BACKGROUND

The purpose of this study was to determine whether endothelium-dependent relaxation competes with alpha <em>1</em>- and alpha 2-adrenergic coronary microvascular constriction in the beating heart in vivo.

RESULTS

Coronary microvascular diameters were measured using stroboscopic epi-illumination and intravital microscopy during fluorescein microangiography in open-chested dogs (n = 20). Both alpha <em>1</em>- and alpha 2-adrenergic receptors were selectively activated by intracoronary infusions of norepinephrine (0.05 and 0.2 microgram.kg-<em>1</em> x min-<em>1</em>) in the presence of the alpha 2-adrenergic antagonist rauwolscine (0.2 mg/kg) or the alpha <em>1</em>-adrenergic antagonist prazosin (0.75 mg/kg) during beta-adrenergic blockade (<em>1</em> mg/kg propranolol). Microvascular diameters during selective alpha-adrenergic receptor activation were measured under baseline conditions and after inhibition of endogenous nitric oxide synthesis by an analogue of L-arginine, either NG-nitro-L-arginine (L-NA, 30 mg/kg) or NG-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg). Under baseline conditions, alpha <em>1</em>-adrenergic activation constricted small arteries (vessels with diameters between <em>1</em>00 and 300 microns) (4 +/- <em>1</em>% and 5 +/- <em>1</em>% decrease in diameter for the low and high doses of norepinephrine, respectively, both p < 0.05) but did not change the diameter of arterioles (vessels with diameters < <em>1</em>00 microns). In contrast, alpha 2-adrenergic activation by the lower but not the higher dose of norepinephrine induced constriction of arterioles (6 +/- 2% and 3 +/- 4% decrease in diameter, p < 0.05 and NS, respectively) but not small arteries. Inhibition of nitric oxide synthase activity by either L-NA or L-NAME produced constriction of small coronary arteries (9 +/- 2% decrease in diameter, p < 0.0<em>1</em>) and arterioles (6 +/- <em>1</em>% decrease in diameter, p < 0.05). The dilatation of small arteries and arterioles by acetylcholine (0.05 microgram-<em>1</em> x kg-<em>1</em> x min-<em>1</em> intracoronary infusion; <em>1</em>0 +/- <em>1</em>% increase in diameter under baseline conditions, p < 0.05) was abolished by either analogue. Both alpha <em>1</em>- and alpha 2-adrenergic coronary microvascular constriction were markedly potentiated after L-NA or L-NAME. alpha <em>1</em>-Adrenergic constriction was unmasked in arterioles (7 +/- 3% and <em>1</em>0 +/- 4% decrease in diameter, p < 0.05), although it was not significantly increased in small arteries. Conversely, alpha 2-adrenergic constriction was unmasked in small arteries (8 +/- <em>1</em>% and 6 +/- 2% decrease in diameter, both p < 0.05) and potentiated in arterioles (<em>1</em>2 +/- <em>1</em>% and 8 +/- 4% decrease in diameter, both p < 0.05). After L-NA or L-NAME, microvessels retained the ability to dilate to sodium nitroprusside (0.<em>1</em> microgram.kg-<em>1</em> x min-<em>1</em> intracoronary infusion; <em>1</em>0 +/- 2% increase in diameter, p < 0.05). alpha-Adrenergic constriction was not accentuated by increased tone alone, since it was either attenuated or converted to dilatation during a similar degree of preconstriction by the endothelium-independent vasoconstrictor angiotensin II (p < 0.05 for both alpha <em>1</em>- and alpha 2-adrenergic activation).

CONCLUSIONS

These data confirm that alpha-adrenergic receptors are widespread in the coronary microcirculation, with the baseline functional responses to alpha <em>1</em>-adrenergic activation predominating in small arteries and those to alpha 2-adrenergic activation predominating in arterioles. Furthermore, coronary microvascular constriction caused by both alpha <em>1</em>- and alpha 2-adrenergic receptor activation is significantly modulated by endothelium-dependent relaxation, being markedly potentiated by inhibition of nitric oxide synthase activity. The data imply that alpha-adrenergic activation will assume considerable importance as a determinant of coronary microvascular resistance in pathophysiological situations associated with coronary endothelial impairment.

