In the past few years, the classical concept of the renin-<em>angiotensin</em> system (RAS) has experienced substantial conceptual changes. The identification of: the renin/prorenin receptor; the <em>angiotensin</em>-converting enzyme homologue, ACE2, as an <em>angiotensin</em> peptide-processing enzyme and a virus receptor for severe acute respiratory syndrome, the Mas as a receptor for <em>angiotensin</em> (<em>1</em>-<em>7</em>) [Ang(<em>1</em>-<em>7</em>)], and the possibility of signaling through ACE have contributed to switch our understanding of the RAS from the classical limited-proteolysis linear cascade to a cascade with multiple mediators, multiple receptors and multifunctional enzymes. With regard to Ang(<em>1</em>-<em>7</em>), the identification of ACE2 and of Mas as a receptor implicated in its actions contributed to decisively establish this heptapeptide as a biologically active member of the RAS cascade. In this review, we will focus on the recent findings related to the ACE2-Ang(<em>1</em>-<em>7</em>)-Mas axis and, in particular, on its putative role as an ACE-Ang II-AT(<em>1</em>) receptor counter-regulatory axis within the RAS.

BACKGROUND

We investigated the effects of candesartan (an angiotensin II antagonist) alone, enalapril alone, and their combination on exercise tolerance, ventricular function, quality of life (QOL), neurohormone levels, and tolerability in congestive heart failure (CHF).

RESULTS

Seven hundred sixty-eight patients in New York Heart Association functional class (NYHA-FC) II to IV with ejection fraction (EF) <0.40 and a 6-minute walk distance (6MWD) <500 m received either candesartan (4, 8, or 16 mg), candesartan (4 or 8 mg) plus 20 mg of enalapril, or 20 mg of enalapril for 43 weeks. There were no differences among groups with regard to 6MWD, NYHA-FC, or QOL. EF increased (P=NS) more with candesartan-plus-enalapril therapy (0.025+/-0.004) than with candesartan alone (0.015+/-0.004) or enalapril alone(0.015+/-0.005). End-diastolic (EDV) and end-systolic (ESV) volumes increased less with combination therapy (EDV 8+/-4 mL; ESV 1+/-4 mL; P<0.01) than with candesartan alone (EDV 27+/-4 mL; ESV 18+/-3 mL) or enalapril alone (EDV 23+/-7 mL; ESV 14+/-6 mL). Blood pressure decreased with combination therapy (6+/-1/4+/-1 mm Hg) compared with candesartan or enalapril alone (P<0.05). Aldosterone decreased (P<0.05) with combination therapy (23.2+/-5.3 pg/mL) at 17 but not 43 weeks compared with candesartan (0.7+/-7.8 pg/mL) or enalapril (-0.8+/-11. 3 pg/mL). Brain natriuretic peptide decreased with combination therapy (5.8+/-2.7 pmol/L; P<0.01) compared with candesartan (4. 4+/-3.8 pmol/L) and enalapril alone (4.0+/-5.0 pmol/L).

CONCLUSIONS

Candesartan alone was as effective, safe, and tolerable as enalapril. The combination of candesartan and enalapril was more beneficial for preventing left ventricular remodeling than either candesartan or enalapril alone.

We investigated in Lewis normotensive rats the effect of coronary artery ligation on the expression of cardiac <em>angiotensin</em>-converting enzymes (ACE and ACE 2) and <em>angiotensin</em> II type-<em>1</em> receptors (AT<em>1</em>a-R) 28 days after myocardial infarction. Losartan, olmesartan, or the vehicle (isotonic saline) was administered via osmotic minipumps for 28 days after coronary artery ligation or sham operation. Coronary artery ligation caused left ventricular dysfunction and cardiac hypertrophy. These changes were associated with increased plasma concentrations of <em>angiotensin</em> I, <em>angiotensin</em> II, <em>angiotensin</em>-(<em>1</em>-<em>7</em>), and serum aldosterone, and reduced AT<em>1</em>a-R mRNA. Cardiac ACE and ACE 2 mRNAs did not change. Both <em>angiotensin</em> II antagonists attenuated cardiac hypertrophy; olmesartan improved ventricular contractility. Blockade of the AT<em>1</em>a-R was accompanied by a further increase in plasma concentrations of the <em>angiotensins</em> and reduced serum aldosterone levels. Both losartan and olmesartan completely reversed the reduction in cardiac AT<em>1</em>a-R mRNA observed after coronary artery ligation while augmenting ACE 2 mRNA by approximately 3-fold. Coadministration of PD<em>1</em>233<em>1</em>9 did not abate the increase in ACE 2 mRNA induced by losartan. ACE 2 mRNA correlated significantly with <em>angiotensin</em> II, <em>angiotensin</em>-(<em>1</em>-<em>7</em>), and <em>angiotensin</em> I levels. These results provide evidence for an effect of <em>angiotensin</em> II blockade on cardiac ACE 2 mRNA that may be due to direct blockade of AT<em>1</em>a receptors or a modulatory effect of increased <em>angiotensin</em>-(<em>1</em>-<em>7</em>).

