<em>Angiotensin</em> II (ANG II) contributes to cardiac remodeling, hypertrophy, and left ventricular dysfunction. ANG II stimulation of the ANG type <em>1</em> receptor (AT(<em>1</em>)R) generates reactive oxygen species via NADPH oxidase, which facilitates this hypertrophy and remodeling. This investigation sought to determine whether cardiac oxidative stress and cellular remodeling could be attenuated by in vivo AT(<em>1</em>)R blockade (AT(<em>1</em>)B) (valsartan) or superoxide dismutase/catalase mimetic (tempol) treatment in a rodent model of chronically elevated tissue levels of ANG II, the transgenic (mRen2) 2<em>7</em> rat (Ren2). Ren2 rats overexpress the mouse renin transgene with resultant hypertension, insulin resistance, proteinuria, and cardiovascular damage. Young (6-<em>7</em> wk old) male Ren2 and age-matched Sprague-Dawley rats were treated with valsartan (30 mg/kg), tempol (<em>1</em> mmol/l), or placebo for 3 wk. Heart tissue NADPH oxidase (NOX) activity and immunohistochemical analysis of subunits NOX2, Rac<em>1</em>, and p22(phox), heart tissue malondialdehyde, and insulin-stimulated protein kinase B (Akt) activation were measured. Structural changes were assessed with cine MRI, transmission electron microscopy, and light microscopy. Increases in septal wall thickness and altered systolic function (cine MRI) were associated with perivascular fibrosis and increased mitochondria in Ren2 on light and transmission electron microscopy (P < 0.05). AT(<em>1</em>)B, but not tempol, reduced blood pressure (P < 0.05); significant improvements were seen with both AT(<em>1</em>)B and tempol on NOX activity, subunit expression, malondialdehyde, and insulin-mediated activation/phosphorylation of Akt (each P < 0.05). Collectively, these data suggest cardiac oxidative stress-induced structural and functional changes are driven, in part, by AT(<em>1</em>)R-mediated increases in NADPH oxidase activity.

OBJECTIVE

To evaluate the contribution of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] and prostaglandins to the acute and long-term antihypertensive actions of captopril in mild-to-moderate essential hypertensive patients.

METHODS

Blood pressure, cardiac rate and the plasma concentrations of <em>angiotensin</em> I (Ang I), <em>angiotensin</em> II (Ang II), Ang-(<em>1</em>-<em>7</em>), prostaglandin E2 and 6-keto prostaglandin F<em>1</em> alpha (the breakdown product of prostacyclin) were determined in the peripheral venous blood of 24 essential hypertensive subjects before and 3 h after administration of 50 mg captopril. Eleven of 24 patients completed a 6-month treatment period with captopril monotherapy (50 mg twice a day). The hemodynamic and hormonal response produced by a last 50 mg dose of captopril was determined once again in the <em>1</em><em>1</em> subjects who maintained blood pressure control with captopril monotherapy for 6 months.

RESULTS

The fall in blood pressure produced 3 h after drug intake was comparable for the first and the last 50 mg captopril dose. Although the first response to captopril increased plasma levels of Ang I only, the response to the last dose of the drug (6 months after) caused significantly higher levels of Ang I and Ang-(<em>1</em>-<em>7</em>). Neither acute nor chronic therapy with captopril had a significant effect on plasma concentrations of Ang II. Although plasma levels of prostaglandin E2 and 6-keto prostaglandin F<em>1</em> alpha were not modified by a first exposure to captopril, the concentrations of 6-keto prostaglandin F<em>1</em> alpha but not prostaglandin E2 rose significantly in subjects treated with the inhibitor for 6 months. A negative correlation was also demonstrated between diastolic blood pressure and plasma Ang-(<em>1</em>-<em>7</em>) levels in the <em>1</em><em>1</em> essential hypertensive subjects in whom blood pressure was controlled with captopril monotherapy.

CONCLUSIONS

Inhibition of <em>angiotensin</em> converting enzyme with captopril had a significant effect on blood pressure that was not directly accounted for by a suppression of plasma Ang II levels. Continuous therapy with captopril unmasked a contribution of Ang-(<em>1</em>-<em>7</em>) and prostacyclin to the antihypertensive actions of this drug.

The renin-<em>angiotensin</em> system (RAS) is one of the best-studied enzyme-neuropeptide systems in the brain and can serve as a model for the action of peptides on neuronal function in general. It is now well established that the brain has its own intrinsic RAS with all its components present in the central nervous system. The RAS generates a family of bioactive <em>angiotensin</em> peptides with variable biological and neurobiological activities. These include <em>angiotensin</em>-(<em>1</em>-8) [Ang II], <em>angiotensin</em>-(3-8) [Ang IV], and <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)]. These neuroactive forms of <em>angiotensin</em> act through specific receptors. Only Ang II acts through two different high-specific receptors, termed AT<em>1</em> and AT2. Neuronal AT<em>1</em> receptors mediate the stimulatory actions of Ang II on blood pressure, water and salt intake, and the secretion of vasopressin. In contrast, neuronal AT2 receptors have been implicated in the stimulation of apoptosis and as being antagonistic to AT<em>1</em> receptors. Among the many potential effects mediated by stimulation of AT2 are neuronal regeneration after injury and the inhibition of pathological growth. Ang-(<em>1</em>-<em>7</em>) mediates its antihypertensive effects by stimulating the synthesis and release of vasodilator prostaglandins and nitric oxide and by potentiating the hypotensive effects of bradykinin. New data concerning the roles of Ang IV and Ang-(<em>1</em>-<em>7</em>) in cognition also support the existence of complex site-specific interactions between multiple <em>angiotensins</em> and multiple receptors in the mediation of important central functions of the RAS. Thus, the RAS of the brain is involved not only in the regulation of blood pressure, but also in the modulation of multiple additional functions in the brain, including processes of sensory information, learning, and memory, and the regulation of emotional responses.

