Natalia Alenina

Lrp5 functions in bone to regulate bone mass

The human skeleton is affected by mutations in low-density lipoprotein receptor-related protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with osteocyte-specific expression of inducible Lrp5 mutations that cause high and low bone mass phenotypes in humans. We found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also induced an Lrp5 mutation in cells that form the appendicular skeleton but not in cells that form the axial skeleton; we observed that bone properties were altered in the limb but not in the spine. These data indicate that Lrp5 signaling functions locally, and they suggest that increasing LRP5 signaling in mature bone cells may be a strategy for treating human disorders associated with low bone mass, such as osteoporosis.

Growth retardation and altered autonomic control in mice lacking brain serotonin

Serotonin synthesis in mammals is initiated by 2 distinct tryptophan hydroxylases (TPH), TPH1 and TPH2. By genetically ablating TPH2, we created mice (Tph2−/−) that lack serotonin in the central nervous system. Surprisingly, these mice can be born and survive until adulthood. However, depletion of serotonin signaling in the brain leads to growth retardation and 50% lethality in the first 4 weeks of postnatal life. Telemetric monitoring revealed more extended daytime sleep, suppressed respiration, altered body temperature control, and decreased blood pressure (BP) and heart rate (HR) during nighttime in Tph2−/− mice. Moreover, Tph2−/− females, despite being fertile and producing milk, exhibit impaired maternal care leading to poor survival of their pups. These data confirm that the majority of central serotonin is generated by TPH2. TPH2-derived serotonin is involved in the regulation of behavior and autonomic pathways but is not essential for adult life.

Impairment of In Vitro and In Vivo Heart Function in Angiotensin-(1-7) Receptor Mas Knockout Mice

In this study we investigated the effects of the genetic deletion of the angiotensin (Ang)-(1-7) receptor Mas on heart function. Localization of Mas in the mouse heart was evaluated by binding of rhodamine-labeled Ang-(1-7). Cardiac function was examined using isolated heart preparations. Echocardiography was used to confirm the results obtained with isolated heart studies. To elucidate the possible mechanisms involved in the cardiac phenotype observed in Mas−/− mice, whole-cell calcium currents in cardiomyocytes and the expression of collagen types I, III, and VI and fibronectin were analyzed. Ang-(1-7) binding showed that Mas is localized in cardiomyocytes of the mouse heart. Isolated heart techniques revealed that Mas-deficient mice present a lower systolic tension (average: 1.4±0.09 versus 2.1±0.03 g in Mas+/+ mice), ±dT/dt, and heart rate. A significantly higher coronary vessel resistance was also observed in Mas-deficient mice. Echocardiography revealed that hearts of Mas-deficient mice showed a significantly decreased fractional shortening, posterior wall thickness in systole and left ventricle end-diastolic dimension, and a higher left ventricle end-systolic dimension. A markedly lower global ventricular function, as defined by a higher myocardial performance index, was observed. A higher delayed time to the peak of calcium current was also observed. The changes in cardiac function could be partially explained by a marked change in collagen expression to a profibrotic profile in Mas-deficient mice. These results indicate that Ang-(1-7)-Mas axis plays a key role in the maintenance of the structure and function of the heart.

Mas Deficiency in FVB/N Mice Produces Marked Changes in Lipid and Glycemic Metabolism

OBJECTIVE— Metabolic syndrome is characterized by the variable coexistence of obesity, hyperinsulinemia, insulin resistance, dyslipidemia, and hypertension. It is well known that angiotensin (Ang) II is importantly involved in the metabolic syndrome. However, the role of the vasodilator Ang-(1-7)/Mas axis is not known. The aim of this study was to evaluate the effect of genetic deletion of the G protein–coupled receptor, Mas, in the lipidic and glycemic metabolism in FVB/N mice. RESEARCH DESIGN AND METHODS— Plasma lipid, insulin, and cytokine concentrations were measured in FVB/N Mas-deficient and wild-type mice. A glucose tolerance test was performed by intraperitoneally injecting d-glucose into overnight-fasted mice. An insulin sensitivity test was performed by intraperitoneal injection of insulin. Uptake of 2-deoxy-[3H]glucose by adipocytes was used to determine the rate of glucose transport; adipose tissue GLUT4 was quantified by Western blot. Gene expression of transforming growth factor (TGF)-β, type 1 Ang II receptor, and angiotensinogen (AGT) were measured by real-time PCR. RESULTS— Despite normal body weight, Mas-knockout (Mas-KO) mice presented dyslipidemia, increased levels of insulin and leptin, and an ∼50% increase in abdominal fat mass. In addition, Mas gene–deleted mice presented glucose intolerance and reduced insulin sensitivity as well as a decrease in insulin-stimulated glucose uptake by adipocytes and decreased GLUT4 in adipose tissue. Mas−/− presented increased muscle triglycerides, while liver triglyceride levels were normal. Expression of TGF-β and AGT genes was higher in Mas-KO animals in comparison with controls. CONCLUSIONS— These results show that Mas deficiency in FVB/N mice leads to dramatic changes in glucose and lipid metabolisms, inducing a metabolic syndrome–like state.

