The heptapeptide <em>angiotensin</em>-(<em>1</em>-<em>7</em>) is a circulating biologically active product of the renin-<em>angiotensin</em> system. In this study, we evaluated the role of the vascular endothelium in the formation of <em>angiotensin</em>-(<em>1</em>-<em>7</em>). Metabolism of <em>1</em>25I-<em>angiotensin</em> I was investigated using confluent cultured bovine and human aortic and umbilical vein endothelial cells. The fetal calf serum-supplemented medium was replaced by serum-free medium containing 0.2% bovine serum albumin. One hour later, this medium was replaced by serum-free medium containing <em>1</em>25I-<em>angiotensin</em> I. After incubation of <em>1</em>25I-<em>angiotensin</em> I for various intervals at 3<em>7</em> degrees C, the medium was collected and analyzed for formed products by high-performance liquid chromatography. Products of <em>angiotensin</em> I metabolism were identified by comparison of their retention times with those of radiolabeled standards. The contribution of proteases released into the medium was evaluated by incubation of <em>1</em>25I-<em>angiotensin</em> I with medium previously incubated for <em>1</em> hour with endothelial cells. Incubation of <em>1</em>25I-<em>angiotensin</em> I with bovine and human endothelial cells produced a time-dependent generation of <em>1</em>25I-<em>angiotensin</em>-(<em>1</em>-<em>7</em>) greater than <em>1</em>25I-<em>angiotensin</em> II greater than <em>1</em>25I-<em>angiotensin</em>-(<em>1</em>-4). Generation of <em>angiotensin</em> peptides was not due to the presence of proteases in the medium. When human umbilical endothelial cells were incubated in the presence of the <em>angiotensin</em> converting enzyme inhibitor enalaprilat (<em>1</em> microM), generation of <em>angiotensin</em> II was undetectable. In contrast, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) production increased by an average of 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

The renin-<em>angiotensin</em> system contributes to atherosclerotic lesion formation. <em>Angiotensin</em>-converting enzyme 2 (ACE2) catabolizes <em>angiotensin</em> II (Ang II) to <em>angiotensin</em> <em>1</em>-<em>7</em> (Ang-(<em>1</em>-<em>7</em>)) to limit effects of the renin-<em>angiotensin</em> system. The purpose of this study was to define the role of ACE2 in atherosclerosis.

RESULTS

Male Ace2(-/y) mice in an low-density lipoprotein receptor-deficient background were fed a high-fat diet for 3 months. ACE2 deficiency increased atherosclerotic area (Ace2(+/y), <em>1</em><em>7</em> ± <em>1</em>; Ace2(-/y), 23 ± 2 mm(2), P < 0.002). This increase was blunted by losartan. To determine whether leukocytic ACE2 influenced atherosclerosis, irradiated low-density lipoprotein receptor-deficient male mice were repopulated with bone marrow-derived cells from Ace2(+/y) or Ace2(-/y) mice and fed a high-fat diet for 3 months. ACE2 deficiency in bone marrow-derived cells increased atherosclerotic area (Ace2(+/y), <em>1</em>.6 ± 0.3; Ace2(-/y), 2.8 ± 0.3 mm(2); P < 0.05). Macrophages from Ace2(-/y) mice exhibited increased Ang II secretion and elevated expression of inflammatory cytokines. Conditioned media from mouse peritoneal macrophages of Ace2(-/y) mice increased monocyte adhesion to human umbilical vein endothelial cells. Incubation of human umbilical vein endothelial cells with Ang II promoted monocyte adhesion, which was blocked by Ang-(<em>1</em>-<em>7</em>). Coinfusion of Ang-(<em>1</em>-<em>7</em>) with Ang II reduced atherosclerosis.

CONCLUSIONS

These results demonstrate that ACE2 deficiency in bone marrow-derived cells promotes atherosclerosis through regulation of Ang II/Ang-(<em>1</em>-<em>7</em>) peptides.

Respiratory, circulatory, and renal failure are among the gravest features of COVID-<em>1</em>9 and are associated with a very high mortality rate. A common denominator of all affected organs is the expression of <em>angiotensin</em>-converting enzyme 2 (ACE2), a protease responsible for the conversion of <em>Angiotensin</em> <em>1</em>-8 (Ang II) to <em>Angiotensin</em> <em>1</em>-<em>7</em> (Ang <em>1</em>-<em>7</em>). Ang <em>1</em>-<em>7</em> acts on these tissues and in other target organs via Mas receptor (MasR), where it exerts beneficial effects, including vasodilation and suppression of inflammation and fibrosis, along an attenuation of cardiac and vascular remodeling. Unfortunately, ACE2 also serves as the binding receptor of SARS viral spike glycoprotein, enabling its attachment to host cells, with subsequent viral internalization and replication. Although numerous reports have linked the devastating organ injuries to viral homing and attachment to organ-specific cells widely expressing ACE2, little attention has been given to ACE-2 expressed by the immune system. Herein we outline potential adverse effects of SARS-CoV2 on macrophages and dendritic cells, key cells of the immune system expressing ACE2. Specifically, we propose a new hypothesis that, while macrophages play an important role in antiviral defense mechanisms, in the case of SARS-CoV, they may also serve as a Trojan horse, enabling viral anchoring specifically within the pulmonary parenchyma. It is tempting to assume that diverse expression of ACE2 in macrophages among individuals might govern the severity of SARS-CoV-2 infection. Moreover, reallocation of viral-containing macrophages migrating out of the lung to other tissues is theoretically plausible in the context of viral spread with the involvement of other organs.

Keywords: ACE2; SARS-CoV-2; acute respiratory distress syndrome; defense; lung; macrophages; reservoir.

ACE (<em>angiotensin</em>-converting enzyme) 2 is expressed in the heart and kidney and metabolizes Ang (<em>angiotensin</em>) II to Ang-(<em>1</em>-<em>7</em>) a peptide that acts via the Ang-(<em>1</em>-<em>7</em>) or mas receptor. The aim of the present study was to assess the effect of Ang-(<em>1</em>-<em>7</em>) on blood pressure and cardiac remodelling in a rat model of renal mass ablation. Male SD (Sprague-Dawley) rats underwent STNx (subtotal nephrectomy) and were treated for <em>1</em>0 days with vehicle, the ACE inhibitor ramipril (oral <em>1</em> mg·kg(-<em>1</em>) of body weight·day(-<em>1</em>)) or Ang-(<em>1</em>-<em>7</em>) (subcutaneous 24 μg·kg(-<em>1</em>) of body weight·h(-<em>1</em>)) (all n = <em>1</em>5 per group). A control group (n = <em>1</em>0) of sham-operated rats were also studied. STNx rats were hypertensive (P<0.0<em>1</em>) with renal impairment (P<0.00<em>1</em>), cardiac hypertrophy (P<0.00<em>1</em>) and fibrosis (P<0.05), and increased cardiac ACE (P<0.00<em>1</em>) and ACE2 activity (P<0.05). Ramipril reduced blood pressure (P<0.0<em>1</em>), improved cardiac hypertrophy (P<0.00<em>1</em>) and inhibited cardiac ACE (P<0.00<em>1</em>). By contrast, Ang-(<em>1</em>-<em>7</em>) infusion in STNx was associated with further increases in blood pressure (P<0.05), cardiac hypertrophy (P<0.05) and fibrosis (P<0.0<em>1</em>). Ang-(<em>1</em>-<em>7</em>) infusion also increased cardiac ACE activity (P<0.00<em>1</em>) and reduced cardiac ACE2 activity (P<0.05) compared with STNx-vehicle rats. Our results add to the increasing evidence that Ang-(<em>1</em>-<em>7</em>) may have deleterious cardiovascular effects in kidney failure and highlight the need for further in vivo studies of the ACE2/Ang-(<em>1</em>-<em>7</em>)/mas receptor axis in kidney disease.

