Robert W. Regenhardt

Cerebroprotection by angiotensin‐(1–7) in endothelin‐1‐induced ischaemic stroke

Activation of angiotensin‐converting enzyme 2 (ACE2), production of angiotensin‐(1–7) [Ang‐(1–7)] and stimulation of the Ang‐(1–7) receptor Mas exert beneficial actions in various peripheral cardiovascular diseases, largely through opposition of the deleterious effects of angiotensin II via its type 1 receptor. Here we considered the possibility that Ang‐(1–7) may exert beneficial effects against CNS damage and neurological deficits produced by cerebral ischaemic stroke. We determined the effects of central administration of Ang‐(1–7) or pharmacological activation of ACE2 on the cerebral damage and behavioural deficits elicited by endothelin‐1 (ET‐1)‐induced middle cerebral artery occlusion (MCAO), a model of cerebral ischaemia. The results of the present study demonstrated that intracerebroventricular infusion of either Ang‐(1–7) or an ACE2 activator, diminazine aceturate (DIZE), prior to and following ET‐1‐induced MCAO significantly attenuated the cerebral infarct size and neurological deficits measured 72 h after the insult. These beneficial actions of Ang‐(1–7) and DIZE were reversed by co‐intracerebroventricular administration of the Mas receptor inhibitor, A‐779. Neither the Ang‐(1–7) nor the DIZE treatments altered the reduction in cerebral blood flow elicited by ET‐1. Lastly, intracerebroventricular administration of Ang‐(1–7) significantly reduced the increase in inducible nitric oxide synthase mRNA expression within the cerebral infarct that occurs following ET‐1‐induced MCAO. This is the first demonstration of cerebroprotective properties of the ACE2–Ang‐(1–7)–Mas axis during ischaemic stroke, and suggests that the mechanism of the Ang‐(1–7) protective action includes blunting of inducible nitric oxide synthase expression.

Anti-inflammatory effects of angiotensin-(1-7) in ischemic stroke

Previously we demonstrated that central administration of angiotensin-(1-7) [Ang-(1-7)] into rats elicits significant cerebroprotection against ischemic stroke elicited by endothelin-1 induced middle cerebral artery occlusion. Ang-(1-7), acting via its receptor Mas, reduced cerebral infarct size, and rats exhibited improved performance on neurological exams. These beneficial actions of Ang-(1-7) were not due to inhibition of the effects of endothelin-1 on cerebral vasoconstriction or effects on cerebral blood flow, and so we considered other potential mechanisms. Here we investigated the possibility that the Ang-(1-7)-induced cerebroprotection involves an anti-inflammatory effect, since stroke-induced cerebral damage includes an excessive intracerebral inflammatory response. Our quantitative RT-PCR analyses revealed that central Ang-(1-7) treatment attenuates the increased expression of mRNAs for inducible nitric oxide synthase (iNOS), several pro-inflammatory cytokines and cluster of differentiation molecule 11b (microglial marker) within the cerebral cortex following endothelin-1 induced stroke. Western blotting confirmed similar changes in iNOS protein expression in the cerebral cortex. In support of these observations, immunostaining revealed the presence of immunoreactive Mas on activated microglia within the cerebral cortical infarct zone, and in vitro experiments demonstrated that lipopolysaccharide-induced increases in nitric oxide production in glial cultures are attenuated by Ang-(1-7) acting via Mas. Collectively these findings demonstrate an anti-inflammatory action of Ang-(1-7) in the brain, and suggest that the cerebroprotective action of this peptide in ischemic stroke may involve effects on nitric oxide generation by microglia.

The angiotensin type 2 receptor agonist Compound 21 elicits cerebroprotection in endothelin-1 induced ischemic stroke

Evidence indicates that angiotensin II type 2 receptors (AT2R) exert cerebroprotective actions during stroke. A selective non-peptide AT2R agonist, Compound 21 (C21), has been shown to exert beneficial effects in models of cardiac and renal disease, as well as hemorrhagic stroke. Here, we hypothesize that C21 may exert beneficial effects against cerebral damage and neurological deficits produced by ischemic stroke. We determined the effects of central and peripheral administration of C21 on the cerebral damage and neurological deficits in rats elicited by endothelin-1 induced middle cerebral artery occlusion (MCAO), a model of cerebral ischemia. Rats infused centrally (intracerebroventricular) with C21 before endothelin-1 induced MCAO exhibited significant reductions in cerebral infarct size and the neurological deficits produced by cerebral ischemia. Similar cerebroprotection was obtained in rats injected systemically (intraperitoneal) with C21 either before or after endothelin-1 induced MCAO. The protective effects of C21 were reversed by central administration of an AT2R inhibitor, PD123319. While C21 did not alter cerebral blood flow at the doses used here, peripheral post-stroke administration of this agent significantly attenuated the MCAO-induced increases in inducible nitric oxide synthase, chemokine (C-C) motif ligand 2 and C-C chemokine receptor type 2 mRNAs in the cerebral cortex, indicating that the cerebroprotective action is associated with an anti-inflammatory effect. These results strengthen the view that AT2R agonists may have potential therapeutic value in ischemic stroke, and provide the first evidence of cerebroprotection induced by systemic post stroke administration of a selective AT2R agonist.

