsilateral hemisphere (cortex and striatum/subcortex). Furthermore, we did not see nearly the same magnitude of increase in IL6 expression due to stroke looking at the entire hemisphere. We propose that the effects in the stroke region are diluted when excess healthy tissue is used in the analysis. As much of our early attention was given to iNOS, we verified that this change in iNOS gene expression also existed at the protein level. Protein expression at 24 h post stroke was significantly increased, and this increase was significantly blunted by Ang (1 7) treatment. In future experiments, it will be valuable to verify changes in protein translation for other genes with altered mRNA transcription due to An g (1 7) treatment. We made several interesting observations with the Mas co localization study. Interestingly Mas immunoreactivity does not appear to co localize with GFAP immunoreactivity. There is evidence in the literature to suggest that astroglia m ay have Mas, however these studies were performed in cultured astrocytes and brain slices from other brain regions 216, 239 We cannot conclude with certainty that astrocytes do not have Mas, but we do feel that since the protein levels are below the detection threshold of immunostaining, astrocytes may not be as important in this Mas mediated mechanism. Al so, Mas staining is observed not only on the cellular membrane, but also in the nucleus and cyt osol. This abundant presence of Mas in the nucleus is consistent
65 with its original discovery as an oncogene 240 perhaps being involved in cell cycle regulation. To control for any changes in Mas localization during stro ke, we also performed a co localization study on tissue that was collected from animals 24 h after MCAO. Mas immunoreactivity was present on neurons in the penumbra, as well as microglia within the ischemic core. Studying cerebral tissue from a rat that un derwent stroke confirmed that infiltrating neutrophils were devoid of Mas. We encountered several challenges while designing our in vitro experiments. Ang (1 7) is broken down relatively rapidly in our cell culture system as measured by HPLC analysis (T able 2 3). Therefore, we designed a model with multiple treatments of a low dose given 12 h before LPS, at the time of LPS, and 12 h after LPS. This treatment model demonstrated that Ang (1 7) could blunt the LPS induced increase in NO 2 We interpret this as a proof of principle that there is a direct effect on glia. This study provides further support that activating the ACE2/Ang (1 7)/Mas axis has a therapeutic effect during stroke 218 It also demonstrates for the fir st time an anti inflammatory effect of Ang (1 7) in CNS tissue. This supports the theory that activation of the ACE2/Ang (1 7)/Mas axis could prove to be a novel therapeutic target for other diseases where the underlying pathology involves inflammation, s uch as other cardiovascular diseases, type 2 diabetes, chronic kidney disease, and cancer 241 There is more evidence accumulating that the activation of this axis has anti inflammatory effects in many tissues, such as the heart 242 aortic valve 243 joints 244 and kidneys 245 Since we demonstrate a central effe ct here, it also may prove to be therapeutic for other 241 Further
66 studies will be needed to determine other potential direct effects of Ang (1 7) a nd also the intracellular signaling involved.
67 Table 2 1 PCR array data showing fold changes in gene expression in ipsilateral (right) cerebrocortical tissue 6 h after endothelin 1 (ET 1) induced middle cerebral artery occlusion (MCAO) stroke compared to sham controls (0.9% saline instead of ET 1). Gene 6 Hours aCSF+ET 1 Fold Change Ang (1 7)+ET 1 Fold Change Heme oxygenase 9 5 Interleukin 1 5 3 Interleukin 6 24 4 Myosin light polypeptide 2 6 2 Pentraxin related gene 4 4 Only those genes that showed a fold change from MCA O greater than 4 are presented. Seven days of Ang (1 7) (1.1 nM; 0.5 L/h) treatment prior to MCAO blunts the increase s in IL1 IL6, heme oxygenase and myosin light polypeptide 2 when compared to control animals treated with artificial cerebrospinal fluid (aCSF; 0.5 L/h). RNA samples were pooled (N=5) for each aCSF+sham, aCSF+ET 1, and Ang (1 7)+ET 1 groups so that 3 ar rays were used as described in the methods. Table 2 2. PCR array data showing fold changes in gene expression in ipsilateral (right) cerebrocortical tissue 24 h after ET 1 induced MCAO stroke compared to sham controls. Gene 24 Hours aCSF+ET 1 Fold Cha nge Ang (1 7)+ET 1 Fold Change Heme oxygenase 34 40 Interleukin 6 45 38 Inducible NO synthase 60 8 Plasminogen activator 12 12 Pentraxin related gene 72 42 Only those genes that showed a fold change from MCAO greater than 4 are presented. Seven days of Ang (1 7) (1.1 nM; 0.5 L/h) treatment prior to MCAO blunts the increases IL6, iNOS, and pentraxin related gene when compared to control animals treated with aCSF (0.5 L/h). RNA samples were pooled (N=5) for each aCSF+sham, aCSF+ET 1, and Ang (1 7)+ET 1 groups so that 3 arrays were used as described in the methods.
68 Table 2 3 Degradation of Ang (1 7) in media of primary mixed glial cultures. Cells cultured as described in the Methods. Ang (1 7) Start Conc, M Ave Conc Time 0 h Ave Conc Time 14 h Ave Co nc Time 24 h Ave Conc Time 48 h 100 112 82 69 5 10 14 2 8 0.04 1 1 0.41 0.39 0.00 Averages (N=2) of media samples were calculated from HPLC analysis at different time points after Ang (1 7) was added to the media.
69 Figure 2 1 Intracerebral pretreatment with Ang (1 7) reduces infarct size 24 h after ET 1 induced MCAO. Rats were pretreated via the ICV route with Ang (1 7) (1.1 nM; 0.5 1 induced MCAO as described in the methods. A control group of rats also underwent sham MCAO with 0.9% saline injection instead of ET 1 (n=5). Brains were removed for TTC staining 24 h after MCAO. B ar graphs are means + SEM showing the percentage infarcted grey matter in each treatment group. 1/aCSF. Below are representative brain sections showing infarcted (white) and non infarceted (red) grey ma tter.
70 Figure 2 2. Gene expression in ipsilateral (right) cerebrocortical tissue 6 h after ET 1 induced MCAO. Rats underwent sham or ET 1 induced MCAO in the absence or presence of seven days infusion of Ang (1 7) (1.1 nM; 0.5 and expression of the indicated genes in the ipsilateral cerebral cortex was assessed by qRT PCR as detailed in the Methods. Bar graphs are means + SEM. N=6 11/group. *P<0.05 to ET 1/aCSF.
71 Figure 2 3. Gene expression of NOS isozymes and pro inflammatory cytokines in ipsilateral (right) cerebrocortical tissue 24 h after ET 1 induced MCAO. Rats u nderwent sham or ET 1 induced MCAO in the absence or presence of seven days infusion of Ang (1 7) (1.1 nM; 0.5 route. At 24 h post MCAO rats were euthanized and expression of the indicated genes in the ipsilateral cerebral cortex was assessed by qRT PCR as detailed in the Methods. Bar graphs are means + SEM. N=7 12/group. *P<0. 1/aCSF.
72 Figure 2 4. Gene expression of cell markers and migratory factors in ipsilateral (right) cerebrocortical tissu e 24 h after ET 1 induced MCAO. Rats underwent sham or ET 1 induced MCAO in the absence or presence of seven days infusion of Ang (1 7) (1.1 nM; 0.5 post MCAO rats were euthanized and expression of the indicated genes in the ipsilateral cerebral cortex was assessed by qRT PCR as detailed in the Methods. Bar graphs are means + SEM. N=7 12/group. *P<0.0 5 compared to 1/aCSF.
73 Figure 2 5. Levels of iNOS protein in ipsilateral (right) cerebrocortical tissue 24 h after ET 1 induced MCAO stroke or sham procedure. Rats underwent sham or ET 1 induced MCAO in the absence or presence of seven days infusion of Ang (1 7) (1.1 nM; 0.5 MCAO rats were euthanized and expression of iNOS protein in the ipsilateral cerebral cortex was assessed by Western blotting as detailed in the Methods. Bar graphs show levels of iNOS protein under each treatment condition, normalized against cofilin. Data are means + SEM. N=7 12/group. *P<0.05 1/aCSF. Also shown are representative iNOS protein bands from each treatment, plus from a positive control (mix ed cultured glial cells treated with LPS).
74 Figure 2 6. Previous page. Cellular localization of immunoreactive Mas in normal rat cerebral cortex and 24 h after ET 1 induced MCAO. Representative fluorescence micrographs taken at 40x magnification. A, B, C, D, and E ) Panels from normal rat cerebral cortex and show respective co localization of Mas (M; red fluorescence) with either NeuN (N), Ox 42 (Ox), Reca (Re), GFAP (G), or MPO [all green fluorescence]. F, G, and H ) Panels from cortex 24 h after MCAO and sh ow respective co localization of Mas (M; red fluorescence) with either NeuN (N), Ox 42 (Ox) or MPO [all green fluorescence]. NeuN images (F) were taken in the penumbra. Ox 42 (G) and MPO (H) images were taken in the core of the stroke. Arrows indicate co l ocalization of Mas with neurons (NeuN), microglia (Ox 42) or endothelial cells (Reca). Panels I and J confirm Mas antibody specificity, showing no staining in knockout mouse brain tissue (KO, panel I) and staining in wildtype tissue (WT, panel J). N=3.
