Mas Receptor Expression Research Articles

The expression of angiotensin-converting enzyme 2–angiotensin-(1–7)–Mas receptor axis are upregulated after acute cerebral ischemic stroke in rats

There is now unequivocal evidence that the angiotensin-converting enzyme 2(ACE2)–Ang-(1–7)–Mas axis is a key component of the renin–angiotensin system (RAS) cascade, which is closely correlated with ischemic insult occurrence. Our previous studies demonstrated that the Ang-(1–7), was an active member of the brain RAS. However, the ACE2–Ang-(1–7)–Mas axis expression after cerebral ischemic injury are currently unclear. In the present study, we investigated the time course of ACE2–Ang-(1–7) and Mas receptor expression in the acute stage of cerebral ischemic stroke. The content of Ang-(1–7) 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-(1–7) at 6–48h compared with sham operation group following focal ischemic stroke (12h: 7.276±0.320ng/ml vs. 2.466±0.410ng/ml, serum; 1.024±0.056ng/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 (19.167±2.858 vs. 7.833±2.483) (P<0.05). The evidence collected in our present study will indicate that, ACE2–Ang-(1–7)–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.

Abstract 633: Aerobic Training Induces Time-course Changes Of Ace2-Ang(1-7)-Mas Receptor Axis And Reactive Oxygen Species In The Heart Of Spontaneously Hypertensive Rats

Objective: Since we showed recently that T causes time-dependent changes on brain RAS and oxidative stress of hypertensive rats, we sought now to investigate the sequential effects of T on ACE2-Ang(1-7)-MasR axis and reactive oxygen species (ROS) production in the left ventricle (LV) of SHRs. Methods: Male SHR and WKY aged 12 weeks underwent maximal exercise test (ET) and were allocated to T (50-60% of maximum exercise capacity, 1 h/day, 5 days/week) or sedentary (S) groups for 8 weeks. Subgroups of rats were cannulated at weeks 0 (S0), 1 (T1), 2 (T2), 4 (T4) and 8 (S8 and T8) for measurement of resting pressure (AP) and heart rate (HR), followed by saline perfusion and harvesting of the heart. ACE2 and MasR expression (Western Blotting) and ROS production (Dihydroethidium staining) were measured in the LV. Results: At the beginning of protocols SHR vs WKY exhibited higher MAP and HR (169±1 mmHg, 351±9 b/min, corresponding to 44% and 7% increases), elevated MasR (1,22±0,03 vs 1,00± 0,01 AU) but similar ACE2 expression (1,06±0,04 AU). ROS production was higher in SHR vs WKY (78±4 x 103 vs 66±1 x 103 AU). T increased treadmill performance (+0,87±0.10 km/h) and decreased resting HR (-15% at T4-T8) in both groups; MAP was only reduced in SHR (-10% at T8). T caused no change in the WKY group but increased ACE2 expression in the SHR (~1.14±0.03 au, T1-T4 P&lt;0.05 vs WKY), with a biphasic response in Mas receptor expression: an early fall and a late increase (1,02±0,07 AU at T2-T4 and 1,20±0,04 AU at T8, respectively, P&lt;0.05). In the SHR, T also caused an early normalization in ROS production (65±1 x 103 AU at T2) which was maintained up to T8. Again T did not change ROS production in the WKY group. Conclusions: Augmented ACE2 expression and reduced ROS production in the heart of the SHR are early responses to training, which are followed by a late increase in MasR expression. Data suggest that the prompt normalization of ROS production is mediated by ACE2-induced reduction of angiotensin II content in the LV of the trained SHR.

The Effects of Angiotensin II and Angiotensin-(1–7) in the Rostral Ventrolateral Medulla of Rats on Stress-Induced Hypertension

