Florian Gembardt

G-protein-coupled receptor mas is a physiological antagonist of the angiotensin II type 1 receptor

Background—We previously identified the G-protein–coupled receptor Mas, encoded by the Mas proto-oncogene, as an endogenous receptor for the heptapeptide angiotensin-(1-7); however, the receptor is also suggested to be involved in actions of angiotensin II. We therefore tested whether this could be mediated indirectly through an interaction with the angiotensin II type 1 receptor, AT1. Methods and Results—In transfected mammalian cells, Mas was not activated by angiotensin II; however, AT1 receptor–mediated, angiotensin II–induced production of inositol phosphates and mobilization of intracellular Ca2+ was diminished by 50% after coexpression of Mas, despite a concomitant increase in angiotensin II binding capacity. Mas and the AT1 receptor formed a constitutive hetero-oligomeric complex that was unaffected by the presence of agonists or antagonists of the 2 receptors. In vivo, Mas acts as an antagonist of the AT1 receptor; mice lacking the Mas gene show enhanced angiotensin II–mediated vasoconstriction in mesenteric microvessels. Conclusions—These results demonstrate that Mas can hetero-oligomerize with the AT1 receptor and by so doing inhibit the actions of angiotensin II. This is a novel demonstration that a G-protein–coupled receptor acts as a physiological antagonist of a previously characterized receptor. Consequently, the AT1-Mas complex could be of great importance as a target for pharmacological intervention in cardiovascular diseases.

Organ-specific distribution of ACE2 mRNA and correlating peptidase activity in rodents

n Abstractn n Biochemical analysis revealed that angiotensin-converting enzyme related carboxy-peptidase (ACE2) cleaves angiotensin (Ang) II to Ang-(1–7), a heptapeptide identified as an endogenous ligand for the G protein-coupled receptor Mas. No data are currently available that systematically describe ACE2 distribution and activity in rodents. Therefore, we analyzed the ACE2 expression in different tissues of mice and rats on mRNA (RNase protection assay) and protein levels (immunohistochemistry, ACE2 activity, western blot). Although ACE2 mRNA in both investigated species showed the highest expression in the ileum, the mouse organ exceeded rat ACE2, as also demonstrated in the kidney and colon. Corresponding to mRNA, ACE2 activity was highest in the ileum and mouse kidney but weak in the rat kidney, which was also confirmed by immunohistochemistry. Contrary to mRNA, we found weak activity in the lung of both species. Our data demonstrate a tissue- and species-specific pattern for ACE2 under physiological conditions.n n

The SGLT2 inhibitor empagliflozin ameliorates early features of diabetic nephropathy in BTBR ob/ob type 2 diabetic mice with and without hypertension

Diabetic nephropathy is the leading cause of end-stage renal disease in humans in the Western world. The recent development of Na+-glucose cotransporter 2 (SGLT2) inhibitors offers a new antidiabetic therapy via enhanced glucose excretion. Whether this strategy exerts beneficial effects on the development of type 2 diabetic nephropathy is still largely unclear. We investigated the effects of the specific SGLT2 inhibitor empagliflozin in BTBR.Cg-Lep/WiscJ (BTBR ob/ob) mice, which spontaneously develop type 2 diabetic nephropathy. In the first experiment, BTBR ob/ob mice received either a diet containing 300 ppm empagliflozin or equicaloric placebo chow for 12 wk. In the second experiment, BTBR ob/ob mice received 1 μg·kg body wt(-1)·day(-1) ANG II to induce arterial hypertension and were separated into the same two diet groups for 6 wk. In both experiments, empagliflozin treatment enhanced glucosuria, thereby lowering blood glucose. Independently of hypertension, empagliflozin reduced albuminuria in diabetic mice. However, empagliflozin treatment affected diabetes-related glomerular hypertrophy, markers of renal inflammation, and mesangial matrix expansion only in BTBR ob/ob mice without hypertension. In summary, empagliflozin demonstrated significant antihyperglycemic effects, differentially ameliorating early features of diabetic nephropathy in BTBR ob/ob mice with and without hypertension.

