BACKGROUND

The intracardiac conversion rate of angiotensin (Ang) I to Ang II and the expression of angiotensin converting enzyme (ACE) mRNA are amplified in rat hearts with left ventricular hypertrophy (LVH). To examine whether the accelerated intracardiac Ang II generation in LVH is related to an induction of cardiac ACE, we studied localization and function of cardiac ACE in hypertrophied rat hearts using specific ACE inhibitors.

RESULTS

Cardiac ACE was localized and quantified in hearts from male Wistar rats with LVH due to chronic experimental aortic stenosis and from control rats. With the ACE inhibitor 125I-351A, a derivative of lisinopril, as a radioligand on coronal sections of LVH and control hearts, in vitro autoradiography demonstrated ACE binding in aorta, coronary arteries, atria, and ventricles of both groups. Quantitative analyses revealed that ACE density (counts per minute per cross-sectional area of tissue) was twofold higher within the myocardium of hypertrophied left ventricles compared with controls (p < 0.005). Quantitative morphometry demonstrated a modest increase in the fractional volume of myocytes as well as capillary volume without an increase in the fractional volume of endothelial cells in left ventricular tissue from aortic stenosis rats. These data suggest that an increase in endothelial cell volume per se cannot alone account for the observed doubling of ACE density and support an upregulation of ACE production in hypertrophied tissue. The role of cardiac ACE in intracardiac conversion of Ang I to Ang II and its specific inhibition was studied in isolated, isovolumic beating, buffer-perfused LVH and control hearts. Biochemical conversion rates as well as functional changes in response to 3 x 10(-7) M Ang I were examined in the absence or presence of the ACE inhibitor enalaprilat (4 x 10(-6) M). After a brief stabilization period, groups of LVH and control hearts were subjected to the following infusion protocols: 15 minutes of vehicle followed by 30 minutes of Ang I plus vehicle, 15 minutes of enalaprilat followed by 30 minutes of Ang I plus enalaprilat (enal/Ang I), or 45 minutes of vehicle only to allow comparison with a time control. Intracardiac Ang I-to-Ang II conversion rate was fourfold higher in LVH than in control hearts (p < 0.05). Infusion of enalaprilat reduced the intracardiac Ang I-to-Ang II conversion rate in LVH hearts by 70% (p < 0.05 versus Ang I). At similar levels of constant coronary flow per gram, Ang I increased coronary perfusion pressure by 23 +/- 5 mm Hg (p < 0.01 versus vehicle) in LVH hearts and by 36 +/- 10 mm Hg (p < 0.005 versus vehicle) in control hearts. When enalaprilat was infused with Ang I, the increase in perfusion pressure was limited to 5 +/- 5 mm Hg (NS versus vehicle) in LVH hearts and 12 +/- 3 mm Hg (p < 0.05 versus vehicle) in control hearts and was significantly lower than in hearts infused with Ang I only (p < 0.05 in LVH and p < 0.05 in control hearts, respectively). Systolic function was not affected by either infusion protocol. In contrast, Ang I infusion was associated with diastolic dysfunction. In LVH hearts, left ventricular end-diastolic pressure (LVEDP) increased from 10 +/- 1 mm Hg at baseline to 25 +/- 2 mm Hg at the end of the Ang I infusion (p < 0.001 versus vehicle), which was inhibited by infusion of enalaprilat. In control hearts, there was a lesser increase in LVEDP from 10 +/- 1 mm Hg to 15 +/- 1 mm Hg in response to Ang I (p < 0.05 versus LVH). Control hearts treated with enalaprilat with Ang I displayed no increase in LVEDP:

CONCLUSIONS

These observations indicate that ACE protein is increased within the myocardium of LVH hearts, extending recent findings of increased cardiac ACE activity and mRNA levels in this model of pressure-overload LVH in the rat. Blockade of the enzyme by an ACE inhibitor decreases intracardiac Ang I-to-Ang II conversion rate and prevents the functional changes of Ang I-to-Ang II activation

Our long-term goal is to use <em>angiotensin</em> converting enzyme (ACE) inhibitors to mitigate the increase in lung collagen synthesis that is induced by irradiation to the lung, which could result from accidental exposure or radiological terrorism. Rats (WAG/RijCmcr) were given a single dose of <em>1</em>3 Gy (dose rate of <em>1</em>.43 Gy/min) of X-irradiation to the thorax. Three structurally-different ACE inhibitors, captopril, enalapril and fosinopril were provided in drinking water beginning <em>1</em> week after irradiation. Rats that survived acute pneumonitis (at 6-<em>1</em>2 weeks) were evaluated monthly for synthesis of lung collagen. Other endpoints included breathing rate, wet to dry lung weight ratio, and analysis of lung structure. Treatment with captopril (<em>1</em>45-20<em>7</em> mg/m(2)/day) or enalapril (<em>1</em>9-28 mg/m(2)/day), but not fosinopril (<em>1</em>9-28 mg/m(2)/day), decreased morbidity from acute pneumonitis. Lung collagen in the surviving irradiated rats was increased over that of controls by <em>7</em> months after irradiation. This increase in collagen synthesis was not observed in rats treated with any of the three ACE inhibitors. Analysis of the lung morphology at <em>7</em> months supports the efficacy of ACE inhibitors against radiation-induced fibrosis. The effectiveness of fosinopril against fibrosis, but not against acute pneumonitis, suggests that pulmonary fibrosis may not be a simple consequence of injury during acute pneumonitis. In summary, three structurally-different ACE inhibitors mitigate the increase in collagen synthesis <em>7</em> months following irradiation of the whole thorax and do so, even when therapy is started one week after irradiation.