Previously we demonstrated that kidney concentration and urinary excretion of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) are increased during normal pregnancy in rats. Since this finding may reflect local kidney production of <em>angiotensin</em>-(<em>1</em>-<em>7</em>), we determined the immunocytochemical distribution of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and its newly described processing enzyme, ACE2, in kidneys of virgin and <em>1</em>9-day-pregnant Sprague-Dawley rats. Sprague-Dawley rats were killed at the <em>1</em>9th day of pregnancy, and tissues were prepared for immunocytochemical by using a polyclonal antibody to <em>angiotensin</em>- (<em>1</em>-<em>7</em>) or a monoclonal antibody to ACE2. <em>Angiotensin</em>-(<em>1</em>-<em>7</em>) immunostaining was predominantly localized to the renal tubules traversing both the inner cortex and outer medulla. ACE2 immunostaining was localized throughout the cortex and outer medulla and was visualized in the renal tubules of both virgin and pregnant rats. The quantification of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and ACE2 immunocytochemical staining showed that in pregnant animals, the intensity of the staining increased by 56% and <em>1</em><em>1</em><em>7</em>%, respectively (P<0.05). This first demonstration of the immunocytochemical distribution of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and ACE2 in kidneys of pregnant rats shows that pregnancy increases <em>angiotensin</em>-(<em>1</em>-<em>7</em>) immunocytochemical expression in association with increased ACE2 intensity of staining. The findings suggest that ACE2 may contribute to the local production and overexpression of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) in the kidney during pregnancy.

The novel peptide, <em>angiotensin</em> (ANG)-(<em>1</em>-<em>1</em>2), elicits a systemic pressor response and vasoconstriction. These effects are blocked by ANG converting enzyme (ACE) inhibitors or AT(<em>1</em>) receptor antagonists, suggesting a role as an ANG II precursor. However, ANG-(<em>1</em>-<em>1</em>2) can serve as a substrate for either ANG II or ANG-(<em>1</em>-<em>7</em>) formation, depending on the local tissue enzymes. Although levels of ANG-(<em>1</em>-<em>1</em>2) are higher than ANG I or ANG II in brain, the role and processing of this peptide for autonomic control of heart rate (HR) has yet to be considered. Thus we examined the effects of nucleus tractus solitarii (NTS) microinjection of ANG-(<em>1</em>-<em>1</em>2) on baroreflex sensitivity for control of HR, resting arterial pressure (AP) and HR, and indexes of sympathovagal balance in urethane/chloralose anesthetized Sprague-Dawley rats. NTS injection of ANG-(<em>1</em>-<em>1</em>2) (<em>1</em>44 fmol/<em>1</em>20 nl) significantly impaired the evoked baroreflex sensitivity to increases in AP [n = <em>7</em>; <em>1</em>.06 +/- 0.06 baseline vs. 0.44 +/- 0.0<em>7</em> ms/mmHg after ANG-(<em>1</em>-<em>1</em>2)], reduced the vagal component of spontaneous baroreflex sensitivity and HR variability, and elicited a transient depressor response (P < 0.05). NTS pretreatment with an AT(<em>1</em>) receptor antagonist or ACE inhibitor prevented ANG-(<em>1</em>-<em>1</em>2)-mediated autonomic and depressor responses. ANG-(<em>1</em>-<em>1</em>2) immunostaining was observed in cells within the NTS of Sprague-Dawley rats, providing a potential intracellular source for the peptide. However, acute NTS injection of an ANG-(<em>1</em>-<em>1</em>2) antibody did not alter resting baroreflex sensitivity, AP, or HR in these animals. Collectively, these findings suggest that exogenous ANG-(<em>1</em>-<em>1</em>2) is processed to ANG II for cardiovascular actions at AT(<em>1</em>) receptors within the NTS. The lack of acute endogenous ANG-(<em>1</em>-<em>1</em>2) tone for cardiovascular regulation in Sprague-Dawley rats contrasts with chronic immunoneutralization in hypertensive rats, suggesting that ANG-(<em>1</em>-<em>1</em>2) may be activated only under hypertensive conditions.

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-<em>angiotensin</em> system composed by <em>angiotensin</em>-converting enzyme 2, <em>angiotensin</em>-(<em>1</em>-<em>7</em>), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic <em>angiotensin</em>-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested <em>1</em>-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-<em>7</em>-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule <em>angiotensin</em>-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (<em>1</em> mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro<em>7</em>-Ang-(<em>1</em>-<em>7</em>), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that <em>angiotensin</em>-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

OBJECTIVE

Recently, recombinant <em>angiotensin</em>-converting enzyme 2 was shown to protect mice from acute lung injury, an effect attributed to reduced bioavailability of <em>angiotensin</em> II. Since <em>angiotensin</em>-converting enzyme 2 metabolizes <em>angiotensin</em> II to <em>angiotensin</em>-(<em>1</em>-<em>7</em>), we hypothesized that this effect is alternatively mediated by <em>angiotensin</em>-(<em>1</em>-<em>7</em>) and activation of its receptor(s).

METHODS

To test this hypothesis, we investigated the effects of intravenously infused <em>angiotensin</em>-(<em>1</em>-<em>7</em>) in three experimental models of acute lung injury.

METHODS

Animal research laboratory.

METHODS

Male Sprague-Dawley rats, Balb/c mice, and C5<em>7</em>Bl6/J mice.

METHODS

Angiotensin-(<em>1</em>-<em>7</em>) was administered with ventilator- or acid aspiration-induced lung injury in mice or 30 minutes after oleic acid infusion in rats. In vitro, the effect of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) on transendothelial electrical resistance of human pulmonary microvascular endothelial cells was analyzed.

