Adam P. Mecca

Protection from angiotensin II‐induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats

Angiotensin converting enzyme 2 (ACE2), a newly discovered member of the renin–angiotensin system (RAS), is a potential therapeutic target for the control of cardiovascular disease owing to its key role in the formation of vasoprotective peptides from angiotensin II. The aim of the present study was to evaluate whether overexpression of ACE2 could protect the heart from angiotensin II‐induced hypertrophy and fibrosis. Lentiviral vector encoding mouse ACE2 (lenti‐mACE2) or GFP was injected intracardially in 5‐day‐old Sprague–Dawley rats. This resulted in expression of mACE2 in cardiac tissue for the duration of the study. Infusion of 200 ng kg−1 min−1 angiotensin II for 4 weeks resulted in an 80 mmHg increase in systolic blood pressure, a significant increase in the heart weight to body weight ratio (HW : BW), and marked myocardial fibrosis in control rats. Transduction with lenti‐mACE2 resulted in significant attenuation of the increased HW : BW and myocardial fibrosis induced by angiotensin II infusion. These observations demonstrate that ACE2 overexpression results in protective effects on angiotensin II‐induced cardiac hypertrophy and fibrosis.

Therapeutic Implications of the Vasoprotective Axis of the Renin-Angiotensin System in Cardiovascular Diseases

The recent discovery of angiotensin-converting enzyme 2 (ACE2) and the Mas receptor has resulted in the recognition of a counterregulatory, ACE2/Ang-(1-7)/Mas, axis within the renin-angiotensin system (RAS). Any disturbance in the balance between this and the ACE/AngII/AT1 receptor axis is suggested to lead to the development and progression of cardiovascular disease (CVD). Therefore, activation of the ACE2/Ang-(1-7)/Mas axis has been an obvious target for CVD therapeutics. In this review, we will focus on the current status of the RAS, highlight evidence for the existence of the ACE2/Ang-(1-7)/Mas axis, and discuss, the role of this axis in the pathophysiology of the cardiovascular, renal, pulmonary and central nervous systems and its potential for future CVD therapeutics.

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.

Effects of central and peripheral injections of apelin on fluid intake and cardiovascular parameters in rats

Previous studies have indicated that apelin, a novel peptide suggested to have some actions related to angiotensin peptides, has either a dipsogenic or an antidipsogenic effect and either increases or decreases blood pressure. The present study attempts to provide replication or understanding of these disparate effects. Neither central (lateral or third cerebral ventricle) nor peripheral (intravenous) administration of apelin induced water intake in sated rats, nor did it decrease water intake in deprived rats. It also had no effect on sodium appetite. Peripherally injected apelin had a hypotensive action in anesthetized rats, but had no consistent effect in awake, unrestrained rats. We conclude that apelin does not have reliable or robust effects on fluid intake or blood pressure in Sprague-Dawley rats under normal conditions, but discuss the possibility for a role of apelin in fluid homeostasis in selected physiological states.

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.

Candesartan pretreatment is cerebroprotective in a rat model of endothelin‐1‐induced middle cerebral artery occlusion

Endogenous levels of angiotensin II (Ang II) are increased in the cortex and hypothalamus following stroke, and Ang II type 1 receptor blockers (ARBs) have been shown to attenuate the deleterious effects in animal stroke models using middle cerebral artery (MCA) intraluminal occlusion procedures. However, the endothelin‐1 (ET‐1)‐induced middle cerebral artery occlusion (MCAO) model of cerebral ischaemia is thought to more closely mimic the temporal events of an embolic stroke. This method provides rapid occlusion of the MCA and a gradual reperfusion that lasts for 16–22 h. The aim of the present study was to evaluate whether systemic administration of an ARB prior to ET‐1‐induced MCAO would provide cerebroprotection during this model of ischaemic stroke. Injection of 3 μl of 80 μm ET‐1 adjacent to the MCA resulted in complete occlusion of the vessel that resolved over a period of 30–40 min. Following ET‐1‐induced MCAO, rats had significant neurological impairment, as well as an infarct that consisted of 30% of the ipsilateral grey matter. Systemic pretreatment with 0.2 mg kg−1 day−1 candesartan for 7 days attenuated both the infarct size and the neurological deficits caused by ET‐1‐induced MCAO without altering blood pressure. This study confirms the cerebroprotective properties of ARBs during ischaemic stroke and validates the ET‐1‐induced MCAO model for examination of the role of the brain renin–angiotensin system in ischaemic stroke.

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.

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated

Efficacy of 3,5-dibromo-L-phenylalanine in rat models of stroke, seizures and sensorimotor gating deficit.

70 billion in the United States in 2009. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area. In the early 1990s, endothelin-1 (ET-1) was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.