76 Figure 2 7 Ang (1 7) blunts lipopolysaccharide (LPS) induced increases in nitrite in the media of primary mixed glial cultures. Cells cultured as described in the Methods were treated as follows. Contro l solution (PBS), 24 h; LPS (10 ng/mL), 24 h; LPS (24 h) + Ang (1 7) (0.1 nmol/mL), 24h. LPS (24 h) + Ang (1 7) + A779 (1.0 nmol/mL). Note that Ang (1 7) +/ A779 were administered 12 h before LPS, at the time of LPS treatment, and 12 h after LPS. Grow th media were sampled 24 h after LPS treatment for analysis of nitrite (NO 2 ) via the Greiss reaction. A) Bar graphs are means + SEM showing the levels of NO 2 under each treatment condition. N=7 for control and N=10 for other treatment groups. P<0.05 comp ared to control. P<0.05 compared to LPS. B and C) Fluorescence micrographs demonstrate the presence of immunoreactive GFAP (panel B) and immunoreactive Iba 1 (panel C) in the cultures used here, indicating the respective presence of astroglia and microglia.
77 CHAPTER 3 ANGIOTENSIN (1 7) HAS CEREBROPROTEC TIVE POTENTIAL IN HE MORRHAGIC STROKE AND INCREASES SURVIVAL OF STROKE PRONE SPONTANEOUSLY HYPERTENSIVE RATS Introduction In the United States, stroke is the fourth leading cause of death and a major cause of serious, long term disability 220, 246 There are two types of stroke: ischemic, where blood flow to the brain is reduced, and hemorrhagic, where persistent hypertension leads to vessel thinning and intracerebral bleeding. Very few therapies exist for both types, and the therapies that do exist can only be used in certain candidate patients. Hemorrhagic stroke, in particular, accounts for 10 20% of all strokes in Western populations 45 with a disproportionate affinity for Asians and African Americans 5 Patients with hemorrhagic strokes that are most commonly induced by persistent hypertension have poor prognoses, often muc h worse than ischemic strokes of similar size. Furthermore, up to 30% of patients with ischemic stroke undergo hemorrhagic transformation 6 and many patients with ischemic strokes develop symptomatic hematomas followi ng thrombolysis 54 Both types of stroke are multifactorial, yet mounting evidence indicates that the renin angiotensin system (RAS) is highly involved 165, 167 171, 17 3 175, 177, 185, 186, 191, 235 The RAS has been implicated in many cardiovascular diseases. Primarily known for the effects of the angiotensin converting enzyme/angiotensin II/angiotensin type 1 receptor (ACE/Ang II/AT1R) axis, the RAS has been a target for the development of many potential therapies, including preventing the deleterious effects that lead to CNS damage following cerebral ischemia 247 Numerous studies have shown that blocking the actions of Ang II at AT1R via angiotensin receptor blockers (ARBs) decreases
78 cortical/subcortical infarct size and the ensuing neurological deficits in animal models of stroke 168, 171, 173 Human cl inical trials have also shown that ARBs reduce cardiovascular risk and improve stroke prevention 167, 185, 186, 222 Another axis of the RAS, the a ngiotensin converting enzyme 2/angiotensin (1 7)/Mas (ACE2/Ang (1 7)/Mas) axis, is receiving more attention in the literature for its therapeutic potential in cardiovascular disease and stroke diseases 137, 143, 223 We demonstrated that intracerebroventricular treatment of Ang (1 7) is cerebroprotective in endothelin 1 induced middle cerebral artery occlusion. Central Ang (1 7) decreased the size of the formance on neurological exams, and did not have any effect on cerebral blood flow 218 Furthermore, we showed that this peptide has anti inflammatory effects in the brain during stroke, preventing an over activation of microglia and blunting pathological increases in pro inflammatory cytokines and iNOS ( Chapter 2 ). Given Ang (1 inflammatory effects, our aim here to determine if it also has any cerebroprotective effects in hemorrhagic stroke. Inflammation has a strong role in the mechanism of injury in hemorrhagic stroke. The inflammatory response begins soon after hemorrhage and peaks several days later in humans and animal models 58, 101, 114, 115 The infiltration of neutrophils occurs within 2 days, and the activation of microglia may continue for 1 month 114, 116 Evidence supports a role of microglia in hemorrhagic stroke pathology because inhibiting their activation reduces damage 117, 118, 120 Furthermore, there is believed to be a str ong association between inflammation and edema, as evidenced by data that correlates plasma tumor necrosis factor alpha concentrations in patients with
79 hemorrhagic stroke to the degree of brain edema 123 For these e xperiments, we used stroke prone Sponataneously Hypertensive Rats (spSHR). When fed a high salt diet, these rats spontaneously develop extremely high blood pressure resulting in hemorrhagic stroke and death. Here we show for the first time a cerebroprotect ive effect of Ang (1 7) in spSHR, resulting in increased survival. Methods Ethical Approval and Animals For the experiments described here, we used a total of 90 male stroke prone Spontaneously Hypertensive Rats (spSHR) and 13 male Wistar Kyoto (WKY) rats purchased from Charles River Farms (Wilmington, MA, USA). All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee. In addition, the principles governing the care and treatment of animals, as stated in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication no. 85 323, revised 1996) and adopted by the American Physiological Society, were followed at all times during this study. Rats had ad l ibitum access to water and were housed in a well ventilated, specific pathogen free, temperature controlled environment (24 1C; 12 h 12 h light dark cycle). Rats were obtained at 35 days of age (weaning), and the spSHR were immediately started on a 4.0% high sodium diet (Research Diets). WKY rats were kept on standard chow. Implantation of Intracranial ICV Cannulae and Osmotic Pumps ICV treatments were initiated when rats were 49 days old, except for the experiments involving direct blood pressure measur ement (see below). Rats were anesthetized with a mixture of O 2 (1.5 L/min) and 4% isoflurane, placed in a Kopf stereotaxic frame, and anesthesia was maintained for the duration of the surgeries using
80 an O 2 /isoflurane (2%) mixture delivered through a nose c one attached to the frame. Animals then underwent an intracranial ICV surgery essentially as detailed previously 176, 218 This involved implantation into the left cerebroventricle (1.3 mm posterior t o bregma, 1.5 mm lateral to bregma, and down through the cranium flush with the skull surface) of a stainless steel cannula (kit 1, ALZET, Cupertino, CA) coupled via vinyl tubing to a 6 week osmotic pump (model 2006, ALZET, Cupertino, CA) 197, 218, 248 Osmotic pumps were implanted subcutaneously between the shoulder blades and were used to infuse Ang (1 7) the Mas antagonist A779 (mixed in same pump) or control artificial cere brospinal fluid (aCSF) into the left lateral cerebral ventricle. Infusion started at the time of ICV cannula placement and lasted for 6 weeks, until rats were 91 days old. Following this surgery, the wound was closed and each rat was administered an analge sic agent (buprenorphine; 0.05 mg/kg sc) before waking. Several studies were done, including a survival study, a study where animals were euthanized just before treatment ended (90 d old), and a study where animals were euthanized after the first spSHR die d (108 d). Hemorrhage Number and Severity Assessment To compare the hemorrhages at the time of death, the brains were removed immediately after the rats were found dead. The whole brain for each rat was then photographed. Next, serial 2 mm sections were ma de throughout the entire brain to scan the subcortical regions. These sections were also photographed. The photographs were then analyzed by a research assistant who was blinded to the treatment groups. For each animal, the number of brain hemorrhages was counted. In addition, the severity of each hemorrhage was graded on a scale of 1 to 4, where 1 is minor and 4 most severe 249, 250 Then the sum of the severity scores was calculated to give a glo bal
81 using the whole brain and subcortex using the 2 mm sections. For the 2 mm sections, each section was counted, with no attempt to distinguish if a hemorrhage in one sect ion had been counted in a previous section. We believe that this is the most objective and consistent way to count, as if a hemorrhage was counted more than once it would represent greater severity. Hermorrhage Volume For hemorrhage volume and hemoglobin c ontent analysis 251 animals were euthanized at 108 days of age (when the first animal began to show signs of stroke) and transcardially perfused with normal saline. Brains were then taken from the skull, cerebellums an d brainstems removed and discarded, and the remainder of the brain dissected into hemispheres. The right hemisphere was homogenized in 3 mL of PBS for 30 s and sonicated on ice for 1 min. 200 uL aliquots (15 fold dilution) were made. To prepare a standard curve, incremental volumes of homologous blood (0, 3, 6, 9, 12 uL) was added to 200 uL aliquots of homogenates from age matched WKY rats. These 200 uL aliquots were homogenized for 10 s and sonicated on ice for 20 s. Samples were then centrifuged at 13000 rpm for 30 min to allow collection of the hemoglobin (Sigma) and allowed to stand for 15 min at room temperature. The OD at 540 nm was then measured with a spectrophotome ter. The concentrations of hemoglobin were then calculated using the standard curve of known volumes of blood. Then the dilution factor was used to calculate the amount of blood in the entire hemisphere. Brain Water Content For water content analysis, anim als were euthanized at 108 days of age and
82 transcardially perfused with normal saline. Brains were then taken from the skull, cerebellums and brainstems removed and discarded, and the remainder of the brain dissected into hemispheres. The left hemisphere w as weighed upon removal and again after 24 hours in a dryer oven to calculate the % water weight with the following formula: 100 x (wet weight dry weight) / wet weight 251 253 Vid eo Tracking and Locomotor Activity Rats were habituated to a circular open field (diameter 55 cm, height 42 cm) that was black in color. For acclimation, they were placed in the field for 10 min/day for three consecutive days before measurements were taken Locomotor activity was monitored for 20 min on the fourth day (age 108 days old) and analyzed using Noldus Ethovision Software (Wageningen, The Netherlands) 254 Immobility and rotation frequency were calculate d. Immobility is the time that the running average mobility is below the immobile threshold. It is calculated by calculat ing the changed area of the rat indicating movement Rota tion Frequency is the number of turns (either clockwise or counterclockwise) of 360 degrees. As rats are place in a circular field, we also interpret this result as a measure of activity. Sunflower Seed Task A sunflower seed eating task 176, 218, 255 wa s used that provides an index of fine motor function to assess the level of neurological deficits. In this test, the number of shell pieces that the rats leave after consuming 5 seeds is recorded. Rats with less neurological impairment open the five seeds more efficiently by breaking the shell into fewer pieces to accomplish the task. This task was performed from age 56 to 102 days old to monitor sensorimotor function.