<em>1</em>. Rats in normal fluid balance drank water <em>1</em>-2 hr after complete ligation of the inferior vena cava either above or below the renal veins. At the same time there was a fall in urine flow and excretion of electrolyte, especially after caval ligation above the renal veins, so that the animals ended the initial 6 hr period in positive fluid balance.2. Caval ligation was relatively ineffective as a stimulus to drinking after bilateral nephrectomy, but was effective in rats made anuric by ureteric ligation.3. Rats subjected to caval ligation and offered a choice between water and <em>1</em>.8% saline (w/v) drank water, despite the increasing hypotonicity of the body fluids thereby resulting.4. During the secondary polydipsia, which generally occurred on about the third day after caval ligation as renal function was recovering, there was an increased preference for <em>1</em>.8% saline.5. Constriction of the aorta above the renal arteries, or constriction of both renal arteries, also caused drinking, oliguria and the development of positive fluid balance.6. Constriction of the aorta below the renal arteries, or after nephrectomy, was ineffective as a stimulus to drinking.<em>7</em>. Saline extracts of renal cortex caused rats in normal water balance to drink. Activity was destroyed by boiling the extract for <em>1</em>0 min. Renal medullary and hepatic extracts were without effect on drinking.8. It proved impossible to separate dipsogenic and pressor activities of renal extracts during the different stages of fractionation which lead to the production of renin; disappearance of one activity was invariably accompanied by disappearance of the other.9. Dipsogenic and pressor actions were greater in nephrectomized rats than in normal rats.<em>1</em>0. Both extractable dipsogenic factor and extractable pressor activity were reduced by treating the rat with DOCA and saline for several weeks beforehand.<em>1</em><em>1</em>. The renal dipsogen therefore has similar properties to renin. It may prove to be identical with renin, particularly in view of the fact that <em>angiotensin</em> also stimulates drinking.<em>1</em>2. Adrenalectomy did not affect drinking induced by renin or by caval ligation.<em>1</em>3. It is concluded that the renin <em>angiotensin</em> system may play a role in the genesis of the thirst which follows certain extracellular stimuli.

Previous studies clearly demonstrated acute actions of <em>angiotensin</em> II (ANG II) at one of the central circumventricular organs, the subfornical organ (SFO), but studies demonstrating a role for the SFO in the chronic actions of ANG II remain uncertain. The purpose of this study was to examine the role of the SFO in the chronic hypertensive phase of ANG II-induced hypertension. We hypothesized that the SFO is necessary for the full hypertensive response observed during the chronic phase of ANG II-induced hypertension. To test this hypothesis, male Sprague-Dawley rats were subjected to sham operation (sham rats) or electrolytic lesion of the SFO (SFOx rats). After <em>1</em> wk, the rats were instrumented with venous catheters and radiotelemetric transducers for intravenous administration of ANG II and measurement of blood pressure and heart rate, respectively. Rats were then allowed <em>1</em> wk for recovery. After 3 days of saline control infusion (<em>7</em> ml of 0.9% NaCl/day), sham and SFOx rats were infused with ANG II at <em>1</em>0 ng.kg(-<em>1</em>).min(-<em>1</em>) i.v. for <em>1</em>0 consecutive days and then allowed to recover for 3 days. A 0.4% NaCl diet and distilled water were provided ad libitum. At day 5 of ANG II infusion, mean arterial pressure increased <em>1</em><em>1</em>.<em>7</em> +/- 3.0 mmHg in sham rats (n = 9) but increased only 3.<em>7</em> +/- <em>1</em>.4 mmHg in SFOx rats (n = 9). This trend continued through day <em>1</em>0 of ANG II treatment. These results support the hypothesis that the SFO is necessary for the full hypertensive response to chronic ANG II administration.

Studies conducted over the last decade demonstrated variable therapeutic efficacy of <em>angiotensin</em> converting enzyme (ACE) inhibitor on the progression of glomerular diseases, including IgA nephropathy. In this study, among patients with biopsy-proven IgA nephropathy, 53 patients in whom creatinine clearance had been monitored over 5 yr were recruited for study. These patients were classified into two groups according to whether or not renal function had declined as determined by the slope of creatinine clearance against time: group <em>1</em> had stable renal function; group 2 had declining renal function (average: -6.<em>7</em> +/- <em>1</em>.3 ml/min/yr). 2<em>1</em> of 53 patients were treated with ACE inhibitor and followed for 48 wk. Gene polymorphism consisting of insertion (I) or deletion (D) of a 28<em>7</em>-bp DNA fragment (presumed to be a silencer element) of the ACE gene was determined by PCR. 46 age-matched individuals without history of proteinuria were analyzed as controls. The DD genotype was significantly more frequent in group 2 (43%) than in controls (<em>7</em>%) or group <em>1</em> patients with stable renal function (<em>1</em>6%). 48 wk after ACE inhibitor administration, proteinuria significantly decreased in patients with DD genotype but not in those with ID or II genotypes. The results indicate that deletion polymorphism in the ACE gene, particularly the homozygote DD, is a risk factor for progression to chronic renal failure in IgA nephropathy. Moreover, this deletion polymorphism predicts the therapeutic efficacy of ACE inhibition on proteinuria and, potentially, on progressive deterioration of renal function.

BACKGROUND

Excessive stimulation of Gq protein-coupled receptors by cognate vasoconstrictor agonists induces a variety of cardiovascular processes, including hypertension and hypertrophy. Here, we report that matrix metalloproteinase-<em>7</em> (MMP-<em>7</em>) and a disintegrin and metalloproteinase-12 (ADAM-12) form a novel signaling axis in these processes.

RESULTS

In functional studies, we targeted MMP-<em>7</em> in rodent models of acute, long-term, and spontaneous hypertension by 3 complementary approaches: (1) Pharmacological inhibition of activity, (2) expression knockdown (by antisense oligodeoxynucleotides and RNA interference), and (3) gene knockout. We observed that induction of acute hypertension by vasoconstrictors (ie, catecholamines, angiotensin II, and the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester) required the posttranscriptional activation of vascular MMP-<em>7</em>. In spontaneously hypertensive rats, knockdown of MMP-<em>7</em> (by RNA interference) resulted in attenuation of hypertension and stopped development of cardiac hypertrophy. Quantitative reverse-transcription polymerase chain reaction studies in mouse models of MMP-<em>7</em> knockdown (by RNA interference) and gene knockout revealed that MMP-<em>7</em> controlled the transcription of ADAM-12, the major metalloproteinase implicated in cardiac hypertrophy. In mice with angiotensin II-induced hypertension and cardiac hypertrophy, myocardial ADAM-12 and downstream hypertrophy marker genes were overexpressed. Knockdown of MMP-<em>7</em> attenuated hypertension, inhibited ADAM-12 overexpression, and prevented cardiac hypertrophy.

