Acute and chronic angiotensin-(1-7) restores vasodilation and reduces oxidative stress in mesenteric arteries of salt-fed rats

This study determined the effect of ANG-(1-7) on salt-induced suppression of endothelium-dependent vasodilatation in the mesenteric arteries of male Sprague-Dawley rats. Chronic intravenous infusion of ANG-(1-7), oral administration of the nonpeptide mas receptor agonist AVE-0991, and acute preincubation of the arteries with ANG-(1-7) and AVE-0991 all restored vasodilator responses to both ACh and histamine that were absent in the arteries of rats fed a high-salt (4% NaCl) diet. The protective effects of ANG-(1-7) and AVE-0991 were inhibited by acute or chronic administration of the mas receptor antagonist A-779, the ANG II type 2 (AT(2)) receptor blocker PD-123319, or N-nitro-l-arginine methyl ester, but not the ANG II type 1 receptor antagonist losartan. Preincubation with the antioxidant tempol or the nitric oxide (NO) donor diethylenetriamine NONOate and acute and chronic administration of the AT(2) receptor agonist CGP-42112 mimicked the protective effect of ANG-(1-7) to restore vascular relaxation. Acute preincubation with ANG-(1-7) and chronic infusion of ANG-(1-7) ameliorated the elevated superoxide levels in rats fed a high-salt diet, but the expression of Cu/Zn SOD and Mn SOD enzyme proteins in the vessel wall was unaffected by ANG-(1-7) infusion. These results indicate that both acute and chronic systemic administration of ANG-(1-7) or AVE-0991 restore endothelium-dependent vascular relaxation in salt-fed Sprague-Dawley rats by reducing vascular oxidant stress and enhancing NO availability via mas and AT(2) receptors. These findings suggest a therapeutic potential for mas/AT(2) receptor activation in preventing the vascular oxidant stress and endothelial dysfunction associated with elevated dietary salt intake.     

Evidence that the vasodilator angiotensin-(1-7)-Mas axis plays an important role in erectile function

The vasodilator/antiproliferative peptide angiotensin-(1-7) [ANG-(1-7)] is released into the corpus cavernosum sinuses, but its role in erectile function has yet to be defined. In this study, we sought to determine whether ANG-(1-7) and its receptor Mas play a role in erectile function. The ANG-(1-7) receptor Mas was immunolocalized in rat corpus cavernosum by confocal microscopy. Infusion of ANG-(1-7) into corpus cavernosum at a rate of 15.5 pmol x kg(-1) x min(-1) potentiated the elevation of the corpus cavernosum pressure induced by electrical stimulation of the major pelvic ganglion (MPG) in rats. The facilitatory effect of ANG-(1-7) was completely blunted by the specific ANG-(1-7) receptor blocker A-779 and N(omega)-nitro-L-arginine methyl ester. Nitric oxide (NO) release in the corpus cavernosum was evaluated with the fluorescent dye 4-amino-5 methylamino-2',7'-difluorofluorescein diacetate. Electrical stimulated-release of NO in rat corpus cavernosum was potentiated by ANG-(1-7). Furthermore, incubation of rat and mouse corpus cavernosum strips with ANG-(1-7) at 10 nmol/l resulted in an increase of NO release. This effect was completely abolished in mas-deficient mice. More importantly, genetic deletion of Mas resulted in compromised erectile function as demonstrated by penile fibrosis and severely depressed response to electrical stimulation of the MPG. Furthermore, the attenuated erectile function of DOCA-salt hypertensive rats was fully restored by ANG-(1-7) administration. Together these data provide strong evidence for a key role of the ANG-(1-7)-Mas axis in erectile function.     