beta-Arrestins are cytosolic proteins that mediate homologous desensitization of G protein-coupled receptors (GPCRs) by binding to agonist-occupied receptors and by uncoupling them from heterotrimeric G proteins. The recent finding that beta-arrestins bind to some mitogen-activated protein (MAP) kinases has suggested that they might also function as scaffolds for GPCR-stimulated MAP kinase activation. To define the role of beta-arrestins in the regulation of ERK MAP kinases, we examined the effect of beta-arrestin overexpression on ERK<em>1</em>/2 activation and nuclear signaling in COS-<em>7</em> cells expressing <em>angiotensin</em> II type <em>1</em>a receptors (AT<em>1</em>aRs). Expression of either beta-arrestin<em>1</em> or beta-arrestin2 reduced <em>angiotensin</em>-stimulated phosphatidylinositol hydrolysis but paradoxically increased <em>angiotensin</em>-stimulated ERK<em>1</em>/2 phosphorylation. The increase in ERK<em>1</em>/2 phosphorylation in beta-arrestin-expressing cells correlated with activation of a beta-arrestin-bound pool of ERK2. The beta-arrestin-dependent increase in ERK<em>1</em>/2 phosphorylation was accompanied by a significant reduction in ERK<em>1</em>/2-mediated, Elk<em>1</em>-driven transcription of a luciferase reporter. Analysis of the cellular distribution of phospho-ERK<em>1</em>/2 by confocal immunofluorescence microscopy and cellular fractionation revealed that overexpression of beta-arrestin resulted in a significant increase in the cytosolic pool of phospho-ERK<em>1</em>/2 and a corresponding decrease in the nuclear pool of phospho-ERK<em>1</em>/2 following <em>angiotensin</em> stimulation. beta-Arrestin overexpression resulted in formation of a cytoplasmic pool of beta-arrestin-bound phospho-ERK, decreased nuclear translocation of phospho-ERK<em>1</em>/2, and inhibition of Elk<em>1</em>-driven luciferase transcription even when ERK<em>1</em>/2 was activated by overexpression of cRaf-<em>1</em> in the absence of AT<em>1</em>aR stimulation. These data demonstrate that beta-arrestins facilitate GPCR-mediated ERK activation but inhibit ERK-dependent transcription by binding to phospho-ERK<em>1</em>/2, leading to its retention in the cytosol.

The renin-<em>angiotensin</em> system (RAS) is a key player in the control of the cardiovascular system and hydroelectrolyte balance, with an influence on organs and functions throughout the body. The classical view of this system saw it as a sequence of many enzymatic steps that culminate in the production of a single biologically active metabolite, the octapeptide <em>angiotensin</em> (ANG) II, by the <em>angiotensin</em> converting enzyme (ACE). The past two decades have revealed new functions for some of the intermediate products, beyond their roles as substrates along the classical route. They may be processed in alternative ways by enzymes such as the ACE homolog ACE2. One effect is to establish a second axis through ACE2/ANG-(<em>1</em>-<em>7</em>)/MAS, whose end point is the metabolite ANG-(<em>1</em>-<em>7</em>). ACE2 and other enzymes can form ANG-(<em>1</em>-<em>7</em>) directly or indirectly from either the decapeptide ANG I or from ANG II. In many cases, this second axis appears to counteract or modulate the effects of the classical axis. ANG-(<em>1</em>-<em>7</em>) itself acts on the receptor MAS to influence a range of mechanisms in the heart, kidney, brain, and other tissues. This review highlights the current knowledge about the roles of ANG-(<em>1</em>-<em>7</em>) in physiology and disease, with particular emphasis on the brain.

Renin-<em>angiotensin</em> system (RAS) signaling and <em>angiotensin</em>-converting enzyme 2 (ACE2) have been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS). We postulated that repleting ACE2 using GSK258688<em>1</em>, a recombinant form of human <em>angiotensin</em>-converting enzyme 2 (rhACE2), could attenuate acute lung injury.

We conducted a two-part phase II trial comprising an open-label intrapatient dose escalation and a randomized, double-blind, placebo-controlled phase in ten intensive care units in North America. Patients were between the ages of <em>1</em>8 and 80 years, had an American-European Consensus Criteria consensus diagnosis of ARDS, and had been mechanically ventilated for less than <em>7</em>2 h. In part A, open-label GSK258688<em>1</em> was administered at doses from 0.<em>1</em> mg/kg to 0.8 mg/kg to assess safety, pharmacokinetics, and pharmacodynamics. Following review of data from part A, a randomized, double-blind, placebo-controlled investigation of twice-daily doses of GSK258688<em>1</em> (0.4 mg/kg) for 3 days was conducted (part B). Biomarkers, physiological assessments, and clinical endpoints were collected over the dosing period and during follow-up.

Dose escalation in part A was well-tolerated without clinically significant hemodynamic changes. Part B was terminated after 39 of the planned 60 patients following a planned futility analysis. Angiotensin II levels decreased rapidly following infusion of GSK258688<em>1</em>, whereas <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and <em>angiotensin</em>-(<em>1</em>-5) levels increased and remained elevated for 48 h. Surfactant protein D concentrations were increased, whereas there was a trend for a decrease in interleukin-6 concentrations in rhACE2-treated subjects compared with placebo. No significant differences were noted in ratio of partial pressure of arterial oxygen to fraction of inspired oxygen, oxygenation index, or Sequential Organ Failure Assessment score.

GSK258688<em>1</em> was well-tolerated in patients with ARDS, and the rapid modulation of RAS peptides suggests target engagement, although the study was not powered to detect changes in acute physiology or clinical outcomes.

ClinicalTrials.gov, NCT0<em>1</em>59<em>7</em>635 . Registered on 26 January 20<em>1</em>2.

BACKGROUND

Angiotensin-converting enzyme 2 (ACE2) is a pleiotropic monocarboxypeptidase capable of metabolizing several peptide substrates. We hypothesized that ACE2 is a negative regulator of angiotensin II (Ang II)-mediated signaling and its adverse effects on the cardiovascular system.

