Malnutrition affects up to one billion people in the world and is a major cause of mortality. In many cases, malnutrition is associated with diarrhoea and intestinal inflammation, further contributing to morbidity and death. The mechanisms by which unbalanced dietary nutrients affect intestinal homeostasis are largely unknown. Here we report that deficiency in murine angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (Ace2), which encodes a key regulatory enzyme of the renin-angiotensin system (RAS), results in highly increased susceptibility to intestinal inflammation induced by epithelial damage. The RAS is known to be involved in acute lung failure, cardiovascular functions and SARS infections. Mechanistically, ACE2 has a RAS-independent function, regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the ecology of the gut microbiome. Transplantation of the altered microbiota from Ace2 mutant mice into germ-free wild-type hosts was able to transmit the increased propensity to develop severe colitis. ACE2-dependent changes in epithelial immunity and the gut microbiota can be directly regulated by the dietary amino acid tryptophan. Our results identify ACE2 as a key regulator of dietary amino acid homeostasis, innate immunity, gut microbial ecology, and transmissible susceptibility to colitis. These results provide a molecular explanation for how amino acid malnutrition can cause intestinal inflammation and diarrhoea. DOI: https://doi.org/10.1038/nature11228Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-65968 Originally published at: Hashimoto, Tatsuo; Perlot, Thomas; Rehman, Ateequr; Trichereau, Jean; Ishiguro, Hiroaki; Paolino, Magdalena; Sigl, Verena; Hanada, Toshikatsu; Hanada, Reiko; Lipinski, Simone; Wild, Birgit; Camargo, Simone M R; Singer, Dustin; Richter, Andreas; Kuba, Keiji; Fukamizu, Akiyoshi; Schreiber, Stefan; Clevers, Hans; Verrey, Francois; Rosenstiel, Philip; Penninger, Josef M (2012 Supplementary Fig. 4), indicating that these effects are independent of the classical RAS system. Whether ACE inhibition might alter the phenotype of Agtr1a -/-Ace2 -/y mice needs to be further examined. In addition to cleaving AngII, ACE2 exhibits catalytic activity towards a second peptide system, Apelin 15 .However, DSS-induced colitis was not altered in mice carrying genetic mutations in Apelin ( Supplementary Fig. 5) or its receptor Apj (Supplementary Fig. 6). Thus, the catalytic activity of ACE2, essential for its function in the RAS and Apelin cleavage, has no overt role in DSS-induced intestinal inflammation.It had been reported that the RAS can control immune functions 16 . However, in unchallenged Ace2 mutant mice, we did not observe any apparent differences in immune cell populations of the colon and small intestine (insert: "not shown"?). Fig. 10a,b) nor did it affect apoptosis rates of intestinal epithelial cells ( Supplementary Fig. 10a,c).ACE levels were slightly, albeit not significa...