The aim of this study was to evaluate the <em>angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>) effects in isolated mouse hearts. The hearts of male C5<em>7</em>BL/6J and knockout mice for the Ang-(<em>1</em>-<em>7</em>) receptor Mas were perfused by the Langendorff method. After a basal period, the hearts were perfused for 20 minutes with Krebs-Ringer solution (KRS) alone (control) or KRS containing Ang-(<em>1</em>-<em>7</em>) (0.22 pmol/L), the Mas antagonist A-<em>7</em><em>7</em>9 (<em>1</em><em>1</em>5 nmol/L), the <em>angiotensin</em> type <em>1</em> receptor antagonist losartan (2.2 micromol/L), or the <em>angiotensin</em> type 2 receptor antagonist PD<em>1</em>233<em>1</em>9 (<em>1</em>30 nmol/L). To evaluate the involvement of Ang receptors, prostaglandins, and nitric oxide in the Ang-(<em>1</em>-<em>7</em>) effects, the hearts were perfused for 20 to 30 minutes with KRS containing either A-<em>7</em><em>7</em>9, losartan, PD<em>1</em>233<em>1</em>9, indomethacin, or NG-nitro-L-arginine methyl ester (L-NAME) alone or in association with subsequent Ang-(<em>1</em>-<em>7</em>) perfusion. In addition, hearts from Mas-knockout mice were perfused for 20 minutes with KRS containing Ang-(<em>1</em>-<em>7</em>) (0.22 pmol/L) and losartan. Ang-(<em>1</em>-<em>7</em>) alone did not change the perfusion pressure. Strikingly, in the presence of losartan, 0.22 pmol/L Ang-(<em>1</em>-<em>7</em>) induced a significant decrease in perfusion pressure, which was blocked by A-<em>7</em><em>7</em>9, indomethacin, and L-NAME. Furthermore, this effect was not observed in Mas-knockout mice. In contrast, in the presence of PD<em>1</em>233<em>1</em>9, Ang-(<em>1</em>-<em>7</em>) produced a significant increase in perfusion pressure. This change was not modified by the addition of A-<em>7</em><em>7</em>9. Losartan reduced but did not abolish this effect. Our results suggest that Ang-(<em>1</em>-<em>7</em>) produces complex vascular effects in isolated, perfused mouse hearts involving interaction of its receptor with <em>angiotensin</em> type <em>1</em>- and type 2-related mechanisms, leading to the release of prostaglandins and nitric oxide.

Vascular remodeling is central to the pathophysiology of hypertension and atherosclerosis. Recent evidence suggests that vasoconstrictive substances, such as <em>angiotensin</em> II (AII), may function as a vascular smooth muscle growth promoting substance. To explore the role of the counterregulatory hormone, atrial natriuretic polypeptide (ANP) in this process, we examined the effect of ANP (alpha-rat ANP [<em>1</em>-28]) on the growth characteristics of cultured rat aortic smooth muscle (RASM) cells. ANP (<em>1</em>0(-<em>7</em>) M) significantly suppressed the proliferative effect of <em>1</em>% and 5% serum as measured by 3H-thymidine incorporation and cell number, confirming ANP as an antimitogenic factor. In quiescent RASM cells, ANP (<em>1</em>0(-<em>7</em>), <em>1</em>0(-6) M) significantly suppressed the basal incorporations of 3H-uridine and leucine by 50 and 30%, respectively. ANP (<em>1</em>0(-<em>7</em>), <em>1</em>0(-6) M) also suppressed AII-induced RNA and protein syntheses (by 30-40%) with the concomitant reduction of the cell size. Furthermore, ANP also significantly attenuated the increase of 3H-uridine and leucine incorporations caused by transforming growth factor-beta (4 x <em>1</em>0(-<em>1</em><em>1</em>), 4 x <em>1</em>0(-<em>1</em>0) M), a potent hypertrophic factor. These results indicate that ANP possesses an antihypertrophic action on vascular smooth muscle cells. Down-regulation of protein kinase C by 24-h treatment with phorbol <em>1</em>2,<em>1</em>3-dibutyrate did not inhibit ANP-induced suppression on 3H-uridine incorporation. Based on the observation that ANP was more potent than a ring-deleted analogue of ANP on inhibiting 3H-uridine incorporation, we conclude that the ANP's inhibitory effect is primarily mediated via the activation of a guanylate cyclase-linked ANP receptor(s). Indeed 8-bromo cGMP mimicked the antihypertrophic action of ANP. Accordingly, we speculate that in addition to its vasorelaxant and natriuretic effects, the antihypertrophic action of ANP observed in the present study may serve as an additional compensatory mechanism of ANP in hypertension.

Obesity is increasing in prevalence and is strongly associated with metabolic and cardiovascular disorders. The renin-<em>angiotensin</em> system (RAS) has emerged as a key pathogenic mechanism for these disorders; <em>angiotensin</em> (Ang)-converting enzyme 2 (ACE2) negatively regulates RAS by metabolizing Ang II into Ang <em>1</em>-<em>7</em>. We studied the role of ACE2 in obesity-mediated cardiac dysfunction. ACE2 null (ACE2KO) and wild-type (WT) mice were fed a high-fat diet (HFD) or a control diet and studied at 6 months of age. Loss of ACE2 resulted in decreased weight gain but increased glucose intolerance, epicardial adipose tissue (EAT) inflammation, and polarization of macrophages into a proinflammatory phenotype in response to HFD. Similarly, human EAT in patients with obesity and heart failure displayed a proinflammatory macrophage phenotype. Exacerbated EAT inflammation in ACE2KO-HFD mice was associated with decreased myocardial adiponectin, decreased phosphorylation of AMPK, increased cardiac steatosis and lipotoxicity, and myocardial insulin resistance, which worsened heart function. Ang <em>1</em>-<em>7</em> (24 µg/kg/h) administered to ACE2KO-HFD mice resulted in ameliorated EAT inflammation and reduced cardiac steatosis and lipotoxicity, resulting in normalization of heart failure. In conclusion, ACE2 plays a novel role in heart disease associated with obesity wherein ACE2 negatively regulates obesity-induced EAT inflammation and cardiac insulin resistance.