The purpose of this study was to evaluate the time-course effect of a 36-h treatment with ACTH (<em>1</em>0(-8) M), transforming growth factor-beta<em>1</em> (TGFbeta<em>1</em>; <em>1</em>0(-<em>1</em>0) M), <em>angiotensin</em> II (AngII; <em>1</em>0 (-<em>7</em>) M), and insulin-like growth factor I (IGF-I; <em>1</em>0(-8) M) on the steroidogenic capacity of bovine adrenocortical cells (BAC) and on messenger RNA (mRNA) levels of ACTH receptor, cytochrome P450c<em>1</em><em>7</em>, 3beta-hydroxysteroid dehydrogenase (3betaHSD), steroidogenic acute regulatory protein (StAR), and StAR protein. ACTH and IGF-I enhanced, in a time-dependent manner, the acute 2-h ACTH-induced cortisol production, whereas TGFbeta <em>1</em> and AngII markedly reduced it. ACTH, IGF-I, and AngII increased ACTH receptor mRNA, but the opposite was observed after TGFbeta<em>1</em> treatment. ACTH and IGF-I increased P450c<em>1</em><em>7</em> and 3betaHSD mRNAs, whereas AngII and TGFbeta<em>1</em> had the opposite effects. However, the effects of the four peptides on ACTH-induced cortisol production appeared before any significant alterations of the mRNA levels occurred. The most marked and rapid effect of the four peptides was on StAR mRNA. The stimulatory effect of ACTH was seen within <em>1</em>.5 h, peaked at 4-6 h, and declined thereafter, but at the end of the 36-h pretreatment, the levels of StAR mRNA and protein were higher than those in control cells. IGF-I also enhanced StAR mRNA levels within <em>1</em>.5 h, and these levels remained fairly constant. The effects of AngII on StAR mRNA expression were biphasic, with a peak within <em>1</em>.5-3 h, followed by a rapid decline to almost undetectable levels of both mRNA and protein. TGFbeta<em>1</em> had no significant effect during the first 3 h, but thereafter StAR mRNA declined, and at the end of the experiment the StAR mRNA and protein were almost undetectable. Similar results were observed when cells were treated with ACTH plus TGFbeta<em>1</em>. A 2-h acute ACTH stimulation at the end of the 36-h pretreatment caused a higher increase in StAR mRNA and protein in ACTH- or IGF-I-pretreated cells than in control cells, which, in turn, had higher levels than cells pretreated with TGFbeta<em>1</em>, ACTH plus TGFbeta<em>1</em>, or AngII. These results and the fact that the stimulatory (IGF-I) or inhibitory (AngII and TGFbeta<em>1</em>) effects on ACTH-induced cortisol production were more pronounced than those on the ability of cells to transform pregnenolone into cortisol strongly suggest that regulation of StAR expression is one of the main factors, but not the only one, involved in the positive (IGF-I) or negative (TGFbeta<em>1</em> and AngII) regulation of BAC for ACTH steroidogenic responsiveness. A high correlation between steady state mRNA level and acute ACTH-induced cortisol production favors this conclusion.

Renal hemodynamics increase dramatically during pregnancy, and pressor responsiveness to exogenous administration of vasoconstrictors is attenuated. We investigated whether or not vasodilatory prostaglandins mediate these phenomena. Trained, chronically instrumented, conscious pregnant rats were used. Control values of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were elevated at midgestation (P less than 0.0<em>1</em> and P = 0.05 from prepregnant means, respectively), and effective renal vascular resistance was decreased (P = 0.05). Indomethacin (4.5-6.5 mg/kg body weight [BW]) failed to decrease renal hemodynamics at this stage of pregnancy; in fact, it raised GFR somewhat further (P less than 0.05). Systemic pressor responsiveness to bolus administration of norepinephrine and <em>angiotensin</em> II (AII) was significantly attenuated by at least gestational day 20. Neither indomethacin (<em>7</em> mg/kg BW) or meclofenamate (6 mg/kg BW) affected the refractory response. The renal vasculature was also relatively unresponsive to an intravenous infusion of AII (5 ng X kg-<em>1</em> X min-<em>1</em>) during late gestation (day <em>1</em>9); in particular, the fall in ERPF in response to AII (<em>1</em>6 +/- 3%) was markedly less than that observed in the prepregnant condition (34 +/- 3%; P less than 0.05). Indomethacin (6 mg/kg BW) failed to restore this blunted response, and further attenuation was evident, despite the presence of the inhibitor (gestational day 2<em>1</em>). We conclude that vasodilatory prostaglandins do not appear to mediate the rise in renal hemodynamics, and the attenuation of the systemic and renal pressor responsiveness observed during pregnancy, insofar as these phenomena were unaffected by acute cyclooxygenase inhibition in unstressed, conscious rats.

Statins exert beneficial effects in chronically damaged tissues. <em>Angiotensin</em> II (ANG II) participates in liver fibrogenesis by inducing oxidative stress, inflammation, and transforming growth factor-beta<em>1</em> (TGF-beta<em>1</em>) expression. We investigate whether atorvastatin modulates ANG II-induced pathogenic effects in the liver. Male Wistar rats were infused with saline or ANG II (<em>1</em>00 ng kg(-<em>1</em>) min(-<em>1</em>)) for 4 wk through a subcutaneous osmotic pump. Rats received either vehicle or atorvastatin (5 mg kg(-<em>1</em>) day(-<em>1</em>)) by gavage. ANG II infusion resulted in infiltration of inflammatory cells (CD43 immunostaining), oxidative stress (4-hydroxynonenal), hepatic stellate cells (HSC) activation (smooth muscle alpha-actin), increased intercellular adhesion molecule (ICAM-<em>1</em>), and interleukin-6 hepatic gene expression (quantitative PCR). These effects were markedly blunted in rats receiving atorvastatin. The beneficial effects of atorvastatin were confirmed in an additional model of acute liver injury (carbon tetrachloride administration). We next explored whether the beneficial effects of atorvastatin on ANG II-induced actions are also reproduced at the cellular level. We studied HSC, a cell type with inflammatory and fibrogenic properties. ANG II (<em>1</em>0(-8)M) stimulated cell proliferation, proinflammatory actions (NF-kappaB activation, ICAM-<em>1</em> expression, interleukin-8 secretion) as well as expression of procollagen-alpha(<em>1</em>(I)) and TGF-beta<em>1</em>. All of these effects were reduced in the presence of atorvastatin (<em>1</em>0(-<em>7</em>)M). These results indicate that atorvastatin attenuates the pathogenic events induced by ANG II in the liver both in vivo and in vitro. Therefore, statins could have beneficial effects in conditions characterized by hepatic inflammation.