Direct Angiotensin II Type 2 Receptor Stimulation Acts Anti-Inflammatory Through Epoxyeicosatrienoic Acid and Inhibition of Nuclear Factor κB

Angiotensin II type 2 (AT2) receptors can be regarded as an endogenous repair system, because the AT2 receptor is upregulated in tissue damage and mediates tissue protection. A potential therapeutic use of this system has only recently come within reach through synthesis of the first selective, orally active, nonpeptide AT2 receptor agonist, compound 21 (C21; dissociation constant for AT2 receptor: 0.4 nM; dissociation constant for angiotensin II type 1 receptor: >10 000 nM). This study tested AT2 receptor stimulation with C21 as a potential future therapeutic approach for the inhibition of proinflammatory cytokines and of nuclear factor &kgr;B. C21 dose-dependently (1 nM to 1 &mgr;mol/L) reduced tumor necrosis factor-&agr;–induced interleukin 6 levels in primary human and murine dermal fibroblasts. AT2 receptor specificity was controlled for by inhibition with the AT2 receptor antagonist PD123319 and by the absence of effects in AT2 receptor–deficient cells. AT2 receptor–coupled signaling leading to reduced interleukin 6 levels involved inhibition of nuclear factor &kgr;B, activation of protein phosphatases, and synthesis of epoxyeicosatrienoic acid. Inhibition of interleukin 6 promoter activity by C21 was comparable in strength to inhibition by hydrocortisone. C21 also reduced monocyte chemoattractant protein 1 and tumor necrosis factor-&agr; in vitro and in bleomycin-induced toxic cutaneous inflammation in vivo. This study is the first to show the anti-inflammatory effects of direct AT2 receptor stimulation in vitro and in vivo by the orally active, nonpeptide AT2 receptor agonist C21. These data suggest that pharmacological AT2 receptor stimulation may be an orally applicable future therapeutic approach in pathological settings requiring the reduction of interleukin 6 or inhibition of nuclear factor &kgr;B.

Evidence for a Functional Interaction of the Angiotensin-(1–7) Receptor Mas With AT1 and AT2 Receptors in the Mouse Heart

The aim of this study was to evaluate the angiotensin (Ang)-(1–7) effects in isolated mouse hearts. The hearts of male C57BL/6J and knockout mice for the Ang-(1–7) receptor Mas were perfused by the Langendorff method. After a basal period, the hearts were perfused for 20 minutes with Krebs-Ringer solution (KRS) alone (control) or KRS containing Ang-(1–7) (0.22 pmol/L), the Mas antagonist A-779 (115 nmol/L), the angiotensin type 1 receptor antagonist losartan (2.2 &mgr;mol/L), or the angiotensin type 2 receptor antagonist PD123319 (130 nmol/L). To evaluate the involvement of Ang receptors, prostaglandins, and nitric oxide in the Ang-(1–7) effects, the hearts were perfused for 20 to 30 minutes with KRS containing either A-779, losartan, PD123319, indomethacin, or NG-nitro-l-arginine methyl ester (l-NAME) alone or in association with subsequent Ang-(1–7) perfusion. In addition, hearts from Mas-knockout mice were perfused for 20 minutes with KRS containing Ang-(1–7) (0.22 pmol/L) and losartan. Ang-(1–7) alone did not change the perfusion pressure. Strikingly, in the presence of losartan, 0.22 pmol/L Ang-(1–7) induced a significant decrease in perfusion pressure, which was blocked by A-779, indomethacin, and l-NAME. Furthermore, this effect was not observed in Mas-knockout mice. In contrast, in the presence of PD123319, Ang-(1–7) produced a significant increase in perfusion pressure. This change was not modified by the addition of A-779. Losartan reduced but did not abolish this effect. Our results suggest that Ang-(1–7) produces complex vascular effects in isolated, perfused mouse hearts involving interaction of its receptor with angiotensin type 1- and type 2-related mechanisms, leading to the release of prostaglandins and nitric oxide.