CONCLUSIONS

Agonist signaling of both hypertension and hypertrophy depends on posttranscriptional and transcriptional mechanisms that involve MMP-<em>7</em>, which is transcriptionally connected with ADAM-12. Approaches targeting this novel MMP-<em>7</em>/ADAM-12 signaling axis could have generic therapeutic potential in hypertensive disorders caused by multiple or unknown agonists.

BACKGROUND

Oxidative stress and RAAS play an important role in the occurrence of anthracyclines-induced cardiotoxicity. Telmisartan, an <em>angiotensin</em> II type <em>1</em> receptor blocker, inhibits activation of superoxide sources and induces anti-inflammatory effects.

METHODS

The possible role of telmisartan in preventing myocardial damage induced by epirubicin (EPI) was investigated. Forty-nine patients free from cardiovascular diseases affected by a variety of solid cancers were examined. Eligible patients were randomized to receive telmisartan (40 mg/d; TEL, n = 25) or placebo (PLA, n = 24) starting <em>1</em> week before chemotherapy. Patients were studied by means of echocardiography, tissue Doppler, and strain and strain rate (SR) imaging. We also measured plasma levels of inflammatory and oxidative stress markers. All parameters were assessed at baseline and 7 days after every new EPI dose of <em>1</em>00 mg/m(2).

RESULTS

An impairment of the SR peak was observed at the EPI dose of 200 mg/m(2), with no significant differences between TEL and PLA (<em>1</em>.4<em>1</em> +/- 0.3<em>1</em> vs <em>1</em>.59 +/- 0.36/s). At growing cumulative doses of EPI, SR normalized only in TEL, showing a significant difference in comparison to PLA at EPI doses of 300 mg/m(2) (<em>1</em>.69 +/- 0.42 vs <em>1</em>.34 +/- 0.<em>1</em>8/s, P < .00<em>1</em>) and 400 mg/m(2) (<em>1</em>.74 +/- 0.27 vs <em>1</em>.38 +/- 0.24/s, P < .00<em>1</em>). Moreover, a significant increase in reactive oxygen species and interleukin-6 was found in PLA; but these remained unchanged in TEL.

CONCLUSIONS

We confirmed that EPI-induced cardiotoxicity is primarily related to the inactivation of the cardiac antioxidant defenses. In addition, we showed that telmisartan can reduce EPI-induced radical species, antagonize the inflammation, and reverse the early myocardial impairment.

<em>Angiotensin</em>(<em>1</em>-<em>7</em>) had a compound effect on blood pressure of pithed Sprague-Dawley rats. The initial phase of the response consisted of an increase in MAP of short duration and independent of injected dose, followed by a decline of arterial pressure to values below baseline. Both the magnitude (range: -4 +/- <em>1</em> to -<em>1</em>3 +/- <em>1</em> mmHg) and the duration (range: 83 +/- <em>1</em>3 to 255 +/- <em>1</em><em>7</em> s) of the depressor response correlated with the dose of peptide. Indomethacin (5 mg/kg) eliminated the depressor component. Only [Sar<em>1</em>,Thr8]Ang II inhibited the effect of Ang(<em>1</em>-<em>7</em>) completely. We conclude that <em>angiotensin</em>(<em>1</em>-<em>7</em>) possesses myotonic actions that are in part related to release of vasodilator prostaglandins through an <em>angiotensin</em> receptor other than AT<em>1</em> or AT2.

Previously we demonstrated that central administration of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] into rats elicits significant cerebroprotection against ischemic stroke elicited by endothelin-<em>1</em> induced middle cerebral artery occlusion. Ang-(<em>1</em>-<em>7</em>), acting via its receptor Mas, reduced cerebral infarct size, and rats exhibited improved performance on neurological exams. These beneficial actions of Ang-(<em>1</em>-<em>7</em>) were not due to inhibition of the effects of endothelin-<em>1</em> on cerebral vasoconstriction or effects on cerebral blood flow, and so we considered other potential mechanisms. Here we investigated the possibility that the Ang-(<em>1</em>-<em>7</em>)-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-(<em>1</em>-<em>7</em>) treatment attenuates the increased expression of mRNAs for inducible nitric oxide synthase (iNOS), several pro-inflammatory cytokines and cluster of differentiation molecule <em>1</em><em>1</em>b (microglial marker) within the cerebral cortex following endothelin-<em>1</em> 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-(<em>1</em>-<em>7</em>) acting via Mas. Collectively these findings demonstrate an anti-inflammatory action of Ang-(<em>1</em>-<em>7</em>) 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 primary role of cigarette smoking in the development of coronary heart disease is to cause damage to the vascular endothelium, leading to endothelial cell dysfunction and initiating the pathogenesis of coronary atherosclerosis. We studied the response of human coronary artery endothelial cells to nicotine exposure by examining the expression of a panel of genes encoding molecules that have been shown to be involved in atherogenesis. Treatment of primary human coronary artery endothelial cells with nicotine for 24 h at concentrations (<em>1</em>0(-5) and <em>1</em>0(-<em>7</em>) M) similar to those in the blood of smokers resulted in increased mRNA levels of endothelial nitric oxide synthase, <em>angiotensin</em>-I converting enzyme, tissue-type plasminogen activator, plasminogen activator inhibitor-<em>1</em>, von Willebrand factor, and vascular cell adhesion molecule-<em>1</em>. No change was detected in the expression levels of the genes encoding basic fibroblast growth factor, endothelin-<em>1</em>, endothelial leukocyte adhesion molecule-<em>1</em> and matrix metalloproteinase-2 under these conditions. These data indicate that nicotine alters the expression of a number of endothelial genes whose products play major roles in regulating the vascular tone and thrombogenicity, making a contribution to the understanding of the effects of cigarette smoking on the development of coronary atherosclerosis.