Centrally administered angiotensin-(1–7) increases the survival of stroke-prone spontaneously hypertensive rats

What is the central question of this study? Activation of angiotensin‐converting enzyme 2, resulting in production of angiotensin‐(1–7) and stimulation of its receptor, Mas, exerts beneficial actions in a number cardiovascular diseases, including ischaemic stroke. A potential beneficial role for angiotensin‐(1–7) in haemorrhagic stroke has not previously been reported. What is the main finding and its importance? Central administration of angiotensin‐(1–7) into stroke‐prone spontaneously hypertensive rats, a model of haemorrhagic stroke, increases lifespan and improves the neurological status of these rats, as well as decreasing microglial numbers in the striatum (implying attenuation of cerebral inflammation). These actions of angiotensin‐(1–7) have not previously been reported and identify this peptide as a potential new therapeutic target in haemorrhagic stroke.

Activation of the Neuroprotective Angiotensin-Converting Enzyme 2 in Rat Ischemic Stroke

The angiotensin-converting enzyme 2/angiotensin-(1–7)/Mas axis represents a promising target for inducing stroke neuroprotection. Here, we 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. Among 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 4 hours post stroke, but rebounded to reach higher than baseline levels 3 days post stroke. Treatment after 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 after stroke suggest that it may be a favorable therapeutic target. Indeed, significant neuroprotection resulted from poststroke 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.

Neuroprotective Mechanisms of the ACE2–Angiotensin-(1-7)–Mas Axis in Stroke

The discovery of beneficial neuroprotective effects of the angiotensin converting enzyme 2–angiotensin-(1-7)–Mas axis [ACE2–Ang-(1-7)–Mas] in ischemic and hemorrhagic stroke has spurred interest in a more complete characterization of its mechanisms of action. Here, we summarize findings that describe the protective role of the ACE2–Ang-(1-7)–Mas axis in stroke, along with a focused discussion on the potential mechanisms of neuroprotective effects of Ang-(1-7) in stroke. The latter incorporates evidence describing the actions of Ang-(1-7) to counter the deleterious effects of angiotensin II (AngII) via its type 1 receptor, including anti-inflammatory, anti-oxidant, vasodilatory, and angiogenic effects, and the role of altered kinase–phosphatase signaling. Interactions of Mas with other receptors, including bradykinin receptors and AngII type 2 receptors are also considered. A more complete understanding of the mechanisms of action of Ang-(1-7) to elicit neuroprotection will serve as an essential step toward research into potential targeted therapeutics in the clinical setting.

Clinical Imaging Factors Associated With Infarct Progression in Patients With Ischemic Stroke During Transfer for Mechanical Thrombectomy

Importance When transferred from a referring hospital (RH) to a thrombectomy-capable stroke center (TCSC), patients with initially favorable imaging profiles (Alberta Stroke Program Early CT Score [ASPECTS] ≥6) often demonstrate infarct progression significant enough to make them ineligible for mechanical thrombectomy at arrival. In rapidly evolving stroke care networks, the question of the need for vascular imaging at the RHs remains unsolved, resulting in an important amount of futile transfers for thrombectomy. Objective To examine the clinical imaging factors associated with unfavorable imaging profile evolution for thrombectomy in patients with ischemic stroke initially transferred to non-TCSCs. Design, Setting, and Participants Data from patients transferred from 1 of 30 RHs in our regional stroke network and presenting at our TCSC from January 1, 2010, to January 1, 2016, were retrospectively analyzed. Consecutive patients with acute ischemic stroke initially admitted to a non–thrombectomy-capable RH and transferred to our center for which a RH computed tomography (CT) and a CT angiography (CTA) at arrival were available for review. Main Outcomes and Measures ASPECTS were evaluated. The adequacy of leptomeningeal collateral blood flow was rated as no or poor, decreased, adequate, or augmented per the adapted Maas scale. The main outcome was an ASPECTS decay, defined as an initial ASPECTS of 6 or higher worsening between RH and TCSC CTs to a score of less than 6 (making the patient less likely to derive clinical benefit from thrombectomy at arrival). Results A total of 316 patients were included in the analysis (mean [SD] age, 70.3 [14.2] years; 137 [43.4%] female). In multivariable models, higher National Institutes of Health Stroke Score, lower baseline ASPECTSs, and no or poor collateral blood vessel status were associated with ASPECTS decay, with collateral blood vessel status demonstrating the highest adjusted odds ratio of 5.14 (95% CI, 2.20-12.70; P < .001). Similar results were found after stratification by vessel occlusion level. Conclusions and Relevance In patients with ischemic stroke transferred for thrombectomy, poor collateral blood flow and stroke clinical severity are the main determinants of ASPECTS decay. Our findings suggest that in certain subgroups vascular imaging, including collateral assessment, can play a crucial role in determining the benefits of transfer for thrombectomy.

