Angiotensin-(1-7) in human follicular fluid correlates with oocyte maturation.

STUDY QUESTION: Do angiotensin (Ang)-(1-7) levels in human ovarian follicular fluid (FF) correlate with the number and proportion of mature oocytes obtained for IVF? SUMMARY ANSWER: The present study shows for the first time that Ang-(1-7) levels in human FF correlate with the proportion of mature oocytes collected upon ovarian stimulation for IVF. WHAT IS KNOWN ALREADY: Ang-(1-7) is an active peptide of the renin-angiotensin system that stimulates oocyte maturation in isolated rabbit and rat ovaries. However, its role in human ovulation remains unexplored. STUDY DESIGN, SIZE, DURATION: This was a prospective cohort study including 64 participants from a single IVF center. Sample size was calculated to achieve a statistical power of 80% in detecting 20% differences in the proportion of mature oocytes between groups. The participants were enrolled in the study during six consecutive months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Plasma samples were obtained from all subjects at Day 21 of the last menstrual cycle before starting pituitary blockade and controlled ovarian stimulation (COS). Plasma and FF samples were quickly mixed with a protease inhibitor cocktail and stored at -80°C. Ang-(1-7) was quantified in plasma and FF samples by a highly sensitive and specific radioimmunoassay, which was preceded by solid phase extraction, speed vacuum concentration and sample reconstitution in assay buffer. FF Ang-(1-7) levels were stratified into tertiles and the patients of each tertile were compared for COS/IVF outcomes using Kruskal-Wallis ANOVA. Multiple regression analysis was used to adjust correlations for potential confounders. The mRNA encoding for Mas, a receptor for Ang-(1-7), was investigated by real-time PCR in luteinized granulosa cells purified from the FF. MAIN RESULTS AND THE ROLE OF CHANCE: There was a four-fold increase in plasma Ang-(1-7) after ovulation induction (median 160.9 vs 41.4 pg/ml, P < 0.0001). FF Ang-(1-7) levels were similar to (169.9 pg/ml) but did not correlate with plasma Ang-(1-7) levels (r = -0.05, P = 0.665). Patients at the highest FF Ang-(1-7) tertile had a higher proportion of mature oocytes compared to patients at the lower FF Ang-(1-7) tertile (median 100% vs 70%, P < 0.01). There was a linear correlation between FF Ang-(1-7) and the proportion of mature oocytes (r = 0.380, P < 0.01), which remained significant after adjustment for age and duration of infertility (r = 0.447, P < 0.001). The luteinized granulosa cells expressed Mas receptor mRNA, which was positively correlated to the number of mature oocytes in women with more than three mature oocytes retrieved (r = 0.42, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This is an observational study, therefore, no causal relationship can be established between Ang-(1-7) and human oocyte maturation. Mas protein expression was not quantified due to limited availability of granulosa cells. WIDER IMPLICATIONS OF THE FINDINGS: Since this peptide promotes oocyte maturation in other species, it deserves further investigation as a potential maturation factor to human oocytes. STUDY FUNDING AND COMPETING INTEREST(S): Research supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). The authors have nothing to disclose.

Mechanisms of the anti-inflammatory actions of the angiotensin type 1 receptor antagonist losartan in experimental models of arthritis.

Angiotensin (Ang) II and its AT1 receptors have been implicated in the pathogenesis of rheumatoid arthritis. Activation of the counter-regulatory Ang-(1-7)-Mas receptor axis may contribute to some of the effects of AT1 receptor blockers (ARBs). In this study, we have used losartan, an ARB, to investigate the role of and the mechanisms by which AT1 receptors participated in two experimental models of arthritis: antigen-induced arthritis (AIA) in mice and adjuvant-induced arthritis (AdIA) in rats. Treatment with losartan decreased neutrophil recruitment, hypernociception and the production of TNF-α, IL-1ß and chemokine (C-X-C motif) ligand 1 in mice subjected to AIA. Histopathological analysis showed significant reduction of tissue injury and inflammation and decreased proteoglycan loss. In addition to decreasing cytokine production, losartan directly reduced leukocyte rolling and adhesion. Anti-inflammatory effects of losartan were not associated to Mas receptor activation and/or Ang-(1-7) production. Anti-inflammatory effects were reproduced in rats subjected to AdIA. This study shows that ARBs have potent anti-inflammatory effects in animal models of arthritis. Mechanistically, reduction of leukocyte accumulation and of joint damage was associated with local inhibition of cytokine production and direct inhibition of leukocyte-endothelium interactions. The anti-inflammatory actions of losartan were accompanied by functional improvement of the joint, as seen by reduced joint hypernociception. These findings support the use of ARBs for the treatment of human arthritis and provide potential mechanisms for the anti-inflammatory actions of these compounds.

Angiotensin-converting enzyme 2 activation improves endothelial function.