Cardiac phenotypic modulation and remodeling appear to be involved in the pathophysiology of cardiac hypertrophy and heart failure. We undertook this study to examine whether <em>angiotensin</em> II (Ang II) in vivo, independent of blood pressure, contributes to cardiac phenotypic modulation and remodeling. A low dose (200 ng/kg per minute) of Ang II was continuously infused into rats by osmotic minipump for 24 hours or 3 or <em>7</em> days to examine the effects on the expression of cardiac phenotype-related or fibrosis-related genes. This Ang II dose caused a small and gradual increase in blood pressure over <em>7</em> days. Left ventricular mRNAs for skeletal alpha-actin, beta-myosin heavy chain, atrial natriuretic polypeptide, and fibronectin were already increased by 6.9-, <em>1</em>.8-, 4.8-, and <em>1</em>.5-fold, respectively, after 24 hours of Ang II infusion and by 6.9-, 3.3-, <em>7</em>.5-, and 2.5-fold, respectively, after 3 days, whereas ventricular alpha-myosin heavy chain and smooth muscle alpha-actin mRNAs were not significantly altered by Ang II infusion. Ventricular transforming growth factor-beta <em>1</em> and types I and III collagen mRNA levels did not increase at 24 hours and began to increase by <em>1</em>.4-, 2.8-, and 2.<em>1</em>-fold, respectively, at 3 days. An increase in left ventricular weight occurred 3 days after Ang II infusion. Treatment with TCV-<em>1</em><em>1</em>6 (3 mg/kg per day), a nonpeptide selective <em>angiotensin</em> type <em>1</em> receptor antagonist, completely inhibited the above-mentioned Ang II-induced increases in ventricular gene expressions and weight. Hydralazine (<em>1</em>0 mg/kg per day), which completely normalized blood pressure, did not block cardiac hypertrophy or increased cardiac gene expressions by Ang II.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

Previous concepts regarding the pathways involved in the generation of <em>angiotensin</em> II (Ang II) have been challenged by studies showing the existence of a peptide acting as an endogenous antagonist of Ang II. The discovery that <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] opposes the pressor, proliferative, profibrotic, and prothrombotic actions mediated by Ang II has contributed to the realization that the renin-<em>angiotensin</em> system is composed of two opposing arms: the pressor arm constituted by the enzyme <em>angiotensin</em>-converting enzyme (ACE), Ang II as the product, and the Ang II type <em>1</em> (AT<em>1</em>) receptor as the main protein mediating the biological actions of Ang II; the second arm is composed of the monocarboxypeptidase <em>angiotensin</em>-converting enzyme 2 (ACE2), Ang-(<em>1</em>-<em>7</em>) produced through hydrolysis of Ang II, and the Mas receptor as the protein conveying the vasodilator, antiproliferative, antifibrotic, and antithrombotic effects of Ang-(<em>1</em>-<em>7</em>).

RESULTS

Experimental and clinical studies demonstrate a role for the Ang-(<em>1</em>-<em>7</em>)/ACE2/Mas axis in the evolution of hypertension, the regulation of renal function, and the progression of renal disease including diabetic nephropathy. Additional evidence suggests that a reduction in the expression and activity of this vasodepressor component may be a critical factor in mediating the progression of cardiovascular disease.

CONCLUSIONS

Further research on the contribution of the Ang-(<em>1</em>-<em>7</em>)/ACE2/Mas axis to cardiovascular pathology will lead to the development of new pharmacological approaches resulting in the design of molecular or genetic means to increase the expression of ACE2, allow for increased tissue levels of Ang-(<em>1</em>-<em>7</em>), or both.

We investigated the processing enzymes involved in the formation of circulating <em>angiotensin</em>-(<em>1</em>-<em>7</em>) after intravenous administration of <em>angiotensin</em> I to conscious spontaneously hypertensive and Wistar-Kyoto rats. Immunoreactive products, including <em>angiotensin</em> I, <em>angiotensin</em> II, and <em>angiotensin</em>-(<em>1</em>-<em>7</em>), were measured in arterial blood by three specific radioimmunoassays. <em>Angiotensin</em> I infusion (2 nmol) induced a rapid increase in immunoreactive <em>angiotensin</em> II and <em>angiotensin</em>-(<em>1</em>-<em>7</em>). Pretreatment with the <em>angiotensin</em> converting enzyme inhibitor enalaprilat (2 mg/kg) eliminated <em>angiotensin</em> II formation and augmented circulating levels of <em>angiotensin</em> I and <em>angiotensin</em>-(<em>1</em>-<em>7</em>) in spontaneously hypertensive and Wistar-Kyoto rats. The elevated levels of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) in enalaprilat-treated rats were blocked by concurrent treatment with the neutral endopeptidase (EC 3.4.24.<em>1</em><em>1</em>) inhibitor SCH 39,3<em>7</em>0 (<em>1</em>5 mg/kg) in both strains. Administration of SCH 39,3<em>7</em>0 alone decreased <em>angiotensin</em>-(<em>1</em>-<em>7</em>) levels in spontaneously hypertensive rats, whereas <em>angiotensin</em> II levels increased in both strains (p less than 0.0<em>1</em>). Comparisons of the metabolism of <em>angiotensin</em> I in the two rat strains showed increased formation of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) in spontaneously hypertensive rats not given any of the enzyme inhibitors. In addition, levels of <em>angiotensin</em> I were higher after administration of SCH 39,3<em>7</em>0 in hypertensive rats. These novel findings reveal that neutral endopeptidase EC 3.4.24.<em>1</em><em>1</em> participates in the conversion of <em>angiotensin</em> I to <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and in the metabolism of <em>angiotensin</em> II in the circulation of both spontaneously hypertensive and Wistar-Kyoto rats. Our results suggest that neutral endopeptidase EC 3.4.24.<em>1</em><em>1</em> is a major enzymatic constituent of the circulating renin-<em>angiotensin</em> system.