The mechanisms by which <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] exerts its beneficial effects on end-organ damage associated with diabetes and hypertension are not well understood. The purpose of this study was A) to compare the effects of apocynin with Ang-(<em>1</em>-<em>7</em>) on renal vascular dysfunction and NADPH oxidase activity in a combined model of diabetes and hypertension and B) to further determine whether chronic treatment with Ang-(<em>1</em>-<em>7</em>) can modulate renal catalase, and peroxisome proliferator activated receptor- gamma (PPAR-gamma) levels in streptozotocin-induced diabetes in both normotensive Wistar Kyoto rats (WKY) and in spontaneously hypertensive rats (SHR). Apocynin or Ang-(<em>1</em>-<em>7</em>) treatment for one month starting at the onset of diabetes similarly attenuated elevation of renal NADPH oxidase activity in the diabetic SHR kidney and reduced the degree of proteinuria and hyperglycemia, but had little or modest effect on reducing mean arterial pressure. Both drugs also attenuated the diabetes-induced increase in renal vascular responsiveness to endothelin-<em>1</em>. Induction of diabetes in WKY and SHR animals resulted in significantly reduced renal catalase activity and in PPAR-gamma mRNA and protein levels. Treatment with Ang-(<em>1</em>-<em>7</em>) significantly prevented diabetes-induced reduction in catalase activity and the reduction in PPAR-gamma mRNA and protein levels in both animal models. Taken together, these data suggest that activation of Ang-(<em>1</em>-<em>7</em>)-mediated signaling could be an effective way to prevent the elevation of NADPH oxidase activity and inhibition of PPAR-gamma and catalase activities in diabetes and/or hypertension.

Stimulation of MAS oncogene receptor (MAS) or <em>angiotensin</em> (Ang) receptor type 2 (AT2) may be novel therapeutic options for neonatal chronic lung disease (CLD) by counterbalancing the adverse effects of the potent vasoconstrictor <em>angiotensin</em> II, consisting of arterial hypertension (PAH)-induced right ventricular hypertrophy (RVH) and pulmonary inflammation. We determined the cardiopulmonary effects in neonatal rats with CLD of daily treatment during continuous exposure to <em>1</em>00% oxygen for <em>1</em>0 days with specific ligands for MAS [cyclic Ang-(<em>1</em>-<em>7</em>); <em>1</em>0-50 μg·kg(-<em>1</em>)·day(-<em>1</em>)] and AT2 [dKcAng-(<em>1</em>-<em>7</em>); 5-20 μg·kg(-<em>1</em>)·day(-<em>1</em>)]. Parameters investigated included lung and heart histopathology, fibrin deposition, vascular leakage, and differential mRNA expression in the lungs of key genes involved in the renin-<em>angiotensin</em> system, inflammation, coagulation, and alveolar development. We investigated the role of nitric oxide synthase inhibition with N(ω)-nitro-l-arginine methyl ester (25 mg·kg(-<em>1</em>)·day(-<em>1</em>)) during AT2 agonist treatment. Prophylactic treatment with agonists for MAS or AT2 for <em>1</em>0 days diminished cardiopulmonary injury by reducing alveolar septum thickness and medial wall thickness of small arterioles and preventing RVH. Both agonists attenuated the pulmonary influx of inflammatory cells, including macrophages (via AT2) and neutrophils (via MAS) but did not reduce alveolar enlargement and vascular alveolar leakage. The AT2 agonist attenuated hyperoxia-induced fibrin deposition. In conclusion, stimulation of MAS or AT2 attenuates cardiopulmonary injury by reducing pulmonary inflammation and preventing PAH-induced RVH but does not affect alveolar and vascular development in neonatal rats with experimental CLD. The beneficial effects of AT2 activation on experimental CLD were mediated via a NOS-independent mechanism.

A novel assay was developed for evaluation of mouse <em>angiotensin</em>-converting enzyme (ACE) 2 and recombinant human ACE2 (rACE2) activity. Using surface-enhanced laser desorption/ionization time of flight mass spectrometry (MS) with ProteinChip Array technology, ACE<em>1</em> and ACE2 activity could be measured using natural peptide substrates. Plasma from C5<em>7</em>BL/6 mice, kidney from wild-type and ACE2 knockout mice, and rACE2 were used for assay validation. Plasma or tissue extracts were incubated with <em>angiotensin</em> I (Ang I; <em>1</em>296 m/z) or <em>angiotensin</em> II (Ang II; <em>1</em>045 m/z). Reaction mixtures were spotted onto the ProteinChips WCX2 and peptides detected using surface-enhanced laser desorption/ionization time of flight MS. MS peaks for the substrates, Ang I and Ang II, and the generated peptides, Ang (<em>1</em>-<em>7</em>) and Ang (<em>1</em>-9), were monitored. The ACE2 inhibitor MLN 4<em>7</em>60 (0.0<em>1</em> to <em>1</em>00 micromol/L) significantly inhibited rACE2 activity (IC50=3 nmol/L). Ang II was preferably cleaved by rACE2 (km=5 mumol/L), whereas Ang I was not a good substrate for rACE2. There was no detectable ACE2 activity in plasma. Assay specificity was validated in a model of ACE2 gene deletion. In kidney extract from ACE2-deficient mice, there was no generation of Ang (<em>1</em>-<em>7</em>) from Ang II. However, Ang (<em>1</em>-<em>7</em>) was produced when Ang I was used as a substrate. In conclusion, we developed a specific and sensitive assay for ACE2 activity, which used the natural endogenous peptide substrate Ang II. This approach allows for the rapid screening for ACE2, which has applications in drug testing, high-throughput enzymatic assays, and identification of novel substrates/inhibitors of the renin-<em>angiotensin</em> system.