Angiotensin-(1-7) inhibits growth of cardiac myocytes through activation of the mas receptor

Peptide hormones such as ANG II and endothelin contribute to cardiac remodeling after myocardial infarction by stimulating myocyte hypertrophy and myofibroblast proliferation. In contrast, angiotensin-(1-7) [ANG-(1-7)] infusion after myocardial infarction reduced myocyte size and attenuated ventricular dysfunction and remodeling. We measured the effect of ANG-(1-7) on protein and DNA synthesis in cultured neonatal rat myocytes to assess the role of the heptapeptide in cell growth. ANG-(1-7) significantly attenuated either fetal bovine serum- or endothelin-1-stimulated [(3)H]leucine incorporation into myocytes with no effect on [(3)H]thymidine incorporation. [d-Ala(7)]-ANG-(1-7), the selective ANG type 1-7 (AT(1-7)) receptor antagonist, blocked the ANG-(1-7)-mediated reduction in protein synthesis in cardiac myocytes, whereas the AT(1) and AT(2) angiotensin peptide receptors were ineffective. Serum-stimulated ERK1/ERK2 mitogen-activated protein kinase activity was significantly decreased by ANG-(1-7) in myocytes, a response that was also blocked by [d-Ala(7)]-ANG-(1-7). Both rat heart and cardiac myocytes express the mRNA for the mas receptor, and a 59-kDa immunoreactive protein was identified in both extracts of rat heart and cultured myocytes by Western blot hybridization with the use of an antibody to mas, an ANG-(1-7) receptor. Transfection of cultured myocytes with an antisense oligonucleotide to the mas receptor blocked the ANG-(1-7)-mediated inhibition of serum-stimulated MAPK activation, whereas a sense oligonucleotide was ineffective. These results suggest that ANG-(1-7) reduces the growth of cardiomyocytes through activation of the mas receptor. Because ANG-(1-7) is elevated after treatment with angiotensin-converting enzyme inhibitors or AT(1) receptor blockers, ANG-(1-7) may contribute to their beneficial effects on cardiac dysfunction and ventricular remodeling after myocardial infarction.     

Angiotensin-(1--7) in the ovine fetus

In the adult animal, ANG-(1-7) may counterbalance some effects of ANG II. Its effects in the fetus are unknown. Basal ANG-(1-7), ANG I, ANG II, and renin concentrations were measured in plasma from ovine fetuses and their mothers (n = 10) at 111 days of gestation. In the fetus, concentrations of ANG I, ANG-(1-7), and ANG II were 86 +/- 21, 13 +/- 2, and 14 +/- 2 fmol/ml, respectively. In the ewe, concentrations of ANG I were significantly lower (20 +/- 4 fmol/ml, P < 0.05) as were concentrations of ANG-(1-7) (2.9 +/- 0.6 fmol/ml), whereas ANG II concentrations were not different (10 +/- 1 fmol/ml). Plasma renin concentrations were higher in the fetus (4.8 +/- 1.1 pmol ANG I x ml(-1) x h(-1)) than in the ewe (0.9 +/- 0.2 pmol x ml(-1) x h(-1), P < 0.05). Infusion of ANG-(1-7) (approximately 9 microg/h) for a 3-day period caused a significant increase in plasma concentrations of ANG-(1-7) reaching a maximum of 448 +/- 146 fmol/ml on day 3 of infusion. Plasma levels of ANG I and II as well as renin were unchanged by the infusion. Urine flow rate, glomerular filtration rate, and fetal arterial blood pressure did not change and were not different than values in fetuses receiving a saline infusion for 3 days (n = 5). However, the osmolality of amniotic and allantoic fluid was significantly higher in fetuses that received ANG-(1-7). Also, compared with the saline-infused animals, mRNA expression levels of renin, the AT(1) receptor, and AT(2) receptor were elevated in kidneys of fetuses that received infusions of ANG-(1-7). Infusion of an ANG-(1-7) antagonist ([D-Ala(7)]-ANG-(1-7), 20 microg/h) for 3 days had no effect on fetal blood pressure or renal function. In conclusion, although infusion of ANG-(1-7) did not affect fetal urine flow rate, glomerular filtration rate, or blood pressure, changes in fetal fluids and gene expression indicate that ANG-(1-7) may play a role in the fetal kidney.     