OBJECTIVE

<em>Angiotensin</em> peptides play a central role in cardiovascular physiology and pathology. Among these peptides, <em>angiotensin</em> II (Ang II) has been investigated most intensively. However, further <em>angiotensin</em> peptides such as Ang <em>1</em>-<em>7</em>, Ang III, and Ang IV also contribute to vascular regulation, and may elicit additional, different, or even opposite effects to Ang II. Here, we describe a novel Ang II-related, strong vasoconstrictive substance in plasma from healthy humans and end-stage renal failure patients.

RESULTS

Chromatographic purification and structural analysis by matrix-assisted laser desorption/ionisation time-of-flight/time-of-flight (MALDI-TOF/TOF) revealed an <em>angiotensin</em> octapeptide with the sequence Ala-Arg-Val-Tyr-Ile-His-Pro-Phe, which differs from Ang II in Ala<em>1</em> instead of Asp<em>1</em>. Des[Asp<em>1</em>]-[Ala<em>1</em>]-Ang II, in the following named <em>Angiotensin</em> A (Ang A), is most likely generated enzymatically. In the presence of mononuclear leukocytes, Ang II is converted to Ang A by decarboxylation of Asp<em>1</em>. Ang A has the same affinity to the AT<em>1</em> receptor as Ang II, but a higher affinity to the AT2 receptor. In the isolated perfused rat kidney, Ang A revealed a smaller vasoconstrictive effect than Ang II, which was not modified in the presence of the AT2 receptor antagonist PD <em>1</em>233<em>1</em>9, suggesting a lower intrinsic activity at the AT<em>1</em> receptor. Ang II and Ang A concentrations in plasma of healthy subjects and end-stage renal failure patients were determined by matrix-assisted laser desorption/ionisation mass-analysis, because conventional enzyme immunoassay for Ang II quantification did not distinguish between Ang II and Ang A. In healthy subjects, Ang A concentrations were less than 20% of the Ang II concentrations, but the ratio Ang A/Ang II was higher in end-stage renal failure patients.

CONCLUSIONS

Ang A is a novel human strong vasoconstrictive angiotensin-derived peptide, most likely generated by enzymatic transformation through mononuclear leukocyte-derived aspartate decarboxylase. Plasma Ang A concentration is increased in end-stage renal failure. Because of its stronger agonism at the AT2 receptor, Ang A may modulate the harmful effects of Ang II.

The COVID-<em>1</em>9 pandemic is an emerging threat to global public health. While our current understanding of COVID-<em>1</em>9 pathogenesis is limited, a better understanding will help us develop efficacious treatment and prevention strategies for COVID-<em>1</em>9. One potential therapeutic target is <em>angiotensin</em> converting enzyme 2 (ACE2). ACE2 primarily catalyzes the conversion of <em>angiotensin</em> I (Ang I) to a nonapeptide <em>angiotensin</em> or the conversion of <em>angiotensin</em> II (Ang II) to <em>angiotensin</em> <em>1</em>-<em>7</em> (Ang <em>1</em>-<em>7</em>) and has direct effects on cardiac function and multiple organs via counter-regulation of the renin-<em>angiotensin</em> system (RAS). Significant to COVID-<em>1</em>9, ACE2 is postulated to serve as a major entry receptor for SARS-CoV-2 in human cells, as it does for SARS-CoV. Many infected individuals develop COVID-<em>1</em>9 with fever, cough, and shortness of breath that can progress to pneumonia. Disease progression promotes the activation of immune cells, platelets, and coagulation pathways that can lead to multiple organ failure and death. ACE2 is expressed by epithelial cells of the lungs at high level, a major target of the disease, as seen in post-mortem lung tissue of patients who died with COVID-<em>1</em>9, which reveals diffuse alveolar damage with cellular fibromyxoid exudates bilaterally. Comparatively, ACE2 is expressed at low level by vascular endothelial cells of the heart and kidney but may also be targeted by the virus in severe COVID-<em>1</em>9 cases. Interestingly, SARS-CoV-2 infection downregulates ACE2 expression, which may also play a critical pathogenic role in COVID-<em>1</em>9. Importantly, targeting ACE2/Ang <em>1</em>-<em>7</em> axis and blocking ACE2 interaction with the S protein of SARS-CoV-2 to curtail SARS-CoV-2 infection are becoming very attractive therapeutics potential for treatment and prevention of COVID-<em>1</em>9. Here, we will discuss the following subtopics: <em>1</em>) ACE2 as a receptor of SARS-CoV-2; 2) clinical and pathological features of COVID-<em>1</em>9; 3) role of ACE2 in the infection and pathogenesis of SARS; 4) potential pathogenic role of ACE2 in COVID-<em>1</em>9; 5) animal models for pathological studies and therapeutics; and 6) therapeutics development for COVID-<em>1</em>9.

<strong class="sub-title"> Keywords: </strong> ACE2; COVID-<em>1</em>9; and animal model; pathogenesis; spike protein.