We have shown that angiotensin II (Ang II) and angiotensin-(1–7) [Ang-(1–7)] increased arterial blood pressure (BP) via glutamate release when microinjected into the rostral ventrolateral medulla (RVLM) in normotensive rats (control). In the present study, we tested the hypothesis that Ang II and Ang-(1–7) in the RVLM are differentially activated in stress-induced hypertension (SIH) by comparing the effects of microinjection of Ang II, Ang-(1–7), and their receptor antagonists on BP and amino acid release in SIH and control rats. We found that Ang II had greater pressor effect, and more excitatory (glutamate) and less inhibitory (taurine and γ-aminobutyric acid) amino acid release in SIH than in control animals. Losartan, a selective AT1 receptor (AT1R) antagonist, decreased mean BP in SIH but not in control rats. PD123319, a selective AT2 receptor (AT2R) antagonist, increased mean BP in control but not in SIH rats. However, Ang-(1–7) and its selective Mas receptor antagonist Ang779 evoked similar effects on BP and amino acid release in both SIH and control rats. Furthermore, we found that in the RVLM, AT1R, ACE protein expression (western blot) and ACE mRNA (real-time PCR) were significantly higher, whereas AT2R protein, ACE2 mRNA and protein expression were significantly lower in SIH than in control rats. Mas receptor expression was similar in the two groups. The results support our hypothesis and demonstrate that upregulation of Ang II by AT1R, not Ang-(1–7), system in the RVLM causes hypertension in SIH rats by increasing excitatory and suppressing inhibitory amino acid release.

Endothelin-1 downregulates Mas receptor expression in human cardiomyocytes

Endothelin-1 (ET-1) and the renin-angiotensin system (RAS) are involved in the pathogenesis of cardiac dysfunction. The Mas receptor is a functional binding site for angiotensin (Ang)‑(1-7), which is now considered a critical component of the RAS and exerts cardioprotective effects. To the best of our knowledge, the present study aimed to examine, for the first time, the effects of ET-1 on Mas expression in cultured human cardiomyocytes. Human cardiomyocytes were treated with ET-1 at different concentrations (1, 5, 10, 20 and 30 nM) for varied time periods (0.5, 1.5, 3, 4.5 or 6 h) with or without the transcription inhibitor actinomycin D, endothelin A (ETA) receptor blocker BQ123 and ETB receptor blocker BQ788, or different kinase inhibitors. ET-1 decreased the Mas mRNA level in a statistically significant dose- and time-dependent manner within 4.5 h, which was reflected in the dose-dependent downregulation of Mas promoter activity, Mas protein levels and Ang-(1-7) binding on the cell membrane. Actinomycin D (1 mg/ml), BQ123 (1 µM), p38 mitogen-activated protein kinase (MAPK) siRNA and inhibitor PD169316 (25 µM), completely eliminated the inhibitory effects of ET-1 on Mas expression in human cardiomyocytes. In conclusion, the present study demonstrated that ET-1 downregulates Mas expression at the transcription level in human cardiomyocytes via the ETA receptor by a p38 MAPK‑dependent mechanism. This study provides novel insights into the function of ET-1 and the Ang‑(1-7)/Mas axis in cardiac pathophysiology.

The components of the angiotensin-(1-7) system are differentially expressed during follicular wave in cattle

This study was based on the hypothesis that some components of the angiotensin-(1-7) (Ang-(1-7)) system are differentially expressed during follicular development and can be involved in the follicular health/atresia transition in bovine. The largest (F1) and second largest follicles (F2) were collected from cows before (Day 2), during (Day 3), or after (Day 4) the expected moment of follicular deviation. In the second experiment, F1 was induced to atresia through intrafollicular injection of fulvestrant (estrogen receptor-antagonist) and, in both experiments, mRNA expression of the Mas receptor, ACE2, NEP, and PEP was evaluated in the granulosa and theca cells. The mRNA expression of Mas receptor was upregulated in the granulosa cells of F2 after the establishment of follicular deviation, while PEP mRNA increased during and after the deviation process. The mRNA expression of ACE2 was upregulated in the granulosa cells of F1 during and after the follicular deviation. The mRNA expression of NEP was not regulated in F1 and F2. Mas receptor expression increased in the F1 induced to atresia. mRNA for Mas receptor, ACE2, and PEP are differentially expressed in granulosa cells throughout follicular development and the Mas receptor can be involved with the establishment of follicular dominance.