RESULTS

Ang II infusion (1.5 mg x kg(-1) x d(-1)) for 14 days resulted in worsening cardiac fibrosis and pathological hypertrophy in ACE2 knockout (Ace2(-/y)) mice compared with wild-type (WT) mice. Daily treatment of Ang II-infused wild-type mice with recombinant human ACE2 (rhACE2; 2 mg x kg(-1) x d(-1) IP) blunted the hypertrophic response and expression of hypertrophy markers and reduced Ang II-induced superoxide production. Ang II-mediated myocardial fibrosis and expression of procollagen type I alpha 1, procollagen type III alpha 1, transforming growth factor-beta1, and fibronectin were also suppressed by rhACE2. Ang II-induced diastolic dysfunction was inhibited by rhACE2 in association with reduced plasma and myocardial Ang II and increased plasma Ang 1-7 levels. rhACE2 treatment inhibited Ang II-mediated activation of protein kinase C-alpha and protein kinase C-beta1 protein levels and phosphorylation of the extracellular signal-regulated 1/2, Janus kinase 2, and signal transducer and activator of transcription 3 signaling pathways in wild-type mice. A subpressor dose of Ang II (0.15 mg . kg(-1) . d(-1)) resulted in a milder phenotype that was strikingly attenuated by rhACE2 (2 mg x kg(-1) x d(-1) IP). In adult ventricular cardiomyocytes and cardiofibroblasts, Ang II-mediated superoxide generation, collagen production, and extracellular signal-regulated 1/2 signaling were inhibited by rhACE2 in an Ang 1-7-dependent manner. Importantly, rhACE2 partially prevented the development of dilated cardiomyopathy in pressure-overloaded wild-type mice.

CONCLUSIONS

Elevated Ang II induced hypertension, myocardial hypertrophy, fibrosis, and diastolic dysfunction, which were exacerbated by ACE2 deficiency, whereas rhACE2 attenuated Ang II- and pressure-overload-induced adverse myocardial remodeling. Hence, ACE2 is an important negative regulator of Ang II-induced heart disease and suppresses adverse myocardial remodeling.

<strong class="sub-title"> Aims/hypothesis: </strong> Coronavirus disease-20<em>1</em>9 (COVID-<em>1</em>9) is a life-threatening infection caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus. Diabetes has rapidly emerged as a major comorbidity for COVID-<em>1</em>9 severity. However, the phenotypic characteristics of diabetes in COVID-<em>1</em>9 patients are unknown.

<strong class="sub-title"> Methods: </strong> We conducted a nationwide multicentre observational study in people with diabetes hospitalised for COVID-<em>1</em>9 in 53 French centres in the period <em>1</em>0-3<em>1</em> March 2020. The primary outcome combined tracheal intubation for mechanical ventilation and/or death within <em>7</em> days of admission. Age- and sex-adjusted multivariable logistic regressions were performed to assess the prognostic value of clinical and biological features with the endpoint. ORs are reported for a <em>1</em> SD increase after standardisation.

<strong class="sub-title"> Results: </strong> The current analysis focused on <em>1</em>3<em>1</em><em>7</em> participants: 64.9% men, mean age 69.8 ± <em>1</em>3.0 years, median BMI 28.4 (25th-<em>7</em>5th percentile: 25.0-32.<em>7</em>) kg/m²; with a predominance of type 2 diabetes (88.5%). Microvascular and macrovascular diabetic complications were found in 46.8% and 40.8% of cases, respectively. The primary outcome was encountered in 29.0% (95% CI 26.6, 3<em>1</em>.5) of participants, while <em>1</em>0.6% (9.0, <em>1</em>2.4) died and <em>1</em>8.0% (<em>1</em>6.0, 20.2) were discharged on day <em>7</em>. In univariate analysis, characteristics prior to admission significantly associated with the primary outcome were sex, BMI and previous treatment with renin-angiotensin-aldosterone system (RAAS) blockers, but not age, type of diabetes, HbA_<em>1</em>c, diabetic complications or glucose-lowering therapies. In multivariable analyses with covariates prior to admission, only BMI remained positively associated with the primary outcome (OR <em>1</em>.28 [<em>1</em>.<em>1</em>0, <em>1</em>.4<em>7</em>]). On admission, dyspnoea (OR 2.<em>1</em>0 [<em>1</em>.3<em>1</em>, 3.35]), as well as lymphocyte count (OR 0.6<em>7</em> [0.50, 0.88]), C-reactive protein (OR <em>1</em>.93 [<em>1</em>.43, 2.59]) and AST (OR 2.23 [<em>1</em>.<em>7</em>0, 2.93]) levels were independent predictors of the primary outcome. Finally, age (OR 2.48 [<em>1</em>.<em>7</em>4, 3.53]), treated obstructive sleep apnoea (OR 2.80 [<em>1</em>.46, 5.38]), and microvascular (OR 2.<em>1</em>4 [<em>1</em>.<em>1</em>6, 3.94]) and macrovascular complications (OR 2.54 [<em>1</em>.44, 4.50]) were independently associated with the risk of death on day <em>7</em>.

<strong class="sub-title"> Conclusions/interpretations: </strong> In people with diabetes hospitalised for COVID-<em>1</em>9, BMI, but not long-term glucose control, was positively and independently associated with tracheal intubation and/or death within <em>7</em> days.

<strong class="sub-title"> Trial registration: </strong> clinicaltrials.gov <a href="http://clinicaltrials.gov/show/NCT04324<em>7</em>36" title="See in ClinicalTrials.gov">NCT04324<em>7</em>36</a>.