We designed our studies to determine whether blood pressure is elevated in obese Zucker rats compared with lean control rats and to test the importance of the renin-<em>angiotensin</em> and adrenergic nervous systems in long-term blood pressure control in this genetic model of obesity. We monitored mean arterial pressure 24 hours per day using computerized methods in <em>1</em>3- to <em>1</em>4-week-old lean and obese Zucker rats maintained on a fixed, normal sodium intake (3.3 mmol/d). Mean arterial pressure (average of 5 days) was higher in obese (<em>1</em>00 +/- <em>1</em> mm Hg) than in lean (86 +/- <em>1</em>) rats. Although control plasma renin activity was lower in obese than in lean rats (3.66 +/- 0.<em>1</em>5 versus 5.48 +/- 0.<em>1</em><em>1</em> ng <em>angiotensin</em> I/mL per hour), blood pressure sensitivity to exogenous <em>angiotensin</em> II was greater in obese than in lean rats. Blockade of endogenous <em>angiotensin</em> II receptors with losartan (<em>1</em>0 mg/kg per day) for <em>7</em> days also caused a greater decrease in blood pressure in obese (36 +/- 2 mm Hg, n = 6) than in lean (25 +/- <em>1</em>, n = 5) rats. However, combined alpha- and beta-adrenergic blockade with terazosin (<em>1</em>0 mg/kg per day) and propranolol (<em>1</em>0 mg/kg per day), respectively, for 8 days caused only modest decreases in blood pressure in obese (9 +/- 3 mm Hg, n = 8) and lean (4 +/- 2, n = 6) rats, despite effective alpha- and beta-adrenergic blockade. These results suggest that increased arterial pressure in obese Zucker rats depends in part on <em>angiotensin</em> II. However, additional mechanisms may also contribute to increased blood pressure in obese Zucker rats.

Studies have demonstrated that local <em>angiotensin</em> II (Ang II) generation is enhanced in repairing kidney and that ACE inhibition or AT(<em>1</em>) receptor blockade attenuates renal fibrosis. The localization of ACE and Ang II receptors and their relationship to collagen synthesis in the injured kidney, however, remain uncertain. Using a rat model of renal injury with subsequent fibrosis created with chronic elevations in circulating aldosterone (ALDO), we examined the distribution and binding density of ACE and Ang II receptors in repairing kidneys, as well as their anatomic relationship to transforming growth factor-beta<em>1</em> (TGF-beta<em>1</em>) mRNA, type I collagen mRNA, collagen accumulation, and myofibroblasts. Two groups of animals (n=<em>7</em> in each group) were studied: (<em>1</em>) normal rats served as controls, and (2) uninephrectomized rats received ALDO (0.<em>7</em>5 microg/h SC) and <em>1</em>% NaCl in drinking water for 6 weeks. Compared with control rats, in ALDO-treated rats we found (<em>1</em>) significantly (P<0.0<em>1</em>) increased blood pressure, reduced plasma renin activity, and increased plasma creatinine levels, (2) diffuse fibrosis in both renal cortex and medulla, (3) abundant myofibroblasts at these sites of fibrosis, (4) significantly increased (P<0.0<em>1</em>) binding density of ACE and Ang II receptors (60% AT(<em>1</em>), 40% AT(2)) at the sites of fibrosis, and (5) markedly increased (P<0.0<em>1</em>) expression of TGF-beta<em>1</em> and type I collagen mRNAs at these same sites. Thus, in this rat model of renal repair, the enhanced expression of ACE, Ang II receptors, and TGF-beta<em>1</em> is associated with renal fibrosis. Ang II generated at the sites of repair appears to have autocrine/paracrine functions in the regulation of renal fibrous tissue formation alone or through its stimulation of TGF-beta<em>1</em> synthesis.

<em>Angiotensin</em> II (Ang II) has been implicated in the pathogenesis of the vascular injury associated with hypertension and diabetes mellitus. Increased vascular permeability is an important early manifestation of endothelial dysfunction and the pathogenesis of atherosclerosis. How Ang II contributes to endothelial dysfunction and promotes an increase in vascular permeability is unknown but is classically attributed to its pressor actions. We demonstrate that human vascular smooth muscle cells express abundant mRNA for vascular permeability/endothelial growth factor. Vascular permeability factor is a 34- to 42-kD glycoprotein that markedly increases vascular endothelial permeability and is a potent endothelial mitogen. Ang II potently induced a concentration-dependent (maximal, <em>1</em>0(-<em>7</em>) mol/L) and time-dependent increase in vascular permeability factor mRNA expression by human vascular smooth muscle cells that was maximal after 3 hours and diminished by 24 hours. Ang II-induced vascular permeability factor mRNA expression by human vascular smooth muscle cells was inhibited by the specific Ang II receptor antagonist losartan (DuP <em>7</em>53), confirming that this is an Ang II receptor subtype <em>1</em>-mediated event. These results describe a new action of Ang II on human vascular smooth muscle, notably the induction of vascular permeability factor mRNA expression. The wide spectrum and potent activity of vascular permeability factor suggest a novel mechanism whereby Ang II could locally and directly influence the permeability, growth, and function of the vascular endothelium independent of changes in hemodynamics.

Compelling evidence has emerged pointing to the interaction of oxidative stress and renal interstitial inflammation and their mutual contribution to the pathogenesis of hypertension in experimental animals. Renal interstitial inflammation in spontaneously hypertensive rats (SHR) is accompanied by and largely due to activation of redox-sensitive, proinflammatory nuclear transcription factor-kappaB (NF-kappaB). Therefore, the present study was designed to test the hypothesis that long-term inhibition of NF-kappaB, beginning early in the course of the disease, may attenuate renal interstitial inflammation and hypertension in SHR. To this end, we administered the reputed NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) (<em>1</em>00 mg/kg daily intraperitoneally) to SHR from <em>7</em> to 25 weeks of age and compared the results with vehicle-treated SHR. Vehicle-treated and PDTC-treated Wistar Kyoto (WKY) rats served as controls. The untreated SHR exhibited a significant rise in arterial pressure; increased NF-kappaB activation, elevated intercellular adhesion molecule (ICAM)-<em>1</em> and in situ mRNA macrophage chemoattractant molecule-<em>1</em> (MCP-<em>1</em>) expressions; and interstitial accumulation of lymphocytes, macrophages, and <em>angiotensin</em>-II-positive cells. PDTC administration prevented the rise in blood pressure, and normalized renal cortical NF-kappaB activity as well as ICAM-<em>1</em> and MCP-<em>1</em> expressions. This was accompanied by a significant reduction in infiltration of immune cells, <em>angiotensin</em> II-expressing cells, and renal tissue malondialdehyde content to values that matched those found in the control WKY rats. Results suggest that NF-kappaB-driven intrarenal inflammatory reactivity play a major role in the pathogenesis of hypertension in the SHR.