Beneficial Effects of the Activation of the Angiotensin-(1–7) Mas Receptor in a Murine Model of Adriamycin-Induced Nephropathy

Angiotensin-(1–7) [Ang-(1–7)] is a biologically active heptapeptide that may counterbalance the physiological actions of angiotensin II (Ang II) within the renin-angiotensin system (RAS). Here, we evaluated whether activation of the Mas receptor with the oral agonist, AVE 0991, would have renoprotective effects in a model of adriamycin (ADR)-induced nephropathy. We also evaluated whether the Mas receptor contributed for the protective effects of treatment with AT1 receptor blockers. ADR (10 mg/kg) induced significant renal injury and dysfunction that was maximal at day 14 after injection. Treatment with the Mas receptor agonist AVE 0991 improved renal function parameters, reduced urinary protein loss and attenuated histological changes. Renoprotection was associated with reduction in urinary levels of TGF-β. Similar renoprotection was observed after treatment with the AT1 receptor antagonist, Losartan. AT1 and Mas receptor mRNA levels dropped after ADR administration and treatment with losartan reestablished the expression of Mas receptor and increased the expression of ACE2. ADR-induced nephropathy was similar in wild type (Mas+/+) and Mas knockout (Mas −/−) mice, suggesting there was no endogenous role for Mas receptor activation. However, treatment with Losartan was able to reduce renal injury only in Mas+/+, but not in Mas −/− mice. Therefore, these findings suggest that exogenous activation of the Mas receptor protects from ADR-induced nephropathy and contributes to the beneficial effects of AT1 receptor blockade. Medications which target specifically the ACE2/Ang-(1–7)/Mas axis may offer new therapeutic opportunities to treat human nephropathies.

Fetal betamethasone exposure attenuates angiotensin-(1-7)-Mas receptor expression in the dorsal medulla of adult sheep

Glucocorticoids including betamethasone (BM) are routinely administered to women entering into early preterm labor to facilitate fetal lung development and decrease infant mortality; however, fetal steroid exposure may lead to deleterious long term consequences. In a sheep model of fetal programming, BM-exposed (BMX) offspring exhibit elevated mean arterial pressure (MAP) and decreased baroreflex sensitivity (BRS) for control of heart rate by 0.5-years of age associated with changes in the circulating and renal renin-angiotensin systems (RAS). In the brain solitary tract nucleus, angiotensin (Ang) II actions through the AT1 receptor oppose the beneficial actions of Ang-(1-7) at the Mas receptor for BRS regulation. Therefore, we examined Ang peptides, angiotensinogen (Aogen), and receptor expression in this brain region of exposed and control offspring of 0.5- and 1.8-years of age. Mas protein expression was significantly lower (>40%) in the dorsal medulla of BMX animals at both ages; however, AT1 receptor expression was not changed. BMX offspring exhibited a higher ratio of Ang II to Ang-(1-7) (2.30±0.36 versus 0.99±0.28; p<0.01) and Ang II to Ang I at 0.5-years. Although total Aogen was unchanged, Ang I-intact Aogen was lower in 0.5-year BMX animals (0.78±0.06 vs. 1.94±0.41; p<0.05) suggesting a greater degree of enzymatic processing of the precursor protein in exposed animals. We conclude that in utero BM exposure promotes an imbalance in the central RAS pathways of Ang II and Ang-(1-7) that may contribute to the elevated MAP and lower BRS in this model.

Angiotensin-(1–7) in the Rostral Ventrolateral Medulla Modulates Enhanced Cardiac Sympathetic Afferent Reflex and Sympathetic Activation in Renovascular Hypertensive Rats

Enhancement of the cardiac sympathetic afferent reflex (CSAR) contributes to sympathetic excitation in hypertension. The aim of the present study was to determine whether angiotensin (Ang)-(1-7) in the rostral ventrolateral medulla (RVLM) modulated the enhanced CSAR and sympathetic activation, and the signaling pathways that mediated these effects in the 2-kidney, 1-clip renovascular hypertension model. Cardiac sympathetic afferent reflex was evaluated using renal sympathetic nerve activity and mean arterial pressure responses to epicardial capsaicin application in anesthetized sinoaortic-denervated and cervical-vagotomized rats. RVLM microinjection of Ang-(1-7) induced greater increases in renal sympathetic nerve activity and mean arterial pressure, and greater enhancement in CSAR in 2-kidney, 1-clip rats than in sham-operated rats, which was blocked by Mas receptor antagonist A-779, adenylyl cyclase inhibitors SQ22536 and MDL-12,330A, and protein kinase A inhibitors rp-adenosine-3',5'-cyclic monophosphorothionate and H-89. Mas receptor expression in RVLM was increased in 2-kidney, 1-clip rats. Treatment with A-779, SQ22536, MDL-12,330A, rp-adenosine-3',5'-cyclic monophosphorothionate, or H-89 in RVLM inhibited CSAR and decreased renal sympathetic nerve activity and mean arterial pressure in 2-kidney, 1-clip rats, whereas cAMP analogue dibutyryl-cAMP had the opposite effects. Ang-(1-7) in RVLM increased, whereas A-779 decreased the cAMP level and the epicardial capsaicin application-induced increases in the cAMP level in RVLM. These results indicate that Ang-(1-7) in the RVLM enhances the CSAR and increases the sympathetic outflow and blood pressure via Mas receptor activation. The increased endogenous Ang-(1-7) and Mas receptor activity in RVLM contributes to the enhanced CSAR and sympathetic activation in renovascular hypertension, and the cAMP-protein kinase A pathway is involved in these Ang-(1-7)-mediated effects in the RVLM.