83 Morris Water Maze Rats underwent the Morris Water Maze task as previously described 256 Briefly, each was placed in a 160 cm diameter black pool filled to a depth of 30 cm with clear water maintained at room temperature. Rats were required to locate a submerged platform using visual cues placed around the pool. They were placed in the pool at one of four different locations (N, S, E, W) and given 90 s to locate a 10 cm sided square platform submerged 2 cm below the surface of the water. Three trials of 5 d each were conducted, starting at 70, 77, a nd 8 4 days of age, respectively. For the first 4 d, the latency (s) to find the platform was recorded. On the 5 th day, a probe trial was conducted in which rats were placed in the pool for 90 s without the platform. The time spent in the quandrant of the pool that previously had the platform was recorded. Mas Co localization Immunohistochemistry For the Mas immunohistochemistry, control spSHR were euthanized at 90 days of age. The brains were fresh frozen in Tissue Finetech, Torran ce, CA), as we previously determined the Mas antibody to work better with fresh frozen tissue ( Chapter 2 ). Mas was co stained with NeuN, GFAP, RECA, and OX 42, which are respective specific markers for neurons, astroglia, endothelial cells and microglia. P rocedures were as follows. Brain sections were cut from the 2 mm coronal section block at 5um and air dried at room temperature overnight. OCT was removed in a wash of 1X TBS for 5 minutes. Slides were drained and wiped, then blocked for 1 hour in 2% horse serum diluted in 1X TBS. Slides were wiped and blocking buffer was replaced with antibody cocktails (rabbit anti Mas [1:400] combined with either mouse anti NeuN [1:100], anti GFAP [1:100], anti OX 42 [1:100], anti RECA [1:100] or anti MPO [1:50] diluted in antibody diluent (Invitrogen, Carlsbad, CA), and
84 incubated overnight at 4C. Following two 5min washes in TBS, Alexafluor donkey anti rabbit 594 and donkey anti mouse 488 were added to the slides, both at 1:500. After a 45 minute room temp incubation, slides were again double washed in TBS. Sections were then post fixed for 5 minutes in 10% neural buffered formalin, washed twice more, then wiped dry before mounting in DAPI vectashield (Vector Labs, Burlingame, CA). Furthermore, to verify Mas antibody s pecificity, it was tested in brain tissue from a Mas knockout mouse. There was no positive staining present in the knockout brain tissue, while staining was present in tissue from the wild type. Stereology and Immunostaining For stereology experiments, an imals were euthanized just before the pumps ran out of treatment at 90 days of age. These animals were perfused transcardially (normal saline followed by 10% formalin), and the brains were removed. The brains were postfixed in 10% formalin for 24 h, and th en cryoprotected in 30% sucrose in 0.1M phosphate buffer Brains were then frozen in dry ice and stored at 80C. Systematic uniform random sampling was achieved by exhaustively sectioning brains coronally on a calibrate caudal extent of the striatum, beginning just caudal to the olfactory bulbs and ending at the rostral hippocampus. A registered 1 in 15 series of sections was obtained for each animal and separate series were randomly assigned for processing to detect total striatal microglia (IBA 1) or neurons (NeuN). Sections were collected into cold 0.1M TBS and stored at 4C until stained immunohistochemically. Free floating tissue sections were washed several t imes in Tris buffered saline (TBS; 100 mM Tris HCL, 150 mM NaCl, pH 7.5), pre incubated in blocking solution containing 3% normal donkey serum (NDS) and 0.3% Triton X 100 in TBS for 1 hour, and incubated in blocking solution with the addition of
85 the approp riate primary antibodies for 72 hrs at 4C. Specific antibody combinations and dilutions used were: rabbit anti IBA 1 (Wako 019 19741, 1:500) and mouse anti NeuN (Millipore MAB377, 1:1000). After primary incubation, sections were washed in 0.1M TBS, and i ncubated in TBS containing 3% NDS and appropriate secondary antibodies (Alexa 488 and 555; Molecular Probes, 1:300). Secondary incubations were performed for 2 h at room temperature in the dark. After secondary incubation, sections were washed in 0.1M TBS, and mounted onto Superfrost++ slides. Coverslips were applied with ProLong Gold (Invitrogen), sealed with clear fingernail polish, and stored in the dark at 4C until analysis. Quantification was done using the optical fractionator method 257, 258 implemented using a Zeiss AxioImager.M2 epiflourfescent microscope, equipped with the appropriate filter sets, a CCD camera, and a computer driven X, Y and Z Ludl motorized stage controlled with Ste reoInvestigator software (MBF Bioscience, Williston, VT). Initial z plane analyses confirmed that each antibody penetrated the full section thickness. The boundaries of the striatum were delineated at low power magnification (2.5X) in a systematically rand th section), that extended rostrally from the emergence of the striatum in the corpus callosum and ended caudally at the disappearance of the striatum when the rostral hippocampus appeared. Cell estimates were derived from a minimum of 9 sections (per label). The motorized stage of the microscope was moved in evenly spaced x y intervals under the computer control, surveying the regions of interest in each section according to a systematic random sampling s cheme ( see Table 3 1 for sampling details ). Section thickness was measured at each sampling site using a 40X oil immersion objective
86 (with 1.4 numerical aperture) and quantification was confined to a 20 m optical disector height which was positioned 3 m below the surface of the tissue. Cells were counted at each site if its top most nucleus first came into focus with the optical disector, provided it did not encroach on the exclusion lines of the counti ng frame 257, 259 The total number of IBA 1 and NeuN immunopositive cells in the stratum were estimated using the optical fractionator method 258 in whi ch the product of the cells counted in a known, uniformly random sample of the region of interest is multiplied by the reciprocal of the sampling fraction. Additional details, including stereological sampling parameters are provided in Table 3 1. The preci sion of the stereological estimates was determined by estimating the coefficients of error (CE) using methods previously described 257 Equations used to fication m=1, as the areas defined for the cell counts changed smoothly from the rostral entrance and caudal conclusion of the striatum. qRT PCR For RNA, rats were euthanized just before the pumps ran out of treatment at 90 days of age. Fresh brains were t aken from the skull, the cerebellum and olfactory bulbs were removed, and the remaining tissue was flash frozen in liquid nitrogen and saved at 80C. Total RNA was extracted using TRIzol (Invirogen, etc) and purified using an RNeasy Plus kit (Qiagen, Vale ncia, CA, USA). Isolated RNA underwent DNAse I treatment to remove genomic DNA Extracted RNA was reverse transcribed with a high capacity cDNA reverse transcription kit (Bio Rad Laboratories, Hercules, CA) and then analyzed via quantitative real time reve rse transcriptase polymerase chain reaction (qRT PCR) in a PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA) as detailed by us previously 218, 225 Oligonucleotide primers and Taq Ma n
87 1 were obtained from Applied Biosystems (Carlsbad, California). Data were normalized to GAPDH expression. Direct Blood Pressure For direct blood pressure measurements, radiotelemet ry transducers were implanted at 49 days of age. To allow time for recovery, the ICV cannulae and mini osmotic pumps were not implanted until 63 days of age (procedure described above). For the telemetry transducer implantation, rats were anesthetized with a mixture of O 2 (1 L/min) and isoflurane (4%). Rat telemetry transducers (TA11PA C40, DSI) were implanted into the abdominal aorta, as described by us previously 176, 260 Rats were administered the an algesic (buprenorphine; 0.05 mg kg S.C.) following surgery, and were left to recover for 14 days before the implantation of subcutaneous mini osmotic pumps. Data were collected every 2 3 days from age 49 d to age 72 d, when the sample sizes started to ge t small due to fewer animals surviving. Indirect Blood Pressure For indirect blood pressure measurements, we implanted the mini osmotic pumps at 49 days of age to remain consistent with our other experiments. Blood pressure was recorded via an indirect ta il cuff plethysmography method as described previously 197, 218 Briefly, blood pressure was non invasively measured by determining the tail blood volume with a volume pressure recording sensor and a n occlusion tail cuff (CODA System, Kent Scientific, Torrington, CT). Rats were placed in the restraint and warmed on the warming plate for about 3 min until external temperature is 30 C. The pressure cuff was then cycled 5 times to acclimatize the rat fol lowed by 15 cycles to collect blood pressure data. Data were collected every 2 5 days from age 70 d to age