Complex and bidirectional interactions between the renin-<em>angiotensin</em> system (RAS) and autonomic nervous system have been well established for cardiovascular regulation under both physiological and pathophysiological conditions. Most research to date has focused on deleterious effects of components of the vasoconstrictor arm of the RAS on cardiovascular autonomic control, such as renin, <em>angiotensin</em> II, and aldosterone. The recent discovery of prorenin and the prorenin receptor have further increased our understanding of RAS interactions in autonomic brain regions. Therapies targeting these RAS components, such as <em>angiotensin</em>-converting enzyme (ACE) inhibitors and <em>angiotensin</em> receptor blockers, are commonly used for treatment of hypertension and cardiovascular diseases, with blood pressure-lowering effects attributed in part to sympathetic inhibition and parasympathetic facilitation. In addition, a vasodilatory arm of the RAS has emerged that includes <em>angiotensin</em>-(<em>1</em>-<em>7</em>), ACE2, and alamandine, and promotes beneficial effects on blood pressure in part by reducing sympathetic activity and improving arterial baroreceptor reflex function in animal models. The role of the vasodilatory arm of the RAS in cardiovascular autonomic regulation in clinical populations, however, has yet to be determined. This review will summarize recent developments in autonomic mechanisms involved in the effects of the RAS on cardiovascular regulation, with a focus on newly discovered pathways and therapeutic targets for this hormone system.

Accumulating evidence suggests that the <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)], is an active member of the brain renin-<em>angiotensin</em> system (RAS). We evaluated the possibility that intracerebroventricular (ICV, lateral ventricle) infusion of exogenous Ang-(<em>1</em>-<em>7</em>) could participate in the potentiation of bradykinin (BK) release and the kinin receptor expression in ischemic brain parenchyma after focal cerebral ischemia-reperfusion in rats. The middle cerebral artery occlusion (MCAO) and sham-operated models were prepared, continuously administrated with Ang-(<em>1</em>-<em>7</em>) or artificial cerebrospinal fluid (aCSF) by implanted Alzet osmotic minipumps into lateral cerebral ventricle after reperfusion in male Sprague-Dawley (SD) rats. Experimental animals were divided into sham-operated group (sham+aCSF), aCSF treatment group (MCAO+aCSF) and Ang-(<em>1</em>-<em>7</em>) treatment groups [MCAO+Ang-(<em>1</em>-<em>7</em>)] at low (<em>1</em> pmol/0.5 microl/h), medium (<em>1</em>00 pmol/0.5 microl/h) or high (<em>1</em>0 nmol/0.5 microl/h) dose levels. Cerebral infarction resulted in a significant increase of BK formation from 3 h to 6 h compared with sham-operated group after reperfusion, whereas medium- and high-dose Ang-(<em>1</em>-<em>7</em>) infusion markedly enhanced BK levels from 6 h to 48 h after reperfusion. Medium- and high-dose Ang-(<em>1</em>-<em>7</em>) infusion markedly increased kinin B(2) receptor mRNA and protein expression, whereas only high-dose Ang-(<em>1</em>-<em>7</em>) infusion induced upregulating the expression of B(<em>1</em>) receptor. Low-dose Ang-(<em>1</em>-<em>7</em>) infusion did not modify both the kinin B(<em>1</em>) and B(2) receptor expression compared with aCSF treatment group after focal cerebral ischemia-reperfusion at each time point. The finding might indicate complex interactions between Ang-(<em>1</em>-<em>7</em>) and kallikrein-kinin system in the CNS after focal cerebral ischemia-reperfusion in rats.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single-stranded RNA virus that causes the potentially lethal Covid-19 respiratory tract infection. It does so by binding to host cell angiotensin converting enzyme 2 (ACE2) receptors, leading to endocytosis with the receptor, and subsequently using the host cell's machinery to replicate copies of itself and invade new cells. The extent of the spread of infection in the body is dependent on the pattern of ACE2 expression and overreaction of the immune system. Additionally, by inducing an imbalance in the renin-angiotensin-aldosterone system (RAAS) and the loss of ACE2 would favour the progression of inflammatory and thrombotic processes in the lungs. No drug or vaccine has yet been approved to treat human coronaviruses. Hundreds of clinical trials on existing approved drugs from different classes acting on a multitude of targets in the virus life cycle are ongoing to examine potential effectiveness for the prevention and treatment of the infection. This review summarizes the SARS-CoV-2 virus life cycle in the host cell and provides a biological and pathological point of view for repurposed and experimental drugs for this novel coronavirus. The viral lifecycle provides potential targets for drug therapy.

Keywords: ACE2; Ang (1-7); Covid-19; Immunomodulators; Remdesivir; SARS-CoV-2.

