Michael J. Katovich

Brain Microglial Cytokines in Neurogenic Hypertension

Accumulating evidence indicates a key role of inflammation in hypertension and cardiovascular disorders. However, the role of inflammatory processes in neurogenic hypertension remains to be determined. Thus, our objective in the present study was to test the hypothesis that activation of microglial cells and the generation of proinflammatory cytokines in the paraventricular nucleus (PVN) contribute to neurogenic hypertension. Intracerebroventricular infusion of minocycline, an anti-inflammatory antibiotic, caused a significant attenuation of mean arterial pressure, cardiac hypertrophy, and plasma norepinephrine induced by chronic angiotensin II infusion. This was associated with decreases in the numbers of activated microglia and mRNAs for interleukin (IL) 1&bgr;, IL-6, and tumor necrosis factor-&agr;, and an increase in the mRNA for IL-10 in the PVN. Overexpression of IL-10 induced by recombinant adenoassociated virus-mediated gene transfer in the PVN mimicked the antihypertensive effects of minocycline. Furthermore, acute application of a proinflammatory cytokine, IL-1&bgr;, into the left ventricle or the PVN in normal rats resulted in a significant increase in mean arterial pressure. Collectively, this indicates that angiotensin II induced hypertension involves activation of microglia and increases in proinflammatory cytokines in the PVN. These data have significant implications on the development of innovative therapeutic strategies for the control of neurogenic hypertension.

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.

Evidence for angiotensin-converting enzyme 2 as a therapeutic target for the prevention of pulmonary hypertension.

RATIONALE It has been proposed that an activated renin angiotensin system (RAS) causes an imbalance between the vasoconstrictive and vasodilator mechanisms involving the pulmonary circulation leading to the development of pulmonary hypertension (PH). Recent studies have indicated that angiotensin-converting enzyme 2 (ACE2), a member of the vasoprotective axis of the RAS, plays a regulatory role in lung pathophysiology, including pulmonary fibrosis and acute lung disease. Based on these observations, we propose the hypothesis that activation of endogenous ACE2 can shift the balance from the vasoconstrictive, proliferative axis (ACE-Ang II-AT1R) to the vasoprotective axis [ACE2-Ang-(1-7)-Mas] of the RAS, resulting in the prevention of PH. OBJECTIVES We have taken advantage of a recently discovered synthetic activator of ACE2, XNT (1-[(2-dimethylamino) ethylamino]-4-(hydroxymethyl)-7-[(4-methylphenyl) sulfonyl oxy]-9H-xanthene-9-one), to study its effects on monocrotaline-induced PH in rats to support this hypothesis. METHODS The cardiopulmonary effects of XNT were evaluated in monocrotaline-induced PH rat model. MEASUREMENTS AND MAIN RESULTS A single subcutaneous treatment of monocrotaline in rats resulted in elevated right ventricular systolic pressure, right ventricular hypertrophy, increased pulmonary vessel wall thickness, and interstitial fibrosis. These changes were associated with increases in the mRNA levels of renin, ACE, angiotensinogen, AT1 receptors, and proinflammatory cytokines. All these features of PH were prevented in these monocrotaline-treated rats by chronic treatment with XNT. In addition, XNT caused an increase in the antiinflammatory cytokine, IL-10. CONCLUSIONS These observations provide conceptual support that activation of ACE2 by a small molecule can be a therapeutically relevant approach for treating and controlling PH.

Structure-Based Identification of Small-Molecule Angiotensin-Converting Enzyme 2 Activators as Novel Antihypertensive Agents