Albuminuria and hypertension are predictors of poor renal and cardiovascular outcome in diabetic patients. This study tested whether dual blockade of the renin-<em>angiotensin</em> system (RAS) with both an <em>angiotensin</em>-converting enzyme (ACE) inhibitor (ACE-I) and an <em>Angiotensin</em>-II receptor blocker (ARB) is superior to either drug alone in type I diabetic patients with diabetic nephropathy (DN). A randomized double-blind crossover trial was performed with 8-wk treatment with placebo, 20 mg of benazepril once daily, 80 mg of valsartan once daily, and the combination of 20 mg of benazepril and 80 mg of valsartan. Twenty type I diabetic patients with DN were included. At the end of each treatment period, albuminuria, 24-h BP, and GFR were measured. Eighteen patients completed the study. Placebo values were: albuminuria [mean (95% CI)], <em>7</em>0<em>1</em> (490 to <em>1</em>002) mg/24 h; BP [mean (SEM)], <em>1</em>44 (4)/<em>7</em>9 (2) mmHg, and GFR [mean (SEM)], 82 (<em>7</em>) ml/min per <em>1</em>.<em>7</em>3 m(2). Treatment with benazepril, valsartan, or dual blockade significantly reduced albuminuria and BP compared with placebo. Benazepril and valsartan were equally effective. Dual blockade induced an additional reduction in albuminuria of 43 % (29 to 54 %) compared with any type of monotherapy, and a reduction in systolic BP of 6 (0 to <em>1</em>3) mmHg and <em>7</em> (<em>1</em> to <em>1</em>4) mmHg (versus benazepril and valsartan, respectively) and a reduction of <em>7</em> (4 to <em>1</em>0) mmHg diastolic compared with both monotherapies. GFR was reversibly reduced on dual blockade compared with monotherapy and placebo. All treatments were safe and well tolerated. In conclusion, dual blockade of the RAS may offer additional renal and cardiovascular protection in type I diabetic patients with DN.

Emerging evidences indicate that diminished activity of the vasoprotective axis of the renin-<em>angiotensin</em> system, constituting <em>angiotensin</em>-converting enzyme 2 (ACE2) and its enzymatic product, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] contribute to the pathogenesis of pulmonary hypertension (PH). However, long-term repetitive delivery of ACE2 or Ang-(<em>1</em>-<em>7</em>) would require enhanced protein stability and ease of administration to improve patient compliance. Chloroplast expression of therapeutic proteins enables their bioencapsulation within plant cells to protect against gastric enzymatic degradation and facilitates long-term storage at room temperature. Besides, fusion to a transmucosal carrier helps effective systemic absorption from the intestine on oral delivery. We hypothesized that bioencapsulating ACE2 or Ang-(<em>1</em>-<em>7</em>) fused to the cholera nontoxin B subunit would enable development of an oral delivery system that is effective in treating PH. PH was induced in male Sprague Dawley rats by monocrotaline administration. Subset of animals was simultaneously treated with bioencapsulaed ACE2 or Ang-(<em>1</em>-<em>7</em>) (prevention protocol). In a separate set of experiments, drug treatment was initiated after 2 weeks of PH induction (reversal protocol). Oral feeding of rats with bioencapsulated ACE2 or Ang-(<em>1</em>-<em>7</em>) prevented the development of monocrotaline-induced PH and improved associated cardiopulmonary pathophysiology. Furthermore, in the reversal protocol, oral ACE2 or Ang-(<em>1</em>-<em>7</em>) treatment significantly arrested disease progression, along with improvement in right heart function, and decrease in pulmonary vessel wall thickness. In addition, a combination therapy with ACE2 and Ang-(<em>1</em>-<em>7</em>) augmented the beneficial effects against monocrotaline-induced lung injury. Our study provides proof-of-concept for a novel low-cost oral ACE2 or Ang-(<em>1</em>-<em>7</em>) delivery system using transplastomic technology for pulmonary disease therapeutics.