RESULTS

Infusion of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) starting 30 minutes after oleic acid administration protected rats from acute lung injury as evident by reduced lung edema, myeloperoxidase activity, histological lung injury score, and pulmonary vascular resistance while systemic arterial pressure was stabilized. Such effects were largely reproduced by the nonpeptidic <em>angiotensin</em>-(<em>1</em>-<em>7</em>) analog AVE099<em>1</em>. Infusion of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) was equally protective in murine models of ventilator- or acid aspiration-induced lung injury. In the oleic acid model, the two distinct <em>angiotensin</em>-(<em>1</em>-<em>7</em>) receptor blockers A<em>7</em><em>7</em>9 and D-Pro-<em>angiotensin</em>-(<em>1</em>-<em>7</em>) reversed the normalizing effects of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) on systemic and pulmonary hemodynamics, but only D-Pro-<em>angiotensin</em>-(<em>1</em>-<em>7</em>) blocked the protection from lung edema and protein leak, whereas A<em>7</em><em>7</em>9 restored the infiltration of neutrophils. Rats were also protected from acute lung injury by the AT<em>1</em> antagonist irbesartan; however, this effect was again blocked by A<em>7</em><em>7</em>9 and D-Pro-<em>angiotensin</em>-(<em>1</em>-<em>7</em>). In vitro, <em>angiotensin</em>-(<em>1</em>-<em>7</em>) protected pulmonary microvascular endothelial cells from thrombin-induced barrier failure, yet D-Pro-<em>angiotensin</em>-(<em>1</em>-<em>7</em>) or NO synthase inhibition blocked this effect.

CONCLUSIONS

Angiotensin-(<em>1</em>-<em>7</em>) or its analogs attenuate the key features of acute lung injury and may present a promising therapeutic strategy for the treatment of this disease.

Diastolic heart failure (DHF) has become a social burden; however, evidences leading to its therapeutic strategy are lacking. This study investigated effects of addition of <em>angiotensin</em> II type <em>1</em> receptor blocker (ARB) to <em>angiotensin</em>-converting enzyme inhibitor (ACEI) at advanced stage of DHF in hypertensive rats. Dahl salt-sensitive rats fed 8% NaCl diet from age <em>7</em> weeks served as DHF model, and those fed a normal chow served as control. The DHF model rats were arbitrarily assigned to 3 treatment regimens at age <em>1</em><em>7</em> weeks: ACEI (temocapril 0.4 mg/kg per day), combination of ACEI (temocapril 0.2 mg/kg per day) with ARB (olmesartan 0.3 mg/kg per day), or placebo. At age <em>1</em><em>7</em> weeks, this model represents progressive ventricular hypertrophy and fibrosis, relaxation abnormality, and myocardial stiffening. Data were collected at age 20 weeks. As compared with the monotherapy with ACEI, the addition of ARB induced more prominent suppression of ventricular hypertrophy and fibrosis, leading to suppression of myocardial stiffening, improvement of relaxation, and inhibition of hemodynamic deterioration. Such benefits were associated with greater decreases in reactive oxygen species (ROS) generation, macrophage infiltration, and gene expression of transforming growth factor (TGF)-beta(<em>1</em>) and interleukin (IL)-<em>1</em>beta, but not with changes in gene expression of monocyte chemoattractant protein (MCP)-<em>1</em> and tumor necrosis factor (TNF)-alpha. Thus, ARB added to ACEI provides more benefits as compared with ACEI alone in DHF when initiated at an advanced stage. The additive effects are likely provided through more prominent suppression of ROS generation and inflammatory changes without effects on expression of MCP-<em>1</em> and TNF-alpha.