Endothelial dysfunction through genetic deletion or inhibition of the G protein-coupled receptor Mas: a new target to improve endothelial function.

Background Endothelial dysfunction is an initial step in the pathogenesis of cardiovascular diseases. Since we previously identified the G protein-coupled receptor Mas as a receptor for angiotensin (Ang)-(1–7), a heptapeptide with endothelium-dependent vasorelaxant properties, we investigated whether alterations on the Ang-(1–7)/Mas axis alter endothelial function. Results Ang-(1–7)-mediated relaxation of murine wild-type mesenteric arteries was equally impaired in both wild-type arteries pretreated with the Ang-(1–7) receptor blocker, A779, and arteries isolated from Mas-deficient mice. Importantly, the response to the endothelium-dependent vasorelaxant, bradykinin (BK), and acetylcholine (ACh) effects were comparably inhibited, while endothelium-independent vessel relaxation by sodium nitroprusside was unaltered in these vessels. Hypothesizing endothelial dysfunction, we proved the in-vivo relevance of the ex-vivo findings investigating mesenteric properties after 1 week of minipump infusion of A779 in wild-type mice. Both BK- and ACh-induced relaxation were significantly impaired in wild-type vessels of pretreated animals. A779-induced impairment of endothelial function was confirmed in vitro, since BK-mediated nitric oxide (NO) release was increased by Ang-(1–7) and blunted by A779 pretreatment in primary human endothelial cell cultures. Conclusions Our data highlight a pivotal role for the receptor Mas in preserving normal vascular relaxation. Consequently, Mas agonists arise as a promising tool in the treatment of cardiovascular diseases characterized by endothelial dysfunction.

Angiotensin-(1–7) stimulates hematopoietic progenitor cells in vitro and in vivo

This study demonstrates that the angiotensin II metabolite Ang-(1-7) stimulates the proliferation of hematopoietic progenitor cells and promotes their engraftment in a xenograft model, suggesting that the renin-angiotensin system is a regulator of blood cell formation. See related perspective article on page 745. Effects of angiotensin (Ang)-(1–7), an AngII metabolite, on bone marrow-derived hematopoietic cells were studied. We identified Ang-(1–7) to stimulate proliferation of human CD34+ and mononuclear cells in vitro. Under in vivo conditions, we monitored proliferation and differentiation of human cord blood mononuclear cells in NOD/SCID mice. Ang-(1–7) stimulated differentially human cells in bone marrow and accumulated them in the spleen. The number of HLA-I+ and CD34+ cells in the bone marrow was increased 42-fold and 600-fold, respectively. These results indicate a decisive impact of Ang-(1–7) on hematopoiesis and its promising therapeutic potential in diseases requiring progenitor stimulation.

Successive Action of Meprin A and Neprilysin Catabolizes B-Type Natriuretic Peptide

Natriuretic peptides such as B-type natriuretic peptide (BNP) are important cardioprotective hormones with essential functions in sodium excretion, water balance and blood pressure regulation. Consequently, the catabolism of these peptides is in the focus of clinical research. In previous studies, we demonstrated that BNP, in contrast to the structurally related atrial and C-type natriuretic peptide, was not hydrolyzed by neprilysin (NEP). Because membrane preparations of several organs of NEP-knockout mice rapidly degrade BNP, the aim of this study was to identify BNP-catabolizing peptidases responsible for this fast clearance. Using kidney membranes of wild-type and NEP-knockout mice, as well as several peptidase inhibitors, we monitored the catabolism of BNP and analyzed its degradation products. We identified meprin A, a multimeric metalloprotease expressed in the brush borders of kidney proximal tubules, to initially truncate mouse BNP in the N terminus to mBNP7-32, a BNP metabolite with conserved biological activity. Consequently, in vivo experiments with the meprin inhibitor actinonin successfully elevated plasma BNP concentration in rats. We further demonstrated that the generation of mBNP7-32 is the prerequisite to catabolize BNP and identified NEP as the peptidase degrading the truncated BNP. Thus, the cooperative, successive action of the 2 transmembranal peptidases meprin A and NEP is crucial for rapid renal BNP inactivation. Therefore, the inhibition of meprin A could be a potent tool for increasing circulating BNP levels.