BACKGROUND

<em>Angiotensin</em>-converting enzyme (ACE) 2 (ACE2) is expressed mainly in the heart and kidney and forms <em>angiotensin</em>-<em>1</em>-<em>7</em> from <em>angiotensin</em> II. ACE2 might act in a counterregulatory manner to ACE. There is little information about renal ACE and ACE2 expression in human diabetic nephropathy.

METHODS

Cross-sectional study.

METHODS

Kidney tissue from 20 patients with type 2 diabetes and overt nephropathy and 20 healthy kidney donors.

METHODS

Diabetes status.

METHODS

Renal expression of ACE and ACE2 assessed by means of immunohistochemistry and in situ hybridization. Correlation between ACE and ACE2 expression and levels of various biochemical parameters.

RESULTS

Decreased ACE2 and increased ACE expression in both the tubulointerstitium and glomeruli resulted in a significant (P < 0.00<em>1</em>) increase in ACE/ACE2 ratio in patients with diabetes with overt nephropathy compared with controls, although ACE messenger RNA in the tubulointerstitium did not significantly increase. ACE/ACE2 ratio correlated positively with values for mean blood pressure, fasting blood glucose, serum creatinine, proteinuria, and hemoglobin A(<em>1</em>c) and inversely with estimated glomerular filtration rate (P < 0.00<em>1</em>).

CONCLUSIONS

Inclusion of small number of human renal biopsy specimens with structural distortion of cortical tissue.

CONCLUSIONS

The high ACE/ACE2 ratio in kidneys of patients with type 2 diabetes with overt nephropathy may contribute to renal injury.

In this study, the effects of inhibition of aldosterone on regression of existing hypertension-related glomerulosclerosis were investigated. Adult male Sprague Dawley rats (220 to 250 g) underwent 5/6 nephrectomy (Nx). Severity of glomerulosclerosis was assessed by renal biopsy 8 wk later, and rats were divided into four groups with equal biopsy sclerosis and then randomized by group to 4-wk treatments as follows: Control with no further treatment (CONT; n = 6); spironolactone (SP) alone (200 mg/kg per d, by gavage, n = 6); or SP combined with nonspecific triple antihypertensive drugs (TRX; reserpine, hydralazine, and hydrochlorothiazide in drinking water; SP+TRX, n = <em>7</em>) or with <em>angiotensin</em> type <em>1</em> receptor antagonist (AT<em>1</em>RA; losartan in drinking water; SP+AT<em>1</em>RA, n = 8). When the rats were killed <em>1</em>2 wk after Nx, autopsy glomerulosclerosis index (SI; 0 to 4+ scale) was compared with biopsy SI in the same rats. Systolic BP was increased at 8 wk after Nx and continued to increase at <em>1</em>2 wk after Nx in the CONT and SP groups but not in SP+TRX- or SP+AT<em>1</em>RA-treated rats. Serum creatinine at <em>1</em>2 wk was significantly decreased in all SP-treated groups versus CONT. CONT rats had on average a <em>1</em>5<em>7</em>% increase in SI from biopsy to killing at <em>1</em>2 wk, compared with only 84% increase in SP rats, with regression of SI in some rats. The effects on glomerulosclerosis by SP were further enhanced (when systolic BP was controlled by TRX or by AT<em>1</em>RA). It is concluded that inhibition of aldosterone by SP not only slows development of glomerulosclerosis but also induces regression in some rats of existing glomerulosclerosis.

BACKGROUND

Aldosterone has been suggested to play a role in the initiation and progression of diabetic nephropathy. Currently recommended treatment with angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers [renin-angiotensin system (RAS) blockade] does not suppress circulating aldosterone sufficiently. We therefore aimed to evaluate the short-term effect of aldosterone antagonism with spironolactone on albuminuria and blood pressure in diabetic nephropathy.

METHODS

Twenty Caucasian type 1 diabetic patients with persistent macroalbuminuria despite antihypertensive treatment, including RAS blockade, completed this double-masked, randomized cross-over trial. Patients were treated in random order with spironolactone 25 mg once daily and matched placebo for two months, respectively, on top of usual antihypertensive treatment. After each treatment period albuminuria, 24-hour blood pressure, and glomerular filtration rate (GFR) were determined.

RESULTS

Spironolactone on top of usual antihypertensive treatment induced a 30% (95% CI 17 to 41) reduction in albuminuria from [geometric mean (95% CI)] 831 (624 to 1106) mg/24-hour on placebo treatment (P < 0.001), and a reduction in fractional albumin clearance of 35% (20 to 46, P < 0.001). Twenty-four-hour blood pressure showed an insignificant reduction of [mean reduction (95% CI)] 8 (-1 to 17)/3 (-0.2 to 7) mm Hg (P < 0.10). There was an insignificant reversible reduction in GFR during treatment with spironolactone. On spironolactone treatment, one patient was excluded due to hyperkalemia (plasma potassium 5.7 mmol/L) and one due to orthostatic dizziness. Otherwise treatment was well tolerated.