88 129 d, when the sample sizes started to get small due to fewer animals surviving. Body weight Body weight was recorded using an analog Taconic Rat Sc ale Model YG 700 (Evanston, IL) just before rats were euthanized at 108 days old. Corticosterone At 108 days of age blood was collected prior to euthanasia. Serum was separated for corticosterone analysis using an enzyme immunoassay kit (K014 H1, Arbor Ass ays) as described by the manufacturer. Creatinine and BUN At 108 days of age blood was collected prior to euthanasia. Serum was separated for creatinine and BUN analyses. A University of Florida veterinary laboratory performed these standard analyses. Kid ney and Heart Pathology Rats were euthanized at 90 days of age and perfused transcardially (normal saline followed by 10% formalin). Kidneys and hearts were removed and stored in 10% formalin until sectioned. Kidney sections were stained with H&E and PAS, and h eart sections were stained with H&E and trichrome by the histology core at the University of Florida. Pathology was assessed in blinded fashion by a licensed veterinary pathologist. Glomerular and tubular alterations were evaluated by a lesion scoring system developed by our collaborator 261 Specifically, glomeruli were evaluated for the glomerular fibrosis, membranoproliferative changes, degr ee of hypercellularity, thickening of entire slide. Lesion scores were assigned as follows: 0 = no lesions, 1 = less than 10%,
89 2 = 10 to 25%, 3 = 25 to 50%, and 4 = gre ater than 50% of total slide area affected. Similarly, tubular lesions were scored from across the entire section, including interstitial fibrosis, cellular infiltrates, and tubular atrophy with thickening of the basement membrane. Lesion scores were assig ned as follows: 0 = no lesions, 1 = <25%, 2 = 25 to 50%, and 3 = >50% of total slide area affected. Heart lesions were scored from across the entire section, including degenerative myofiber changes, interstitial fibrosis, and cellular infiltrates 262 The severity of microscopic lesions observed was graded based on the degree and extent of tissue damage using a four point scale of absent (0), minimal (1), mild (2), moderate (3), and marked (4). Minimal (grade 1) lesi ons involved less than 10% of the heart section. Mild (grade 2) lesions involved 11% 40% of the heart section. Moderate (grade 3) lesions involved 41% 80% of the heart section. Marked (grade 4) lesions involved greater than 81% of the heart section. Chemic als Ang (1 7) and A779 were purchased from Bachem Bioscience (Torrance, CA, USA). ET 1 was from American Peptide Company, Inc (Sunnyvale, CA, USA). Rabbit anti Ang (1 7) Mas receptor antibody was from Alomone Labs (Jerusalem, Israel). Mouse anti rat RECA 1 was from AbD Serotec (Raleigh, NC). Mouse anti MPO, mouse anti GFAP, rabbit anti iNOS (ab15323), rabbit anti Cofilin (ab11062) and goat anti rabbit IgG HRP (ab6721) antibodies were from Abcam (Cambridge, MA). Mouse anti OX 42 antibody was from BD Bioscie nces (San Jose, CA). Mouse anti NeuN was from Millipore (Bedford, MA). Rabbit anti Iba 1 (01919741) was from Wako (Richmond, VA). Alexafluor donkey anti rabbit 594 and anti mouse 488 were from Molecular Probes [Invitrogen] (Carlsbad, CA, USA). Vectashield mounting medium with DAPI ( 4',6 diamidino 2 phenylindole ) was from Vector Labs (Burlingame, CA, USA). All other
90 chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA). Data Analysis Data are expressed as means SEM. Statistical signific ance was evaluated, as specified in the figure legends, with the use of a one way ANOVA, two way row matched ANOVA, Newman Keuls test, t test or log rank as appropriate. Differences were considered significant at p<0.05. Results Ang (1 7) Tr eatment Improves Survival of Stroke prone Spontaneously Hypertensive Rats In previous studies, we demonstrated that Ang (1 7), administered ICV, had cerebroprotective effects in ischemic stroke 218 Furthermore, our st udies investigating underlying mechanism s showed that this cerebroprotection was likely due to an anti inflammatory effect of Ang (1 7) ( Chapter 2 ). Therefore, we were moti vated to examine any therapeutic effects of Ang (1 7) in hemorrhagic stroke, as infl ammation and edema contribute to its pathophysiology 58, 101, 114, 115 Here we show that 6 weeks of central Ang (1 7) treatment, infused from 49 to 91 days of age, increases the survival of stroke prone Spontaneously Hypertensive Rats (spSHR) (Figure 3 1). The median survival is increased from 108 days in control aCSF treated spSHR to 154 days in Ang (1 7) treated spSHR. Furthermore, this effect is reversed when the Mas antagonist A779 is co administered with Ang (1 7), indicating that this effect on survival is Mas dependent Ang (1 7) Treatment Decreases the Number and Severity of Hemorrhages in the Subcortex/Striatum but not in the Cortex at the Time of Deat h of spSHR Observational studies of spSHR have shown that they exhibit several cortical and subcortical hemorrhages when they die and that these hemorrhages are considered their primary cause of death in most cases 249, 250 Here, both cortical and subcortical
91 hemorrhages were observed in brains removed from spSHR immediately after death (Figure 3 2A, 3 2 B). Ang (1 7) did not have an effect on the number or severity of hemorrhages counted in the co rtex (Figure 3 2C, 3 2 E) in these brains. There was, however, a significant decrease in the number of hemorrhages in the subcortex/striatum and a trend toward decreasing the severity of the hemorrhages in these regions (Figure 3 2D, 3 2 F). Ang (1 7) Trea tment has no Effect on Brain Hemorrhage Volume ( Hemoglobin Content ) or Water Content To more thoroughly investigate the severity of hemorrhages and edema, total hemorrhage volume ( brain hemoglobin content) and water content. There was no effect of Ang (1 7 ) on total hemorrhage volume when the rats were euthanized at 108 days old. This is likely because significant hemorrhagic strokes had yet to occur when rats that spSH R hemoglobin content was not significantly different from the hemorrhage volume of the control WKY rats. In addition, Ang (1 7) treatment did not have an effect on water content of the brains when rats were euthanized at 108 days old. Similar to the hemorr hage volume data, the water content of spSHR brains was not different from that of the control WKY brains, suggesting that edema and other pathology had not yet occurred to a significant degree when rats were euthanized. Ang (1 7) Treatment Improves the Ne urological Status of spSHR, but has no Effect on Visuospatial Memory The most obvious signs of neurological disease in spSHR are lethargy and general sensorimotor dysfunction 249 We therefore chose to measure spontan eous movement using video trac king software and a sunflower seed eating task 218 The video tracking software was used at 108 days of age. Ang (1 7) decreased the mean
92 immobility of spSHR, or the duration of time that r ats were immobile (Figure 3 4A). This suggests that the rats treated with Ang (1 7) demonstrated less lethargic behavior. Also, Ang (1 7) increased the rotation frequency within a circular arena, indicating that spontaneous movement is increased (Figure 3 4B). The sunflower seed eating task was performed repeatedly to track general sensorimotor function from 56 to 102 days of age. A significant improvement in the efficiency to shell seeds was observed in the Ang (1 7) treated spSHR when analyzing both curve s by repeated ANOVA with several time points showing very large differences (Figure 3 4C). They left fewer shell pieces behind, showing that they could better manipulate the seeds to eat them. Despite these improvements in neurological function observed in the Ang (1 7) treated spSHR, no improvements were observed in visuospatial memory as measured by the Morris water maze task (Figure 3 5A, 5B) Cellular Localization of Immunoreactive Mas in spSHR Striatum Having shown that Ang (1 7) has therapeutic potent ial in hemorrhagic stroke, we sought to examine potential mechanisms for these effects. We used immunostaining to investigate both the cell types that expressed the Ang (1 7) receptor Mas and the cellular location of this receptor in the spSHR striatum to learn what cell types could be involved. Our results were in accordance with our previous study of Sprague Dawley cortical tissue ( Chapter 2 ). Mas immunoreactivity is present in neurons (anti NeuN, Figure 3 6A, 6B) micro glia/macrophages (anti OX 42, Figur e 3 6C, 6D), and endothelial cells (anti RECA, Figure 3 6E, 6F) However, Mas did not co localize with astroglia (anti GFAP, Figure 3 6G, 6H). It should be noted that we confirmed the specificity of the Mas antibody using Mas knockout mouse brain tissue. T here is no staining in the knockout, where staining is obvious in the wild type (Figure 3 6I, 6J).