<strong class="sub-title"> Keywords: </strong> BMI; COVID-<em>1</em>9; Death; Diabetes; HbA<em>1</em>c; Hypertension; Mechanical ventilation.

BACKGROUND

Tumor necrosis factor-alpha (TNF-alpha) and angiotensin II (Ang II) are implicated in the development and further progression of heart failure, which might be, at least in part, mediated by the production of reactive oxygen species (ROS). However, the cause and consequences of this agonist-mediated ROS production in cardiac myocytes have not been well defined. Recently, we demonstrated that increased ROS production was associated with mitochondrial DNA (mtDNA) damage and dysfunction in failing hearts. We thus investigated whether the direct exposure of cardiac myocytes to TNF-alpha and Ang II in vitro could induce mtDNA damage via production of ROS.

RESULTS

TNF-alpha increased ROS production within cultured neonatal rat ventricular myocytes after 1 hour, as assessed by 2',7'-dichlorofluorescin diacetate fluorescence microscopy. TNF-alpha also decreased mtDNA copy number by Southern blot analysis in association with complex III activity, which was prevented in the presence of the antioxidant alpha-tocopherol. A direct exposure of myocytes to H2O2 caused a similar decrease in mtDNA copy number. In contrast, Ang II did not affect mtDNA copy number, despite the similar increase in ROS production. TNF-alpha-mediated ROS production and a decrease in mtDNA copy number were inhibited by the sphingomyelinase inhibitor D609. Furthermore, N-acetylsphingosine (C2-ceramide), a synthetic cell-permeable ceramide analogue, increased myocyte ROS production, suggesting that TNF-alpha-mediated ROS production and subsequent mtDNA damage were mediated by the sphingomyelin-ceramide signaling pathway.

CONCLUSIONS

The intimate link between TNF-alpha, ROS, and mtDNA damage might play an important role in myocardial remodeling and failure.

<em>Angiotensin</em>-converting enzyme 2 (ACE2) is a newly discovered carboxy-peptidase responsible for the formation of vasodilatory peptides such as <em>angiotensin</em>-(<em>1</em>-<em>7</em>). We hypothesized that ACE2 is part of the brain renin-<em>angiotensin</em> system, and its expression is regulated by the other elements of this system. ACE2 immunostaining was performed in transgenic mouse brain sections from neuron-specific enolase-AT(<em>1</em>A) (overexpressing AT(<em>1</em>A) receptors), R(+)A(+) (overexpressing <em>angiotensin</em>ogen and renin), and control (nontransgenic littermates) mice. Results show that ACE2 staining is widely distributed throughout the brain. Using cell-type-specific antibodies, we observed that ACE2 staining is present in the cytoplasm of neuronal cell bodies but not in glial cells. In the subfornical organ, an area lacking the blood-brain barrier and sensitive to blood-borne <em>angiotensin</em> II, ACE2 was significantly increased in transgenic mice. Interestingly, ACE2 mRNA and protein expression were inversely correlated in the nucleus of tractus solitarius/dorsal motor nucleus of the vagus and the ventrolateral medulla, when comparing transgenic to nontransgenic mice. These results suggest that ACE2 is localized to the cytoplasm of neuronal cells in the brain and that ACE2 levels appear highly regulated by other components of the renin-<em>angiotensin</em> system, confirming its involvement in this system. Moreover, ACE2 expression in brain structures involved in the control of cardiovascular function suggests that the carboxypeptidase may have a role in the central regulation of blood pressure and diseases involving the autonomic nervous system, such as hypertension.

By binding to agonist-activated G protein-coupled receptors (GPCRs), beta-arrestins mediate homologous receptor desensitization and endocytosis via clathrin-coated pits. Recent data suggest that beta-arrestins also contribute to GPCR signaling by acting as scaffolds for components of the ERK mitogen-activated protein kinase cascade. Because of these dual functions, we hypothesized that the stability of the receptor-beta-arrestin interaction might affect the mechanism and functional consequences of GPCR-stimulated ERK activation. In transfected COS-<em>7</em> cells, we found that <em>angiotensin</em> AT<em>1</em>a and vasopressin V2 receptors, which form stable receptor-beta-arrestin complexes, activated a beta-arrestin-bound pool of ERK2 more efficiently than alpha <em>1</em>b and beta2 adrenergic receptors, which form transient receptor-beta-arrestin complexes. We next studied chimeric receptors in which the pattern of beta-arrestin binding was reversed by exchanging the C-terminal tails of the beta2 and V2 receptors. The ability of the V2 beta 2 and beta 2V2 chimeras to activate beta-arrestin-bound ERK2 corresponded to the pattern of beta-arrestin binding, suggesting that the stability of the receptor-beta-arrestin complex determined the mechanism of ERK2 activation. Analysis of covalently cross-linked detergent lysates and cellular fractionation revealed that wild type V2 receptors generated a larger pool of cytosolic phospho-ERK<em>1</em>/2 and less nuclear phospho-ERK<em>1</em>/2 than the chimeric V2 beta 2 receptor, consistent with the cytosolic retention of beta-arrestin-bound ERK. In stably transfected HEK-293 cells, the V2 beta 2 receptor increased ERK<em>1</em>/2-mediated, Elk-<em>1</em>-driven transcription of a luciferase reporter to a greater extent than the wild type V2 receptor. Furthermore, the V2 beta 2, but not the V2 receptor, was capable of eliciting a mitogenic response. These data suggest that the C-terminal tail of a GPCR, by determining the stability of the receptor-beta-arrestin complex, controls the extent of beta-arrestin-bound ERK activation, and influences both the subcellular localization of activated ERK and the physiologic consequences of ERK activation.