Molecular Changes of the Angiotensin Converting Enzyme 2 Pathway in Myocardial Tissue from Heart Failure Patients Undergoing Heart Transplantation

Background: The angiotensin-converting enzyme homologue ACE2 and the peptide angiotensin 1-7 (Ang 1-7) have antifibrotic effects, but it is not understood whether they have a protective role in cardiac remodeling. We aimed to analyze the Ang 1-7 Mas receptor and ACE2 expresion levels and their relationship with fibrotic and remodeling factors in human heart failure (HF) and compared them with non-pathological human hearts.Methods: This was a human case-control study in which two groups of samples were analyzed. Controls were organ donors without cardiovascular pathology, but whose heart could not be used for transplantation. Cases were patients with end-stage HF and left ventricle samples were harvested right after the heart transplantation surgery. They were either kept with liquid nitrogen until their analysis or were processed for paraffin embedding. We quantified mRNA expression by real time PCR and ACE2 protein levels by western blot. The collagen deposition was stained by sirius red and quantified with morphometric analysis.Results: Expression of the MMP3 stromelysin mRNA was only detectable in 10 out of 33 pathological samples and in 1 out of 13 control samples. Expression of components of the ACE2 pathway and fibrotic factors were higher in patients with detectable MMP3 expression than in patients with non-detectable MMP3. No differences were found between patients with MMP3 expression or no MMP3 expression regarding etiology, functional class, ventricle dilatation or medication.Conclusions: Only a subset of myocardiums from HF patients is active in the remodeling process at the moment of heart transplantation, as indicated by the MMP3 expression and the higher levels of fibrotic factors. Angiotensin 1-7 Mas receptor expression and ACE2 protein levels are increased in this myocardium subset, suggesting a role for the ACE2 in this process. Overall, we encountered molecular differences in transplanted hearts from patients with similar clinical characteristics.

Captopril intake decreases body weight gain via angiotensin-(1–7)

Angiotensin-(1–7) [Ang-(1–7)] plays a beneficial role in cardiovascular physiology by providing a counterbalance to the function of angiotensin II (Ang II). Although Ang II has been shown to be an adipokine secreted by adipocyte and affect lipid metabolism, the role of Ang-(1–7) in adipose tissue remains to be clarified. The aim of the present study was to investigate whether Ang-(1–7) affects lipid metabolism in adipose tissue. Ang-(1–7) increased glycerol release from primary adipocytes in a dose-dependent manner. A lipolytic effect of Ang-(1–7) was attenuated by pretreatment with A-779, a Mas receptor blocker and with an inhibitor of phosphoinositol 3-kinase (PI3K), or eNOS. However, losartan and PD123319 did not cause any change in Ang-(1–7)-induced lipolysis. Ang-(1–7)-induced lipolysis had an addictive effect with isoproterenol. In normal rats, chronic intake of captopril for 4 wks decreased body weight gain and the amount of adipose tissue and increased plasma Ang-(1–7) level. These effects were attenuated by administration of A-779. The levels of Mas receptor and phosphorylation of hormone-sensitive lipase (p-HSL) were significantly increased by treatment with captopril and these captopril-mediated effects were attenuated by the administration of A-779. There was no difference in diameter of adipocytes among sham, captopril- and captopril+A-779-treated groups. The similar effects of captopril on body weight, expression of Mas receptor, and p-HSL were observed in Ang-(1–7)-treated rats. These results suggest that captopril intake decreased body weight gain partly through Ang-(1–7)/Mas receptor/PI3K pathway.