Coronaviruses (CoVs) possess an enveloped, single, positive-stranded RNA genome which encodes for four membrane proteins, namely spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins 3-5 [<em>1</em>]. With regard to pathogenicity, S proteins are essential for viral entry into host cells [2, 3]. SARS-CoV binds to the <em>angiotensin</em>-converting enzyme (ACE)2 which is present on nonimmune cells, such as respiratory and intestinal epithelial cells, endothelial cells, kidney cells (renal tubules) and cerebral neurons and immune cells, such as alveolar monocytes/macrophages [4-6]. Of note, CD209L or liver/lymph node special intercellular adhesion molecule-3-grabbing non-integrin (SIGN) and dendritic cell (DC)-SIGN are alternative receptors for SARS-CoV but with lower affinity [<em>7</em>]. In the case of MERS-CoV, S proteins bind to the host cell receptor dipeptidyl peptidase 4 (DPP4 or CD26) which is broadly expressed on intestinal, alveolar, renal, hepatic and prostate cells as well as on activated leukocytes [8]. Then, viruses replicate in target cells with release of mature virions, which, in turn, invade new target cells [9]. Evidence has been provided that SARSCoV proteins are cleaved into two subunits, S<em>1</em> and S2, respectively, and the amino acids 3<em>1</em>8-5<em>1</em>0 of the S<em>1</em> represent the receptor-binding domain (RBD) which binds to ACE2 [<em>1</em>0, <em>1</em><em>1</em>]. Quite importantly, in the context of RBD there is the receptor-binding motif (RBM) (amino acids 424- 494), which accounts for complete binding to ACE2 [<em>1</em><em>1</em>]. Moreover, by means of two residues at positions 4<em>7</em>9 and 48<em>7</em> RBD allows virus progression and tropism [<em>1</em>0, <em>1</em><em>1</em>]. In the case of MERSCoV, its RBM binds to DPP4 with residues 484-56<em>7</em>, thus, suggesting that its RBD differs from that of SARS-CoV [<em>1</em>2, <em>1</em>3]. In a very recent paper, Wan and associates [<em>1</em>4] have investigated the receptor recognition by COVID-<em>1</em>9 (a new term to indicate the 20<em>1</em>9-nCoV in Wuhan) on the bases of structural studies. In this respect, the sequence of COVID-<em>1</em>9 RBM is similar to that of SARSCoV, thus, implicating that ACE2 may represent the binding receptors for COVID-<em>1</em>9. Furthermore, gln493 residue of COVID-<em>1</em>9 RBM seems to allow interaction with human ACE2, thus, suggesting the ability of this virus to infect human cells. According, to Wan and associates structural analysis [<em>1</em>4], COVID-<em>1</em>9 binds to human ACE2 with a lesser efficiency than human SARS-CoV (2002) but with higher affinity than human SARS-CoV (2003). Furthermore, same authors predicted that a single mutation at the 50<em>1</em> position may enhance the COVID-<em>1</em>9 RBD binding capacity to human ACE2 and this evolution should be monitored in infected patients [<em>1</em>4]. These predictive findings by Wan and associates [<em>1</em>4] are confirmed by two contemporary studies by Letko and Muster [<em>1</em>5] and Peng and associates [<em>1</em>6]. In particular, the report by Peng and associates [<em>1</em>6], points out the possible origin of COVID-<em>1</em>9 from bats [<em>1</em>6]. From a pathogenic point of view, evidence has been provided that binding of S2 to ACE2 receptor leads to its down-regulation with subsequent lung damage in the course of SARS-CoV infection [<em>1</em><em>7</em>]. Down-regulation of ACE2 causes excessive production of <em>angiotensin</em> (ANG) II by the related enzyme ACE with stimulation of ANG type <em>1</em>a receptor (AT<em>1</em>R) and enhanced lung vascular permeability [<em>1</em>8]. In particular, same authors have reported that recombinant ACE2 could attenuate severe acute lung injury in mice [<em>1</em>8]. Moreover, Battle and associates [<em>1</em>9] also proposed to use already available recombinant ACE2 for intercepting COVID-<em>1</em>9 and attenuating infection. In the previous paragraphs, the presence of ACE2 on immune cells has been pointed out and, by analogy to epithelial cells, this receptor may also be down-regulated following viral entry. Therefore, in CoV-infected animal models and in infected humans further investigations are required to clarify a possible reduced expression of ACE2 on immune cells. In fact, in the course of SARS-CoV infection, a number of immune disorders have been detected. Three reports have demonstrated the ability of CoV to inhibit interferon (IFN)- production in the course of SARS acting as IFN antagonist [20-22]. In senescent Balb/c mice, depletion of T lymphocytes is associated to more severe interstitial pneumonitis and delayed clearance of SARS-CoV, thus, suggesting a protective role played by these cells [23]. In this connection, both SARS-CoV and MERS-CoV have been shown to induce T cell apoptosis, thus, aggravating the clinical course of disease [24, 25]. Quite interestingly, memory CD8+ T cells specific for SARS-CoV M and N proteins have been detected up to <em>1</em><em>1</em> years post-infection [26]. As far as humoral immune responsiveness is concerned, evidence has been provided that S<em>1</em> subunit from MERS-CoV is highly immunogenic in mice [2<em>7</em>]. Moreover, monoclonal antibodies have been shown to be highly neutralizing against MERS-CoV replication and endowed with post exposure effectiveness in susceptible mice [28, 29]. Human neutralizing antibodies have also been isolated from a recovered patient, thus, suggesting the role of humoral immunity in the control of the persistence of CoV in the host [30]. In particular, IgG response occurs early in infection and its prolonged production may serve for virus clearance during recovery also in view of the absence of viremia in convalescent sera from SARS patients [3<em>1</em>]. According to current literature, severity of COVID-<em>1</em>9 infection correlates with lymphopenia and patients who died from COVID-<em>1</em>9 had lower lymphocyte counts when compared to survivors [32, 33]. These data suggest that lymphocyte-mediated anti-viral activity is poorly effective against COVID-<em>1</em>9. Despite lymphopenia, evidence for an exaggerate release of proinflammatory cytokines [interleukin (IL)-<em>1</em> and IL-6] has been reported in the course acute respiratory syndrome in COVID<em>1</em>9 infected patients, thus, aggravating the clinical course of disease [34]. As recently reported, during COVID-<em>1</em>9 pandemic in both Italy and China higher frequency of fatalities have been observed in the frail elderly population with previous comorbidities [35]. It is well known that decline of immunity occurs in ageing and, therefore, COVID-<em>1</em>9 may gain easier access to the respiratory tract in frail elderly patients [36]. There is evidence that ACE2 protects from severe acute lung failure and operates as a negative regulator of the renin-<em>angiotensin</em> system (RAS) [<em>1</em>8, 3<em>7</em>]. It is well known that ANG II via activation of the AT<em>1</em>R promotes detrimental effects on the host, such as, vasoconstriction, reactive oxygen species generation, inflammation and matrix remodelling [38]. ACE2 counterbalances the noxious effects exhibited by ANG II and AT<em>1</em>R via activation of AT2R which arrests cell growth, inflammation and fibrosis [39]. In this framework, Gurwitz [40] proposed to use AT<em>1</em>R blockers, such as losartan, as a potential treatment of COVID-<em>1</em>9 infection. In fact, losartan as well as olmesartan, used for treating hypertension in patients, were able to increase ACE2 expression after 28 days treatment of rats with myocardial infarction [4<em>1</em>]. Then, Gurwitz suggests to evaluate severity of symptoms in COVID-<em>1</em>9 infected patients under previous chronic treatment with AT<em>1</em>R blockers in comparison to COVID-<em>1</em>9 infected patients who did not take AT<em>1</em>R blockers [40]. Quite interestingly, <em>7</em>5% of aged COVID-<em>1</em>9 infected patients admitted to Italian hospitals had hypertension [unpublished data]. However, the putative effects of ACE-2 down-regulation on the cardiovascular system in the course of COVID-<em>1</em>9 pandemic need more intensive studies. Taken together, these evidences suggest that CoV-induced down-regulation of ACE2 activates RAS with collateral damage to organs, such as lungs, in the course of SARS-related pneumonia. Then, putative therapeutic measures aimed at increasing ACE2 levels on respiratory epithelial cells should be taken into serious consideration. Quite interestingly, over the past few years, three key papers have demonstrated the ability of a polyphenol, resveratrol (RES), to experimentally deactivate the RAS system in maternal and post-weaning high fat diet, arterial ageing and high fat diet, respectively [42-44]. In all these experimental models, RES led to an increase of ACE2 with reduction of organ damage, such as liver steatosis and aorta media thickness and decrease of adipose tissue mass, respectively. As far as the mechanism of action of RES is concerned, this polyphenol is able to activate sirtuin (Sirt)<em>1</em> [45-4<em>7</em>]. In turn, Sirt<em>1</em> down-regulates AT<em>1</em>R expression via ACE2 up-regulation [43, 48]. Of importance, Lin and associates [48] have demonstrated the ability of RES to in vitro inhibit MERS-CoV infection of Vero E6 cells, thus, prolonging cell survival in virtue of an anti-apoptotic mechanism. These findings suggest a direct antiviral effect exerted by RES. It would be very interestingly to evaluate the direct effects of RES on COVID-<em>1</em>9, in vitro. The data above discussed strongly suggest, that RES, as an activators of ACE2, should be investigated in animal models of CoV-induced severe pneumonia, also taking into account the antioxidant, anti-inflammatory and immunomodulating effects exerted by polyphenols [49]. Then, successful animal studies may pave the way for RES-based human trials in COVID-infected patients. Note added in proof During the reviewing process of this perspective other related papers have been published. Hanff and associates [50] have discussed the possible association between COVID-<em>1</em>9-associated cardiovascular mortality and dysregulation of the Renin <em>Angiotensin</em> System (RAS). From a pharmacologic point of view, RAS inhibition leads to upregulation of ACE2, thus, attenuating acute respiratory syndrome and myocarditis in COVID-<em>1</em>9-infected patients. Conversely, increase in ACE2 expression may facilitate the access into the host of COVID-<em>1</em>9, thus, aggravating the clinical picture. Such a dilemma would be solved by clinical trials based on RAS blockade or initiation and monitoring related effects. Contemporarily, Danser and associates [5<em>1</em>] claim that there is no evidence to stop RAS blockers in the course of COVID-<em>1</em>9 infection. In fact, there are no available data which support that ACE inhibitors or ANG II type I receptor blockers increase COVID-<em>1</em>9 infection via its binding to ACE2. Finally, Kuster and associates [52] write that there are no data on the strict relationship between ACE2 activity and SARS-CoV2 mortality. Moreover, in the SARSCoV2, cells expressing ACE2 were not attacked by the virus, while cells lacking ACE2 were bound by the SARS-CoV2 virus [53]. These findings suggest that also in the case of RES effects on COVID-<em>1</em>9 infection, the dual role of ACE2 should be taken into serious consideration.