Angiotensin-converting enzyme 2 (ACE2) is a key renin-angiotensin system enzyme involved in balancing the adverse effects of angiotensin II on the cardiovascular system, and its overexpression by gene transfer is beneficial in cardiovascular disease. Therefore, our objectives were 2-fold: to identify compounds that enhance ACE2 activity using a novel conformation-based rational drug discovery strategy and to evaluate whether such compounds reverse hypertension-induced pathophysiologies. We used a unique virtual screening approach. In vitro assays revealed 2 compounds (a xanthenone and resorcinolnaphthalein) that enhanced ACE2 activity in a dose-dependent manner. Acute in vivo administration of the xanthenone resulted in a dose-dependent transient and robust decrease in blood pressure (at 10 mg/kg, spontaneously hypertensive rats decreased 71±9 mm Hg and Wistar-Kyoto rats decreased 21±8 mm Hg; P<0.05). Chronic infusion of the xanthenone (120 μg/day) resulted in a modest decrease in the spontaneously hypertensive rat blood pressure (17 mm Hg; 2-way ANOVA; P<0.05), whereas it had no effect in Wistar-Kyoto rats. Strikingly, the decrease in blood pressure was also associated with improvements in cardiac function and reversal of myocardial, perivascular, and renal fibrosis in the spontaneously hypertensive rats. We conclude that structure-based screening can help identify compounds that activate ACE2, decrease blood pressure, and reverse tissue remodeling. Administration of ACE2 activators may be a valid strategy for antihypertensive therapy.

The angiotensin-converting enzyme 2/angiogenesis-(1-7)/Mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension.

RATIONALE An activated vasoconstrictive, proliferative, and fibrotic axis of the renin angiotensin system (angiotensin-converting enzyme [ACE]/angiotensin [Ang]II/AngII type 1 receptor) has been implicated in the pathophysiology of pulmonary fibrosis (PF) and pulmonary hypertension (PH). The recent discovery of a counterregulatory axis of the renin angiotensin system composed of ACE2/Ang-(1-7)/Mas has led us to examine the role of this vasoprotective axis on such disorders. OBJECTIVES We hypothesized that Ang-(1-7) treatment would exert protective effects against PF and PH. METHODS Lentiviral packaged Ang-(1-7) fusion gene or ACE2 cDNA was intratracheally administered into the lungs of male Sprague Dawley rats. Two weeks after gene transfer, animals received bleomycin (2.5 mg/kg). In a subsequent study, animals were administered monocrotaline (MCT, 50 mg/kg). MEASUREMENTS AND MAIN RESULTS In the PF study, bleomycin administration resulted in a significant increase in right ventricular systolic pressure, which was associated with the development of right ventricular hypertrophy. The lungs of these animals also exhibited excessive collagen deposition, decreased expression of ACE and ACE2, increased mRNA levels for transforming growth factor β and other proinflammatory cytokines, and increased protein levels of the AT₁R. Overexpression of Ang-(1-7) significantly prevented all the above-mentioned pathophysiological conditions. Similar protective effects were also obtained with ACE2 overexpression. In the PH study, rats injected with MCT developed elevated right ventricular systolic pressure, right ventricular hypertrophy, right ventricular fibrosis, and pulmonary vascular remodeling, all of which were attenuated by Ang-(1-7) overexpression. Blockade of the Mas receptor abolished the beneficial effects of Ang-(1-7) against MCT-induced PH. CONCLUSIONS Our observations demonstrate a cardiopulmonary protective role for the ACE2/Ang-(1-7)/Mas axis in the treatment of lung disorders.

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.

Cardiac Overexpression of Angiotensin Converting Enzyme 2 Protects the Heart From Ischemia-Induced Pathophysiology

Angiotensin converting enzyme 2 (ACE2) has been linked to cardiac dysfunction and hypertension-induced cardiac pathophysiology. In this study, we used a gene overexpression approach to investigate the role of ACE2 in cardiac function and remodeling after myocardial infarction. Rats received an intracardiac injection of 4.5×108 lentivirus containing ACE2 cDNA, followed by permanent coronary artery ligation (CAL) of the left anterior descending artery. At 24 hours and 6 weeks after surgery, cardiac functions, viability, and pathophysiology were assessed by MRI) and by histological analysis. At 24 hours post-CAL, left ventricular (LV) anterior wall motion was stunted to the same extent in control CAL and lenti-ACE2–treated CAL rats. However lenti-ACE2–treated CAL rats showed a 60% reduction in delayed contrast-enhanced LV volume after gadodiamide injection, indicating early ischemic protection of myocardium by ACE2. At 6 weeks after CAL, lenti-ACE2 rats demonstrated a complete rescue of cardiac output, a 41% rescue of ejection fraction, a 44% rescue in contractility, a 37% rescue in motion, and a 53% rescue in LV anterior (infracted) wall thinning compared with control CAL rats. No changes were observed in the LV posterior (noninfarcted) wall other than an 81% rescue in motion produced by ACE2 in CAL rats. Finally, infarct size measured by 2,3,5-triphenyl-tetrazolium chloride staining was not significantly different between the ligated groups. These observations demonstrate that cardiac overexpression of ACE2 exerts protective influence on the heart during myocardial infarction by preserving cardiac functions, LV wall motion and contractility, and by attenuating LV wall thinning.

