Neuroprotection by Post-stroke Activation of the ACE2-Ang-(1-7)-Mas Axis

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DESCRIPTION (provided by applicant): As the 4th leading cause of death in the United States, stroke affects nearly every American in some way. Despite extensive research, there are few therapies for treating stroke after onset, and there are no approved treatments to specifically counteract the inflammatory stroke damage that occurs within the hours and days after the ischemic event. This study is designed to test a recently discovered target for the treatment of acute ischemic stroke. Over the past several decades, it has become apparent that over-activation of the classical renin angiotensin system (RAS), which includes angiotensin II binding to its type 1 receptor, is detrimental in stroke. More recently, a protective axis of the RAS, the angiotensin converting enzyme 2 - angiotensin-(1-7) - Mas [ACE2-Ang-(1-7)-Mas] axis, has been shown to have opposite effects that are protective in stroke. In this study, this protective axis will be targeted using methods that are clinically-feasible and at post-stroke time points that mimic what an actual stroke patient might receive following stroke onset. In the first specific aim, several animal models of stroke will be used to test the neuroprotective effects of diminazene aceturate (DIZE), an activator of the ACE2. Efficacy will be evaluated in rodent stroke models, with post-stroke administration of different doses of DIZE at +4 hrs, +24 hrs, and +48 hrs following stroke, with and without concurrent administration of the Mas receptor antagonist A-779. At one day and three days after the stroke is induced, brains will be analyzed to determine the extent of the protective effects of this drug treatment. The importance of the ACE2 enzyme within this axis will be evaluated utilizing the Cre/lox system in mice to test the specific hypothesis that ACE2 overexpression in stroke will result in neuroprotection. Endpoint measures for this aim include infarct size, neurological function, brain levels of Ang-(1-7), and markers of inflammation. In the second specific aim, experiments have been designed to better understand the mechanisms by which DIZE induces neuroprotection in stroke. In particular, they will test whether DIZE induces neuroprotection by 1) increasing levels of cerebral blood flow, and 2) mediating activation of inflammatory and anti-inflammatory microglia. Cerebral blood flow will be measured using MRI during baseline and stroke conditions to evaluate whether DIZE induces protection in a flow dependent manner. Antibody labeling will facilitate visualization of microglial subtype activation following stroke. The endpoint measures to determine the effectiveness of post-stroke DIZE administrations on these processes include MRI measures of cerebral blood flow and relative levels of microglial subtype activation. These studies will have a positive impact and clinical relevance since they will inform future animal studies and human clinical trials regarding this protective axis as therapeutic target for the acute treatment of ischemic stroke.