The cardiac expression of Mas receptor is responsive to different physiological and pathological stimuli

The Mas protooncogene encodes a G protein-coupled receptor that has been described as a functional receptor for the cardioprotective fragment of the renin-angiotensin system (RAS), Angiotensin (Ang)-(1-7). The aim of this current study was to evaluate the responsiveness of Mas expression in hearts during different physiological and pathological conditions in rats. Physical training was considered a physiological condition, while isoproterenol-induced hypertrophy, myocardial infarction and DOCA-salt model of hypertension were used as pathological models of heart injury. The expression of Mas was analyzed by western blotting. Although swim-trained rats presented significant cardiac hypertrophy, our physical training protocol was unable to induce changes in the expression of Mas. On the other hand, cardiac hypertrophy and damage elicited by isoproterenol treatment led to a reduction in Mas expression. Myocardial infarction also significantly decreased the expression of Mas after 21 days of myocardial ischemia. Additionally, Mas expression levels were increased in hearts of DOCA-salt rats. Our present data indicate that Mas expression is responsive to different pathological stimuli, thereby suggesting that Mas receptor is involved in the homeostasis of the heart, as well as in the establishment and progression of cardiac diseases.

Role of endothelium on the abnormal Angiotensin-mediated vascular functions in epileptic rats

Epidemiological studies have found that the risk for cardiovascular disease is increased in patients with epilepsy. The Renin Angiontensin System (RAS), an important player in vascular tone control, is also involved in many neurological disorders, including seizures and epilepsy. Although it has been reported that Angiotensin II (Ang II) release and Angiotensin receptors expression are altered in many cerebral areas in patients/animal models with neurological disorders, there are no data on the vascular function. We evaluated Ang I and Ang II-mediated vascular responses and to correlate their contractile responses to the pres- ence of endothelium and the protein levels of components of the RAS (AT1, AT2, Mas and ACE) in aorta isolated from genetically epileptic rats (WAR strain). The major finding was that the vascular contractile response induced by Ang I and Ang II is endothelium-dependent. Ang II induced contractions in aortas from Wistar rats either with intact endothelium (E+) (1.16 ± 0.04 g, n = 6) and endothelium-denuded (E-) (1.24 ± 0.04 g, n = 6). Maximum contractile response (ME) induced by Ang I was lower in Wistar E+ (0.45 ± 0.03 g, n = 6) compared with Wistar E- (1.13 ± 0.08 g, n = 6). Ang I and Ang II failed to induce contraction in WAR E+, whereas the ME induced by Ang I in WAR E- was lower (0.52 ± 0.04 g, n = 11) than in the Wistar. ME induced by Ang II in aortas from WAR was also lower (0.40 ± 0.03 g, n = 11) compared with Wistar. AT1 receptor expression in both E+ WAR and Wistar was lower than in both E- WAR and Wistar. AT2 and Mas receptor expression was higher in Wistar E- and E+ as compared to WAR E- and E+. ACE expression was higher in both E+ WAR and Wistar, but it was lower in both E- WAR and Wistar. Endothelium impairs the contractile response induced by Angiotensin in WAR, suggesting that endothelial relaxing factors play important role on the aorta contraction.

Chronic exercise modulates RAS components and improves balance between pro- and anti-inflammatory cytokines in the brain of SHR

Recently, exercise has been recommended as a part of lifestyle modification for all hypertensive patients; however, the precise mechanisms of its effects on hypertension are largely unknown. Therefore, this study aimed to investigate the mechanisms within the brain that can influence exercise-induced effects in an animal model of human essential hypertension. Young normotensive WKY rats and SHR were given moderate-intensity exercise for 16weeks. Blood pressure was measured bi-weekly by tail-cuff method. Animals were then euthanized; paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM), important cardiovascular regulatory centers in the brain, were collected and analyzed by real-time RT-PCR, Western blot, EIA, and fluorescent microscopy. Exercise of 16-week duration attenuated systolic, diastolic, and mean arterial pressure in SHR. Sedentary SHR exhibited increased pro-inflammatory cytokines (PICs) and decreased anti-inflammatory IL-10 levels in the PVN and RVLM. Furthermore, SHR(sed) rats exhibited elevated levels of ACE, AT1R, and decreased levels of ACE2 and receptor Mas in the PVN and RVLM. Chronic exercise not only prevented the increase in PICs (TNF-α, IL-1β), ACE, and AT1R protein expression in the brain of SHR, but also dramatically upregulated IL-10, ACE2, and Mas receptor expression in SHR. In addition, these changes were associated with reduced plasma AngII levels, reduced neuronal activity, reduced NADPH-oxidase subunit gp91(phox) and inducible NO synthase in trained SHRs indicating reduced oxidative stress. These results suggest that chronic exercise not only attenuates PICs and the vasoconstrictor axis of the RAS but also improves the anti-inflammatory defense mechanisms and vasoprotective axis of the RAS in the brain, which, at least in part, explains the blood pressure-lowering effects of exercise in hypertension.