BACKGROUND

Angiotensin II (ATII), a potent vasoconstrictor, causes hypertension, promotes infiltration of myelomonocytic cells into the vessel wall, and stimulates both vascular and inflammatory cell NADPH oxidases. The predominant source of reactive oxygen species, eg, vascular (endothelial, smooth muscle, adventitial) versus phagocytic NADPH oxidase, and the role of myelomonocytic cells in mediating arterial hypertension have not been defined yet.

RESULTS

Angiotensin II (1 mg · kg(-1) · d(-1) for 7 days) increased the number of both CD11b(+)Gr-1(low)F4/80(+) macrophages and CD11b(+)Gr-1(high)F4/80(-) neutrophils in mouse aorta (verified by flow cytometry). Selective ablation of lysozyme M-positive (LysM(+)) myelomonocytic cells by low-dose diphtheria toxin in mice with inducible expression of the diphtheria toxin receptor (LysM(iDTR) mice) reduced the number of monocytes in the circulation and limited ATII-induced infiltration of these cells into the vascular wall, whereas the number of neutrophils was not reduced. Depletion of LysM(+) cells attenuated ATII-induced blood pressure increase (measured by radiotelemetry) and vascular endothelial and smooth muscle dysfunction (assessed by aortic ring relaxation studies) and reduced vascular superoxide formation (measured by chemiluminescence, cytochrome c assay, and oxidative fluorescence microtopography) and the expression of NADPH oxidase subunits gp91(phox) and p67(phox) (assessed by Western blot and mRNA reverse-transcription polymerase chain reaction). Adoptive transfer of wild-type CD11b(+)Gr-1(+) monocytes into depleted LysM(iDTR) mice reestablished ATII-induced vascular dysfunction, oxidative stress, and arterial hypertension, whereas transfer of CD11b(+)Gr-1(+) neutrophils or monocytes from gp91(phox) or ATII receptor type 1 knockout mice did not. CONCLUSIONS- Infiltrating monocytes with a proinflammatory phenotype and macrophages rather than neutrophils appear to be essential for ATII-induced vascular dysfunction and arterial hypertension.

<em>Angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>) is now recognized as a biologically active component of the renin-<em>angiotensin</em> system (RAS). Ang-(<em>1</em>-<em>7</em>) appears to play a central role in the RAS because it exerts a vast array of actions, many of them opposite to those attributed to the main effector peptide of the RAS, Ang II. The discovery of the Ang-converting enzyme (ACE) homolog ACE2 brought to light an important metabolic pathway responsible for Ang-(<em>1</em>-<em>7</em>) synthesis. This enzyme can form Ang-(<em>1</em>-<em>7</em>) from Ang II or less efficiently through hydrolysis of Ang I to Ang-(<em>1</em>-9) with subsequent Ang-(<em>1</em>-<em>7</em>) formation by ACE. In addition, it is now well established that the G protein-coupled receptor Mas is a functional binding site for Ang-(<em>1</em>-<em>7</em>). Thus, the axis formed by ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas appears to represent an endogenous counterregulatory pathway within the RAS, the actions of which are in opposition to the vasoconstrictor/proliferative arm of the RAS consisting of ACE, Ang II, and AT(<em>1</em>) receptor. In this brief review, we will discuss recent findings related to the biological role of the ACE2/Ang-(<em>1</em>-<em>7</em>)/Mas arm in the cardiovascular and renal systems, as well as in metabolism. In addition, we will highlight the potential interactions of Ang-(<em>1</em>-<em>7</em>) and Mas with AT(<em>1</em>) and AT(2) receptors.

New components and functions of the renin-<em>angiotensin</em> system (RAS) are still being unravelled. The classical RAS as it looked in the middle <em>1</em>9<em>7</em>0s consisted of circulating renin, acting on <em>angiotensin</em>ogen to produce <em>angiotensin</em> I, which in turn was converted into <em>angiotensin</em> II (Ang II) by <em>angiotensin</em>-converting enzyme (ACE). Ang II, still considered the main effector of RAS was believed to act only as a circulating hormone via <em>angiotensin</em> receptors, AT<em>1</em> and AT2. Since then, an expanded view of RAS has gradually emerged. Local tissue RAS systems have been identified in most organs. Recently, evidence for an intracellular RAS has been reported. The new expanded view of RAS therefore covers both endocrine, paracrine and intracrine functions. Other peptides of RAS have been shown to have biological actions; <em>angiotensin</em> 2-8 heptapeptide (Ang III) has actions similar to those of Ang II. Further, the <em>angiotensin</em> 3-8 hexapeptide (Ang IV) exerts its actions via insulin-regulated amino peptidase receptors. Finally, <em>angiotensin</em> <em>1</em>-<em>7</em> (Ang <em>1</em>-<em>7</em>) acts via mas receptors. The discovery of another ACE2 was an important complement to this picture. The recent discovery of renin receptors has made our view of RAS unexpectedly complex and multilayered. The importance of RAS in cardiovascular disease has been demonstrated by the clinical benefits of ACE inhibitors and AT<em>1</em> receptor blockers. Great expectations are now generated by the introduction of renin inhibitors. Indeed, RAS regulates much more and diverse physiological functions than previously believed.