Angiotensin metabolites can stimulate receptors of the Mas-related genes family

The Mas protooncogene encodes a G protein-coupled receptor, we identified, also by using the specific angiotensin-(1-7) antagonist A-779, to be associated with intracellular signaling of the angiotensin (Ang) II metabolite Ang-(1-7). Recently, Mas-related genes (Mrg) have been identified coding for the Mrg-receptor family. All family members share high sequence homology to Mas. Most of them are orphan receptors. To proof whether structure similarities of the Mrg receptors with Mas turn them into potential receptors for Ang-(1-7) or other Ang metabolites, we transfected COS or HEK293 cells with an assortment of Mrg receptors and investigated arachidonic acid (AA) release and transcriptional activation by recording serum response factor (SRF) activation after stimulation with Ang II, Ang III, Ang IV, and Ang-(1-7). None of the investigated receptors activated transcription via SRF. Ang-(1-7) stimulated AA release already in control vector-transfected COS cells, indicating the existence of an endogenous receptor (A-779 sensitive). Though less pronounced than for Mas, two of the six studied receptors (MrgD, MRG) initiated significant AA release after stimulation with Ang-(1-7). Interestingly, Mas, MrgD, and MRG mediated Ang IV-stimulated AA release that was highest for Mas. While Ang III activated Mas and MrgX2, Ang II stimulated AA release via Mas and MRG. Thus, we identified other receptors of the Mrg family to respond on Ang-(1-7) stimulation. Furthermore, we describe first an AT1-independent direct Ang IV signaling and show that Ang II and Ang III mediate signaling independent of their specific receptors AT1 and AT2, whereby the receptor specificity differs.

Cardiac phenotype and angiotensin II levels in AT1a, AT1b, and AT2 receptor single, double, and triple knockouts

AIMSnOur aim was to determine the contribution of the three angiotensin (Ang) II receptor subtypes (AT(1a), AT(1b), AT(2)) to coronary responsiveness, cardiac histopathology, and tissue Ang II levels using mice deficient for one, two, or all three Ang II receptors.nnnMETHODS AND RESULTSnHearts of knockout mice and their wild-type controls were collected for histochemistry or perfused according to Langendorff, and kidneys were removed to measure tissue Ang II. Ang II dose-dependently decreased coronary flow (CF) and left ventricular systolic pressure (LVSP), and these effects were absent in all genotypes deficient for AT(1a), independently of AT(1b) and AT(2). The deletion of Ang II receptors had an effect neither on the morphology of medium-sized vessels in the heart nor on the development of fibrosis. However, the lack of both AT(1) subtypes was associated with atrophic changes in the myocardium, a reduced CF and a reduced LVSP. AT(1a) deletion alone, independently of the presence or absence of AT(1b) and/or AT(2), reduced renal Ang II by 50% despite a five-fold rise of plasma Ang II. AT(1b) deletion, on top of AT(1a) deletion (but not alone), further decreased tissue Ang II, while increasing plasma Ang II. In mice deficient for all three Ang II receptors, renal Ang II was located only extracellularly.nnnCONCLUSIONnThe lack of both AT(1) subtypes led to a baseline reduction of CF and LVSP, and the effects of Ang II on CF and LVSP were found to be exclusively mediated via AT(1a). The lack of AT(1a) or AT(1b) does not influence the development or maintenance of normal cardiac morphology, whereas deficiency for both receptors led to atrophic changes in the heart. Renal Ang II levels largely depend on AT(1) binding of extracellularly generated Ang II, and in the absence of all three Ang II receptors, renal Ang II is only located extracellularly.