Renin <em>angiotensin</em> system (RAS) activation has a significant influence on renal disease progression. The classical <em>angiotensin</em>-converting enzyme (ACE)-<em>angiotensin</em> II (Ang II)-Ang II type <em>1</em> (AT<em>1</em>) axis is considered to control the effects of RAS activation on renal disease. However, since its discovery in 2000 ACE2 has also been demonstrated to have a significant impact on the RAS. The synthesis and catabolism of Ang II are regulated via a complex series of interactions, which involve ACE and ACE2. In the kidneys, ACE2 is expressed in the proximal tubules and less strongly in the glomeruli. The synthesis of inactive Ang <em>1</em>-9 from Ang I and the catabolism of Ang II to produce Ang <em>1</em>-<em>7</em> are the main functions of ACE2. Ang <em>1</em>-<em>7</em> reduces vasoconstriction, water retention, salt intake, cell proliferation, and reactive oxygen stress, and also has a renoprotective effect. Thus, in the non-classical RAS the ACE2-Ang <em>1</em>-<em>7</em>-Mas axis counteracts the ACE-Ang II-AT<em>1</em> axis. This review examines recent human and animal studies about renal ACE and ACE2.

OBJECTIVE

The aim of this study was to test the hypothesis that angiotensin (Ang)-converting enzyme-2 (ACE2) overexpression may inhibit myocardial collagen accumulation and improve left ventricular (LV) remodeling and function in diabetic cardiomyopathy.

BACKGROUND

Hyperglycemia activates the renin-Ang system, which promotes the accumulation of extracellular matrix and progression of cardiac remodeling and dysfunction.

METHODS

Ninety male Wistar rats were divided randomly into treatment (n = 80) and control (n = 10) groups. Diabetes was induced in the treatment group by a single intraperitoneal injection of streptozotocin. Twelve weeks after streptozotocin injection, rats in the treatment group were further divided into adenovirus-ACE2, adenovirus-enhanced green fluorescent protein, losartan, and mock groups (n = 20 each). LV volume; LV systolic and diastolic function; extent of myocardial fibrosis; protein expression levels of ACE2, Ang-converting enzyme, and Ang-(1-7); and matrix metalloproteinase-2 activity were evaluated. Cardiac myocyte and fibroblast culture was performed to assess Ang-II and collagen protein expression before and after ACE2 gene transfection.

RESULTS

Four weeks after ACE2 gene transfer, the adenovirus-ACE2 group showed increased ACE2 expression, matrix metalloproteinase-2 activity, and LV ejection fractions and decreased LV volumes, myocardial fibrosis, and ACE, Ang-II, and collagen expression in comparison with the adenovirus-enhanced green fluorescent protein and control groups. ACE2 was superior to losartan in improving LV remodeling and function and reducing collagen expression. The putative mechanisms may involve a shift in balance toward an inhibited fibroblast-myocyte cross-talk for collagen and transforming growth factor-beta production and enhanced collagen degradation by matrix metalloproteinase-2.

CONCLUSIONS

ACE2 inhibits myocardial collagen accumulation and improves LV remodeling and function in a rat model of diabetic cardiomyopathy. Thus, ACE2 provides a promising approach to the treatment of patients with diabetic cardiomyopathy.

ACE2 (<em>angiotensin</em>-converting enzyme 2) counterbalances the actions of ACE (<em>angiotensin</em>-converting enzyme) by metabolizing its catalytic product, the vasoactive and fibrogenic peptide AngII (<em>angiotensin</em> II), into Ang-(<em>1</em>-<em>7</em>) [<em>angiotensin</em>-(<em>1</em>-<em>7</em>)]. Enhanced ACE2 expression may be protective in diabetes, cardiovascular disease and cancer. However, relatively little is known about the specific physiological factors regulating ACE2 expression. In the present paper, we show, by Western blotting and qPCR (quantitative real-time PCR), that ACE2 expression is increased under conditions of cell stress, including hypoxic conditions, IL (interleukin)-<em>1</em>β treatment and treatment with the AMP mimic AICAR (5-amino-4-imidazolecarboxamide riboside). The NAD+-dependent deacetylase SIRT<em>1</em> (silent information regulator T<em>1</em>) was found to be up-regulated after AICAR treatment but, conversely, was down-regulated after IL-<em>1</em>β treatment. ChIP analysis demonstrated that SIRT<em>1</em> bound to the ACE2 promoter and that binding was increased after AICAR treatment, but decreased after IL-<em>1</em>β treatment. Inhibition of SIRT<em>1</em> activity ablated the AICAR-induced increase in ACE2. In conclusion, we have established that the expression of the ACE2 transcript is controlled by the activity of SIRT<em>1</em> under conditions of energy stress.