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.

Similarities between morphine withdrawal in the rat and the menopausal hot flush

Skin temperature, cardiovascular and neuroendocrine responses to morphine withdrawal in the rat were evaluated in an effort to develop a potential animal model for the menopausal hot flush in women. Morphine dependency was produced by s.c. implantation of pellets containing morphine alkaloid. In response to precipitous, naloxone-induced withdrawal, rats showed surges in tail skin temperature (TST) which were similar in magnitude (4.8 to 7.2 degrees C) and duration (60 to 90 min.) to peripheral skin temperature increases reported during menopausal hot flushes. Additionally, a brief period of accelerated heart rate (59%) and a 9-fold hypersecretion of luteinizing hormone (LH) preceded the TST response to morphine withdrawal. These cardiovascular and neuroendocrine responses are observed to precede or coincide with the menopausal hot flush. Additionally, protracted morphine withdrawal subsequent to abstention, resulted in TST instability characterized by spontaneous, high amplitude TST fluctuations. Thus, the alteration in skin temperature, heart rate and LH secretion during precipitated morphine withdrawal in the rat are similar in magnitude, duration and in their temporal relationship to those observed during the hot flush. These data suggest a possible opioid etiology in this vasomotor disturbance. Acute withdrawal in the morphine addicted rats may serve as an animal model by which to study the neural mechanism underlying the menopausal hot flush.

Prevention of Pulmonary Hypertension by Angiotensin-Converting Enzyme 2 Gene Transfer

In spite of recent advancements in the treatment of pulmonary hypertension, successful control has yet to be accomplished. The abundant presence of angiotensin-converting enzyme 2 (ACE2) in the lungs and its impressive effect in the prevention of acute lung injury led us to test the hypothesis that pulmonary overexpression of this enzyme could produce beneficial outcomes against pulmonary hypertension. Monocrotaline (MCT) treatment of mice for 8 weeks resulted in significant increases in right ventricular systolic pressure, right ventricle:left ventricle plus septal weight ratio, and muscularization of pulmonary vessels. Administration of a lentiviral vector containing ACE2, 7 days before MCT treatment prevented the increases in right ventricular systolic pressure (control: 25±1 mm Hg; MCT: 44±5 mm Hg; MCT+ACE2: 26±1 mm Hg; n=6; P<0.05) and right ventricle:left ventricle plus septal weight ratio (control: 0.25±0.01; MCT: 0.31±0.01; MCT+ACE2: 0.26±0.01; n=8; P<0.05). A significant attenuation in muscularization of pulmonary vessels induced by MCT was also observed in animals overexpressing ACE2. These beneficial effects were associated with an increase in the angiotensin II type 2 receptor:angiotensin II type 1 receptor mRNA ratio. Also, pulmonary hypertension–induced increases in proinflammatory cytokines were significantly attenuated by lentiviral vector–containing ACE2 treatment. Furthermore, ACE2 gene transfer in mice after 6 weeks of MCT treatment resulted in a significant reversal of right ventricular systolic pressure. These observations demonstrate that ACE2 overexpression prevents and reverses right ventricular systolic pressure and associated pathophysiology in MCT-induced pulmonary hypertension by a mechanism involving a shift from the vasoconstrictive, proliferative, and fibrotic axes to the vasoprotective axis of the renin-angiotensin system and inhibition of proinflammatory cytokines.