Endothelial dysfunction and elevated blood pressure in MAS gene-deleted mice.

Mas codes for a G protein–coupled receptor that is implicated in angiotensin-(1-7) signaling. We studied the cardiovascular phenotype of Mas-deficient mice backcrossed onto the FVB/N genetic background using telemetry and found that they exhibit higher blood pressures compared with controls. These Mas−/− mice also had impaired endothelial function, decreased NO production, and lower endothelial NO synthase expression. Reduced nicotinamide-adenine dinucleotide phosphate oxidase catalytic subunit gp91phox protein content determined by Western blotting was higher in Mas−/− mice than in controls, whereas superoxide dismutase and catalase activities were reduced. The superoxide dismutase mimetic, Tempol, decreased blood pressure in Mas−/− mice but had a minimal effect in control mice. Our results show a major cardiovascular phenotype in Mas−/− mice. Mas-deletion results in increased blood pressure, endothelial dysfunction, and an imbalance between NO and reactive oxygen species. Our animals represent a promising model to study angiotensin-(1-7)–mediated cardiovascular effects and to evaluate Mas agonistic compounds as novel cardioprotective and antihypertensive agents based on their beneficial effects on endothelial function.

Discovery and Characterization of Alamandine, a Novel Component of the Renin-Angiotensin System

Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand β-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/β-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders. # Novelty and Significance {#article-title-32}Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand &bgr;-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/&bgr;-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders.

Molecular Mechanisms Involved in the Angiotensin-(1-7)/Mas Signaling Pathway in Cardiomyocytes

Recently there has been growing evidence suggesting that beneficial effects of angiotensin-(1-7) [Ang-(1-7)] in the heart are mediated by its receptor Mas. However, the signaling pathways involved in these effects in cardiomyocytes are unknown. Here, we investigated the involvement of the Ang-(1-7)/Mas axis in NO generation and Ca2+ handling in adult ventricular myocytes using a combination of molecular biology, intracellular Ca2+ imaging, and confocal microscopy. Acute Ang-(1-7) treatment (10 nmol/L) leads to NO production and activates endothelial NO synthase and Akt in cardiomyocytes. Ang-(1-7)–dependent NO raise was abolished by pretreatment with A-779 (1 &mgr;mol/L). To confirm that Ang-(1-7) action is mediated by Mas, we used cardiomyocytes isolated from Mas-deficient mice. In Mas-deficient cardiomyocytes, Ang-(1-7) failed to increase NO levels. Moreover, Mas-ablation was accompanied by significant alterations in the proteins involved in the regulation of endothelial NO synthase activity, indicating that endothelial NO synthase and its binding partners are important effectors of the Mas-mediated pathway in cardiomyocytes. We then investigated the role of the Ang-(1-7)/Mas axis on Ca2+ signaling. Cardiomyocytes treated with 10 nmol/L of Ang-(1-7) did not show changes in Ca2+-transient parameters such as peak Ca2+ transients and kinetics of decay. Nevertheless, cardiomyocytes from Mas-deficient mice presented reduced peak and slower [Ca2+]i transients when compared with wild-type cardiomyocytes. Lower Ca2+ ATPase of the sarcoplasmic reticulum expression levels accompanied the reduced Ca2+ transient in Mas-deficient cardiomyocytes. Therefore, chronic Mas-deficiency leads to impaired Ca2+ handling in cardiomyocytes. Collectively, these observations reveal a key role for the Ang-(1-7)/Mas axis as a modulator of cardiomyocyte function.

The endothelium-dependent vasodilator effect of the nonpeptide Ang(1-7) mimic AVE 0991 is abolished in the aorta of mas-knockout mice.

Recently, we demonstrated that the endothelium-dependent vasodilator effect of angiotensin(1-7) 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(1-7) in this vessel, in this work we studied the mechanism underlying the vasorelaxant effect of AVE 0991, a nonpeptide mimic of the effects of Ang(1-7), using wild-type and Mas-deficient mice. Ang(1-7) and AVE 0991 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(1-7) and AVE 0991 was completely blocked by 2 specific Ang(1-7) receptor antagonists, A-779 and D-Pro7-Ang(1-7), and by inhibition of NO synthase with L-NAME. Moreover, in aortic rings from Mas-deficient mice, the vasodilator effect of both Ang(1-7) and AVE 0991 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 42112A was not altered in Mas-deficient mice. Our results show that Ang(1-7) and AVE 0991 produced an NO-dependent vasodilator effect in the mouse aorta that is mediated by the G protein-coupled receptor Mas.