APJ is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin, was identified recently. They are highly expressed in the cardiovascular system, suggesting that APJ is important in the regulation of blood pressure. To investigate the physiological functions of APJ, we have generated mice lacking the gene encoding APJ. The base-line blood pressure of APJ-deficient mice is equivalent to that of wild-type mice in the steady state. The administration of apelin transiently decreased the blood pressure of wild-type mice and a hypertensive model animal, a spontaneously hypertensive rat. On the other hand, this hypotensive response to apelin was abolished in APJ-deficient mice. This apelininduced response was inhibited by pretreatment with a nitric-oxide synthase inhibitor, and apelin-induced phosphorylation of endothelial nitric-oxide synthase in lung endothelial cells from APJ-deficient mice disappeared. In addition, APJ-deficient mice showed an increased vasopressor response to the most potent vasoconstrictor angiotensin II, and the base-line blood pressure of double mutant mice homozygous for both APJ and angiotensin-type 1a receptor was significantly elevated compared with that of angiotensintype 1a receptor-deficient mice. These results demonstrate that APJ exerts the hypotensive effect in vivo and plays a counterregulatory role against the pressor action of angiotensin II.A family of G protein-coupled receptors bind a large variety of ligands and plays an essential role for physiological functions in vivo including the maintenance of homeostasis in the cardiovascular system. APJ (a putative receptor protein related to the angiotensin-type 1 receptor (AT1)) 1 is a G protein-coupled receptor that was isolated from human genomic DNA using the polymerase chain reaction (1). The APJ has a 31% amino acid sequence homology with the AT1, but APJ does not display specific binding for angiotensin II, which is the ligand of AT1 and exerts a pressor action in the blood pressure regulation (1). Recently, the endogenous ligand of APJ was identified from bovine stomach, and this peptide was named apelin (for APJ endogenous ligand) (2). APJ and apelin are expressed in several tissues including the cardiovascular and the central nervous systems (3-6), and the structure of APJ and apelin is highly conserved among species, suggesting its important physiological roles.Intravenous administration of apelin suggested a hypotensive effect in rat (5, 7-9). On the other hand, apelin potently contracts human saphenous vein smooth muscle cells in vitro (10), indicating that apelin is a potent vasoconstrictor. Thus, at this moment, the action of apelin in blood pressure regulation is controversial, and it is still unclear whether these actions of apelin are really through APJ because of the absence of specific receptor blocker to clarify the in vivo functions of APJ. Therefore, in this study, by using animal models such as APJ-deficient mice, APJ/AT1a double knock-out mice, and spontaneously hypertens...

The recently identified endogenous peptide apelin and its specific apelin receptor (APJ) are currently being considered as potential regulators in vascular tissue. Previously, we reported apelin mediates phosphorylation of myosin light chain and elicits vasoconstriction in vascular smooth muscle. In this study, physiological roles of the apelin-APJ system were investigated on atherosclerosis. In APJ and apolipoprotein E double-knockout (APJ ؊/؊ ApoE ؊/؊ ) mice fed a high-cholesterol diet, atherosclerotic lesions were dramatically reduced when compared with APJ Apelin receptor (APJ) is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin has been recently identified.1,2 The structures of APJ and apelin are highly conserved among species, and both are highly expressed in the cardiovascular system. 3,4 In the vascular system, APJ and apelin are known to be expressed in endothelium and vascular smooth muscle cells (VSMCs). Histological studies in rat show that the VSMCs of the medial layer of the aorta and pulmonary artery display intense staining for APJ-like immunoreactivity. 4 The vascular actions of apelin-APJ system may be complex. Under physiological conditions, the apelin-APJ system shows transient hypotension. The baseline blood pressure of APJ and angiotensin-type 1 receptor doubleknockout mice was significantly elevated compared with that of angiotensin-type 1 receptor knockout mice, 5 although APJ knockout (APJ Ϫ/Ϫ ) mice did not show any significant changes in cardiovascular parameters. In spontaneously hypertensive rats, APJ and apelin expression in both heart and aorta were markedly depressed compared with Wistar-Kyoto rats. 6 In aortae from type 2 diabetic db/db mice, APJ and apelin expression were

The renin-angiotensin system in the kidney plays a critical role in the regulation of renal hemodynamics and sodium handling through the activation of vascular, glomerular and tubular angiotensin II type 1 (AT1) receptor-mediated signaling. We previously cloned a molecule that specifically bound to the AT1 receptor and modulated AT1 receptor signaling in vitro, which we named ATRAP (for AT1 receptor-associated protein). The purpose of this study is to analyze the renal distribution of ATRAP and to examine whether ATRAP is co-expressed with the AT1 receptor in the mouse kidney. We performed in situ hybridization, Western blot analysis, and immunohistochemistry to investigate the expression of ATRAP mRNA and protein in the mouse kidney. The results of Western blot analysis revealed the ATRAP protein to be abundantly expressed in the kidney. Employing in situ hybridization and immunohistochemistry, we found that both ATRAP mRNA and the protein were widely distributed along the renal tubules from Bowman's capsules to the inner medullary collecting ducts. ATRAP mRNA was also detected in the glomeruli, vasculature, and interstitial cells. In all tubular cells, the ATRAP protein colocalized with the AT1 receptor. Finally, we found that the dietary salt depletion significantly decreased the renal expression of ATRAP as well as AT1 receptor. These findings show ATRAP to be abundantly and broadly distributed in nephron segments where the AT1 receptor is expressed. Furthermore, this is the first report demonstrating a substantial colocalization of ATRAP and AT1 receptor in vivo.