93 Ang (1 7) Decreases Microglia and Increases Neuron Survival in the Striatum Since we previously demonstrated an effect of Ang (1 7) on macrophage/ microglia activation in an ischemic model of stroke and in cell culture ( Chapter 2 ), we aimed to quantify the number of microglia in the striatum in this spSHR model to determine their role in the mechanism of cerebroprotection. We focused on the striatum because i t was the brain region where Ang (1 7) significantly decreased the number of hemorrhages. Using stereology and Iba 1 immunostaining, Ang (1 7) was shown to decrease the number of macrophages/microglia in the striatum (Figure 3 7). Ang (1 7) appeared to blu nt their over activation and migration to this region Furthermore, we also quantified neurons to determine if their survival was affected by Ang (1 7) treatment. Using stereology and NeuN immunostaing, Ang (1 7) was shown to increase the number of neurons in the striatum (Figure 3 7). Ang (1 7) does not Alter the Expression of Genes Related to Inflammation in Whole Brain Homogenate Our previous study investigating anti inflammatory effects of Ang (1 7) in ischemic stroke indicated that the expression of s everal key genes was altered by Ang (1 7) ( Chapter 2 ). GFAP, CD11b, and MCP 1 in spSHR to see if a similar effect could be measured. IL6, were no differences du e to Ang (1 7) treatment (Figure 3 8) Although there was a trend 1 to be decreased by Ang (1 7) treatment, none of the differences were statistically significant.
94 Ang (1 7) has no Effect on Mean Blood Pressure during Infusion in spSHR, b ut Rats Treated with Ang (1 7) have Lower Mean Blood Pressure later in Life after Treatment Ends Effects of central Ang (1 7) on blood pressure would of course affect stroke, so we examined mean arterial pressure two different ways (Figure 3 9) Direct blo od pressure measurements using t elemetry transducers showed no effect of central Ang (1 7) infusion on blood pressure. In another group of spSHR, i ndirect blood pressure measurements using tail cuff plethysmography showed no effect of Ang (1 7) during the infusion. However, this technique allowed longer monitoring of animals after the pumps ran out of treatment. The spSHR that were previously treated with Ang (1 7) had lower blood pressure later in life. Ang (1 7) has no Effect on Body W eight, Corticostero ne Creatinine BUN, or Kidney/Heart Pathology We also aimed to rule out other peripheral effects of Ang (1 7). Body weight was recorded just prior to euthanasia at 108 days old. There was no significant effect of Ang (1 7). Furthermore, spSHR animals did not have different body weights compared to age matched WKY rats. In addition, b lood was collected at 108 days old, and serum was separated for corticosterone, creatinine, and BUN anal ysis There were no significant effects of Ang (1 7) on any of these ana lyses. Again, age matched WKY rats were not significantly different from spSHR either. We also scored the kidney and heart pathology at 90 days of age, as this is more sensitive than creatinine and BUN levels. A blinded licensed veterinary pathologist asse thickening, periglomerular fibrosis, membranoproliferative changes, tubular interstitial fibrosis and cell infiltration, tubular degeneration/regeneration, heart degeneration, and
95 heart fibrosis and inflammation. For eac h metric, spSHR had more pathology that age matched WKY rats, but there was no effect of Ang (1 7). Discussion This present study demonstrates, for the first time, cerebroprotective effects of central Ang (1 7) in spSHR. Perhaps the most convincing evidenc ability to increase the survival of this rat strain. We believe this is a Mas dependent effect, since the survival time in spSHR treated with both Ang (1 7) and A779 (Mas antagonist) is between spSHR treated with aCSF or Ang (1 7) alone Other evidence in support of Ang (1 number of hemorrhages in the striatum/subcortex at the time of death. There was a trend to decrease the number in the cortex, but it did not reach s tatistical significance. It is possible that the Ang (1 7) could better diffuse to the striatum relative to the cortex, as it is infused into the lateral ventricle, which borders the striatum. We were surprised that there were no statistical differences in the hemoglobin content or water content when animals were euthanized at 108 days old. There are several experimental details that may provide an explanation. First, whole brain hemispheres were analyzed. If Ang (1 triatum, the effects may be diluted. Also, the animals were all euthanized at 108 days old, the same age. This is the ideal way to compare groups, but it is possible that not enough hemorrhages and pathology/edema existed in many of the animals. This is su pported by the data as the water content and hemoglobin content in WKY rats are not significantly different from both spSHR groups. There is, however, convincing neurological data at these ages. One of the most common neurological signs in spSHR as the pat hology progresses is lethargy. Central Ang (1 7) treatment reduces immobility and increases rotation frequency in a circular
96 open field designed to measure spontaneous activity. In addition, spSHR treated with Ang (1 7) show improved performance on the sun flower seed eating task when analyzed by repeated ANOVA over a time course which measured global sensorimotor function We further hypothesized that because spSHR experience multiple microhemorrhages and edema as they age 249 visuospatial memory might decline. This was not observed when we subjected rats to the Morris water maze task, however. For each of these neurological exams we did not include a WKY control group, as it has been well documented that WKY are not good controls for spSHR, especially when it comes to behavioral studies 263, 264 We believe these functional improvements add greatly to the evidence supporting a cerebroprotective effect of An g (1 7). Previously, we performed an immunohistochemical co localization study to determine what cell types in the brain of Sprague Dawley rats contain Mas ( C hapter 2 ). We repeated this study with spSHR to confirm that the expression of Mas was the same. Indeed we found Mas immunoreactivity to be present on neurons, microglia, and endothelial cells. This affirmed our hypothesis that inflammation could be involved in the mechanism of cerebroprotection in spSHR. As previously stated, inflammation has a stron g role in the mechanism of injur y in hemorrhagic stroke The inflammatory response begins soon after hemorrhage and peaks several days later in humans and animal models 58, 101, 101, 115 The infiltration of neutrophils occurs within 2 days, and the activation of microglia may continue for 1 month 114, 116 Evidence supports a role of microglia in hemorrh agic stroke pathology because inhibiting their activa tion reduces damage 117, 118, 120 To observe changes in microglia and neurons in the striatum just before treatment ran out at 90 days of age, we used stereological techniques to count
97 cells. As stated, we focused on the striatum because it was this region where Ang (1 7) had the greatest effect to reduce the number of hemorrhages. Ang (1 7) decreases the number of microglia and incr eases the number of surviving neurons, providing further support of a neuroprotective action. Interestingly, we did not observe changes in gene expr ession that corroborated this. In an ischemic model of stroke, we saw Ang (1 7) blunt pathological increases in iNOS, proinflammatory cytokines, and CD11b ( C hapter 2 ). We did not observe any Ang (1 7) effect here when rats were euthanized just before the end of treatment at 90 days old. This may be because not enough pathology had developed to cause measurable c hanges in gene expression at this time point. Alternatively, we have observed that some gene expression differences are not measurable when analyzing the entire brain 218 as we did here. However, we believe this is the only way to perform a gene expression study in this model because the hemorrhages occur throughout the brain. Furthermore, it would be difficult to consistently dissect out just one region, such as the striatum, for analysis. Another major question we con sidered was whether or not centrally administered Ang (1 7) could have any peripheral or systemic effects. We performed two studies to investigate mean arterial pressure. The first measured pressure directly using radiotelemetry transducers. There was no e ffect of Ang (1 7) on blood pressure. The second measured pressure indirectly using tail cuff plethesmography. These animals that did not undergo the invasive telemetry transducer implantation surgery survived longer. Because of this, we could carry out th e experiment after ICV pumps ran out of treatments. As with the direct measurements, we observed no effect of Ang (1 7) during infusion. However, when the curve is separated into treatment and post treatment
98 curves and analyzed separately, the animals that were previously treated with Ang (1 7) have decreased mean arterial pressure later in life after the pumps run out of treatment. This suggests a protective effect of Ang (1 7) that is not measureable later until pathology develops. Perhaps Ang (1 7) is ha ving some kind of effect on blood pressure control regions of the brain by decreasing inflammation there. Further experiments will need to be conducted to answer these questions We also wanted to examine several other endpoints. At 108 days of age, there was no effect of Ang (1 7) on body weight, serum corticosterone, BUN, or creatinine. Lastly, we could not measure any effect of centrally administered Ang (1 7) on the histopathology of the heart or kidneys, although there were differences compared to the control WKY rats. This study provides the first evidence that activating the ACE2/Ang (1 7)/Mas axis may have therapeutic potential in hemorrhagic stroke. Ang (1 7) given ICV is cerebroprotective in spSHR and increases their survival. This corroborates a cerebroprotective effect we have observed in a model of ischemic stroke 218 The stereology data in this study also corroborates that Ang (1 7) has anti inflammatory effects ( Chapter 2 ). We think the findings are robust as survival is improved, physical endpoints are improved, and behavioral endpoints are improved. The interesting observation that animals treated with Ang (1 7) have lower blood pressure later in life after treatment ends merits further experiments. In ad dition, further studies will need to be performed to better understand the intracellular signaling and mechanism of protection of Ang (1 7).