Opposing actions of angiotensins on angiogenesis

Using the murine sponge model of angiogenesis, associated to functional and morphological parameters we have demonstrated opposing actions of angiotensin II (Ang II) and angiotensin-(1-7;Ang-1-7) in modulating fibrovascular tissue growth. Angiogenesis in the implants was assessed at day 7 postimplantation by extracting the hemoglobin content, by determining the outflow rate of sodium fluorescein applied intraimplant and by histological analysis. Furthermore, the proliferative activity of control and angiotensin-treated implants was established using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4 -sulfonyl)2H-tetrazolium)assay. The hemoglobin content in the control implants was 2.4 +/- 0.14 (microg/mg wet weight) versus 3.6 +/- 0.27(Ang II;100 ng) and 0.86 +/- 0.07 Ang-(1-7); 20 ng. Blood flow in the implants as determined by t1/2 values (time taken for the fluorescence to reach 50% of the peak in the systemic circulation) showed that Ang II stimulated angiogenesis, whereas Ang-(1-7) inhibited it. The proliferative activity of the sponge-induced fibrovascular tissue was enhanced by Ang II and diminished by Ang-(1-7). These results show the pro-versus anti-angiogenic effects of these angiotensin molecules, providing evidence for their opposing effects on vascular tissue growth and wound healing in vivo.     

Angiopoietin: a TIE(d) balance in tumor angiogenesis

Angiopoietins (ANG-1 and ANG-2) and their TIE-2 receptor tyrosine kinase have wide-ranging effects on tumor malignancy that includes angiogenesis, inflammation, and vascular extravasation. These multifaceted pathways present a valuable opportunity in developing novel inhibition strategies for cancer treatment. However, the regulatory role of ANG-1 and ANG-2 in tumor angiogenesis remains controversial. There is a complex interplay between complementary yet conflicting roles of both the ANGs in shaping the outcome of angiogenesis. Embryonic vascular development suggests that ANG-1 is crucial in engaging interaction between endothelial and perivascular cells. However, recruitment of perivascular cells by ANG-1 has recently been implicated in its antiangiogenic effect on tumor growth. It is becoming clear that TIE-2 signaling may function in a paracrine and autocrine manner directly on tumor cells because the receptor has been increasingly found in tumor cells. In addition, alpha(5)beta(1) and alpha(v)beta(5) integrins were recently recognized as functional receptors for ANG-1 and ANG-2. Therefore, both the ligands may have wide-ranging functions in cellular activities that affect overall tumor development. Collectively, these TIE-2-dependent and TIE-2-independent activities may account for the conflicting findings of ANG-1 and ANG-2 in tumor angiogenesis. These uncertainties have impeded development of a clear strategy to target this important angiogenic pathway. A better understanding of the molecular basis of ANG-1 and ANG-2 activity in the pathophysiologic regulation of angiogenesis may set the stage for novel therapy targeting this pathway.     

Stromal vascular fraction cells plus sustained release VEGF/Ang-1-PLGA microspheres improve fat graft survival in mice

Autologous fat transplantation is increasingly applied in plastic and reconstructive surgery. Stromal vascular fraction cells (SVFs) combined with angiogenic factors, such as VEGF (vascular endothelial growth factor A) and Ang-1 (angiogenin-1), can improve angiogenesis, which is a critical factor for graft survival. However, direct transplant with such a mixture is insufficient owing to the short half-life of angiogenic factors. In this study, we evaluated whether a double sustained release system of VEGF/ANG-1-PLGA (poly (lactic-co-glycolic acid)) microspheres plus SVFs can improve angiogenesis and graft survival after autologous fat transplantation. VEGF/ANG-1-PLGA-sustained release microspheres were fabricated by a modified double emulsion–solvent evaporation technique. Human aspirated fat was mixed with SVF suspension plus VEGF/ANG-1 sustained release microspheres (Group C), SVF suspension (Group B) alone, or Dulbecco’s modified Eagle’s medium as the control (Group A). Eighteen immunocompromised nude mice were injected with these three mixtures subcutaneously at random positions. After 8 weeks, the mean volume of grafts was greater in the SVFs plus VEGF/ANG-1-PLGA group than in the control and SVFs groups (1.08 ± 0.069 ml vs. 0.62 ± 0.036 ml, and 0.83 ± 0.059 ml, respectively). Histological assessments showed that lower fibrosis, but greater microvascular density in the SVFs plus VEGF/ANG-1-PLGA group than in the other groups, though the SVFs group also had an appropriate capillary density and reduced fibrosis. Our findings indicate that SVFs plus VEGF/ANG-1-PLGA-sustained release microspheres can improve angiogenesis and graft survival after autologous fat transplantation.     