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The SARS-CoV receptor, human <em>angiotensin</em> <em>1</em>-converting enzyme 2 (hACE2), was detected in ciliated airway epithelial cells of human airway tissues derived from nasal or tracheobronchial regions, suggesting that SARS-CoV may infect the proximal airways. To assess infectivity in an in vitro model of human ciliated airway epithelia (HAE) derived from nasal and tracheobronchial airway regions, we generated recombinant SARS-CoV by deletion of open reading frame <em>7</em>a/<em>7</em>b (ORF<em>7</em>a/<em>7</em>b) and insertion of the green fluorescent protein (GFP), resulting in SARS-CoV GFP. SARS-CoV GFP replicated to titers similar to those of wild-type viruses in cell lines. SARS-CoV specifically infected HAE via the apical surface and replicated to titers of <em>1</em>0(<em>7</em>) PFU/ml by 48 h postinfection. Polyclonal antisera directed against hACE2 blocked virus infection and replication, suggesting that hACE2 is the primary receptor for SARS-CoV infection of HAE. SARS-CoV structural proteins and virions localized to ciliated epithelial cells. Infection was highly cytolytic, as infected ciliated cells were necrotic and shed over time onto the luminal surface of the epithelium. SARS-CoV GFP also replicated to a lesser extent in ciliated cell cultures derived from hamster or rhesus monkey airways. Efficient SARS-CoV infection of ciliated cells in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.

Stimulation of the local renin-<em>angiotensin</em> system and apoptosis characterize the diabetic heart. Because IGF-<em>1</em> reduces <em>angiotensin</em> (Ang) II and apoptosis, we tested whether streptozotocin-induced diabetic cardiomyopathy was attenuated in IGF-<em>1</em> transgenic mice (TGM). Diabetes progressively depressed ventricular performance in wild-type mice (WTM) but had no hemodynamic effect on TGM. Myocyte apoptosis measured at <em>7</em> and 30 days after the onset of diabetes was twofold higher in WTM than in TGM. Myocyte necrosis was apparent only at 30 days and was more severe in WTM. Diabetic nontransgenic mice lost 24% of their ventricular myocytes and showed a 28% myocyte hypertrophy; both phenomena were prevented by IGF-<em>1</em>. In diabetic WTM, p53 was increased in myocytes, and this activation of p53 was characterized by upregulation of Bax, <em>angiotensin</em>ogen, Ang type <em>1</em> (AT(<em>1</em>)) receptors, and Ang II. IGF-<em>1</em> overexpression decreased these biochemical responses. In vivo accumulation of the reactive O(2) product nitrotyrosine and the in vitro formation of H(2)O(2)-(.)OH in myocytes were higher in diabetic WTM than TGM. Apoptosis in vitro was detected in myocytes exhibiting high H(2)O(2)-(.)OH fluorescence, and apoptosis in vivo was linked to the presence of nitrotyrosine. H(2)O(2)-(.)OH generation and myocyte apoptosis in vitro were inhibited by the AT(<em>1</em>) blocker losartan and the O(2) scavenger TIRON: In conclusion, IGF-<em>1</em> interferes with the development of diabetic myopathy by attenuating p53 function and Ang II production and thus AT(<em>1</em>) activation. This latter event might be responsible for the decrease in oxidative stress and myocyte death by IGF-<em>1</em>.

The zinc metallopeptidase <em>angiotensin</em>-converting enzyme 2 (ACE2) is the only known human homologue of the key regulator of blood pressure <em>angiotensin</em>-converting enzyme (ACE). Since its discovery in 2000, ACE2 has been implicated in heart function, hypertension and diabetes, with its effects being mediated, in part, through its ability to convert <em>angiotensin</em> II to <em>angiotensin</em>-(<em>1</em>-<em>7</em>). Unexpectedly, ACE2 also serves as the cellular entry point for the severe acute respiratory syndrome (SARS) virus and the enzyme is therefore a prime target for pharmacological intervention on several disease fronts.

OBJECTIVE

Diabetic nephropathy is one of the most common causes of end-stage renal failure. Inhibition of ACE2 function accelerates diabetic kidney injury, whereas renal ACE2 is downregulated in diabetic nephropathy. We examined the ability of human recombinant ACE2 (hrACE2) to slow the progression of diabetic kidney injury.

METHODS

Male 12-week-old diabetic Akita mice (Ins2(WT/C96Y)) and control C57BL/6J mice (Ins2(WT/WT)) were injected daily with placebo or with rhACE2 (2 mg/kg, i.p.) for 4 weeks. Albumin excretion, gene expression, histomorphometry, NADPH oxidase activity, and peptide levels were examined. The effect of hrACE2 on high glucose and angiotensin II (ANG II)-induced changes was also examined in cultured mesangial cells.

RESULTS

Treatment with hrACE2 increased plasma ACE2 activity, normalized blood pressure, and reduced the urinary albumin excretion in Akita Ins2(WT/C96Y) mice in association with a decreased glomerular mesangial matrix expansion and normalization of increased alpha-smooth muscle actin and collagen III expression. Human recombinant ACE2 increased ANG 1-7 levels, lowered ANG II levels, and reduced NADPH oxidase activity. mRNA levels for p47(phox) and NOX2 and protein levels for protein kinase Calpha (PKCalpha) and PKCbeta1 were also normalized by treatment with hrACE2. In vitro, hrACE2 attenuated both high glucose and ANG II-induced oxidative stress and NADPH oxidase activity.

CONCLUSIONS

Treatment with hrACE2 attenuates diabetic kidney injury in the Akita mouse in association with a reduction in blood pressure and a decrease in NADPH oxidase activity. In vitro studies show that the protective effect of hrACE2 is due to reduction in ANG II and an increase in ANG 1-7 signaling.