This study investigates the role of extracellular signal-regulated kinases (ERKs) in <em>angiotensin</em> II (Ang II)-generated intracellular second messengers (cytosolic free Ca2+ concentration, ie, [Ca2+]i, and pHi) and in contraction in isolated vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY) using the selective mitogen-activated protein (MAP)/ERK inhibitor, PD98059. VSMCs from mesenteric arteries were cultured on Matrigel basement membrane matrix. These cells, which exhibit a contractile phenotype, were used to measure [Ca2+]i, pHi, and contractile responses to Ang II (<em>1</em>0(-<em>1</em>2) to <em>1</em>0(-6) mol/L) in the absence and presence of PD98059 (<em>1</em>0(-5) mol/L). [Ca2+]i and pHi were measured by fura-2 and BCECF methodology, respectively, and contraction was determined by photomicroscopy. Ang II-stimulated ERK activity was measured by Western blot analysis using a phospho-specific ERK-<em>1</em>/ERK-2 antibody and by an MAPK enzyme assay. Ang II increased [Ca2+]i and pHi and contracted cells in a dose-dependent manner. Maximum Ang II-elicited contraction was greater (P<0.05) in SHR (4<em>1</em>.9+/-5.<em>1</em>% reduction in cell length relative to basal length) than in WKY (28.<em>1</em>+/-3.0% reduction in cell length relative to basal length). Basal [Ca2+]i, but not basal pHi, was higher in SHR compared with WKY. [Ca2+]i and pHi effects of Ang II were enhanced (P<0.05) in SHR compared with WKY (maximum Ang II-induced response [Emax] of [Ca2+]i, 5<em>7</em>6+/-24 versus 4<em>1</em>3+/-43 nmol/L; Emax of pHi, <em>7</em>.33+/-0.0<em>1</em> versus <em>7</em>.2<em>7</em>+/-0.03, SHR versus WKY). PD98059 decreased the magnitude of contraction and attenuated the augmented Ang II-elicited contractile responses in SHR (Emax,<em>1</em>9. 3+/-3% reduction in cell length relative to basal length). Ang II-stimulated [Ca2+]i (Emax, 294+/-55 nmol/L) and pHi (Emax, <em>7</em>. 2<em>7</em>+/-0.04) effects were significantly reduced by PD98059 in SHR. Ang II-induced ERK activity was significantly greater (P<0.05) in SHR than in WKY. In conclusion, Ang II-stimulated signal transduction and associated VSMC contraction are enhanced in SHR. MAP/ERK inhibition abrogated sustained contraction and normalized Ang II effects in SHR. These data suggest that ERK-dependent signaling pathways influence contraction and that they play a role in vascular hyperresponsiveness in SHR.

Myocardial fibrosis is characterized by significant extracellular matrix (ECM) deposition. The specific cellular mediators that contribute to the development of fibrosis are not well understood. Using a model of fibrosis with <em>Angiotensin</em> II (AngII) infusion, our aim was to characterize the cellular elements involved in the development of myocardial fibrosis. Male C5<em>7</em>Bl/6 and Tie2-GFP mice were given AngII (2.0 mg/kg/min) or saline (control) via mini osmotic pumps for up to <em>7</em> days. Hearts were harvested, weighed and processed for analysis. Cellular infiltration and collagen deposition were quantified. Immunostaining was performed for specific markers of leukocytes (CD45, CD<em>1</em><em>1</em>b), myofibroblasts (SMA), endothelial cells (vWF) and hematopoietic progenitor cells (CD<em>1</em>33). Bone marrow (BM) origin of infiltrating cells was assessed using GFP(+) chimeric animals. Relative qRT-PCR was performed for pro-fibrotic cytokines (transforming growth factor (TGF)-β<em>1</em>, CTGF) as well as the chemokine stromal-derived factor (SDF)-<em>1</em>α. Myocardial-infiltrating cells were grown in vitro. AngII exposure resulted in multifocal myocardial cellular infiltration, which preceded extensive ECM deposition. A limited number of myocardial-infiltrating cells were positive for leukocyte markers but were significantly positive for myofibroblast (SMA) and endothelial cell (vWF) markers. However, using Tie2-GFP mice, where endothelial cells are GFP(+), myocardial-infiltrating cells were not GFP(+). Transcript levels for SDF-<em>1</em>α were significantly elevated at <em>1</em> day of AngII exposure suggesting that hematopoietic progenitor cells may be recruited. This was confirmed by positive CD<em>1</em>33 staining of infiltrating cells and evident GFP(+) cellular infiltration when exposing GFP(+) BM chimeras to AngII. Furthermore, a significant number of CD<em>1</em>33(+)/SMA(+) cells were grown in vitro from the myocardium of AngII-exposed animals (P<0.0<em>1</em>). Myocardial ECM deposition is preceded by the infiltration of the myocardium with hematopoietic cells that express mesenchymal markers. These data suggest that mesenchymal progenitor cells are recruited, and may have a primary role, in the initiation of myocardial fibrosis.

The organic nitrate pentaerythritol tetranitrate is devoid of nitrate tolerance, which has been attributed to the induction of the antioxidant enzyme heme oxygenase (HO)-<em>1</em>. With the present study, we tested whether chronic treatment with pentaerythritol tetranitrate can improve <em>angiotensin</em> II-induced vascular oxidative stress and dysfunction. In contrast to isosorbide-5 mononitrate (<em>7</em>5 mg/kg per day for <em>7</em> days), treatment with pentaerythritol tetranitrate (<em>1</em>5 mg/kg per day for <em>7</em> days) improved the impaired endothelial and smooth muscle function and normalized vascular and cardiac reactive oxygen species production (mitochondria, NADPH oxidase activity, and uncoupled endothelial NO synthase), as assessed by dihydroethidine staining, lucigenin-enhanced chemiluminescence, and quantification of dihydroethidine oxidation products in <em>angiotensin</em> II (<em>1</em> mg/kg per day for <em>7</em> days)-treated rats. The antioxidant features of pentaerythritol tetranitrate were recapitulated in spontaneously hypertensive rats. In addition to an increase in HO-<em>1</em> protein expression, pentaerythritol tetranitrate but not isosorbide-5 mononitrate normalized vascular reactive oxygen species formation and augmented aortic protein levels of the tetrahydrobiopterin-synthesizing enzymes GTP-cyclohydrolase I and dihydrofolate reductase in <em>angiotensin</em> II-treated rats, thereby preventing endothelial NO synthase uncoupling. Haploinsufficiency of HO-<em>1</em> completely abolished the beneficial effects of pentaerythritol tetranitrate in <em>angiotensin</em> II-treated mice, whereas HO-<em>1</em> induction by hemin (25 mg/kg) mimicked the effect of pentaerythritol tetranitrate. Improvement of vascular function in this particular model of arterial hypertension by pentaerythritol tetranitrate largely depends on the induction of the antioxidant enzyme HO-<em>1</em> and identifies pentaerythritol tetranitrate, in contrast to isosorbide-5 mononitrate, as an organic nitrate able to improve rather than to worsen endothelial function.

