The angiotensin converting enzyme 2/angiotensin-(1-7)/Mas axis represents a promising target for inducing stroke neuroprotection. Here, explored stroke-induced changes in expression and activity of endogenous angiotensin converting enzyme 2 and other system components in Sprague Dawley rats. To evaluate the clinical feasibility of treatments that target this axis and that may act in synergy with stroke-induced changes, we also tested the neuroprotective effects of diminazene aceturate, an angiotensin converting enzyme 2 activator, administered systemically post-stroke. Amongst rats that underwent experimental endothelin-1-induced ischemic stroke, angiotensin converting enzyme 2 activity in the cerebral cortex and striatum increased in the 24 hours after stroke. Serum angiotensin converting enzyme 2 activity was decreased within 4h post stroke, but rebounded to reach higher than baseline levels 3d post-stroke. Treatment following stroke with systemically-applied diminazene resulted in decreased infarct volume and improved neurological function without apparent increases in cerebral blood flow. Central infusion of A-779, a Mas receptor antagonist, resulted in larger infarct volumes in diminazene-treated rats, and central infusion of the angiotensin converting enzyme 2 inhibitor MLN-4760 alone worsened neurological function. The dynamic alterations of the protective angiotensin converting enzyme 2 pathway following stroke suggest that it may be a favorable therapeutic target. Indeed, significant neuroprotection resulted from post-stroke angiotensin converting enzyme 2 activation, likely via Mas signaling in a blood flow-independent manner. Our findings suggest that stroke therapeutics that target the angiotensin converting enzyme 2/angiotensin-(1-7)/Mas axis may interact cooperatively with endogenous stroke-induced changes, lending promise to their further study as neuroprotective agents.

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Background: The renin angiotensin system has become the focus of recent interest in stroke research as accumulating evidence indicates that activation of the angiotensin converting enzyme 2/angiotensin-(1-7)/mas (ACE2-Ang-(1-7)-Mas) axis exerts neuroprotective benefit in animal stroke models. Pre- and post-stroke activation of this axis by intracerebroventricular infusion of Ang-(1-7) reduces infarct size and improves neurological function in rats. The recent development of an orally active formulation of Ang-(1-7) in hydroxypropylβ-cyclodextrin [HPβCD/Ang-(1-7)] provides a non-invasive avenue for testing the efficacy of systemic post-stroke administration of Ang-(1-7). We tested the hypothesis that post-stroke oral gavage of HPβCD/Ang-(1-7) exerts neuroprotection in a rat model of ischemic stroke. Methods: Male SD rats underwent ischemic stroke by endothelin-1-induced middle cerebral artery occlusion and were randomly divided into 2 groups of 12 each that received oral gavages of dH2O or HPβCD/Ang-(1-7) (50 ug/kg) at 4, 24, and 48h after stroke, as well as blinded neurological assessments at 4, 24, and 72h after stroke. Immediately after the 72h tests, animals were euthanized and cerebral infarct sizes were assessed by TTC staining. Data are expressed as mean ± SEM with significance inferred at p<0.05. Results: Mean infarct sizes (%) were significantly decreased by post-stroke oral gavage of HPβCD/Ang-(1-7) (32.57±2.74) vs. dH2O (43.65±5.27, Fig 1 ). As compared to deficits at 4h post-stroke, 72h neurologic deficits (Bederson Scale) were significantly improved in HPβCD/Ang-(1-7)-treated rats (4h: 1.67±0.22; 72h: 0.83±0.21) and not in dH2O-treated rats (4h: 1.25±0.22; 72h: 1.17±0.24). Conclusions: Our results suggest that activating the ACE2/Ang-(1-7)/Mas axis by post-stroke administration of an oral formulation of Ang-(1-7) is neuroprotective.

Background: The renin angiotensin system is a promising target for stroke neuroprotection and therapy through activation of angiotensin type II receptors (AT2Rs). Compound 21 (C21), a selective non-peptide AT2R agonist, has been shown to exhibit neuroprotection and improve stroke outcomes in preclinical studies. Stimulation of AT2Rs is believed to counteract the negative effects of angiotensin type 1 receptor and provide distinctive beneficial anti-inflammatory and neurotropic effects. We hypothesized that C21 given after stroke through peripheral injections would have sustained neuroprotective effects in aged rats. Methods: Aged adult male SD rats (18-20 months) underwent ischemic stroke by monofilament middle cerebral artery occlusion (MCAO) and were randomly divided into two groups that received intraperitoneal (IP) injections of either 0.9% NaCl or 0.03mg/kg C21 at reperfusion (90 min), 24h, and 48h after stroke. All animals received blinded neurological exams at 4h, 24h, 72h, 7d, 14d, and 21d post-stroke. Infarct size was assessed by magnetic resonance imaging at 21 days. Results: Post-stroke treatment with C21 significantly improved neurological function, as evidenced by neurological testing using Rotarod and somatosensory dysfunction exams. At 7d and 14d after stroke, C21-treated rats had significantly increased Rotarod times versus saline-treated rats, and at 21d, the somatosensory function was significantly improved as measured by time to removal of paw adhesive. Infarct volume tended to be non-significantly decreased by C21 treatment at 21d post-stroke. Conclusions: Our findings indicate that targeting the renin-angiotensin system, specifically by stimulation of AT2Rs with C21, improves neurological function in aged rats with stroke over a sustained period of 21 days. These findings encourage further research into the renin-angiotensin system and specifically AT2Rs, and offers hope for effective alternatives for treating stroke.