Regulation of angiotensin-(1–7) and angiotensin II type 1 receptor by telmisartan and losartan in adriamycin-induced rat heart failure

To investigate the possible effects of telmisartan and losartan on cardiac function in adriamycin (ADR)-induced heart failure in rats, and to explore the changes in plasma level of angiotensin-(1-7)[Ang-(1-7)] and myocardial expression of angiotensin II type 1/2 receptors (AT(1)R / AT(2)R) and Mas receptor caused by the two drugs. Male Sprague-Dawley rats were randomly divided into 4 groups: the control group, ADR-treated heart failure group (ADR-HF), telmisartan plus ADR-treated group (Tel+ADR) and losartan plus ADR-treated group (Los+ADR). ADR was administrated (2.5 mg/kg, ip, 6 times in 2 weeks). The rats in the Tel+ADR and Los+ADR groups were treated orally with telmisartan (10 mg/kg daily po) and losartan (30 mg/kg daily), respectively, for 6 weeks. The plasma level of Ang-(1-7) was determined using ELISA. The mRNA and protein expression of myocardial Mas receptor, AT(1)R and AT(2)R were measured using RT-PCR and Western blotting, respectively. ADR significantly reduced the plasma level of Ang-(1-7) and the expression of myocardial Mas receptor and myocardial AT(2)R, while significantly increased the expression of myocardial AT(1)R. Treatment with telmisartan and losartan effectively increased the plasma level of Ang-(1-7) and suppressed myocardial AT(1)R expression, but did not influence the expression of Mas receptor and AT(2)R. The protective effects of telmisartan and losartan in ADR-induced heart failure may be partially due to regulation of circulating Ang-(1-7) and myocardial AT(1)R expression.

Swimming training improves the vasodilator effect of angiotensin-(1–7) in the aorta of spontaneously hypertensive rat

endothelial dysfunction plays a critical role in the pathogenesis of hypertension. It is well established that physical training has beneficial effects on the cardiovascular system. We recently reported that angiotensin-(1-7) [Ang-(1-7)] concentration and the Mas receptor expression is increased in the left ventricle of trained spontaneous hypertensive rats (SHR). The vascular effects of Ang-(1-7) in trained animals remain so far unknown. In the present study we investigated the effects of physical training on the vasodilator effect of Ang-(1-7) in the aorta of SHR. normotensive Wistar rats and SHR were subjected to an 8-wk period of 5% overload of body weight swimming training. Changes in isometric tension were recorded on myograph. Western blot was used to investigate Ang-(1-7) receptors expression. in aortas from normotensive rats Ang-(1-7) and ACh induced a concentration-dependent vasodilator effect, which was not modified by the physical training. Vessels from SHR had an impaired vasodilator response to Ang-(1-7) and ACh. The swimming training strongly potentiated the vasodilator effect induced by Ang-(1-7) in SHR, but did not modify the effect of ACh. Interestingly, Mas receptor protein expression was substantially increased by physical training in SHR. In trained SHR, the vasodilator effect of Ang-(1-7) was abrogated by removal of the endothelium and by the selective Ang-(1-7) receptor antagonists A-779 and d-Pro(7)-Ang-(1-7). l-NAME decreased Ang-(1-7) vasodilator response and indomethacin abolished the remaining dilatory response. physical training increased Mas receptors expression in SHR aortas, thereby improving the vasodilator effect of Ang-(1-7) through an endothelium-dependent mechanism involving nitric oxide and prostacyclin.