CONCLUSIONS

Our results suggest that spironolactone treatment on top of recommended antihypertensive treatment reduces blood pressure and may offer additional renoprotection in type 1 diabetic patients with diabetic nephropathy.

Observations that <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [ANG-(<em>1</em>-<em>7</em>)] may oppose the vasoconstrictor actions of <em>angiotensin</em> II (ANG II) prompted an investigation of the effects of the heptapeptide on the maintenance of elevated blood pressure in spontaneously hypertensive rats (SHR). ANG-(<em>1</em>-<em>7</em>) (24 micrograms.kg-<em>1</em>.h-<em>1</em>) was infused into the jugular vein of <em>1</em>3-wk-old SHR (n = 64), Wistar-Kyoto (WKY, n = 50), and Sprague-Dawley (SD, n = <em>1</em>8) rats for 2 wk, with the use of osmotic minipumps. Blood pressure, fluid and electrolyte balance, plasma vasopressin, and urinary excretion of prostaglandin E2 and 6-ketoprostaglandin F<em>1</em> alpha (6-keto-PGF<em>1</em> alpha) were measured at days 2, <em>7</em>, and <em>1</em>2 of the infusion. In SHR, ANG-(<em>1</em>-<em>7</em>) caused a sustained and significant reduction in plasma vasopressin concentration that was associated with an increase in urinary prostaglandin E2 and 6-keto-PGF<em>1</em> alpha excretion at day 2 after the commencement of the infusion. These changes were accompanied by diuresis and natriuresis during the first 3 days of infusion in SHR but not in WKY or SD rats. Direct measurements of arterial pressure confirmed the lowering effect of ANG-(<em>1</em>-<em>7</em>) on systolic pressure of SHR on day 2 of treatment with a restoration of the pressure by days <em>7</em> and <em>1</em>2. These findings, along with our previous demonstration that ANG-(<em>1</em>-<em>7</em>) is an active depressor peptide in the intact animal, suggest that ANG-(<em>1</em>-<em>7</em>) may play a significant role as a vasodepressor system opposing the hemodynamic actions of ANG II in this genetic form of experimental hypertension.

<em>Angiotensin</em> converting enzyme (ACE) inhibitors augment circulating levels of the vasodilator peptide <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] in man and animals. Increased concentrations of the peptide may contribute to the antihypertensive effects associated with ACE inhibitors. The rise in Ang-(<em>1</em>-<em>7</em>) following ACE inhibition may result from increased production of the peptide or inhibition of the metabolism of Ang-(<em>1</em>-<em>7</em>)-similar to that observed for bradykinin. To address the latter possibility, we determined whether Ang-(<em>1</em>-<em>7</em>) is a substrate for ACE in vitro. In a pulmonary membrane preparation, the ACE inhibitor lisinopril attenuated the metabolism of low concentrations of <em>1</em>25I-Ang-(<em>1</em>-<em>7</em>). The primary product of <em>1</em>25I-Ang-(<em>1</em>-<em>7</em>) metabolism was identified as Ang-(<em>1</em>-5). Using affinity-purified ACE from canine lung, HPLC separation and amino acid analysis revealed that ACE functioned as a dipeptidyl carboxypeptidase cleaving Ang-(<em>1</em>-<em>7</em>) to the pentapeptide Ang-(<em>1</em>-5). The ACE inhibitors lisinopril and enalaprilat (<em>1</em> micromol/L), as well as the chelating agents EDTA, o-phenanthroline, and DTT (0.<em>1</em>-<em>1</em> mmol/L) abolished the generation of Ang-(<em>1</em>-5) and did not yield other metabolic products. Ang-(<em>1</em>-5) was not further hydrolyzed by ACE. Kinetic analysis of the hydrolysis of Ang-(<em>1</em>-<em>7</em>) by ACE revealed a substrate affinity of 0.8<em>1</em> micromol/L and maximal velocity of 0.65 micromols min(-<em>1</em>) mg(-<em>1</em>). The calculated turnover constant for the peptide was <em>1</em>.8 sec(-<em>1</em>) with a catalytic efficiency (Kcat/Km) of 2200 sec(-<em>1</em>) mmol/L(-<em>1</em>). These findings suggest that increased levels of Ang-(<em>1</em>-<em>7</em>) following ACE inhibition may be due, in part, to decreased metabolism of the peptide.

Blockade of <em>angiotensin</em> II (Ang II) function during 8 days of oral therapy with lisinopril (20 mg/kg) and losartan (<em>1</em>0 mg/kg) normalized the arterial pressure (<em>1</em><em>1</em>2+/-3/<em>7</em>0+/-3 mm Hg) and raised the plasma concentrations of the vasodilator peptide <em>angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] of 2<em>1</em> male spontaneously hypertensive rats (SHR). Treated animals were then given a <em>1</em>5-minute infusion of either mouse immunoglobulin G<em>1</em> or a specific monoclonal Ang-(<em>1</em>-<em>7</em>) antibody while their blood pressure and heart rate were recorded continuously in the awake state. The concentrations of Ang II and Ang-(<em>1</em>-<em>7</em>) in arterial blood were determined by radioimmunoassay. Infusion of the Ang-(<em>1</em>-<em>7</em>) antibody caused significant elevations in mean arterial pressure that were sustained for the duration of the infusion and were accompanied by transient bradycardia. Although the hemodynamic effects produced by infusion of the Ang-(<em>1</em>-<em>7</em>) antibody had no effect on plasma levels of Ang II, they caused a twofold rise in the plasma concentrations of Ang-(<em>1</em>-<em>7</em>). A pressor response of similar magnitude and characteristics was obtained in a separate group of SHR treated with the combination of lisinopril and losartan for 8 days during an infusion of [Sar<em>1</em>-Thr8]Ang II. The pressor response induced by the administration of this competitive, non-subtype-selective Ang II receptor blocker was not modified by pretreatment of the rats with an <em>angiotensin</em> type-2 (AT2) receptor blocker (PD<em>1</em>233<em>1</em>9). Plasma concentrations of Ang II and Ang-(<em>1</em>-<em>7</em>) were not changed by the administration of [Sar<em>1</em>-Thr8]Ang II either in the absence or in the presence of PD<em>1</em>233<em>1</em>9 pretreatment. These results are the first to indicate an important contribution of Ang-(<em>1</em>-<em>7</em>) in mediating the vasodilator effects caused by combined inhibition of <em>angiotensin</em>-converting enzyme and AT<em>1</em> receptors. The comparable results obtained by administration of [Sar<em>1</em>-Thr8]Ang II suggest that the vasodepressor effects of Ang-(<em>1</em>-<em>7</em>) during the combined treatment is modulated by a non-AT<em>1</em>/AT2 <em>angiotensin</em> subtype receptor.