Exposure of cultured rat aortic vascular smooth muscle (VSM) cells to the Ca2+ ionophore ionomycin produced an increase in extracellular signal-regulated kinase <em>1</em>/2 (ERK<em>1</em>/2) activity that was maximal between 2 and 5 minutes but then declined to basal values within 20 minutes of stimulation. Elevation of [Ca2+]i in VSM cells leads to an even more rapid activation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II); thus, it was postulated that the Ca(2+)-dependent component of ERK<em>1</em>/2 activation was mediated by CaM kinase II. Transient ERK<em>1</em>/2 activation by ionomycin was almost completely abolished by pretreating cells with 30 mumol/L KN-93, a CaM kinase II inhibitor. Treatment of cells with KN-93 did not antagonize the ability of ionomycin to mobilize intracellular Ca2+ but prevented CaM kinase II and ERK<em>1</em>/2 activation with almost identical potencies. Consistent with a role for Ca2+ and calmodulin in intracellular Ca(2+)-induced activation of ERK, cells pretreated with calmodulin inhibitors (W-<em>7</em> or calmidazolium) exhibited an attenuated ERK response to ionomycin. ERK<em>1</em>/2 activation in response to phorbol esters and platelet-derived growth factor were not significantly affected by KN-93, whereas the response to <em>angiotensin</em> II and thrombin were attenuated by 60% and 40%, respectively. Transient expression of wild-type delta 2 CaM kinase II in COS-<em>7</em> cells resulted in increased ERK2 activity, whereas coexpression of wild-type and a kinase-negative mutant resulted in a diminution of this response. These data suggest that regulation of cellular responses by Ca(2+)-dependent pathways in VSM cells may be mediated in part by CaM kinase II-dependent activation of ERK<em>1</em>/2.

We have studied cardiovascular and renal phenotypes in Npr<em>1</em> (genetic determinant of natriuretic peptide receptor-A; NPRA) gene-disrupted mutant mouse model. The baseline systolic arterial pressure (SAP) in 0-copy mutant (-/-) mice (<em>1</em>43 +/- 2 mmHg) was significantly higher than in 2-copy wild-type (+/+) animals (<em>1</em>04 +/- 2 mmHg); however, the SAP in <em>1</em>-copy heterozygotes (+/-) was at an intermediate value (<em>1</em>20 +/- 4 mmHg). To determine whether Npr<em>1</em> gene function affects the renin-<em>angiotensin</em>-aldosterone system (RAAS), we measured the components of RAAS in plasma, kidney, and adrenal gland of 0-copy, <em>1</em>-copy, and 2-copy male mice. Newborn (2 days after the birth) 0-copy pups showed 2.5-fold higher intrarenal renin contents compared with 2-copy wild-type counterparts (0-copy <em>7</em>2 +/- <em>1</em>2 vs. 2-copy 30 +/- <em>7</em> microg ANG I. mg protein(-<em>1</em>). h(-<em>1</em>), respectively). The intrarenal ANG II level in 0-copy pups was also higher than in 2-copy controls (0-copy 33 +/- 5 vs. 2-copy 20 +/- 2 pg/mg protein, respectively). However, both young (3 wk) and adult (<em>1</em>6 wk) 0-copy mutant mice showed a dramatic 50-80% reduction in plasma renin concentrations (PRCs) and in expression of renal renin message compared with 2-copy control animals. In contrast, the adrenal renin content and mRNA expression levels were <em>1</em>.5- to 2-fold higher in 0-copy adult mice than in 2-copy animals. The results suggest that inhibition of renal and systemic RAAS is a compensatory response that prevents greater increases in elevated arterial pressures in adult NPRA null mutant mice. However, the greater renin and ANG II levels seen in 0-copy newborn pups provide evidence that the direct effect of NPRA activation on renin is an inhibitory response.

New studies suggest that vasodilator systems may play an important role in restraining the rise in peripheral vascular resistance associated with the evolution of arterial hypertension. We characterized in conscious dogs the hemodynamic and hormonal effects of 4 weeks of feeding either the nitric oxide synthase inhibitor N omega-nitro-L-arginine (3 mg.kg-<em>1</em>.d-<em>1</em>) or the nitric oxide precursor L-arginine (0.3 mg.kg-<em>1</em>.d-<em>1</em>) during the evolution of two-kidney, one clip hypertension. Inhibition of nitric oxide production elicited a form of hypertension more severe than that produced in placebo-fed two-kidney, one clip dogs. The higher levels of blood pressure were accompanied by lower levels of plasma renin activity and lower <em>angiotensin</em> II concentrations. During the chronic phase of renovascular hypertension, the fall in blood pressure produced by acute systemic injections of lisinopril or losartan was significantly reduced in dogs given the nitric oxide inhibitor. In contrast, chronic administration of L-arginine had no effect on the magnitude of hypertension or on the increases in renin activity and hyper<em>angiotensin</em>emia associated with the evolution of renal hypertension. Likewise, the fall in blood pressure produced by pharmacological blockade of <em>angiotensin</em> II was not different from that recorded in untreated renal hypertensive dogs. The vasodilator component of the blood pressure response due to intravenous injections of <em>angiotensin</em>-(<em>1</em>-<em>7</em>) (<em>1</em> to <em>1</em>00 nmol/kg) was augmented in both untreated and L-arginine-treated two-kidney, one clip hypertensive dogs, but was significantly attenuated in hypertensive dogs fed the nitric oxide synthase inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracranial stenosis is a common etiology for ischemic stroke. Due to limitations of imaging studies, there are limited data on the prevalence of symptomatic and asymptomatic intracranial stenosis. Intracranial stenosis is more prevalent in Asian, Hispanic, and African-American populations. The reported proportion of patients with symptomatic intracranial stenosis among those hospitalized for ischemic cerebral events varies from <em>1</em>% in non-Hispanic whites to as high as 50% in Asian populations. In population-based studies, the estimated prevalence of symptomatic intracranial disease varies from <em>1</em> in <em>1</em>00,000 for whites to <em>1</em>5 in <em>1</em>00,000 in African Americans. A Chinese population-based study reported intracranial stenosis in <em>7</em>% of the population aged more than 40 years. Autopsy studies have noted intracranial atherosclerotic disease in about 23% of population in the 6th decade and 80% of population in the 9th decade of life. <em>Angiotensin</em>-converting enzyme polymorphisms, plasma endostatin/vascular endothelial growth factor ratio, glutathione S-transferase omega-<em>1</em> gene polymorphism, and plasma homocysteine levels are non-modifiable risk factors noted to be associated with intracranial stenosis. Hypertension and serum lipid profile are major modifiable risk factors, whereas sickle cell disease is an uncommon risk factor that can be managed to reduce risk. Associations of intracranial atherosclerosis with diabetes mellitus, metabolic syndrome, Alzheimer's disease, aortic plaques, radiotherapy, and meningitis are less well documented.