Blood Pressure and Penumbral Sustenance in Stroke from Large Vessel Occlusion

The global burden of stroke remains high, and of the various subtypes of stroke, large vessel occlusions (LVOs) account for the largest proportion of stroke-related death and disability. Several randomized controlled trials in 2015 changed the landscape of stroke care worldwide, with endovascular thrombectomy (ET) now the standard of care for all eligible patients. With the proven success of this therapy, there is a renewed focus on penumbral sustenance. In this review, we describe the ischemic penumbra, collateral circulation, autoregulation, and imaging assessment of the penumbra. Blood pressure goals in acute stroke remain controversial, and we review the current data and suggest an approach for induced hypertension in the acute treatment of patients with LVOs. Finally, in addition to reperfusion and enhanced perfusion, efforts focused on developing therapeutic targets that afford neuroprotection and augment neural repair will gain increasing importance. ET has revolutionized stroke care, and future emphasis will be placed on promoting penumbral sustenance, which will increase patient eligibility for this highly effective therapy and reduce overall stroke-related death and disability.

Serum activity of angiotensin converting enzyme 2 is decreased in patients with acute ischemic stroke.

Levels of angiotensin converting enzyme 2 (ACE2), a cardio and neuro-protective carboxypeptidase, are dynamically altered after stroke in preclinical models. We sought to characterize the previously unexplored changes in serum ACE2 activity of stroke patients and the mechanism of these changes. Serum samples were obtained from patients during acute ischemic stroke (n=39), conditions mimicking stroke (stroke-alert, n=23), or from control participants (n=20). Enzyme activity levels were analyzed by fluorometric assay and correlated with clinical variables by regression analyses. Serum ACE2 activity was significantly lower in acute ischemic stroke as compared to both control and stroke-alert patients, followed by an increase to control levels at three days. Serum ACE2 activity significantly correlated with the presence of ischemic stroke after controlling for other factors (P=0.01). Additional associations with ACE2 activity included a positive correlation with systolic blood pressure at presentation in stroke-alert (R2=0.24, P=0.03), while stroke levels showed no correlation (R2=0.01, P=0.50). ACE2 sheddase activity was unchanged between groups. These dynamic changes in serum ACE2 activity in stroke, which concur with preclinical studies, are not likely to be driven primarily by acute changes in blood pressure or sheddase activity. These findings provide new insight for developing therapies targeting this protective system in ischemic stroke.

Immediate Vascular Imaging Needed for Efficient Triage of Patients With Acute Ischemic Stroke Initially Admitted to Nonthrombectomy Centers

Background and Purpose— Current guidelines for endovascular thrombectomy (EVT) used to select patients for transfer to thrombectomy-capable stroke centers (TSC) may result in unnecessary transfers. We sought to determine the impact of simulated baseline vascular imaging on reducing unnecessary transfers and clinical-imaging factors associated with receiving EVT after transfer. Methods— We identified patients with stroke transferred for EVT from 30 referring hospitals between 2010 and 2016 who had a referring hospitals brain computed tomography and repeat imaging on TSC arrival available for review. Initial Alberta Stroke Program Early CT scores and TSC vascular occlusion level were assessed. The main outcome variable was receiving EVT at TSC. Models were simulated to derive optimal triaging parameters for EVT. Results— A total of 508 patients were included in the analysis (mean age, 69±14 years; 42% women). Application at referring hospitals of current guidelines for EVT yielded sensitivity of 92% (95% confidence interval, 0.84–0.96) and specificity of 53% (95% confidence interval, 0.48–0.57) for receiving EVT at TSC. Repeated simulations identified optimal selection criteria for transfer as National Institute of Health Stroke Scale >8 plus baseline vascular imaging (sensitivity=91%; 95% confidence interval, 0.83–0.95; and specificity=80%; 95% confidence interval, 0.75–0.83). Conclusions— Our findings provide quantitative estimates of the claim that implementing vascular imaging at the referring hospitals would result in significantly fewer futile transfers for EVT and a data-driven framework to inform transfer policies.