Novel roles of nuclear angiotensin receptors and signaling mechanisms

The renin-angiotensin system (RAS) constitutes an important hormonal system in the physiological regulation of blood pressure. The dysregulation of the RAS is considered a major influence in the development and progression of cardiovascular disease and other pathologies. Indeed, experimental and clinical evidence indicates that blockade of this system with angiotensin-converting enzyme (ACE) inhibitors or angiotensin type 1 receptor (AT1R) antagonists is an effective therapy to attenuate hypertension and diabetic renal injury, and to improve heart failure. Originally defined as a circulating system, multiple tissues express a complete RAS, and compelling evidence now favors an intracellular system involved in cell signaling and function. Within the kidney, intracellular expression of the three predominant ANG receptor subtypes is evident in the nuclear compartment. The ANG type 1 receptor (AT1R) is coupled to the generation of reactive oxygen species (ROS) through the activation of phosphoinositol-3 kinase (PI3K) and PKC. In contrast, both ANG type 2 (AT2R) and ANG-(1-7) (AT7R) receptors stimulate nitric oxide (NO) formation, which may involve nuclear endothelial NO synthase (eNOS). Moreover, blockade of either ACE2-the enzyme that converts ANG II to ANG-(1-7)-or the AT7 receptor exacerbates the ANG II-ROS response on renal nuclei. Finally, in a model of fetal programmed hypertension, the nuclear ROS response to ANG II is enhanced, while both AT2 and AT7 stimulation of NO is attenuated, suggesting that an imbalance in the intracellular RAS may contribute to the development of programming events. We conclude that a functional intracellular or nuclear RAS may have important implications in the therapeutic approaches to cardiovascular disease.     

Vasodilator effect of angiotensin-(1-7) in mature and sponge-induced neovasculature

Angiotensin-(1-7) (Ang-(1-7)), a peptide constituent of the renin-angiotensin system, has been shown to act as a vasodilator mediator in pre-existing (skin) and newly formed vasculatures (14-day-old sponge implants). Blood flow was determined by the outflow rate of sodium fluorescein applied intradermally or intraimplant and the results were expressed in t(1/2) values (time taken for the fluorescence to reach 50% of the peak in the systemic circulation). We showed that the t(1/2) value was significantly lower (4.1+/-0.46) in the implants compared with the cutaneous vasculature (5.7+/-0.5). Ang-(1-7) 20 ng was able to decrease t(1/2) values in both vasculatures. The specific receptor antagonist, D-Ala7-Ang-(1-7) (A-779), prevented Ang-(1-7)-induced vasodilation and altered the basal vascular tone of the implants. The vasodilator effect was also abolished by nitric oxide (NO) synthase inhibitors in both vasculatures and by indomethacin in the implant. Selective AT(1) and AT(2) receptor antagonists did not alter the vasodilation induced by the peptide. These results establish the vasodilator effect of Ang-(1-7) in the cutaneous and implant vasculature and that the peptide is produced endogenously by the fibrovascular tissue, and suggest that this peptide contributes for the vasodilation found in newly formed vascular beds (wound healing, chronic inflammatory processes and tumors).     

Angiotensin receptors contribute to blood pressure homeostasis in salt-depleted SHR