TCV-<em>1</em><em>1</em>6 [(+/-)-(cyclohexyloxycarbony-loxy)ethyl2-ethoxy-<em>1</em>-[[2' -(<em>1</em>H- tetrazol-5-yl)biphenyl-4-yl]methyl]-<em>1</em>H-benzimidazole-<em>7</em>-carboxylate ], a nonpeptide selective <em>angiotensin</em> II type I receptor (AT<em>1</em> receptor) antagonist, at the dose of 0.<em>1</em>, <em>1</em> or <em>1</em>0 mg kg-<em>1</em> day-<em>1</em>, was orally given to 22-week-old stroke-prone spontaneously hypertensive rats (SHRSP) for <em>1</em>0 weeks (from the age of 22-32 weeks) to examine the effects on gene expression of transforming growth factor-beta <em>1</em> (TGF-beta <em>1</em>) and extracellular matrix proteins in the heart and blood vessels. Tissue messenger RNA (mRNA) was measured by northern blot analysis, with a specific complementary DNA probe. In the heart, left ventricular mRNA levels for fibronectin; types I, III and IV collagen; and laminin were significantly higher in SHRSP than control Wistar-Kyoto rats. In the mesenteric artery and aorta of SHRSP, TGF-beta <em>1</em> mRNA and the mentioned extracellular matrix protein mRNAs were increased compared with Wistar-Kyoto rats. Thus, the expression of various genes was up-regulated in cardiovascular tissues of SHRSP. Treatment of SHRSP with TCV-<em>1</em><em>1</em>6 suppressed the gene expression of the mentioned extracellular matrix proteins and TGF-beta <em>1</em> in both heart and blood vessels in a dose-dependent fashion. Furthermore, TCV-<em>1</em><em>1</em>6 regressed cardiac hypertrophy and lessened the medial hypertrophy of the aorta in SHRSP. These results show that <em>angiotensin</em> AT<em>1</em> receptor antagonist in vivo can inhibit the gene expression of TGF-beta <em>1</em> and extracellular matrix proteins in hypertensive cardiovascular tissues. These effects may contribute to the beneficial effects of AT<em>1</em> receptor antagonist on hypertensive cardiac hypertrophy and vascular thickening.

There is now unequivocal evidence that the <em>angiotensin</em>-converting enzyme 2(ACE2)-Ang-(<em>1</em>-<em>7</em>)-Mas axis is a key component of the renin-<em>angiotensin</em> system (RAS) cascade, which is closely correlated with ischemic insult occurrence. Our previous studies demonstrated that the Ang-(<em>1</em>-<em>7</em>), was an active member of the brain RAS. However, the ACE2-Ang-(<em>1</em>-<em>7</em>)-Mas axis expression after cerebral ischemic injury are currently unclear. In the present study, we investigated the time course of ACE2-Ang-(<em>1</em>-<em>7</em>) and Mas receptor expression in the acute stage of cerebral ischemic stroke. The content of Ang-(<em>1</em>-<em>7</em>) in ischemic tissues and blood serum was measured by specific EIA kits. Real-time PCR and western blot were used to determine messenger RNA (mRNA) and protein levels of the ACE2 and Mas. The cerebral ischemic lesion resulted in a significant increase of regional cerebral and circulating Ang-(<em>1</em>-<em>7</em>) at 6-48 h compared with sham operation group following focal ischemic stroke (<em>1</em>2h: <em>7</em>.2<em>7</em>6±0.320 ng/ml vs. 2.466±0.4<em>1</em>0 ng/ml, serum; <em>1</em>.024±0.056 ng/mg vs. 0.499±0.032, brain) (P<0.05). Both ACE2 and Mas expression were markedly enhanced compared to the control in the ischemic tissues (P<0.05). Mas immunopositive neurons were also seen stronger expression in the ischemic cortex (<em>1</em>9.<em>1</em>6<em>7</em>±2.858 vs. <em>7</em>.833±2.483) (P<0.05). The evidence collected in our present study will indicate that, ACE2-Ang-(<em>1</em>-<em>7</em>)-Mas axis are upregulated after acute ischemic stroke and would play a pivotal role in the regulation of acute neuron injury in ischemic cerebrovascular diseases.

<em>Angiotensin</em> II receptor blockers (ARBs) are widely used for the treatment of hypertension. It is believed that treatment with an ARB increases the level of plasma <em>angiotensin</em> II (Ang II) because of a lack of negative feedback on renin activity. However, Ichikawa (Hypertens Res 200<em>1</em>; 24: 64<em>1</em>-646) reported that long-term treatment of hypertensive patients with olmesartan resulted in a reduction in plasma Ang II level, though the mechanism was not determined. It has been reported that <em>angiotensin</em> <em>1</em>-<em>7</em> (Ang-(<em>1</em>-<em>7</em>)) potentiates the effect of bradykinin and acts as an <em>angiotensin</em>-converting enzyme (ACE) inhibitor. It is known that ACE2, which was discovered as a novel ACE-related carboxypeptidase in 2000, hydrolyzes Ang I to Ang-(<em>1</em>-9) and also Ang II to Ang-(<em>1</em>-<em>7</em>). It has recently been reported that olmesartan increases plasma Ang-(<em>1</em>-<em>7</em>) through an increase in ACE2 expression in rats with myocardial infarction. We hypothesized that over-expression of ACE2 may be related to a reduction in Ang II level and the cardioprotective effect of olmesartan. Administration of 0.5 mg/kg/day of olmesartan for 4 weeks to <em>1</em>2-week-old stroke-prone spontaneously hypertensive rats (SHRSP) significantly reduced blood pressure and left ventricular weight compared to those in SHRSP given a vehicle. Co-administration of olmesartan and (D-Ala<em>7</em>)-Ang-(<em>1</em>-<em>7</em>), a selective Ang-(<em>1</em>-<em>7</em>) antagonist, partially inhibited the effect of olmesartan on blood pressure and left ventricular weight. Interestingly, co-administration of (D-Ala<em>7</em>)-Ang-(<em>1</em>-<em>7</em>) with olmesartan significantly increased the plasma Ang II level (453.2+/-<em>1</em><em>1</em>3.8 pg/ml) compared to olmesartan alone (<em>1</em>44.9+/-2<em>7</em>.0 pg/ml, p<0.05). Moreover, olmesartan significantly increased the cardiac ACE2 expression level compared to that in Wistar Kyoto rats and SHRSP treated with a vehicle. Olmesartan significantly improved cardiovascular remodeling and cardiac nitrite/ nitrate content, but co-administration of olmesartan and (D-Ala<em>7</em>)-Ang-(<em>1</em>-<em>7</em>) partially reversed this anti-remodeling effect and the increase in nitrite/nitrate. These findings suggest that olmesartan may exhibit an ACE inhibitory action in addition to an Ang II receptor blocking action, prevent an increase in Ang II level, and protect cardiovascular remodeling through an increase in cardiac nitric oxide production and endogenous Ang-(<em>1</em>-<em>7</em>) via over-expression of ACE2.