The renin-<em>angiotensin</em> system (RAS) has been implicated in the cardiovascular complications of diabetes. We showed that a high-fructose diet increases blood pressure and plasma <em>angiotensin</em> and impairs glucose tolerance. We investigated the role of <em>angiotensin</em> AT(<em>1</em>a) receptors in the development of fructose-induced cardiovascular and metabolic dysfunction. Male <em>angiotensin</em> AT(<em>1</em>a) knockout (AT<em>1</em>aKO) and wild-type (AT<em>1</em>aWT) mice with arterial telemetric catheters were fed a standard diet or one containing 60% fructose. Fructose increased mean arterial pressure (MAP) in AT<em>1</em>aWT but only during the dark phase (8% increase). In AT<em>1</em>aKO mice, fructose unexpectedly decreased MAP, during both light and dark periods (24 and <em>1</em>3% decrease, respectively). Analytical methods were used to measure systolic arterial pressure (SAP) and pulse interval (PI) variability in time and frequency domains. In fructose-fed AT<em>1</em>aWT mice, there was an increase in SAP variance and its low-frequency (LF) domain (<em>1</em><em>1</em> +/- 3 vs. 23 +/- 4 mmHg(2), variance, and <em>7</em> +/- 2 vs. <em>1</em><em>7</em> +/- 3 mmHg(2), LF, control vs. fructose, P < 0.004). There were no changes in SAP variance in AT<em>1</em>aKO mice. Depressor responses to alpha(<em>1</em>)-adrenergic blockade were augmented in fructose-fed AT<em>1</em>a WT compared with AT<em>1</em>aKO mice. Fructose inhibited glucose tolerance with a greater effect in AT<em>1</em>aWT mice. Fructose increased plasma cholesterol in both groups (P < 0.0<em>1</em>) and reduced ANG II in AT<em>1</em>aKO mice. Results document prominent interactions between genetics and diet with data showing that in the absence of <em>angiotensin</em> AT(<em>1</em>a) receptors, a fructose diet decreased blood pressure.

OBJECTIVE

Doxorubicin is a widely used chemotherapy drug, but its application is associated with cardiotoxicity. Free radical generation and mitochondrial dysfunction are thought to contribute to doxorubicin-induced cardiac failure. Angiotensin-converting enzyme inhibitors are commonly used as cardioprotective agents and have recently been shown in clinical studies to be efficacious in the prevention of anthracycline-induced heart failure. This study evaluated a mechanism for these protective effects by testing the ability of the angiotensin-converting enzyme inhibitor enalapril to preserve mitochondrial function in a model of chronic doxorubicin treatment in rats.

METHODS

Sprague Dawley rats were divided into 3 groups and followed for a total of 10 weeks: (1) control-untreated, (2) doxorubicin treated, and (3) doxorubicin + enalapril treated. Doxorubicin was administered via intraperitoneal injection at weekly intervals from weeks 2 to 7. Enalapril was administered in the drinking water of the doxorubicin + enalapril group for the study duration.

RESULTS

Doxorubicin treatment produced a significant loss in left ventricular contractility (P < .05), decrease in mitochondrial function via impairment of state-3 respiration, decrease in the cytosolic fraction of adenosine triphosphate, and up-regulation of free radical production. Enalapril significantly attenuated the decrease in percent fractional shortening (P < .05) and prevented the doxorubicin-associated reduction in respiratory efficiency and cytosolic adenosine triphosphate content (P < .05). Enalapril also abolished the robust doxorubicin-induced increase in free radical formation.

CONCLUSIONS

Administration of enalapril attenuates doxorubicin-induced cardiac dysfunction via preservation of mitochondrial respiratory efficiency and reduction in doxorubicin-associated free radical generation.

The bradykinin peptide system is a tissue-based system with potent cardiovascular and renal effects. To investigate the regulation of this system, we developed a highly sensitive amino terminal-directed radioimmunoassay that, with high performance liquid chromatography, enables the measurement of bradykinin-(<em>1</em>-<em>7</em>), bradykinin-(<em>1</em>-8), and bradykinin-(<em>1</em>-9). Together with a carboxy terminal-directed radioimmunoassay, we characterized bradykinin peptides in rat kidney and blood. The predominant bradykinin peptides in kidney were bradykinin-(<em>1</em>-9) (approximately <em>1</em>00 fmol/g wet weight of tissue) and bradykinin-(<em>1</em>-<em>7</em>) (approximately <em>7</em>0 fmol/g), with low levels of bradykinin-(<em>1</em>-8) (approximately 8 fmol/g) and bradykinin-(4-9) (approximately <em>1</em>2 fmol/g) detectable; bradykinin-(2-9) and bradykinin-(3-9) were below the limits of detection. In blood, the levels of bradykinin-(<em>1</em>-9) were very low (approximately 2 fmol/ml), and other bradykinin peptides were below the limits of detection. Ile,Ser-bradykinin and Met,Ile,Ser-bradykinin were below the limits of detection in both kidney and blood, indicating that T-kininogen makes no detectable contribution to renal or circulating bradykinin peptides. Administration of the <em>angiotensin</em> converting enzyme inhibitor perindopril was associated with an approximate twofold increase in renal levels of bradykinin-(<em>1</em>-8) and bradykinin-(<em>1</em>-9) and a decrease in the bradykinin-(<em>1</em>-<em>7</em>)/bradykinin-(<em>1</em>-9) ratio. The amino terminal-directed radioimmunoassay was also applied to heart, aorta, brown adipose tissue, adrenal lung, and brain. For these tissues, bradykinin-(<em>1</em>-<em>7</em>) and bradykinin-(<em>1</em>-9) were of similar abundance (<em>1</em>6-340 fmol/g), with lower levels of bradykinin-(<em>1</em>-8). These studies demonstrate that tissue levels of bradykinin peptides are much higher than circulating levels, consistent with their formation at a local tissue site. Of peptides derived from K-kininogen, bradykinin-(<em>1</em>-9) is the predominant bioactive peptide in all tissues, and a major pathway of bradykinin-(<em>1</em>-9) metabolism involves the formation of bradykinin-(<em>1</em>-<em>7</em>). In kidney, <em>angiotensin</em> converting enzyme plays an important role in bradykinin-(<em>1</em>-9) metabolism, and increased bradykinin-(<em>1</em>-9) and bradykinin-(<em>1</em>-8) levels may mediate in part the renal effects of converting enzyme inhibition.