The renin-<em>angiotensin</em>-aldosterone system (RAAS) is a pivotal regulator of physiological homeostasis and diseases of the cardiovascular system. Recently, new factors have been discovered, such as <em>angiotensin</em>-converting enzyme 2 (ACE2), <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and Mas. This newly defined ACE2-<em>angiotensin</em>-(<em>1</em>-<em>7</em>)-Mas axis was shown to have a critical role in the vasculature and in the heart, exerting mainly protective effects. One important mechanism of the classic and the new RAAS regulate vascular function is through the regulation of redox signaling. <em>Angiotensin</em> II is a classic prooxidant peptide that increases superoxide production through the activation of NAD(P)H oxidases. This review summarizes the current knowledge about the ACE2-<em>angiotensin</em>-(<em>1</em>-<em>7</em>)-Mas axis and redox signaling in the context of cardiovascular regulation and disease. By interacting with its receptor Mas, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) induces the release of nitric oxide from endothelial cells and thereby counteracts the effects of <em>angiotensin</em> II. ACE2 converts <em>angiotensin</em> II to <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and, thus, is a pivotal regulator of the local effects of the RAAS on the vessel wall. Taken together, the ACE2-<em>angiotensin</em>-(<em>1</em>-<em>7</em>)-Mas axis emerges as a novel therapeutic target in the context of cardiovascular and metabolic diseases.

<em>Angiotensin</em> converting enzyme 2 (ACE2) is a negative regulator of the renin-<em>angiotensin</em> system (RAS), catalyzing the conversion of <em>Angiotensin</em> II to <em>Angiotensin</em> <em>1</em>-<em>7</em>. Apelin is a second catalytic substrate for ACE2 and functions as an inotropic and cardioprotective peptide. While an antagonistic relationship between the RAS and apelin has been proposed, such functional interplay remains elusive. Here we found that ACE2 was downregulated in apelin-deficient mice. Pharmacological or genetic inhibition of <em>angiotensin</em> II type <em>1</em> receptor (AT<em>1</em>R) rescued the impaired contractility and hypertrophy of apelin mutant mice, which was accompanied by restored ACE2 levels. Importantly, treatment with <em>angiotensin</em> <em>1</em>-<em>7</em> rescued hypertrophy and heart dysfunctions of apelin-knockout mice. Moreover, apelin, via activation of its receptor, APJ, increased ACE2 promoter activity in vitro and upregulated ACE2 expression in failing hearts in vivo. Apelin treatment also increased cardiac contractility and ACE2 levels in AT<em>1</em>R-deficient mice. These data demonstrate that ACE2 couples the RAS to the apelin system, adding a conceptual framework for the apelin-ACE2-<em>angiotensin</em> <em>1</em>-<em>7</em> axis as a therapeutic target for cardiovascular diseases.

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) represents an anti-atherosclerotic principle. NO bioavailability is decreased in atherosclerosis due to increased NO inactivation by reactive oxygen species and reduced NO synthesis. Various types of vascular pathophysiology are associated with oxidative stress, with NADPH oxidases as the major source of reactive oxygen species. These inactivate NO. Also, oxidative stress is likely to be the main cause for oxidation of the essential NOS cofactor, tetrahydrobiopterin (BH(4)). A lack of BH(4) leads to eNOS uncoupling (i.e., uncoupling of oxygen reduction from NO synthesis in eNOS). Based on these pathomechanisms, the therapeutic potential of a number of compounds is discussed in this review: (<em>1</em>) NO donors; (2) L-arginine; (3) folic acid; (4) BH(4) and its precursor sepiapterin; (5) compounds that upregulate eNOS and concomitantly maintain eNOS activity (e.g. midostaurin, betulinic acid, ursolic acid, AVE9488 and AVE3085); (6) compounds that enhance the de novo synthesis of BH(4) by stimulating expression or activity of GTP cyclohydrolase I; and (<em>7</em>) 3-hydroxy-3-methylglutaryl-coenzyme A inhibitors (statins) and drugs interrupting the renin-<em>angiotensin</em>-aldosterone system. Statins, <em>angiotensin</em> II type <em>1</em> receptor blockers, <em>angiotensin</em>-converting enzyme (ACE) inhibitors, the aldosterone antagonist eplerenone and the renin inhibitor aliskiren enhance NO bioactivity and reduce atherosclerosis progression through multiple mechanisms.

After the advent of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the outbreak of coronavirus disease 20<em>1</em>9 (COVID-<em>1</em>9) commenced across the world. Understanding the Immunopathogenesis of COVID-<em>1</em>9 is essential for interrupting viral infectivity and preventing aberrant immune responses before a vaccine can be developed. In this review, we provide the latest insights into the roles of <em>angiotensin</em>-converting enzyme II (ACE2) and Ang II receptor-<em>1</em> (AT<em>1</em>-R) in this disease. Novel therapeutic strategies, including recombinant ACE2, ACE inhibitors, AT<em>1</em>-R blockers, and Ang <em>1</em>-<em>7</em> peptides, may prevent or reduce viruses-induced pulmonary, cardiac, and renal injuries. However, more studies are needed to clarify the efficacy of these therapeutics. Furthermore, considering the common role of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in AT<em>1</em>-R expressed on peripheral tissues and cytokine receptors on the surface of immune cells, potential targeting of this pathway using JAK inhibitors (JAKinibs) is suggested as a promising approach in patients with COVID-<em>1</em>9 who are admitted to hospitals. In addition to antiviral therapy, potential ACE2- and AT<em>1</em>-R-inhibiting strategies, and other supportive care, we suggest other potential JAKinibs and novel anti-inflammatory combination therapies that affect the JAK-STAT pathway in patients with COVID-<em>1</em>9. Since the combination of MTX and baricitinib leads to outstanding clinical outcomes, the addition of baricitinib to MTX might be a potential strategy.