<em>1</em>. This paper reviews studies carried out in our laboratory in which we have used the c-fos functional mapping method, in combination with other methods, to determine the functional organization of central baroreceptor pathways as they operate in the conscious rabbit. 2. First, we showed that periods of induced hypertension or hypotension each result in a specific and reproducible pattern of activation of neurons in the brainstem and forebrain. In particular, hypotension (but not hypertension) results in the activation of catecholamine neurons in the medulla and pons and vasopressin-synthesizing neurons in the hypothalamus. 3. The activation of medullary cell groups in response to induced hypertension or hypotension in the conscious rabbit is almost entirely dependent on inputs from arterial baroreceptors, while the activation of hypothalamic vasopressin-synthesising neurons in response to hypotension is largely dependent on baroreceptors, although an increase in circulating <em>angiotensin</em> also appears to contribute. 4. Discrete groups of neurons in the rostral ventrolateral medulla (RVLM) and A5 area in the pons are the major groups of spinally projecting neurons activated by baroreceptor unloading. In contrast, spinally projecting neurons in the paraventricular nucleus in the hypothalamus appear to be largely unaffected by baroreceptor signals. 5. Direct afferent inputs to RVLM neurons in response to increases or decreases in arterial pressure originate primarily from other medullary nuclei, particularly neurons located in the caudal and intermediate levels of the ventrolateral medulla (CVLM and IVLM), as well as in the nucleus tractus solitarius (NTS). 6. There is also a direct projection from barosensory neurons in the NTS to the CVLM/IVLM region, which is activated by baroreceptor inputs. <em>7</em>. Collectively, the results of our studies in conscious animals indicate that baroreceptor signals reach all levels of the brain. With regard to the baroreceptor reflex control of sympathetic activity, our studies are consistent with previous studies in anesthetized animals, but in addition reveal other previously unrecognized pathways that also contribute to this reflex regulation.

The renin-<em>angiotensin</em> system (RAS) constitutes a key hormonal system in the physiological regulation of blood pressure through peripheral and central mechanisms. Indeed, dysregulation of the RAS is considered a major factor in the development of cardiovascular pathologies, and pharmacological blockade of this system by the inhibition of <em>angiotensin</em>-converting enzyme (ACE) or antagonism of the <em>angiotensin</em> type <em>1</em> receptor (AT<em>1</em>R) offers an effective therapeutic regimen. The RAS is now defined as a system composed of different <em>angiotensin</em> peptides with diverse biological actions mediated by distinct receptor subtypes. The classic RAS comprises the ACE-ANG II-AT<em>1</em>R axis that promotes vasoconstriction; water intake; sodium retention; and increased oxidative stress, fibrosis, cellular growth, and inflammation. In contrast, the nonclassical RAS composed primarily of the ANG II/ANG III-AT2R and the ACE2-ANG-(<em>1</em>-<em>7</em>)-AT<em>7</em>R pathways generally opposes the actions of a stimulated ANG II-AT<em>1</em>R axis. In lieu of the complex and multifunctional aspects of this system, as well as increased concerns on the reproducibility among laboratories, a critical assessment is provided on the current biochemical approaches to characterize and define the various components that ultimately reflect the status of the RAS.

The discovery of <em>angiotensin</em>-converting enzyme 2 (ACE2) in 2000 is an important event in the renin-<em>angiotensin</em> system (RAS) story. This enzyme, an homolog of ACE, hydrolyzes <em>angiotensin</em> (Ang) I to produce Ang-(<em>1</em>-9), which is subsequently converted into Ang-(<em>1</em>-<em>7</em>) by a neutral endopeptidase and ACE. ACE2 releases Ang-(<em>1</em>-<em>7</em>) more efficiently than its catalysis of Ang-(<em>1</em>-9) by cleavage of Pro(<em>7</em>)-Phe(8) bound in Ang II. Thus, the major biologically active product of ACE2 is Ang-(<em>1</em>-<em>7</em>), which is considered to be a beneficial peptide of the RAS cascade in the cardiovascular system. This enzyme has 42% identity with the catalytic domain of ACE, is present in most cardiovascular-relevant tissues, and is an ectoenzyme as ACE. Despite these similarities, ACE2 is distinct from ACE. Since it is a monocarboxypeptidase, it has only <em>1</em> catalytic site and is insensitive to ACE inhibitors. As a result, ACE2 is a central enzyme in balancing vasoconstrictor and proliferative actions of Ang II with vasodilatory and antiproliferative effects of Ang-(<em>1</em>-<em>7</em>). In this review, we will summarize the role of ACE2 in the cardiovascular system and discuss the importance of ACE2-Ang-(<em>1</em>-<em>7</em>) axis in the control of normal cardiovascular physiology and ACE2 as a potential target in the development of novel therapeutic agents for cardiovascular diseases.