99 Table 3 1. Parameters used for stereology. Object Sampling Grid ( M x M) Counting Frame ( M x M) Dissector Height ( M) Mean Object Counted Average CE NeuN+ cells 800 x 800 50 x 50 20 1460 0.04 IBA 1+ cells 800 x 800 50 x 50 20 503 0.05 Total cell numbers were estimated using the formula: N (Total number) = 1 / ssf (section sampling fraction) x 1/asf (area sampling fraction) x 1/hsf (height sampling fraction) x number of immunopositive cells counted. The total number of cells counted for each subject and the sampling grid and counting frame used to generate the asf for each label are provided above. The ssf is the current study equaled in every case. The hsf was calculated using the mean weighted thickness (with section thickness measured at each sampling site). SD=standard deviation ; CE=coefficient of error, m=1 ; N= 6 /group.
100 Figure 3 1. Ang (1 7) treatment improves survival of stroke prone Spontaneously Hypertensive Rats (spSHR). spSHR are fed a 4% sodium diet after weaning (35 days old). 6 wk Ang (1 (1 old; 6 week old pumps ran out of treatment at 91 days old. Animals were monitored and maintained on high salt diet until death. N = 9 for aCSF, Ang (1 7). N = 6 for Ang (1 7) + A779.
101 Figure 3 2. Ang (1 7) treatment decreases the number of hemorrhages in the subcortex/striatum, but not in the cortex at the time of death of spSHR. spSHR are fed a 4% sodium diet after weaning (35 days old). 6 wk Ang (1 7) implanted at 49 days old; 6 w k old pumps ran out of treatment at 91 days old. Animals were monitored and maintained on high salt diet until death, when their brains were removed. A) Representative cortex hemor rhages. B) Representative subcortex/striatum hemorrhages. C and D) Cortex/subcortex hemorrhage number was counted as described in the methods. E and F) Cortex/subcortex severity was assessed from 0 4 for each hemorrhage and these values were summed to calc means + SEM. N=6/group. *P<0.05 compared spSHR aCSF.
102 Figure 3 3. Ang (1 7) has no effect on brain hemorrhage volume or water content. Ang (1 7) (1.1 nM; 0.5 old WKY controls. 6 week pumps ran out of treatment at 91 days old. Rats were euthanized at 108 days old and perfused with sterile saline Brains were removed and dissected into hemispheres. For panels A and B, bar graphs are means + SEM. N=4, 9, and 7 for WKY, spSHR aCSF, and spSHR Ang (1 7), respectively. A) Hemoglobin. The right hemisphere was homogenized and hemoglobin content was used to determine the hemorrhage volume as described in the methods. B) Water content. The left hemisphere was weighed upon removal and again 24 hours later to calculate the % water weight.
103 Figure 3 4. Ang (1 7) treatment improves neurologic al status of spSHR. 6 wk Ang (1 7) week old pumps ran out of treatment at 91 days old. A) Ang (1 7) decreases immobility showing a reversal of lethargy as assessed before euthanasia at 108 days old. Bar graphs are means + SEM. N=9 for spSHR aCSF and 7 for spSHR Ang (1 7). B) Ang (1 7) increases rotation frequency showing an increase in spontaneous movement within the circular arena as assessed before euthanasia at 108 days old. Bar grap hs are means + SEM. N=9 for spSHR aCSF and 7 for spSHR Ang (1 7). C) Ang (1 7) improves performance of spSHR on the sunflower seed eating task allowing them more efficiently shell seeds. Timeline shows that the number of remaining shell pieces after eating 5 seeds is reduced, especially at age 81 days old. Data are means +/ SEM. N=6 per group. *P<0.05 compared to spSHR aCSF.
104 Figure 3 5. Ang (1 7) does not improve performance visuospatial memory as assessed by the Morris Water Maze Task. Ang (1 7) (1.1 nM ; 0.5 were also implanted in 49 day old WKY controls. 6 week pumps ran out of treatment at 91 days old. Trial 1, 2, and 3 were started at 70, 77, and 84 days of age, respect ively. N=6/group. A) Time (s) it takes to find the platform. Three trials were performed, each lasting 4 d. B) Probe trials were conducted on the 5th day of every trial. Rats were placed in the maze and the time (s) spent in the quadrant where the platform was previously located was recorded.
105 Figure 3 6 Cellular localization of immunoreactive Mas in spSHR striatum. Representative fluorescence micrographs taken at 40x magnification. N=3. Panels A, B, C, and D show respective co localization of Mas (M ; red fluorescence) with either NeuN (N), Ox 42 (Ox), Reca (Re), or GFAP (G) [all green fluorescence]. Panels E and F verify that the anti Mas antibody is specific as staining is not present in the knockout mouse striatum (E; KO), but is in the wild type m ouse striatum (F; WT).
106 Figure 3 7. Ang (1 7) decreases activated microglia and increases neuron survival in the striatum just before treatment ends. Ang (1 7) (1.1 nM; 0.5 old spSHR. Rats were euthanized at 90 days old, 1 day before treatment ran out of the 6 wk pumps, and perfused with saline and formalin. Brains were removed and fixed. Stereology was used to determine the number of cells in the striatum as described in the methods. N=3 /group.
107 Figure 3 8. Ang (1 7) does not alter the expression of genes related to inflammation in whole brain homogenate after treatment ends. Ang ( 1 7) (1.1 nM; 0.5 old spSHR. Rats were euthanized at 90 days old, 1 day before treatment ran out of the 6 wk pumps. Brains were removed and flash frozen to measure gene expression as detailed in the methods. Bar graphs are means + SEM. N=5/group. *P<0.05 compared to WKY Control.
108 Figure 3 9. Ang (1 7) administered intracerebroventricularly (ICV) has no effect on mean blood pressure during infusion in spSHR, but rats treated with Ang (1 7) have lower mean blood pressur e later after treatment ends. A) Direct blood pressure. Telemetry transducers implanted into the abdominal aorta at 45 days old. 6 wk Ang (1 7) (1.1 nM; 0.5 implanted at 63 days old. N = 6 per group, Data are Means +/ SEM. B) Indirect blood pressure. 6 wk Ang (1 7) or aCSF pumps implanted at 49 days old (not shown); 6 week old pumps ran out of treatment at 91 days old. Tai l cuff plethysmography used after acclimation. N = 4 8 per group, Data are Means +/ SEM.
109 Figure 3 10. Ang (1 7) has no effect on body weight, corticosterone, creatinine, or BUN. Ang (1 ere implanted in 49 day old WKY controls. 6 week pumps ran out of treatment at 91 days old. Rats were euthanized at 108 days old. Bar graphs are means + SEM. N=4, 9, and 7 for WKY, spSHR aCSF, and spSHR Ang (1 7), respectively. A) Body weight was recorded just prior to euthanasia. B, C, D) Blood was collected and serum was separated for corticosterone, creatinine, and BUN analysis as described in the methods.
110 Figure 3 11. Ang (1 7) has no effect on kidney or heart pathology just before treatment ends. Ang (1 7) (1.1 nM; 0.5 in 49 day old WKY controls. Rats were euthanized at 90 days old, 1 day before treatment ran out of the 6 wk pumps, and perfused with saline and for malin. Tissues were dissected, removed, and fixed as detailed in the methods. Pathology was scored by a licensed veterinary pathologist. Bar graphs are means + SEM. N=4, 9, and 7 for WKY, spSHR aCSF, and spSHR Ang (1 7), respectively. *P<0.05 compared to W KY Control.