Molecular characterization and regulation of the angiotensin-converting enzyme type 2/Angiotensin-(1-7)/MAS receptor axis during the ovulation process in cattle

The objective of this study was to characterize the profiles of Ang-(1-7), MAS receptor, ACE(2), NEP and PEP during the ovulatory process in cattle. For this study, 40 synchronized cows with follicular diameter ≥ 12 mm were ovariectomized at different time-points (0, 3, 6, 12 and 24 h) after i.m. application of gonadotropin-releasing hormone (GnRH) to induce a luteinizing hormone surge. Follicular fluid was collected for measuring Ang-(1-7) by radioimmunoassay. Theca and granulosa cells were isolated from the preovulatory follicles to evaluate the gene expression of MAS receptor, ACE(2), NEP and PEP by qRT-PCR assay. Cross-contamination between theca and granulosa cells was tested by RT-PCR to detect cytochrome P450 aromatase (CYP19A1) and 17α-hydroxylase (CYP17A1) mRNA. Ang-(1-7) levels were constant until 12 h and then increased (p < 0.05) at 24 h after GnRH. Messenger RNA expression of MAS, ACE(2), NEP and PEP was detected in theca and granulosa cells at all time-points after GnRH. In granulosa cells, ACE(2), NEP and PEP were differentially expressed after GnRH treatment (p < 0.05). In conclusion, the Ang-(1-7), MAS receptor, ACE(2), NEP and PEP profiles in preovulatory follicles indicate that Ang-(1-7) plays a role in the regulation of the ovulatory process in cattle.

ACE2 Improves Right Ventricular Function in a Pressure Overload Model

BackgroundRight ventricular (RV) dysfunction is a complication of pulmonary hypertension and portends a poor prognosis. Pharmacological therapies targeting RV function in pulmonary hypertension may reduce symptoms, improve hemodynamics, and potentially increase survival. We hypothesize that recombinant human angiotensin-converting enzyme 2 (rhACE2) will improve RV function in a pressure overload model.ResultsrhACE2 administered at 1.8 mg/kg/day improved RV systolic and diastolic function in pulmonary artery banded mice as measured by in vivo hemodynamics. Specifically, rhACE2 increased RV ejection fraction and decreased RV end diastolic pressure and diastolic time constant (p<0.05). In addition, rhACE2 decreased RV hypertrophy as measured by RV/LV+S ratio (p<0.05). There were no significant negative effects of rhACE2 administration on LV function. rhACE2 had no significant effect on fibrosis as measured by trichrome staining and collagen1α1 expression. In pulmonary artery banded mice, rhACE2 increased Mas receptor expression and normalized connexin 37 expression.ConclusionIn a mouse RV load-stress model of early heart failure, rhACE2 diminished RV hypertrophy and improved RV systolic and diastolic function in association with a marker of intercellular communication. rhACE2 may be a novel treatment for RV failure.

Telmisartan attenuates oxidative stress and renal fibrosis in streptozotocin induced diabetic mice with the alteration of angiotensin-(1–7) mas receptor expression associated with its PPAR-γ agonist action

The beneficial effects of telmisartan on Angiotensin (Ang)-II mediated oxidative stress and renal fibrosis in streptozotocin (STZ)-induced diabetic nephropathy (DN) were studied. Thirty mice were divided into normal (NG), STZ-induced diabetic (DG) and telmisartan-treated diabetic (TG) groups. Compared with NG mice, DG mice showed significant up-regulations of AT-1R, TGF-β1, p-p38MAPK, p-MAPKAPK-2, p-Akt, p47phox, p67phox, gp91phox protein and collagen-III and all of these were significantly reversed in TG mice. The down-regulated protein expression of Ang-(1–7) mas receptor, ACE-2, PPAR-γ and PGC-1α were observed in DG mice and a significant up-regulation effect of telmisartan has been seen in the TG mice. Furthermore, TG mice showed reduced expression of fibronectin, production of superoxide radical as well as renal hypertrophy and fibrosis when compared with DG mice. These findings suggest that Ang-II plays a significant role in DN and telmisartan would be beneficial in reducing oxidative stress and fibrosis in STZ-induced DN.

Activation of the Protective Arm of Renin Angiotensin System (RAS) Corrects the Reparative Dysfunction of Diabetic CD34+ Cells.