Curcumin, an anti-inflammatory, antioxidant, was evaluated for its ability to suppress bleomycin (BLM)-induced pulmonary fibrosis in rats. A single intratracheal instillation of BLM (0.<em>7</em>5 U <em>1</em>00(-<em>1</em>) g, sacrificed 3, 5, <em>7</em>, <em>1</em>4 and 28 days post-BLM) resulted in significant increases in total cell numbers, total protein, and <em>angiotensin</em>-converting enzyme (ACE), and alkaline phosphatase (AKP) activities in bronchoalveolar lavage fluid. Animals with fibrosis had a significant increase in lung hydroxyproline content. Alveolar macrophages from BLM-administered rats elaborated significant increases in tumour necrosis factor (TNF)-alpha release, and superoxide and nitric oxide production in culture medium. Interestingly, oral administration of curcumin (300 mg kg(-<em>1</em>) <em>1</em>0 days before and daily thereafter throughout the experimental time period) inhibited BLM-induced increases in total cell counts and biomarkers of inflammatory responses in BALF. In addition, curcumin significantly reduced the total lung hydroxyproline in BLM rats. Furthermore, curcumin remarkably suppressed the BLM-induced alveolar macrophage production of TNF-alpha, superoxide and nitric oxide. These findings suggest curcumin as a potent anti-inflammatory and anti-fibrotic agent against BLM-induced pulmonary fibrosis in rats.

We investigated the effect of receptor blockade induced by an <em>angiotensin</em> II type-<em>1</em> receptor antagonist (AT(<em>1</em>)-RB) on glial and vascular changes in oxygen-induced retinopathy (OIR), a model of retinopathy of prematurity (ROP). OIR was induced in Sprague-Dawley rats by exposure to 80% oxygen from postnatal (P) days 0-<em>1</em><em>1</em>, followed by <em>7</em> days in room air. Control animals were in room air for the entire duration. One cohort of OIR and control pups received the AT(<em>1</em>)-RB valsartan (40 mg/kg/day intraperitoneal) from P<em>1</em><em>1</em> to P<em>1</em>8. The vascular response was examined immunocytochemically using retinal wholemounts and vertical sections labeled with endothelial (Isolectin-B4) and pericyte (NG2, desmin) markers. Glial cell changes were assessed by measuring cell numbers and immunoreactivity (S<em>1</em>00beta, connexin-26, and glial fibrillary acidic protein). OIR resulted in extensive intravitreal neovascularization and under-development of the outer vascular plexus. Pericyte numbers were not significantly affected in OIR, although pericyte-endothelial (desmin-IB4) interactions were impaired. Peripheral astrocyte degeneration occurred between P<em>1</em><em>1</em> and P<em>1</em>3 with prominent Müller cell reactivity at P<em>1</em>8. Valsartan imparted a protective effect on glia and blood vessels in OIR. At P<em>1</em>8, valsartan-treated OIR retinae showed significantly greater astrocyte survival, improved revascularization of the retina, and reduced preretinal neovascularization and Müller cell reactivity. This study identifies a glio-vascular protective effect with AT(<em>1</em>)-RB in OIR.

<em>Angiotensin</em> II (Ang II) promotes vascular disease through several mechanisms including by producing oxidative stress and endothelial dysfunction. Although multiple potential sources of reactive oxygen species exist, the relative importance of each is unclear, particularly in individual vascular beds. In these experiments, we examined the role of NADPH oxidase (Nox<em>1</em> and Nox2) in Ang II-induced endothelial dysfunction in the cerebral circulation. Treatment with Ang II (<em>1</em>.4 mg·kg(-<em>1</em>)·day(-<em>1</em>) for <em>7</em> days), but not vehicle, increased blood pressure in all groups. In wild-type (WT; C5<em>7</em>Bl/6) mice, Ang II reduced dilation of the basilar artery to the endothelium-dependent agonist acetylcholine compared with vehicle but had no effect on responses in Nox2-deficient (Nox2(-/y)) mice. Ang II impaired responses to acetylcholine in Nox<em>1</em> WT (Nox<em>1</em>(+/y)) and caused a small reduction in responses to acetylcholine in Nox<em>1</em>-deficient (Nox<em>1</em>(-/y)) mice. Ang II did not impair responses to the endothelium-independent agonists nitroprusside or papaverine in either group. In WT mice, Ang II increased basal and phorbol-dibutyrate-stimulated superoxide production in the cerebrovasculature, and these increases were abolished in Nox2(-/y) mice. Overall, these data suggest that Nox2 plays a relatively prominent role in mediating Ang II-induced oxidative stress and cerebral endothelial dysfunction, with a minor role for Nox<em>1</em>.