This study evaluated the contribution of angiotensin peptides acting at various receptor subtypes to the arterial pressure and heart rate of adult 9-wk-old male conscious salt-depleted spontaneously hypertensive rats (SHR). Plasma ANG II and ANG I in salt-depleted SHR were elevated sevenfold compared with peptide levels measured in sodium-replete SHR, whereas plasma ANG-(1-7) was twofold greater in salt-depleted SHR compared with salt-replete SHR. Losartan (32.5 micromol/kg), PD-123319 (0.12 micromol. kg(-1). min(-1)), [d-Ala(7)]ANG-(1-7) (10 and 100 pmol/min), and a polyclonal ANG II antibody (0.08 mg/min) were infused intravenously alone or in combination. Combined blockade of AT(2) and AT((1-7)) receptors significantly increased the blood pressure of losartan-treated SHR (+15 +/- 1 mmHg; P < 0.01); this change did not differ from the blood pressure elevation produced by the sole blockade of AT((1-7)) receptors (15 +/- 4 mmHg). On the other hand, sole blockade of AT(2) receptors in losartan-treated SHR increased mean arterial pressure by 8 +/- 1 mmHg (P < 0.05 vs. 5% dextrose in water as vehicle), and this increase was less than the pressor response produced by blockade of AT((1-7)) receptors alone or combined blockade of AT((1-7)) and AT(2) receptors. The ANG II antibody increased blood pressure to the greatest extent in salt-depleted SHR pretreated with only losartan (+11 +/- 2 mmHg) and to the least extent in salt-depleted SHR previously treated with the combination of losartan, PD-123319, and [d-Ala(7)]ANG-(1-7) (+7 +/- 1 mmHg; P < 0.01). Losartan significantly increased heart rate, whereas other combinations of receptor antagonists or the ANG II antibody did not alter heart rate. Our results demonstrate that ANG II and ANG-(1-7) act through non-AT(1) receptors to oppose the vasoconstrictor actions of ANG II in salt-depleted SHR. Combined blockade of AT(2) and AT((1-7)) receptors and ANG II neutralization by the ANG II antibody reversed as much as 67% of the blood pressure-lowering effect of losartan.     

Angiotensin-(1 7) stimulates the phosphorylation of JAK2, IRS-1 and Akt in rat heart in vivo: role of the AT1 and Mas receptors

Angiotensin (ANG) II exerts a negative modulation on insulin signal transduction that might be involved in the pathogenesis of hypertension and insulin resistance. ANG-(1-7), an endogenous heptapeptide hormone formed by cleavage of ANG I and ANG II, counteracts many actions of ANG II. In the current study, we have explored the role of ANG-(1-7) in the signaling crosstalk that exists between ANG II and insulin. We demonstrated that ANG-(1-7) stimulates the phosphorylation of Janus kinase 2 (JAK2) and insulin receptor substrate (IRS)-1 in rat heart in vivo. This stimulating effect was blocked by administration of the selective ANG type 1 (AT(1)) receptor blocker losartan. In contrast to ANG II, ANG-(1-7) stimulated cardiac Akt phosphorylation, and this stimulation was blunted in presence of the receptor Mas antagonist A-779 or the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. The specific JAK2 inhibitor AG-490 blocked ANG-(1-7)-induced JAK2 and IRS-1 phosphorylation but had no effect on ANG-(1-7)-induced phosphorylation of Akt, indicating that activation of cardiac Akt by ANG-(1-7) appears not to involve the recruitment of JAK2 but proceeds through the receptor Mas and involves PI3K. Acute in vivo insulin-induced cardiac Akt phosphorylation was inhibited by ANG II. Interestingly, coadministration of insulin with an equimolar mixture of ANG II and ANG-(1-7) reverted this inhibitory effect. On the basis of our present results, we postulate that ANG-(1-7) could be a positive physiological contributor to the actions of insulin in heart and that the balance between ANG II and ANG-(1-7) could be relevant for the association among insulin resistance, hypertension, and cardiovascular disease.     

ACE2 and ANG-(1-7) in the rat uterus during early and late gestation

The present study was designed to determine ANG peptide content [ANG I, ANG II, ANG-(1-7)], ACE2 mRNA, and the immunocytochemical distribution of ANG-(1-7) and ACE2 in the uteroembryonic unit during early and late gestation in Sprague-Dawley rats and in a rat model of pregnancy-induced hypertension, the reduced uterine perfusion pressure (RUPP) model. At early pregnancy ANG-(1-7) and ACE2 staining were localized in the primary and secondary decidual zone and luminal and glandular epithelial cells. During late gestation, ANG-(1-7) and ACE2 staining was visualized in the labyrinth placenta and amniotic and yolk sac epithelium. Uterine ANG II concentration at early pregnancy was significantly decreased by 21-55% in the implantation and interimplantation sites compared with virgin rats, whereas ANG-(1-7) levels were maintained at prepregnancy levels. At late gestation, uterine concentrations of ANG I and ANG II were significantly increased (30% and 25%, respectively). In RUPP animals, ANG-(1-7) concentration is significantly reduced in the uterus (181 +/- 16 vs. 372 +/- 74 fmol/g of tissue) and placenta (143 +/- 26 vs. 197 +/- 20 fmol/g of tissue). ACE2 mRNA increased in the uterus of early pregnant compared with virgin rats, yet within the implantation site it was downregulated. At late pregnancy, ACE2 mRNA is elevated by 58% in the uterus and decreased by 59% in RUPP animals. The regulation of ANG-(1-7) and ACE2 in early and late pregnancy supports the hypothesis that ANG-(1-7) and ACE2 may act as a local autocrine/paracrine regulator throughout pregnancy, participating in the early (angiogenesis, apoptosis, and growth) and late (uteroplacental blood flow) events of pregnancy.     