OBJECTIVE

To evaluate the effects of the activation of endogenous angiotensin-converting enzyme 2 (ACE2) using the compound diminazene aceturate (DIZE) in an experimental model of glaucoma in Wistar rats.

METHODS

DIZE (1 mg/kg) was administered daily, either systemically or topically, and the IOP was measured weekly. To examine the role of the Mas receptor in the effects of DIZE, the Ang-(1-7) antagonist A-779 was co-administered. Drainage of the aqueous humor was evaluated by using scintigraphy. The analysis of ACE2 expression by immunohistochemistry and the counting of retinal ganglion cells (RGCs) were performed in histologic sections. Additionally, the nerve fiber structure was evaluated by transmission electron microscopy.

RESULTS

The systemic administration and topical administration (in the form of eye drops) of DIZE increased the ACE2 expression in the eyes and significantly decreased the IOP of glaucomatous rats without changing the blood pressure. Importantly, this IOP-lowering action of DIZE was similar to the effects of dorzolamide. The antiglaucomatous effects of DIZE were blocked by A-779. Histologic analysis revealed that the reduction in the number of RGCs and the increase in the expression of caspase-3 in the RGC layer in glaucomatous animals were prevented by DIZE. This compound also prevented alterations in the cytoplasm of axons in glaucomatous rats. In addition to these neuroprotective effects, DIZE facilitated the drainage of the aqueous humor.

CONCLUSIONS

Our results evidence the pathophysiologic relevance of the ocular ACE2/Ang-(1-7)/Mas axis of the renin-angiotensin system and, importantly, indicate that the activation of intrinsic ACE2 is a potential therapeutic strategy to treat glaucoma.

<em>Angiotensin</em> II (Ang II) and Ang III stimulate aldosterone secretion by adrenal glomerulosa, but the <em>angiotensin</em> receptor subtypes involved and the effects of Ang IV and Ang (<em>1</em>-<em>7</em>) are not clear. In vitro, different <em>angiotensins</em> were added to rat adrenal glomerulosa, and aldosterone concentration in the medium was measured. Ang II-induced aldosterone release was blocked (30.3 ± <em>7</em>.<em>1</em>%) by an Ang II type 2 receptor (AT2R) antagonist, PD<em>1</em>233<em>1</em>9. Candesartan, an Ang II type <em>1</em> receptor (AT<em>1</em>R) antagonist, also blocked Ang II-induced aldosterone release (42.9 ± 4.8%). Coadministration of candesartan and PD<em>1</em>233<em>1</em>9 almost abolished the Ang II-induced aldosterone release. A selective AT2R agonist, CGP42<em>1</em><em>1</em>2, was used to confirm the effects of AT2R. CGP42<em>1</em><em>1</em>2 increased aldosterone secretion, which was almost completely inhibited by PD<em>1</em>233<em>1</em>9. In addition to Ang II, Ang III also induced aldosterone release, which was not blocked by candesartan. However, PD<em>1</em>233<em>1</em>9 blocked 22.4 ± <em>1</em>0.5% of the Ang III-induced aldosterone secretion. Ang IV and Ang (<em>1</em>-<em>7</em>) did not induce adrenal aldosterone secretion. In vivo, both Ang II and Ang III infusion increased plasma aldosterone concentration, but only Ang II elevated blood pressure. Ang IV and Ang (<em>1</em>-<em>7</em>) infusion did not affect blood pressure or aldosterone concentration. In conclusion, this report showed for the first time that AT2R partially mediates Ang III-induced aldosterone release, but not AT<em>1</em>R. Also, over 60% of Ang III-induced aldosterone release may be independent of both AT<em>1</em>R and AT2R. Ang III and AT2R signaling may have a role in the pathophysiology of aldosterone breakthrough.

The investigation of therapeutic actions of <em>angiotensin</em> type <em>1</em> (AT<em>1</em>) receptor antagonists and ACE inhibitors (ACEI) demonstrated complex interactions between the renin-<em>angiotensin</em> system (RAS) and the kallikrein-kinin system (KKS) in several experimental and clinical studies. They are evidenced by the fact that (<em>1</em>) ACE efficiently catabolizes kinins; (2) <em>angiotensin</em>-derivatives such as ANG-(<em>1</em>-<em>7</em>) exert kininlike effects; and (3) kallikrein probably serves as a prorenin-activating enzyme. (4) Several authors have demonstrated experimentally that the protective effects of ACEI are at least partly mediated by a direct potentiation of kinin receptor response on BK stimulation. (5) Furthermore, studies on AT<em>1</em> antagonists, which do not directly influence kinin degradation, and studies on <em>angiotensin</em>-receptor transgenic mice have revealed additional interactions between the RAS and the KKS. There is mounting evidence that an autocrine cascade including kinins, nitric oxide, prostaglandins, and cyclic GMP is involved in at least some of the <em>angiotensin</em> type 2 receptor effects. This review discusses multiple possibilities of cross-talks between the RAS and KKS in vascular and cardiac physiology and pathology after ACE inhibition and AT<em>1</em> receptor blockade.

Effects of <em>angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>), an AngII metabolite, on bone marrow-derived hematopoietic cells were studied. We identified Ang-(<em>1</em>-<em>7</em>) to stimulate proliferation of human CD34(+) and mononuclear cells in vitro. Under in vivo conditions, we monitored proliferation and differentiation of human cord blood mononuclear cells in NOD/SCID mice. Ang-(<em>1</em>-<em>7</em>) stimulated differentially human cells in bone marrow and accumulated them in the spleen. The number of HLA-I(+) and CD34(+) cells in the bone marrow was increased 42-fold and 600-fold, respectively. These results indicate a decisive impact of Ang-(<em>1</em>-<em>7</em>) on hematopoiesis and its promising therapeutic potential in diseases requiring progenitor stimulation.