OBJECTIVE

To examine the association between renin-angiotensin-aldosterone system (RAAS) genes and salt sensitivity of blood pressure (BP).

METHODS

A 7-day low-sodium dietary intervention followed by a 7-day high-sodium dietary intervention was conducted among 1906 participants living in a rural region of north China where habitual sodium intake is high. BP measurements were obtained at baseline and following each intervention using a random-zero sphygmomanometer.

RESULTS

DBP and mean arterial pressure responses increased with the number of rs4524238 A alleles in the angiotensin II receptor type 1 gene. For example, mean DBP responses (95% confidence interval) among those with genotypes G/G, G/A, and A/A were -2.53 (-2.89 to -2.18), -3.49 (-4.13 to -2.86), and -5.78 (-9.51 to -2.06) mmHg, respectively, following the low-sodium intervention (P=0.0008). Carriers of the rare A allele of rs5479 in the hydroxysteroid (11-beta) dehydrogenase 2 gene had decreased DBP responses to low sodium (P=0.00004). Those with the C/A and C/C genotypes had DBP responses of -0.70 (-6.62 to 5.22) and -2.71 (-4.88 to -0.54) mmHg, respectively. X chromosome renin-binding protein gene markers rs1557501 and rs2269372 were associated with SBP response to low sodium in men (P=0.00004 and 0.0001, respectively). SBP responses (95% confidence interval) were -6.13 (-6.68 to -5.58) versus -4.07 (-4.88 to -3.26) and -6.04 (-6.57 to -5.52) versus -3.94 (-4.90 to -2.99) mmHg among men with major versus those with minor alleles of rs1557501 and rs2269372, respectively. Haplotype analyses of these genes supported our single-marker findings.

CONCLUSIONS

We identified renin-angiotensin-aldosterone system variants that were predictive of salt sensitivity in a Han population with habitually high-sodium intake.

Recently, we demonstrated that the endothelium-dependent vasodilator effect of <em>angiotensin</em>(<em>1</em>-<em>7</em>) in the mouse aorta is abolished by genetic deletion of the G protein-coupled receptor encoded by the Mas protooncogene. To circumvent the limitations posed by the possible metabolism of Ang(<em>1</em>-<em>7</em>) in this vessel, in this work we studied the mechanism underlying the vasorelaxant effect of AVE 099<em>1</em>, a nonpeptide mimic of the effects of Ang(<em>1</em>-<em>7</em>), using wild-type and Mas-deficient mice. Ang(<em>1</em>-<em>7</em>) and AVE 099<em>1</em> induced an equipotent concentration-dependent vasodilator effect in aortic rings from wild-type mice that was dependent on the presence of endothelium. The vasodilator effect of Ang(<em>1</em>-<em>7</em>) and AVE 099<em>1</em> was completely blocked by 2 specific Ang(<em>1</em>-<em>7</em>) receptor antagonists, A-<em>7</em><em>7</em>9 and D-Pro-Ang(<em>1</em>-<em>7</em>), and by inhibition of NO synthase with L-NAME. Moreover, in aortic rings from Mas-deficient mice, the vasodilator effect of both Ang(<em>1</em>-<em>7</em>) and AVE 099<em>1</em> was abolished. In contrast, the vasodilator effect of acetylcholine and substance P were preserved in Mas-null mice. In addition, the vasoconstriction effect induced by Ang II was slightly increased, and the vasodilation induced by the AT2 agonist CGP 42<em>1</em><em>1</em>2A was not altered in Mas-deficient mice. Our results show that Ang(<em>1</em>-<em>7</em>) and AVE 099<em>1</em> produced an NO-dependent vasodilator effect in the mouse aorta that is mediated by the G protein-coupled receptor Mas.

Inappropriate activation of the renin-<em>angiotensin</em> system (RAS) exacerbates renal and vascular injury. Accordingly, treatment with global RAS antagonists attenuates cardiovascular risk and slows the progression of proteinuric kidney disease. By reducing BP, RAS inhibitors limit secondary immune activation responding to hemodynamic injury in the target organ. However, RAS activation in hematopoietic cells has immunologic effects that diverge from those of RAS stimulation in the kidney and vasculature. In preclinical studies, activating type <em>1</em> <em>angiotensin</em> (AT<em>1</em>) receptors in T lymphocytes and myeloid cells blunts the polarization of these cells toward proinflammatory phenotypes, protecting the kidney from hypertensive injury and fibrosis. These endogenous functions of immune AT<em>1</em> receptors temper the pathogenic actions of renal and vascular AT<em>1</em> receptors during hypertension. By counteracting the effects of AT<em>1</em> receptor stimulation in the target organ, exogenous administration of AT2 receptor agonists or <em>angiotensin</em> <em>1</em>-<em>7</em> analogs may similarly limit inflammatory injury to the heart and kidney. Moreover, although <em>angiotensin</em> II is the classic effector molecule of the RAS, several RAS enzymes affect immune homeostasis independently of canonic <em>angiotensin</em> II generation. Thus, as reviewed here, multiple components of the RAS signaling cascade influence inflammatory cell phenotype and function with unpredictable and context-specific effects on innate and adaptive immunity.