Cardiovascular phenotype of mice lacking all three subtypes of angiotensin II receptors

Angiotensin II activates two distinct receptors, the angiotensin II receptors type 1 (AT1) and type 2 (AT2). In rodents, two AT1 subtypes were identified (AT1a and AT1b). To determine receptor‐specific functions and possible angiotensin II effects independent of its three known receptors we generated mice deficient in either one of the angiotensin II receptors, in two, or in all three (triple knockouts). Triple knockouts were vital and fertile, but survival was impaired. Hypotension and renal histological abnormalities in triple knockouts were comparable to those in mice lacking both AT1 subtypes. All combinations lacking AT1a were distinguished by reduced heart rate. AT1a deletion impaired the in vivo pressor response to angiotensin II bolus injection, whereas deficiency for AT1b and/or AT2 had no effect. However, the additional lack of AT1b in AT1a‐deficient mice further impaired the vasoconstrictive capacity of angiotensin II. Although general vasoconstrictor properties were not changed, angiotensin II failed to alter blood pressure in triple knockouts, indicating that there are no other receptors involved in direct angiotensin II pressor effects. Our data identify mice deficient in all three angiotensin II receptors as an ideal tool to better understand the structure and function of the reninangiotensin system and to search for angiotensin II effects independent of AT1 and AT2.—Gembardt, F., Heringer‐Walther, S., van Esch, J. H. M., Sterner‐Kock, A., van Veghel, R., Le, T. H., Garrelds, I. M., Coffman, T. M., Danser, A. H. J., Schultheiss, H.‐P., and Walther, T. Cardiovascular phenotype of mice lacking all three subtypes of angiotensin II receptors. FASEB J. 22, 3068–3077 (2008)

Angiotensin-(1-7) protects from experimental acute lung injury.

Objectives:Recently, recombinant angiotensin-converting enzyme 2 was shown to protect mice from acute lung injury, an effect attributed to reduced bioavailability of angiotensin II. Since angiotensin-converting enzyme 2 metabolizes angiotensin II to angiotensin-(1–7), we hypothesized that this effect is alternatively mediated by angiotensin-(1–7) and activation of its receptor(s). Design:To test this hypothesis, we investigated the effects of intravenously infused angiotensin-(1–7) in three experimental models of acute lung injury. Setting:Animal research laboratory. Subjects:Male Sprague-Dawley rats, Balb/c mice, and C57Bl6/J mice. Interventions:Angiotensin-(1–7) was administered with ventilator- or acid aspiration–induced lung injury in mice or 30 minutes after oleic acid infusion in rats. In vitro, the effect of angiotensin-(1–7) on transendothelial electrical resistance of human pulmonary microvascular endothelial cells was analyzed. Measurements and Main Results:Infusion of angiotensin-(1–7) starting 30 minutes after oleic acid administration protected rats from acute lung injury as evident by reduced lung edema, myeloperoxidase activity, histological lung injury score, and pulmonary vascular resistance while systemic arterial pressure was stabilized. Such effects were largely reproduced by the nonpeptidic angiotensin-(1–7) analog AVE0991. Infusion of angiotensin-(1–7) was equally protective in murine models of ventilator- or acid aspiration–induced lung injury. In the oleic acid model, the two distinct angiotensin-(1–7) receptor blockers A779 and D-Pro7-angiotensin-(1–7) reversed the normalizing effects of angiotensin-(1–7) on systemic and pulmonary hemodynamics, but only D-Pro7-angiotensin-(1–7) blocked the protection from lung edema and protein leak, whereas A779 restored the infiltration of neutrophils. Rats were also protected from acute lung injury by the AT1 antagonist irbesartan; however, this effect was again blocked by A779 and D-Pro7-angiotensin-(1–7). In vitro, angiotensin-(1–7) protected pulmonary microvascular endothelial cells from thrombin-induced barrier failure, yet D-Pro7-angiotensin-(1–7) or NO synthase inhibition blocked this effect. Conclusions:Angiotensin-(1–7) or its analogs attenuate the key features of acute lung injury and may present a promising therapeutic strategy for the treatment of this disease.