Prevention of angiotensin II-induced cardiac remodeling by angiotensin-(1-7)

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-angiotensin 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 angiotensin-converting enzyme 2 (ACE2) prevents cardiac remodeling and hypertrophy during chronic infusion of angiotensin II (ANG II). This, coupled with the knowledge that ACE2 is a key enzyme in the formation of ANG-(1-7), led us to hypothesize that chronic infusion of ANG-(1-7) 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-(1-7) 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(7)]-ANG-(1-7) (A779), an antagonist to the reported receptor for ANG-(1-7), there was a tendency to attenuate the antiremodeling effects of ANG-(1-7). Chronic infusion of ANG II, with or without coinfusion of ANG-(1-7), had no effect on ANG II type 1 or type 2 receptor binding in cardiac tissue. Together, these findings indicate an antiremodeling role for ANG-(1-7) in cardiac tissue, which is not mediated through modulation of blood pressure or altered cardiac angiotensin receptor populations and may be at least partially mediated through an ANG-(1-7) receptor.     

Regulation of ACE2 in cardiac myocytes and fibroblasts

Angiotensin-converting enzyme 2 (ACE2) preferentially forms angiotensin-(1-7) [ANG-(1-7)] from ANG II. We showed that cardiac ACE2 is elevated following treatment of coronary artery-ligated rats with AT1 receptor blockers (ARBs). Cardiac myocytes and fibroblasts were isolated from neonatal rats to determine the molecular mechanisms for the ACE2 upregulation by ARB treatment. ANG II significantly reduced ACE2 activity and downregulated ACE2 mRNA in cardiac myocytes, effects blocked by the ARB losartan, indicating that ANG II regulates ACE2. ANG II also reduced ACE2 mRNA in cardiac fibroblasts; however, no enzyme activity was detected, reflecting the limited expression of ACE2 in these cells. Endothelin-1 (ET-1) also significantly reduced myocyte ACE2 mRNA. The reduction in ACE2 mRNA by ANG II or ET-1 was blocked by inhibitors of mitogen-activated protein kinase kinase 1, suggesting that ANG II or ET-1 activates extracellular signal-regulated kinase (ERK) 1/ERK2 to reduce ACE2. Although ACE2 mRNA was not affected by ANG-(1-7), both the ANG II- and ET-1-mediated reductions in ACE2 mRNA were blocked by the heptapeptide. The ANG-(1-7) modulatory effect was prevented by the ANG-(1-7) receptor antagonist [D-Ala7]-ANG-(1-7), indicating that the ANG-(1-7) response was mediated by a specific AT(1-7) receptor. Myocyte treatment with atrial natriuretic peptide (ANP) also reversed the ACE2 mRNA downregulation by ANG II or ET-1, whereas treatment with ANP alone was ineffective. These results indicate that multiple hypertrophic and anti-hypertropic peptides regulate ACE2 production in myocytes, suggesting that ACE2 expression in the heart is dependent upon the compliment and concentration of regulatory molecules.     

ACE2/ANG-(1-7)/Mas pathway in the brain: the axis of good

The last decade has seen the discovery of several new components of the renin-angiotensin system (RAS). Among them, angiotensin converting enzyme-2 (ACE2) and the Mas receptor have forced a reevaluation of the original cascade and led to the emergence of a new arm of the RAS: the ACE2/ANG-(1-7)/Mas axis. Accordingly, the new system is now seen as a balance between a provasoconstrictor, profibrotic, progrowth axis (ACE/ANG-II/AT(1) receptor) and a provasodilatory, antifibrotic, antigrowth arm (ACE2/ANG-(1-7)/Mas receptor). Already, this simplistic vision is evolving and new components are branching out upstream [ANG-(1-12) and (pro)renin receptor] and downstream (angiotensin-IV and other angiotensin peptides) of the classical cascade. In this review, we will summarize the role of the ACE2/ANG-(1-7)/Mas receptor, focusing on the central nervous system with respect to cardiovascular diseases such as hypertension, chronic heart failure, and stroke, as well as neurological diseases. In addition, we will discuss the new pharmacological (antagonists, agonists, activators) and genomic (knockout and transgenic animals) tools that are currently available. Finally, we will review the latest data regarding the various signaling pathways downstream of the Mas receptor.     