The renin-<em>angiotensin</em> system (RAS) is well-recognized as one of the oldest and most important regulators of arterial blood pressure, cardiovascular, and renal function. New frontiers have recently emerged in the RAS research well beyond its classic paradigm as a potent vasoconstrictor, an aldosterone release stimulator, or a sodium-retaining hormone. First, two new members of the RAS have been uncovered, which include the renin/(Pro)renin receptor (PRR) and <em>angiotensin</em>-converting enzyme 2 (ACE2). Recent studies suggest that prorenin may act on the PRR independent of the classical ACE/ANG II/AT<em>1</em> receptor axis, whereas ACE2 may degrade ANG II to generate ANG (<em>1</em>-<em>7</em>), which activates the Mas receptor. Second, there is increasing evidence that ANG II may function as an intracellular peptide to activate intracellular and/or nuclear receptors. Third, currently there is a debate on the relative contribution of systemic versus intrarenal RAS to the physiological regulation of blood pressure and the development of hypertension. The objectives of this article are to review and discuss the new insights and perspectives derived from recent studies using novel transgenic mice that either overexpress or are deficient of one key enzyme, ANG peptide, or receptor of the RAS. This information may help us better understand how ANG II acts, both independently or through interactions with other members of the system, to regulate the kidney function and blood pressure in health and disease.

BACKGROUND

Neurogenic hypertension is characterized by an increase in sympathetic activity and often resistance to drug treatments. We previously reported that it is also associated with a reduction of <em>angiotensin</em>-converting enzyme type 2 (ACE2) and an increase in a disintegrin and metalloprotease <em>1</em><em>7</em> (ADAM<em>1</em><em>7</em>) activity in experimental hypertension. In addition, while multiple cells within the central nervous system have been involved in the development of neurogenic hypertension, the contribution of ADAM<em>1</em><em>7</em> has not been investigated.

OBJECTIVE

To assess the clinical relevance of this ADAM<em>1</em><em>7</em>-mediated ACE2 shedding in hypertensive patients and further identify the cell types and signaling pathways involved in this process.

RESULTS

Using a mass spectrometry-based assay, we identified ACE2 as the main enzyme converting <em>angiotensin</em> II into <em>angiotensin</em>-(<em>1</em>-<em>7</em>) in human cerebrospinal fluid. We also observed an increase in ACE2 activity in the cerebrospinal fluid of hypertensive patients, which was correlated with systolic blood pressure. Moreover, the increased level of tumor necrosis factor-α in those cerebrospinal fluid samples confirmed that ADAM<em>1</em><em>7</em> was upregulated in the brain of hypertensive patients. To further assess the interaction between brain renin-<em>angiotensin</em> system and ADAM<em>1</em><em>7</em>, we generated mice lacking <em>angiotensin</em> II type <em>1</em> receptors specifically on neurons. Our data reveal that despite expression on astrocytes and other cells types in the brain, ADAM<em>1</em><em>7</em> upregulation during deoxycorticosterone acetate-salt hypertension occurs selectively on neurons, and neuronal <em>angiotensin</em> II type <em>1</em> receptors are indispensable to this process. Mechanistically, reactive oxygen species and extracellular signal-regulated kinase were found to mediate ADAM<em>1</em><em>7</em> activation.

CONCLUSIONS

Our data demonstrate that <em>angiotensin</em> II type <em>1</em> receptors promote ADAM<em>1</em><em>7</em>-mediated ACE2 shedding in the brain of hypertensive patients, leading to a loss in compensatory activity during neurogenic hypertension.

The renin-<em>angiotensin</em> system (RAS) in brain is a crucial regulator for physiological homeostasis and diseases of cerebrovascular system, such as ischemic stroke. Overactivation of brain <em>Angiotensin</em>-converting enzyme (ACE) - <em>Angiotensin</em> II (Ang II) - <em>Angiotensin</em> II type <em>1</em> receptor (AT<em>1</em>R) axis was found to be involved in the progress of hypertension, atherosclerosis and thrombogenesis, which increased the susceptibility to ischemic stroke. Besides, brain Ang II levels have been revealed to be increased in ischemic tissues after stroke, and contribute to neural damage through elevating oxidative stress levels and inducing inflammatory response in the ischemic hemisphere via AT<em>1</em>R. In recent years, new components of RAS have been discovered, including ACE2, <em>Angiotensin</em>-(<em>1</em>-<em>7</em>) [Ang-(<em>1</em>-<em>7</em>)] and Mas, which constitute ACE2-Ang-(<em>1</em>-<em>7</em>)-Mas axis. ACE2 converts Ang II to Ang-(<em>1</em>-<em>7</em>), and Ang-(<em>1</em>-<em>7</em>) binds with its receptor Mas, exerting benefical effects in cerebrovascular disease. Through interacting with nitric oxide and bradykinin, Ang-(<em>1</em>-<em>7</em>) could attenuate the development of hypertension and the pathologic progress of atherosclerosis. Besides, its antithrombotic activity also prevents thrombogenic events, which may contribute to reduce the risk of ischemic stroke. In addition, after ischemia insult, ACE2-Ang-(<em>1</em>-<em>7</em>)-Mas has been shown to reduce the cerebral infarct size and improve neurological deficits through its antioxidative and anti-inflammatory effects. Taken together, activation of the ACE2-Ang-(<em>1</em>-<em>7</em>)-Mas axis may become a novel therapeutic target in prevention and treatment of ischemia stroke, which deserves further investigations.

Captopril and enalapril-<em>angiotensin</em>-converting enzyme (ACE) inhibitors-were evaluated for their antioxidative protective action against adriamycin-induced cardiac and hepatic toxicity. Rats were treated with either captopril (<em>1</em>0 mg kg(-<em>1</em>)) or enalapril (2 mg kg(-<em>1</em>)) intragastrically (i.g.) daily for <em>7</em> days before single intraperitoneal (i.p.) injection with adriamycin (<em>1</em>5 mg kg(-<em>1</em>)). The animals were killed 30 h after adriamycin administration. Adriamycin produced significant elevation in thiobarbituric acid reactive substances (TBARS), which is an indicator of lipid peroxidation, and significantly inhibited the activity of superoxide dismutase (SOD) in heart and liver tissues, with a significant rise in the serum levels of glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), creatine kinase isoenzyme (CK-MB) and lactic dehydrogenase (LDH), indicating acute cardiac toxicity. A single injection of adriamycin did not affect the cardiac or hepatic glutathione (GSH) content or cardiac catalase (CAT) activity, but hepatic CAT activity was elevated. Pretreatment with ACE inhibitors significantly reduced the TBARS concentration in both heart and liver and ameliorated the inhibition of cardiac and hepatic SOD activity. In addition, the ACE inhibitors significantly improved the serum levels of GOT, GPT, CK-MB and LDH in adriamycin-treated rats. Thus, these results suggest that captopril and enalapril possess antioxidative potential that may protect the heart against adriamycin-induced acute oxidative toxicity. This protective effect might be mediated, at least in part, by the limitation of culprit free radicals and the amelioration of oxidative stress.