AbstractAbstract 2637 Purpose:RAS plays a vital role in regulating many physiological processes of the vascular system. Angiotensin II (Ang II), a product of angiotensin converting enzyme (ACE), mediates its effects through activation of either the AT1 receptor – to induce vasoconstriction, proliferation, fibrosis, and inflammation – or the AT2 receptor to promote NO generation. The protective arm of RAS involves ACE2, which produces angiotensin-(1-7) [Ang-(1-7)]. Ang-(1-7) activates the MAS receptor to promote vascular health. Because diabetic endothelial progenitor cells are dysfunctional and this limits their utility in autologous cell therapy, we asked whether angiotensin (Ang)-(1-7) could restore the vasoreparative function of diabetic CD34+cells. Methods:Healthy nondiabetic (ND) and diabetic (D) Lin−CD45midCD34+ cells were obtained from peripheral blood mononuclear cells (PB-MNCs) by FACS. The effect of Ang-(1-7) on migration, proliferation, NO bioavailability, reactive oxygen species (ROS) levels and NADPH oxidase activity were evaluated in ND- and D-CD34+ cells. The effect of Ang-(1-7) on the formation of ECFCs from ND- and D-MNCs was evaluated. Ang-(1-7) production by cells was analyzed and the expression of ACE2 and Mas-receptor were assessed by real-time PCR and flow cytometry. Effects of ACE2 activators XNT and DIZE in CD34+ cells were also evaluated. D-CD34+ cells were genetically modified to overexpress Ang-(1-7) by lentiviral Ang-(1-7)-fusion transgene and their function was evaluated in vitro. The effect of transduction on the surface expression of CD133, CD34 and CD309 was assessed. In vivo homing function was assessed in a mouse model of ischemia-reperfusion (I/R). After one week of I/R insult, when retinal capillary damage was appreciable, CD34 cells were intravitreally injected. Neural retinas were harvested after 48 hours and human cells within the mouse vasculature were localized by immunohistochemistry. Results:Migration to SDF1- and VEGF-were impaired in D-CD34+ cells. In contrast, Ang-(1-7)-induced migration in both D-CD34+ and ND-CD34+cells was dependent on Mas receptor expression and eNOS. ROS levels and NADPH oxidase activity were reduced and proliferation and NO bioavailability were restored in D-CD34+ cells by Ang-(1-7). ECFCs from D-MNCs were appeared only in the presence of Ang-(1-7). Migration, NO release and Ang-(1-7) release by ACE2 activators, XNT and DIZE, were significantly decreased in D-CD34+ cells. Ang-(1-7) release and ACE2 expression were decreased in D-cells while Mas-receptor expression was similar that in ND-cells. Ang-(1-7) gene-modified cells showed reduced ROS levels, increased NO bioavailability, enhanced migration to SDF-1 and proliferation. Lentiviral transduction did not alter surface expression of CD133, CD34 and CD309. Ang-(1-7)-overexpression restored the homing efficiency of D-CD34+ cells similar to that of ND-CD34+ cells in vivo. Conclusions:Ang-(1-7) stimulates the vasoreparative functions in CD34+ cells. Ang-(1-7) bypasses the reduced ACE2 seen in diabetic CD34+ cells and restores the vasoreparative potential of these cells by decreasing oxidative stress and normalizing NO bioavailability. Pharmacological strategies that either increase ACE2 or Ang-(1-7) in diabetic CD34+ cells will improve their therapeutic utility for autologous cell therapy in treatment of diabetic complications. Disclosures:No relevant conflicts of interest to declare

Expression of Ang 1–7 receptor (MAS receptor) in aortic tissue during pregnancy‐induced hypertension in rats

Low vasopressor response to angiotensin II is characteristic during normal pregnancy. In preeclampsia (PE), this effect is lost. A novel renin‐angiotensin system component, the Ang 1–7 receptor (MAS receptor) shows vasodilatory, anti‐hypertrophic and pro‐apoptotic actions. It is not well known the role of this receptor during PE. The aim of this work was to study MAS receptor expression in aortic tissue during PE. We used a subrenal aorctic coarctation (SRAC) model in female Wistar rats, measuring blood pressure, proteinuria and pups weight. Aortic tissue samples were homogenized and immunoblotted using an Anti‐MAS antibody. We found hypertension, proteinuria and low‐weight pups in SRAC‐pregnant rats. We also found MAS receptor expression in both, pregnant non‐SRAC and pregnant SRAC groups. In pregnant non‐SRAC rats, MAS was expressed markedly. In pregnant SRAC rats however, we found a lower expression of this receptor. These results suggest that MAS expression is increased in normal pregnancy and decreased expression of this receptor during preeclampsia may indicate a role in the pathophysiology of this complication. Supported by SIP‐IPN 20090153 and 20090396 grants.