An outbreak of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that started in Wuhan, China, at the end of 20<em>1</em>9 has become a global pandemic. Both SARS-CoV-2 and SARS-CoV enter host cells via the <em>angiotensin</em>-converting enzyme 2 (ACE2) receptor, which is expressed in various human organs. We have reviewed previously published studies on SARS and recent studies on SARS-CoV-2 infection, named coronavirus disease 20<em>1</em>9 (COVID-<em>1</em>9) by the World Health Organization (WHO), confirming that many other organs besides the lungs are vulnerable to the virus. ACE2 catalyzes <em>angiotensin</em> II conversion to <em>angiotensin</em>-(<em>1</em>-<em>7</em>), and the ACE2/<em>angiotensin</em>-(<em>1</em>-<em>7</em>)/MAS axis counteracts the negative effects of the renin-<em>angiotensin</em> system (RAS), which plays important roles in maintaining the physiological and pathophysiological balance of the body. In addition to the direct viral effects and inflammatory and immune factors associated with COVID-<em>1</em>9 pathogenesis, ACE2 downregulation and the imbalance between the RAS and ACE2/<em>angiotensin</em>-(<em>1</em>-<em>7</em>)/MAS after infection may also contribute to multiple organ injury in COVID-<em>1</em>9. The SARS-CoV-2 spike glycoprotein, which binds to ACE2, is a potential target for developing specific drugs, antibodies, and vaccines. Restoring the balance between the RAS and ACE2/<em>angiotensin</em>-(<em>1</em>-<em>7</em>)/MAS may help attenuate organ injuries. SARS-CoV-2 enters lung cells via the ACE2 receptor. The cell-free and macrophage-phagocytosed virus can spread to other organs and infect ACE2-expressing cells at local sites, causing multi-organ injury.

<strong class="sub-title"> Keywords: </strong> Angiotensin-converting enzyme 2; COVID-<em>1</em>9; Multi-organ injury; SARS-CoV-2.

OBJECTIVE

<em>Angiotensin</em> converting enzyme (ACE) 2 catalyses the cleavage of <em>angiotensin</em> (Ang) I to Ang <em>1</em>-9 and of Ang II to Ang <em>1</em>-<em>7</em>. ACE2 deficiency impairs cardiac contractility and upregulates hypoxia-induced genes, suggesting a link with myocardial ischaemia. We studied the expression of ACE2 after myocardial infarction (MI) in the rat as well as in human failing hearts.

RESULTS

Rats were killed at days <em>1</em>, 3, and 28 after MI, or treated for 4 weeks with the ACE inhibitor ramipril (<em>1</em> mg/kg). Cardiac gene and protein expression of ACE and ACE2 were assessed by quantitative real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry/activity assays/in vitro autoradiography, respectively. Both ACE (P = 0.022) and ACE2 (P = 0.0<em>1</em>5) mRNA increased in the border/infarct area compared with the viable area at day 3 after MI. By day 28, increases in ACE (P = 0.005) and ACE2 (P = 0.006) mRNA were also seen in the viable myocardium of MI rats compared with myocardium of control rats. ACE2 protein localized to macrophages, vascular endothelium, smooth muscle, and myocytes. Ramipril attenuated cardiac hypertrophy and inhibited cardiac ACE. In contrast, ramipril had no effect on cardiac ACE2 mRNA, which remained elevated in all areas of the MI rat heart. Immunoreactivity of both ACE and ACE2 increased in failing human hearts.

CONCLUSIONS

The increase in ACE2 after MI suggests that it plays an important role in the negative modulation of the renin angiotensin system in the generation and degradation of angiotensin peptides after cardiac injury.

<em>Angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>) is a bioactive component of the renin-<em>angiotensin</em> system that is formed endogenously from either Ang I or Ang II. The first actions described for Ang-(<em>1</em>-<em>7</em>) indicated that the peptide mimicked some of the effects of Ang II, including the release of prostanoids and vasopressin. However, Ang-(<em>1</em>-<em>7</em>) is devoid of vasoconstrictor, central pressor, or thirst-stimulating actions. In fact, new findings reveal depressor, vasodilator, and antihypertensive actions that may be more apparent in hypertensive animals or humans. Thus, the accumulating evidence suggests that Ang-(<em>1</em>-<em>7</em>) may oppose the actions of Ang II either directly or by stimulation of prostaglandins and nitric oxide. These observations are significant because they may explain the effective antihypertensive action of converting enzyme inhibitors in a variety of non-renin-dependent models of experimental and genetic hypertension as well as most forms of human hypertension. In this context, studies in humans and animals showed that the antihypertensive action of converting enzyme inhibitors correlated with increases in plasma levels of Ang-(<em>1</em>-<em>7</em>). In this review, we summarize our knowledge of the mechanisms accounting for the counterregulatory actions of Ang-(<em>1</em>-<em>7</em>) and elaborate on the emerging concept that Ang-(<em>1</em>-<em>7</em>) functions as an antihypertensive peptide within the cascade of the renin-<em>angiotensin</em> system.