Angiotensin-(1-7) binds to specific receptors on cardiac fibroblasts to initiate antifibrotic and antitrophic effects

ANG-(1-7) improves the function of the remodeling heart. Although this peptide is generated directly within the myocardium, the effects of ANG-(1-7) on cardiac fibroblasts that play a critical role in cardiac remodeling are largely unknown. We tested the hypothesis that specific binding of ANG-(1-7) to cardiac fibroblasts regulates cellular functions that are involved in cardiac remodeling. 125I-labeled ANG-(1-7) binding assays identified specific binding sites of ANG-(1-7) on adult rat cardiac fibroblasts (ARCFs) with an affinity of 11.3 nM and a density of 131 fmol/mg protein. At nanomolar concentrations, ANG-(1-7) interacted with specific sites that were distinct from ANG II type 1 and type 2 receptors without increasing cytosolic Ca2+ concentration. At these concentrations, ANG-(1-7) had inhibitory effects on collagen synthesis as assessed by [3H]proline incorporation and decreased mRNA expression of growth factors in ARCFs. These effects of ANG-(1-7) contrasted with effects of ANG II. Pretreatment of ARCFs with ANG-(1-7) inhibited ANG II-induced increases in collagen synthesis and in mRNA expression of growth factors, including endothelin-1 and leukemia inhibitory factor. ANG-(1-7) pretreatment also inhibited the stimulatory effects of conditioned medium from ANG II-treated ARCFs on [3H]leucine incorporation and atrial natriuretic factor mRNA expression, markers of hypertrophy, in cardiomyocytes. Thus ANG-(1-7) interacted with specific receptors on ARCFs to exert potential antifibrotic and antitrophic effects that could reverse ANG II effects. These results suggest that ANG-(1-7) may play an important role in the heart in regulating cardiac remodeling.     

Inhibition of inflammatory angiogenesis by distant subcutaneous tumor in mice

We investigated angiogenesis, inflammatory cells accumulation and endogenous production of cytokines in sponge implants of tumor-bearing mice. Seven days after inoculation of Ehrlich tumor cells (2.5 x 10(6)), sponge discs were implanted subcutaneously in the dorsa of mice to induce the formation of fibrovascular tissue. The implants of tumor-bearing and non tumor-bearing animals were assessed for neovascularization and leukocyte accumulation, together with levels of relevant cytokines, vascular endothelial growth factor VEGF), tumor necrosis factor alpha (TNF-alpha), CXCL1-3/KC and CCL2/JE. In the implants of tumor-bearing animals angiogenesis (assessed by hemoglobin content and VEGF levels in the implants) and leukocyte accumulation (assessed by myeloperoxidase -MPO- and N- acetylglucosaminidase-NAG-enzyme activities) were all significantly less than those in the implants of non tumor-bearing animals. Although the chemokine CXCL1-3/KC was lower in the implants of tumor-bearing animals, the chemokine CCL2/JE was increased in this group. The production of TNF-alpha in the implants was not modified by the presence of the subcutaneous tumor. The combination of the methodologies used in this study has provided a novel approach to investigate the interaction between two distinct proliferating tissues that share common features (angiogenesis, cell recruitment, inflammation) and has shown that the predominant inhibitory effect of a tumor mass over repair process is associated with altered cytokine production.     