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-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 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-pro7-Ang-(1-7), 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 angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

Exercise induces renin-angiotensin system unbalance and high collagen expression in the heart of Mas-deficient mice.

The renin-angiotensin system (RAS) is involved in the cardiac and vascular remodeling associated with cardiovascular diseases. Angiotensin (Ang) II/AT(1) axis is known to promote cardiac hypertrophy and collagen deposition. In contrast, Ang-(1-7)/Mas axis opposes Ang II effects in the heart producing anti-trophic and anti-fibrotic effects. Exercise training is known to induce cardiac remodeling with physiological hypertrophy without fibrosis. We hypothesize that cardiac remodeling induced by chronic exercise depends on the action of Ang-(1-7)/Mas axis. Thus, we evaluated the effect of exercise training on collagen deposition and RAS components in the heart of FVB/N mice lacking Mas receptor (Mas-KO). Male wild-type and Mas-KO mice were subjected to a moderate-intense swimming exercise training for 6 weeks. The left ventricle (LV) of the animals was sectioned and submitted to qRT-PCR and histological analysis. Circulating and tissue angiotensin peptides were measured by RIA. Sedentary Mas-KO presented a higher circulating Ang II/Ang-(1-7) ratio and an increased ACE2 expression in the LV. Physical training induced in Mas-KO and WT a similar cardiac hypertrophy accompanied by a pronounced increase in collagen I and III mRNA expression. Trained Mas-KO and trained WT presented increased Ang-(1-7) in the blood. However, only in trained-WT there was an increase in Ang-(1-7) in the LV. In summary, we showed that deletion of Mas in FVB/N mice produced an unbalance in RAS equilibrium increasing Ang II/AT(1) arm and inducing deleterious cardiac effects as deposition of extracellular matrix proteins. These data indicate that Ang-(1-7)/Mas axis is an important counter-regulatory mechanism in physical training mediate cardiac adaptations.

Angiotensin-(1-7)/Mas axis integrity is required for the expression of object recognition memory.

It has been shown that the brain has its own intrinsic renin-angiotensin system (RAS) and angiotensin-(1-7) (Ang-(1-7)) is particularly interesting, because it appears to counterbalance most of the Ang II effects. Ang-(1-7) exerts its biological function through activation of the G-protein-coupled receptor Mas. Interestingly, hippocampus is one of the regions with higher expression of Mas. However, the role of Ang-(1-7)/Mas axis in hippocampus-dependent memories is still poorly understood. Here we demonstrated that Mas ablation, as well as the blockade of Mas in the CA1-hippocampus, impaired object recognition memory (ORM). We also demonstrated that the blockade of Ang II receptors AT1, but not AT2, recovers ORM impairment of Mas-deficient mice. Considering that high concentrations of Ang-(1-7) may activate AT1 receptors, nonspecifically, we evaluate the levels of Ang-(1-7) and its main precursors Ang I and Ang II in the hippocampus of Mas-deficient mice. The Ang I and Ang II levels are unaltered in the whole hipocampus of MasKo. However, Ang-(1-7) concentration is increased in the whole hippocampus of MasKo mice, as well as in the CA1 area. Taken together, our findings suggest that the functionality of the Ang-(1-7)/Mas axis is essential for normal ORM processing.

Transgenic angiotensin-converting enzyme 2 overexpression in vessels of SHRSP rats reduces blood pressure and improves endothelial function.

Rat models of hypertension, eg, spontaneously hypertensive stroke-prone rats (SHRSP), display reduced angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression compared with control animals. The aim of this study was to investigate the role of ACE2 in the pathogenesis of hypertension in these models. Therefore, we generated transgenic rats on a SHRSP genetic background expressing the human ACE2 in vascular smooth muscle cells by the use of the SM22 promoter, called SHRSP-ACE2. In these transgenic rats vascular smooth muscle expression of human ACE2 was confirmed by RNase protection, real-time RT-PCR, and ACE2 activity assays. Transgene expression leads to significantly increased circulating levels of angiotensin-(1-7), a prominent product of ACE2. Mean arterial blood pressure was reduced in SHRSP-ACE2 compared to SHRSP rats, and the vasoconstrictive response to intraarterial administration of angiotensin II was attenuated. The latter effect was abolished by previous administration of an ACE2 inhibitor. To evaluate the endothelial function in vivo, endothelium-dependent and endothelium-independent agents such as acetylcholine and sodium nitroprusside, respectively, were applied to the descending thoracic aorta and blood pressure was monitored. Endothelial function turned out to be significantly improved in SHRSP-ACE2 rats compared to SHRSP. These data demonstrate that vascular ACE2 overexpression in SHRSP reduces hypertension probably by locally degrading angiotensin II and improving endothelial function. Thus, activation of the ACE2/angiotensin-(1-7) axis may be a novel therapeutic strategy in hypertension.