We have previously demonstrated the participation of reactive oxygen species (ROS) in the positive inotropic effect of a physiological concentration of <em>Angiotensin</em> II (Ang II, <em>1</em> nM). The objective of the present work was to evaluate the role and source of ROS generation in the positive inotropic effect produced by an equipotent concentration of endothelin-<em>1</em> (ET-<em>1</em>, 0.4 nM). Isolated cat ventricular myocytes were used to measure sarcomere shortening with a video-camera, superoxide anion (()O(2)(-)) with chemiluminescence, and ROS production and intracellular pH (pH(i)) with epifluorescence. The ET-<em>1</em>-induced positive inotropic effect (40.4+/-3.<em>1</em>%, n=<em>1</em>0, p<0.05) was associated to an increase in ROS production (<em>1</em>05+/-29 fluorescence units above control, n=6, p<0.05). ET-<em>1</em> also induced an increase in ()O(2)(-) production that was inhibited by the NADPH oxidase blocker, apocynin, and by the blockers of mitochondrial ATP-sensitive K(+) channels (mK(ATP)), glibenclamide and 5 hydroxydecanoic acid. The ET-<em>1</em>-induced positive inotropic effect was inhibited by apocynin (0.3 mM; 6.3+/-6.6%, n=<em>1</em>3), glibenclamide (50 microM; 8.8+/-3.5%, n=6), 5 hydroxydecanoic acid (500 microM; <em>1</em>4.<em>1</em>+/-8.<em>1</em>, n=9), and by scavenging ROS with MPG (2 mM; 0.92+/-5.6%, n=8). ET-<em>1</em> enhanced proton efflux (J(H)) carried by the Na(+)/H(+) exchanger (NHE) after an acid load, effect that was blocked by MPG. Consistently, the ET-induced positive inotropic effect was also inhibited by the NHE selective blocker HOE642 (5 microM; 9.3<em>7</em>+/-6.0<em>7</em>%, n=<em>7</em>). The data show that the effect of a concentration of ET-<em>1</em> that induces an increase in contractility of about 40% is totally mediated by an intracellular pathway triggered by mitochondrial ROS formation and stimulation of the NHE.

<em>Angiotensin</em> (ANG) II exerts a negative modulation on insulin signal transduction that might be involved in the pathogenesis of hypertension and insulin resistance. ANG-(<em>1</em>-<em>7</em>), an endogenous heptapeptide hormone formed by cleavage of ANG I and ANG II, counteracts many actions of ANG II. In the current study, we have explored the role of ANG-(<em>1</em>-<em>7</em>) in the signaling crosstalk that exists between ANG II and insulin. We demonstrated that ANG-(<em>1</em>-<em>7</em>) stimulates the phosphorylation of Janus kinase 2 (JAK2) and insulin receptor substrate (IRS)-<em>1</em> in rat heart in vivo. This stimulating effect was blocked by administration of the selective ANG type <em>1</em> (AT(<em>1</em>)) receptor blocker losartan. In contrast to ANG II, ANG-(<em>1</em>-<em>7</em>) stimulated cardiac Akt phosphorylation, and this stimulation was blunted in presence of the receptor Mas antagonist A-<em>7</em><em>7</em>9 or the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. The specific JAK2 inhibitor AG-490 blocked ANG-(<em>1</em>-<em>7</em>)-induced JAK2 and IRS-<em>1</em> phosphorylation but had no effect on ANG-(<em>1</em>-<em>7</em>)-induced phosphorylation of Akt, indicating that activation of cardiac Akt by ANG-(<em>1</em>-<em>7</em>) appears not to involve the recruitment of JAK2 but proceeds through the receptor Mas and involves PI3K. Acute in vivo insulin-induced cardiac Akt phosphorylation was inhibited by ANG II. Interestingly, coadministration of insulin with an equimolar mixture of ANG II and ANG-(<em>1</em>-<em>7</em>) reverted this inhibitory effect. On the basis of our present results, we postulate that ANG-(<em>1</em>-<em>7</em>) could be a positive physiological contributor to the actions of insulin in heart and that the balance between ANG II and ANG-(<em>1</em>-<em>7</em>) could be relevant for the association among insulin resistance, hypertension, and cardiovascular disease.

To compare the benefit of <em>angiotensin</em>-converting enzyme inhibition and direct vasodilation on the prognosis of advanced heart failure, <em>1</em><em>1</em><em>7</em> patients evaluated for cardiac transplantation who had severe symptoms and abnormal hemodynamic status at rest were randomized to treatment with either captopril or hydralazine plus isosorbide dinitrate (Hy-C Trial). Comparable hemodynamic effects of the two regimens were sought by titrating vasodilator doses to match the hemodynamic status achieved with nitroprusside and diuretic agents, attempting to achieve a pulmonary capillary wedge pressure of <em>1</em>5 mm Hg and a systemic vascular resistance of <em>1</em>,200 dynes.s.cm-5. Treatment with the alternate vasodilator was started because of poor hemodynamic response or side effects (40% of patients in the captopril group and 22% in the hydralazine group). Adequate hemodynamic response in patients with a serum sodium level less than <em>1</em>35 mg/dl was more likely with hydralazine than with captopril (<em>7</em><em>1</em>% vs. 33%, p = 0.04). Isosorbide dinitrate was prescribed in 88% of the hydralazine-treated patients and 84% of the captopril-treated patients. The hemodynamic improvements from each regimen were equivalent. After 8 +/- <em>7</em> months of follow-up, the actuarial <em>1</em>-year survival rate was 8<em>1</em>% in the captopril-treated patients and 5<em>1</em>% in the hydralazine-treated patients (p = 0.05). The improved survival with captopril resulted from a lower rate of sudden death, which occurred in only 3 of 44 captopril-treated patients compared with <em>1</em><em>7</em> of 60 hydralazine-treated patients (p = 0.0<em>1</em>). In the subset of patients who continued treatment with the initial vasodilator, results were similar to those for the entire treatment group.(ABSTRACT TRUNCATED AT 250 WORDS)