Altered regulation of the rostral ventrolateral medulla in hypertensive obese Zucker rats

Cardiovascular effects of angiotensin-(1-12) [ANG-(1-12)] were studied in the medial nucleus of the tractus solitarius (mNTS) in anesthetized, artificially ventilated, adult male Wistar rats. Microinjections (100 nl) of ANG-(1-12) (0.06 mM) into the mNTS elicited maximum decreases in mean arterial pressure (MAP; 34 ± 5.8 mmHg) and heart rate (HR; 39 ± 3.7 beats/min). Bilateral vagotomy abolished ANG-(1-12)-induced bradycardia. Efferent greater splanchnic nerve activity was decreased by microinjections of ANG-(1-12) into the mNTS. Blockade of ANG type 1 receptors (AT(1)Rs; using ZD-7155 or L-158,809), but not ANG type 2 receptors (AT(2)Rs; using PD-123319), significantly attenuated ANG-(1-12)-induced cardiovascular responses. Simultaneous inhibition of both angiotensin-converting enzyme (ACE; using captopril) and chymase (using chymostatin) completely blocked the effects of ANG-(1-12). Microinjections of A-779 [ANG-(1-7) antagonist] did not attenuate ANG-(1-12)-induced responses. Pressure ejection of ANG-(1-12) (0.06 mM, 2 nl) caused excitation of barosensitive mNTS neurons, which was blocked by prior application of the AT(1)R antagonist. ANG-(1-12)-induced excitation of mNTS neurons was also blocked by prior sequential applications of captopril and chymostatin. These results indicate that 1) microinjections of ANG-(1-12) into the mNTS elicited depressor and bradycardic responses by exciting barosensitive mNTS neurons; 2) the decreases in MAP and HR were mediated via sympathetic and vagus nerves, respectively; 3) AT(1)Rs, but not AT(2)Rs, mediated these actions of ANG-(1-12); 4) the responses were mediated via the conversion of ANG-(1-12) to ANG II and both ACE and chymase were involved in this conversion; and 5) ANG-(1-7) was not one of the metabolites of ANG-(1-12) in the mNTS.     

Angiotensin-(1-7) prevents development of severe hypertension and end-organ damage in spontaneously hypertensive rats treated with L-NAME

We examined the influence of chronic treatment with ANG-(1-7) on development of hypertension and end-organ damage in spontaneously hypertensive rats (SHR) chronically treated with the nitric oxide synthesis inhibitor L-NAME (SHR-L-NAME). L-NAME administered orally (80 mg/l) for 4 wk significantly elevated mean arterial pressure (MAP) compared with SHR controls drinking regular water (269 +/- 10 vs. 196 +/- 6 mmHg). ANG-(1-7) (24 microg x kg(-1) x h(-1)) or captopril (300 mg/l) significantly attenuated the elevation in MAP due to L-NAME (213 +/- 7 and 228 +/- 8 mmHg, respectively), and ANG-(1-7) + captopril completely reversed the L-NAME-dependent increase in MAP (193 +/- 5 mmHg). L-NAME-induced increases in urinary protein were significantly lower in ANG-(1-7)-treated animals (226 +/- 6 vs. 145 +/- 12 mg/day). Captopril was more effective (96 +/- 12 mg/day), and there was no additional effect of captopril + ANG-(1-7) (87 +/- 5 mg/day). The abnormal vascular responsiveness to endothelin-1, carbachol, and sodium nitroprusside in perfused mesenteric vascular bed of SHR-L-NAME was improved by ANG-(1-7) or captopril, with no additive effect of ANG-(1-7) + captopril. In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischemia was significantly better in ANG-(1-7)- or captopril-treated SHR-L-NAME, with additive effects of combined treatment. The beneficial effects of ANG-(1-7) on MAP and cardiac function were inhibited when indomethacin was administered with ANG-(1-7), but indomethacin did not reverse the protective effects on proteinuria or vascular reactivity. The protective effects of the ANG-(1-7) analog AVE-0991 were qualitatively comparable to those of ANG-(1-7) but were not improved over those of captopril alone. Thus, during reduced nitric oxide availability, ANG-(1-7) attenuates development of severe hypertension and end-organ damage; prostaglandins participate in the MAP-lowering and cardioprotective effects of ANG-(1-7); and additive effects of captopril + ANG-(1-7) on MAP, but not proteinuria or endothelial function, suggest common, as well as different, mechanisms of action for the two treatments. Together, the results provide further evidence of a role for ANG-(1-7) in protective effects of angiotensin-converting enzyme inhibition and suggest dissociation of factors influencing MAP and those influencing end-organ damage.