Cardiac remodeling, which typically results from chronic hypertension or following an acute myocardial infarction, is a major risk factor for the development of heart failure and, ultimately, death. The renin-<em>angiotensin</em> system (RAS) has previously been established to play an important role in the progression of cardiac remodeling, and inhibition of a hyperactive RAS provides protection from cardiac remodeling and subsequent heart failure. Our previous studies have demonstrated that overexpression of <em>angiotensin</em>-converting enzyme 2 (ACE2) prevents cardiac remodeling and hypertrophy during chronic infusion of <em>angiotensin</em> II (ANG II). This, coupled with the knowledge that ACE2 is a key enzyme in the formation of ANG-(<em>1</em>-<em>7</em>), led us to hypothesize that chronic infusion of ANG-(<em>1</em>-<em>7</em>) would prevent cardiac remodeling induced by chronic infusion of ANG II. Infusion of ANG II into adult Sprague-Dawley rats resulted in significantly increased blood pressure, myocyte hypertrophy, and midmyocardial interstitial fibrosis. Coinfusion of ANG-(<em>1</em>-<em>7</em>) resulted in significant attenuations of myocyte hypertrophy and interstitial fibrosis, without significant effects on blood pressure. In a subgroup of animals also administered [d-Ala(<em>7</em>)]-ANG-(<em>1</em>-<em>7</em>) (A<em>7</em><em>7</em>9), an antagonist to the reported receptor for ANG-(<em>1</em>-<em>7</em>), there was a tendency to attenuate the antiremodeling effects of ANG-(<em>1</em>-<em>7</em>). Chronic infusion of ANG II, with or without coinfusion of ANG-(<em>1</em>-<em>7</em>), had no effect on ANG II type <em>1</em> or type 2 receptor binding in cardiac tissue. Together, these findings indicate an antiremodeling role for ANG-(<em>1</em>-<em>7</em>) in cardiac tissue, which is not mediated through modulation of blood pressure or altered cardiac <em>angiotensin</em> receptor populations and may be at least partially mediated through an ANG-(<em>1</em>-<em>7</em>) receptor.

Obesity has become an epidemic problem in western societies, contributing to metabolic diseases, hypertension, and cardiovascular disease. Overweight and obesity are frequently associated with increased plasma levels of aldosterone. Recent evidence suggests that human fat is a highly active endocrine tissue. Therefore, we tested the hypothesis that adipocyte secretory products directly stimulate adrenocortical aldosterone secretion. Secretory products from isolated human adipocytes strongly stimulated steroidogenesis in human adrenocortical cells (NCI-H295R) with a predominant effect on mineralocorticoid secretion. Aldosterone secretion increased <em>7</em>-fold during 24 h of incubation. This stimulation was comparable to maximal stimulation of these cells with forskolin (2 x <em>1</em>0(-5) M). On the molecular level, there was a <em>1</em>0-fold increase in the expression of steroid acute regulatory peptide mRNA. This effect was independent of adipose <em>angiotensin</em> II as revealed by the stimulatory effect of fat cell-conditioned medium even in the presence of the <em>angiotensin</em> type <em>1</em> receptor antagonist, valsartan. None of the recently defined adipocytokines accounted for the effect. Mineralocorticoid-stimulating activity was heat sensitive and could be blunted by heating fat cell-conditioned medium to 99 degrees C. Centrifugal filtration based on molecular mass revealed at least two releasing factors: a heat sensitive fraction (molecular mass >50 kDa) representing 60% of total activity, and an inactive fraction (molecular mass <50 kDa). However, the recovery rate increased to 92% when combining these two fractions, indicating the interaction of at least two factors. In conclusion, human adipocytes secrete potent mineralocorticoid-releasing factors, suggesting a direct link between obesity and hypertension.

We recently reported that the activation of nuclear factor-kappaB (NF-kappaB) promotes inflammation in rats harboring both human renin and <em>angiotensin</em>ogen genes (double-transgenic rats [dTGR]). We tested the hypothesis that the antioxidant pyrrolidine dithiocarbamate (PDTC) inhibits NF-kappaB and ameliorates renal and cardiac end-organ damage. dTGR feature hypertension, severe renal and cardiac damage, and a 40% mortality rate at <em>7</em> weeks. Electrophoretic mobility shift assay showed increased NF-kappaB DNA binding activity in hearts and kidneys of dTGR. Chronic PDTC (200 mg/kg SC) treatment decreased blood pressure (<em>1</em>62+/-8 versus <em>1</em>90+/-<em>7</em> mm Hg; P=0.02) in dTGR compared with dTGR controls. The cardiac hypertrophy index was also significantly reduced (4.90+/-0.<em>1</em> versus 5.<em>7</em><em>7</em>+/-0.<em>1</em> mg/g; P<0. 00<em>1</em>). PDTC reduced 24-hour albuminuria by >95% (2.5+/-0.8 versus 5<em>7</em>. <em>1</em>+/-8.<em>7</em> mg/d; P<0.00<em>1</em>) and prevented death. Vascular injury was ameliorated in small renal and cardiac vessels. Electrophoretic mobility shift assay showed that PDTC inhibited NF-kappaB binding activity in heart and kidney, whereas AP-<em>1</em> activity in the kidney was not decreased. dTGR exhibited increased left ventricular c-fos and c-jun mRNA expression. PDTC treatment reduced c-fos but not c-jun mRNA. Immunohistochemistry showed increased p65 NF-kappaB subunit expression in the endothelium and smooth muscle cells of damaged small vessels, as well as infiltrating cells in glomeruli, tubules, and collecting ducts of dTGR. PDTC markedly reduced the immunoreactivity of p65. PDTC also prevented the NF-kappaB-dependent transactivation of the intercellular adhesion molecule ICAM-<em>1</em> and inducible nitric oxide synthase. Monocyte infiltration was markedly increased in dTGR kidneys and hearts. Chronic treatment reduced monocyte/macrophage infiltration by <em>7</em>2% and 64%, respectively. Thus, these results demonstrate that PDTC inhibits NF-kappaB activity, ameliorates inflammation, and protects against <em>angiotensin</em> II-induced end-organ damage.