111 CHAPTER 4 SUMMARY AND CONCLUSI ONS Summary Specific Aim 1: Investigate the Mechanism of the Cerebroprotective Actions of Ang (1 7) in Ischemic Stroke In recent years, mounting evidence indicates that the renin angiotensin system (RAS) is a potential therapeutic target for stroke, as it is highly involved in the processes that induce cerebral damage 165, 175, 177, 191 Much of the work has focused on the angiotensin converting enzyme/angiotensin II/angiotensin type 1 receptor (ACE/Ang II/AT1R) axis of the RAS. This axis contributes to CNS damage following cerebral ischemia 176, 221 Blocking t his axis has been shown to be therapeutic in a number of animals and human studies 168, 171, 173 167, 185, 186, 222 A lesser known axis of the RAS, the angiotensin converting enzyme 2/angiotensin (1 7)/Mas (ACE2/Ang (1 7)/Mas) axis, has recently begun receiving more attention in the literature. There is accumulating evidence that activation of this axis exerts beneficial actions in several cardiovascular diseases, including hypertension, hypertension related pathology, pulmonary hypertension, myocardial infarction, and heart failure 143, 223 137, 223 136, 222 224 In the brain, Ang (1 7) is primarily generated by the action of ACE2 on Ang II, and its effects are mediated by its receptor, Mas 138 In recent studies, we demonstrated that the intracerebral damage and neurological deficits elicited by endothelin 1 (ET 1) induced middle cerebral artery occlusion (MCAO), a model of ischemic s troke, are significantly reduced by intracerebroventricular (ICV) administration of either exogenous Ang (1 7) or an activator of ACE2, prior to and during the stroke period 218 Since we previously demonstrated a cere broprotective effect of Ang (1 7) treatment during ET 1 induced MCAO, our aim here was to uncover the mechanism of this protection. This would further support the rationale for activating the ACE2/Ang (1 7)/Mas axis for stroke treatment and prevention. Fur thermore, we believed that uncovering this mechanism would lead us to consider other diseases that may benefit from a treatment involving manipulation of the ACE2/Ang (1 7)/Mas axis. In previous
112 studies, we observed no effects of Ang (1 7) on cerebral bloo d flow, percent vessel constriction, or systemic blood pressure (BP) 218 Therefore, we hypothesized that Ang (1 7) would modify the expression of certain genes that may play a role in mediating the observed cerebroprot ective effects. Furthermore, we hypothesized that Mas would be present on certain cell types, indicating which cells are of potential importance in mediating the cerebroprotection. In this study a PCR array was used to identify several markers of inflammat ion that were increased in stroke and blunted by Ang (1 7) therapy. It has previously been shown that damage due to stroke is a result of excessive inflammation. The findings from the array and changes in the expression of other genes, namely inducible nit ric oxide synthase (iNOS) interleukin 1 4 (CXCR4), and a marker for monocytes/microglia (CD11b), were verified by quantitative reverse transcriptase real time PCR (qRT PCR). Each was upregulated in stroke and significantly blunted by intracranial Ang (1 7) infusion. A western blot was used to show that the changes in iNOS also existed at the protein level. To uncover which cell types were involved, we performed a co localization study with the Ang (1 7) receptor Mas and different cell markers. Microglia, endothelial c ells, and neurons were shown to express Mas. Furthermore, we showed that Ang (1 7) has a Mas dependent in vitro effect to blunt nitric oxide production in a mixed glia culture system. These findings indicate for the first time an anti inflammatory property of Ang (1 7) treatment in the CNS and provide a potential mechanism of cerebroprotection. Specific Aim 2: Determine whether Ang (1 7) also Exerts Cerebroprotective Actions in Hemorrhagic Stroke There are two types of stroke: ischemic, where blood flow to the brain is reduced, and hemorrhagic, where persistent hypertension leads to vessel thinning and
113 intracerebral bleeding 45 Both types of stroke are multifactorial, yet mounting evidence indicates that the renin an giotensin system (RAS) is highly involved as described above 165, 167 171, 173 175, 177, 185, 186, 191, 235 The peptide Ang (1 7) has been shown to be therapeutic in many cardiovascular disease states, including ischemic stroke In Aim 1, we demonstrated that this peptide had an in vivo anti inflammatory effect during MCAO in rats and an in vitro anti inflammatory effect in a mixed glia culture system. There is much overlap in the pathophysiology of ischemic and hemorrhagic stro ke, especially regarding the role of excessive inflammation. Inflammation has a strong role in the mechanism of injury in hemorrhagic stroke. The inflammatory response begins soon after hemorrhage and peaks several days later in humans and animal models 58, 101, 114, 115 The infiltration of neutrophils occurs within 2 days, and the activation of microglia may continue for up to 1 month 114, 116 Evidence supports a role of microglia in hemorrhagic stroke pathology because inhibiting their activation reduces damage 117, 118, 120 Further more, there is believed to be a strong association between inflammation and edema, as evidenced by data that correlates plasma tumor necrosis factor alpha concentrations in patients with hemorrhagic stroke to the degree of brain edema 123 Given Ang (1 inflammatory effects, our aim here was to determine if it also has any cerebroprotective effects in hemorrhagic stroke. For these experiments, we used stroke pr one Sponataneously Hypertensive Rats (spSHR). When fed a high salt diet from the time they are weaned, these rats will spontaneously develop extremely high blood pressure resulting in hemorrhagic stroke and death. We administered Ang (1 7) centrally for si x weeks starting at 49 days of age. Ang (1 7) increased the survival of spSHR and
114 decreased the number of hemorrhages in the striatum. Furthermore, spSHR treated with Ang (1 7) also showed less lethargy and improved neurological status as measured by a sun flower seed eating task. There was no effect on brain water content, however. We confirmed the presence of the Ang (1 7) receptor Mas on neurons, microglia, and endothelial cells and showed that Ang (1 7) decreases the number of microglia and increases the number of neurons striatum through the use of stereology. There was no effect of Ang (1 7) on markers of peripheral disease, such as kidney pathology, heart pathology, body weight, corticosterone levels, or extent of hypertension. Interestingly, although BP was not altered by Ang (1 7) during its administration, spSHR given Ang (1 7) showed lower BP later in life after the treatment stopped. That the kidneys and hearts are not altered by Ang (1 7) supports that the actions of this peptide are primarily cen tral. These findings indicate for the first time Ang (1 7) has cerebroprotective actions in hemorrhagic stroke and can increase the survival of spSHR. Discussion The results of these studies provide further insight into the mechanism of cerebroprotection o f activation of the ACE2/Ang (1 7)/Mas axis of the RAS. Previous studies where Ang (1 7) was administered centrally showed increases in bradykinin release, bradykinin receptor stimulation, endothelial nitric oxide synthase (eNOS) activity, and NO productio n 207, 208 These findings raise the possibility of Ang (1 7) having protective vascular effects in stroke, where it increases vasodilation through increasing NO availability. Other studies show that peri pheral Ang (1 7), administered both acutely and chronically, can increase cerebral blood flow 204 206 Despite this, we doubt that the peripheral administration of Ang (1 7) would deliver an effective dose to
115 cross the blood brain barrier (BBB) to have any central effects 265 267 We have shown that our cerebroprotective dose given centrally has no effect on cerebral blood flow, percent vessel constriction, or systemic blood pressure 218 Our data, presented in Chapter 2, demonstrate for the first time an anti inflammatory effect of Ang (1 7) when given centrally. This seems to be the most likely mechanism for cerebroprotection in our model. As stated above, inflammation plays a large role in the pathophysiology of stroke 37 By blunting the expression of iNOS, (1 7) can reduce the excess damage that occurs by an over activated inflammatory response. We showed that neurons, microglia, and endothelial cells have the Ang (1 7) receptor Mas present. Therefore, we proposed that o ne of these cell types is most likely involved. To measure the effects of Ang (1 7) on glia, in the absence of neurons or vessels, we used an LPS insult model with in vitro mixed glial cultures. We showed Ang (1 7) decreased the concentration of released n itrite (NO 2 ), a stable breakdown product of NO. This verifies that Ang (1 7) can have these anti inflammatory effects independent of neurons and blood vessels. Chapter 3 includes data that supports this idea and also poses further questions. Our stereolo gy study indicated that Ang (1 7) decreases numbers of microglia and increases numbers of neurons in the striatum. We believe that Ang (1 7) decreases microglia and allows more neurons to survive. Interestingly, we did not observe an effect of Ang (1 7) on the mRNA of iNOS or other pro inflammatory cytokines (PICs) in the spSHR. This may be due to a technical challenge, where by homogenizing the whole brain there is too much healthy tissue from regions that have not undergone stroke/damage that the effects are diluted. We have previously observed this in the