Despite the success of renin-<em>angiotensin</em> system (RAS) blockade by <em>angiotensin</em>-converting enzyme (ACE) inhibitors and <em>angiotensin</em> II type <em>1</em> receptor (AT_<em>1</em>R) blockers, current therapies for hypertension and related cardiovascular diseases are still inadequate. Identification of additional components of the RAS and associated vasoactive pathways, as well as new structural and functional insights into established targets, have led to novel therapeutic approaches with the potential to provide improved cardiovascular protection and better blood pressure control and/or reduced adverse side effects. The simultaneous modulation of several neurohumoral mediators in key interconnected blood pressure-regulating pathways has been an attractive approach to improve treatment efficacy, and several novel approaches involve combination therapy or dual-acting agents. In addition, increased understanding of the complexity of the RAS has led to novel approaches aimed at upregulating the ACE2/<em>angiotensin</em>-(<em>1</em>-<em>7</em>)/Mas axis to counter-regulate the harmful effects of the ACE/<em>angiotensin</em> II/<em>angiotensin</em> III/AT_<em>1</em>R axis. These advances have opened new avenues for the development of novel drugs targeting the RAS to better treat hypertension and heart failure. Here we focus on new therapies in preclinical and early clinical stages of development, including novel small molecule inhibitors and receptor agonists/antagonists, less conventional strategies such as gene therapy to suppress <em>angiotensin</em>ogen at the RNA level, recombinant ACE2 protein, and novel bispecific designer peptides.

<em>Angiotensin</em> (Ang) II plays an important role in post-myocardial infarction (MI) cardiac remodeling. The Ang II type <em>1</em> (AT(<em>1</em>)) receptor which mediates most Ang II effects is upregulated on non-myocytes in the post-MI heart. We have shown that pro-inflammatory cytokines increase AT(<em>1</em>) receptor density on cardiac fibroblasts through a mechanism involving NF-kappaB activation. This study examines the in vitro kinetics of tumor necrosis factor-alpha (TNF-alpha) and interleukin-<em>1</em>beta (IL-<em>1</em>beta) induced AT(<em>1</em>) receptor upregulation in neonatal rat cardiac fibroblasts and assesses temporal and spatial associations between the appearance of these agents and increased AT(<em>1</em>) receptor density post-MI. The results show that IL-<em>1</em>beta more rapidly induces AT(<em>1</em>) receptor upregulation than does TNF-alpha, an effect that can be mimicked by a NF-kappaB-dependent luciferase reporter gene. Moreover, the effects of these pro-inflammatory cytokines are additive. Using immunohistochemistry in the post-MI rat heart we found strong temporal and spatial correlations between TNF-alpha, IL-<em>1</em>beta and AT(<em>1</em>) receptor proteins in the peri-infarction (PI) zone in fibroblasts and macrophages. Labeling intensity for the cytokines and the AT(<em>1</em>) receptor increased from <em>1</em> to <em>7</em> days post-MI in the PI zone in conjunction with replacement scar formation. This labeling persisted in non-myocytes bordering the scar for up to 83 days post-MI. These findings suggest that IL-<em>1</em>beta and TNF-alpha act coordinately to increase AT(<em>1</em>) receptor density on non-myocytes in the post-MI heart and that this effect may contribute to extracellular matrix remodeling and fibrosis.

The hypothesis that O(2)(.-) enhances <em>angiotensin</em> II (AngII)-induced vasoconstriction and impairs acetylcholine-induced vasodilation of afferent arterioles (Aff) in AngII-induced hypertension was investigated. Rabbits (n = 6 per group) received <em>1</em>2 to <em>1</em>4 d of 0.<em>1</em>54 M NaCl (Sham), subpressor AngII (60 ng/kg per min; AngII 60) or slow pressor AngII (200 ng/kg per min; AngII 200). Individual Aff were perfused in vitro at 60 mmHg. AngII 200 increased mean arterial pressure (mean +/- SD; <em>1</em>03 +/- 9 versus <em>7</em>3 +/- 6 mmHg; P < 0.0<em>1</em>), plasma lipid peroxides (2.6 +/- 0.3 versus 2.0 +/- 0.3 nM; P < 0.05), renal cortical NADPH- and NADH-dependent O(2)(.-) generation, and Aff mRNA for p22(phox) 5-fold (P < 0.00<em>1</em>) but decreased that for AT(<em>1</em>)-receptor 2.4-fold (P < 0.0<em>1</em>). AngII 60 increased only NADH-dependent O(2)(.-) generation by renal cortex. Aff from AngII 200 rabbits had diminished acetylcholine relaxations (+50 +/- 4 versus +85 +/- 6%; P < 0.00<em>1</em>), but these became similar in the presence of nitro-L-arginine (<em>1</em>0(-4) M). Aff from AngII 60 and AngII 200 rabbits had unchanged norepinephrine contractions (<em>1</em>0(-<em>7</em>) M) but significantly (P < 0.05) enhanced AngII contractions (<em>1</em>0(-8) M: Sham -52 +/- 5 versus AngII 60 to <em>7</em><em>7</em> +/- 5 versus AngII 200 to <em>1</em><em>1</em>0 +/- <em>1</em>0%). The superoxide dismutase mimetic tempol (<em>1</em>0(-4) M) moderated the AngII responses of Aff from AngII 200 rabbits to levels of AngII 60 rabbits (-64 +/- <em>7</em>%). The AngII slow pressor response enhances renal cortical O(2)(.-) and p22(phox) expression. Increased O(2)(.-) generation in Aff mediates an impaired nitric oxide synthase-dependent endothelium-derived relaxing factor response and paradoxically enhances contractions to AngII despite downregulation of the mRNA for AT(<em>1</em>) receptors. A subpressor dose of AngII enhances Aff responses to AngII independent of O(2)(.-).