116 MCAO model 218 Another interesting observation related to mechanism of Ang (1 7) action from the spSHR study is that while blood pressure was not affected during 6 w k of Ang (1 7) infusion, the animals that were given Ang (1 7) had lower BP later in life. This raises the possibility that maybe Ang (1 7) had a protective effect where it decreased CNS inflammation/damage that can lead to the development of high BP. Perh aps this occurred at a BP control region of the brain. We think the effect is likely central because we observed no changes from Ang (1 7) on heart/kidney pathology, corticosterone, or body weight. We believe that the primary effects are on microglia, but more work must be done to confirm this. By decreasing the excessive activation/migration of microglia in stroke, there would be less damage. The next step in further understanding the mechanism of this cerebroprotective effect is to study the intracellular signaling responsible for this anti inflammatory effect. If microglia are the primary cells involved, how is Mas signaling regulating their activation/migration? Is there a direct effect on regulating iNOS and PIC expression, or is that an indirect effect of fewer cells migrating to the region of damage? It is also important to consider that there could be multiple sites of action for the cerebroprotective effects. Perhaps in addition to the anti inflammatory effects there is also a vascular effect that we just cannot measure in the ET 1 MCAO model with the dose of Ang (1 7) that we used given centrally. Once more is known about intracellular signaling, we can better learn about commonalities between the stimulation of ACE2/Ang (1 7)/Mas and ACE/Ang II/AT2R and the blockade of ACE/Ang II/AT1R. As mentioned in Chapter 1, there is some overlap that is already understood in the signaling cascades of these RAS axes. A 7 transmembrane domain G protein
117 coupled receptor, the AT2R has 34% sequence homology to the A T1R 268 AT2R stimulation results in the activation of signaling cascades that counteract many of the events mediated by AT1R 269 AT2R stimulation activates several phospha tases, such as MAP Kinase Phosphatase 1 (MPK 1), SHP 1, and PP2A 131, 270 which serve to deactivate ERK1/2, STAT, JAK, and several other AT1R signaling molecules. Increasing these phosphatases can be both G protein dependent and independent. Stimulation of AT2R can also increase NO, cyclic GMP, and bradykinin 271 273 Mas is also a 7 transmembrane domain G protein co upled receptor that similarly antagonizes the AT1R. Mas stimulation can block the phosphorylation of p38MAPK, ERK1/2, and JNK that is induced by AT1R activation 158, 159 Mas signaling also activates SHP 2, which disrupts the AT1R mediated activation of c Src, ERK1/2, and NOX 160 Stimulation of Mas also increases eNOS activity via the phosphatidylinositol 3 kinase (PI3K) /Akt pathway 148 It also causes AA release, production of PGI 2 and potentiates bradykinin signaling 161 164 In summary, both stimulation of AT2R and Mas oppose AT1R, contributing to phosphatase activation, bradykinin receptor activation, and the release of NO. This common pathway of AT1R disruption hints that there may be common mechanisms of cerebroprotection. The effects of Ang II at AT2R are in many ca ses very similar to those of Ang (1 7) at Mas and opposite those of Ang II at AT1R 189 There are many examples of studies where ARBs are cerebroprotective during stroke in both animals and humans 168, 171, 173 167, 185, 186, 222 There is also an example of where an AT2R agonists was ce rebroprotective in stroke 188 Our own data show a novel AT2R agonist to be cerebroprotective in MCAO ( unpublished ). Interestingly, this agonist also had some anti
118 inflammatory effects in vivo, blunting increases i n iNOS and MCP 1 mRNA during stroke that parallel the anti inflammatory effects of Ang (1 7) discussed in Chapter 2. It should be pointed out that developing pharmacotherapy targeted to the CNS can be difficult because the blood brain barrier (BBB) is impe netrable to many small molecules, limiting their activity centrally 265 267 This results in only about 5% of drugs capable of central actions 274 A molecule must meet certain specific requirements to ac ross the BBB. Most must have a molecular weight less than 400 to 500 Da and have high lipophilicity 266 Because Ang (1 7) is 899 Da and hydrophilic, we designed the studies in Chapter 2 and 3 using transcranial central drug delivery directly into the left lateral ventricle. Disadvantages of this design are that this is more invasive and less sustainable long term compared to peripheral administration. This necessitates the exploration of alternative strategies to activating the central ACE2/Ang (1 7)/Mas axis. To increase BBB permeability, several strategies can be utilized. One is the use of medicinal chemistry to alter the molecule, m aking it more lipophilic or enabling it to utilize active transport mechanisms 266 Because increasing lipophilicity increases a his reason, taking advantage of active transport mechanisms is preferred. Another strategy is to disrupt the BBB. This can be accomplished though the use of poorly diffusible osmotic compounds, such as mannitol or lithium, to shrink endothelial cells or th e use of detergents to destabilize membranes. A downside to this approach is that pathology is associated with both neurotoxic plasma proteins and the BBB disrupting compounds themselves 265, 266, 275 Other more recent strategies include the use of transcranial ultrasound and liposome assisted delivery of drugs 276, 277 We are currently undertaking
119 gene therapy a pproaches, as well, utilizing the viral delivery of ACE2 or a secretable form of Ang (1 7). Another more sophisticated treatment strategy we are testing is the engineering of endothelial progenitor cells that over express this secretable form of Ang (1 7). These stem cells can be injected peripherally as they home to the sites of damage after stroke. It should also be mentioned that in stroke there is disruption of the BBB that occurs because of stroke related damage. This may make crossing the BBB easier, allowing the passage of molecules that are administered acutely post stroke that would have otherwise been unable to cross. The difficulty with this approach is that there may be different degrees of BBB disruption in different stroke patients so dosage de cisions may be difficult to make. Within the stroke field, much emphasis is placed on developing acute therapies that are effective after the onset of stroke symptoms. We argue, however, that a preventative approach to stroke is not unrealistic. The major risk factors for stroke, including increasing age and high blood pressure, are well documented. It would be feasible to administer preventative drugs to patients exhibiting high risk. The experiments outlined in Chapters 2 and 3 were designed with a preven tative chronic treatment approach where Ang (1 7) was infused for over 1 wk or 6 wks, respectively. In the ET 1 induced MCAO model of ischemic stroke, Ang (1 7) was given 1 wk before, during, and after stroke until the time of euthanasia. For the spSHR mod el of hemorrhagic stroke, Ang (1 7) was given from 49 to 91 days of age during the development of pathology. In this way, we aimed to most efficiently explore the therapeutic potential of activating the ACE2/Ang (1 7)/Mas axis. That being said, now that we show cerebroprotective effects with these designs, the potential for activating
120 this axis for acute, post stroke therapy should be explored. As mentioned above, this may lessen the difficulty of designing drugs to cross the BBB. Another step that should be made in pushing the translation of these findings to the clinic, is demonstrating the effectiveness of ACE2/Ang (1 7)/Mas axis activation in other models of stroke and in other animals. For ischemic stroke, the intraluminal filament model of MCAO or the photoactivation model could be utilized 278 Large animal models with more heterogeneity in stroke also demonstrate the likelihood of a 279 For hemorrhagic stroke, the collagenase induced model of intracerebral hemorrhage would prove useful. This model also allows for a more controlled, reproducible hemorrhage that may make many of the outcomes easier to measure and conclusions easier to draw 280 After effectiveness in other models is established, clinical trials should be initiated to assess the potential of activating the ACE2/Ang (1 7)/Mas axis in human patients. In conclusion (Figure 4 1), thes e studies have increased the understanding of the mechanism by which activating the ACE2/Ang (1 7)/Mas axis of the RAS is cerebroprotective in ischemic stroke. We demonstrate for the first time an in vivo anti inflammatory property of central Ang (1 7). We also show that Ang (1 7) has anti inflammatory effects in mixed glial cultures lacking neurons and vessels. Furthermore, we have demonstrated that activating this axis also has therapeutic potential in hemorrhagic stroke. It increases the survival of spSH R and improves their neurological status. We also demonstrated in 2 rat strains that Mas is present on microglia, neurons, and endothelial cells. Further work must be done to understand the intracellular signaling that results in these anti inflammatory ef fects, and future studies must confirm
121 the therapeutic potential of activating this axis in other models and ultimately human patients.
122 Figure 4 1. Schematic of proposed mechanism. Both ischemic and hemorrhagic stroke result in excessive inflammation that over activates microglia. More microglia migrate to the area of damage and secrete proinflammatory cytokines which results in increased iNOS expression
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145 BIOGRAPHICAL SKETCH Robert W. Regenhardt was born in Saint Petersburg, Florida, USA in 1985. He graduated valedictorian in 2004 from South Sumter High School in Bushnell, Florida. In 2004, he began undergradu ate studies at the Uni versity of Florida majoring in nutritional sciences and minoring in c hemistry. Two years later, Robert was admitted to medical school through an early acceptance program. He later matriculated into the combined MD PhD program after di scovering a passion for research. His undergraduate honors thesis involved changes in lymphocyte subpopulations within the gut during zinc deficiency in the laboratory of Bobbi Langkamp Henken, Ph.D. He graduated with his B.S. with highest honors after co mpleting his first year of medical school in 2008. In 2009, Robert began his Ph.D. in the laboratory of Colin Sumners, Ph.D. His research interests include the renin angiotensin system, cardiovascular disease, and stroke The title of his doctoral thesis i ACE2/Ang (1 completed in 2012, showing for the first time anti inflammatory properties of Ang (1 7) in ischemic stroke and cerebroprote ctive effects of this peptide in hemorrhagic stroke. He is currently completing the clinical portion of his Medical Doctorate training. In addition to his research interests, Robert has been highly involved in 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. Robert has served in many capacities at this clinic, being Chair for Research and Chair for Behavioral Health. He has also served as president for the local chapter of the American Physician Scientist Association, an organization that he remains actively
146 involved in. Robert aspires to be an effective physician scientist, educator, and healthcare provider.