Objective-Physiological roles of apelin and its specific receptor APJ signaling were investigated in vascular smooth muscle cells (VSMCs). The present study determined whether apelin activates myosin light chain (MLC), a major regulatory event in initiating smooth muscle contraction. Methods and Results-To assess MLC activation, we performed Western blot and immunohistochemical studies using an antibody against the phospho-MLC. In VSMCs, apelin induces the phosphorylation of MLC in a concentrationdependent manner with a peak at 2 minutes. Pretreatment of VSMCs with pertussis toxin abolishes the apelin-induced phosphorylation of MLC. Inhibition of protein kinase C (PKC) with GF-109203X markedly attenuated the apelininduced MLC phosphorylation. In addition, methylisobutyl amiloride, a specific inhibitor of the Na ϩ /H ϩ exchanger (NHE), and KB-R7943, a potent inhibitor for the reverse mode of the Na ϩ /Ca 2ϩ exchanger (NCX), significantly suppressed the action of apelin. In wild-type mice, apelin phosphorylates MLC in vascular tissue, whereas it had no response in APJ-deficient mice by Western blot and immunohistochemistry. Apelin-induced phosphorylation of MLC was accompanied with myosin phosphatase target subunit phosphorylation. Conclusions-These results provide the first evidence to our knowledge for apelin-mediated MLC phosphorylation in vitro and in vivo, which is a potential mechanism of apelin-mediated vasoconstriction. (Arterioscler Thromb Vasc Biol. 2006;26:1267-1272.)Key Words: apelin Ⅲ APJ Ⅲ myosin light chain Ⅲ myosin phosphatase target subunit Ⅲ vasoconstriction A pelin was recently identified from bovine stomach as an endogenous ligand for APJ, a putative receptor protein related to the angiotensin-type 1 receptor (AT1). 1 Despite sharing 31% amino acid sequence homology with AT1, APJ does not display specific binding for angiotensin II. 2 Apelin and APJ are distributed in various tissues including the heart, blood vessels, brain, and gastrointestinal tract, although the physiological role of apelin and APJ is not well understood. [3][4][5][6][7] In the vascular system, apelin and APJ are known to be expressed in endothelium and smooth muscle cells (VSMCs). Histological studies in rat show that the VSMCs of the medial layer of the aorta and pulmonary artery display intense staining for APJ receptor-like immunoreactivity. 7 In spontaneously hypertensive rats, APJ and apelin expression in both heart and aorta were markedly depressed compared with Wistar-Kyoto rats, which suggests a pathophysiological role for APJ and apelin in vascular disease. 8 The action of apelin in blood pressure regulation is controversial. Although the systemic administration of apelin produces transient hypotension in anesthetized 5,9 -11 and conscious rats, 12 a potent vasoconstrictive effect of the peptide has been demonstrated in the isolated human saphenous vein. 13 These results suggest that the apelin-APJ has biphasic effects on blood vessels via the endothelium-mediated vasodilatation and VSMCs-dependent vasoconstrictio...

Angiotensin II (AII) is a multifunctional bioactive peptide, and host renin-angiotensin system (RAS) is closely associated with tumor growth. Recent reports have described that AII is a proangiogenic growth factor, and that Angiotensin II type 1 (AT1) receptor antagonists reduce tumor growth and tumor-associated angiogenesis. In this paper, we investigated the participation of AT1 receptor-signaling in cancer progression using murine Lewis lung carcinoma (LLC) cells, which express AT1 receptor, and AT1a receptor gene-deficient (AT1aÀ/À) mice. When LLC cells were implanted subcutaneously into wild-type (WT) mice, developed tumors showed intensive angiogenesis with an induction of vascular endothelial growth factor (VEGF) a. Compared with WT mice, tumor growth and tumor-associated angiogenesis was reduced in AT1aÀ/À mice with reduced expression of VEGFa. In AT1aÀ/À mice, administration of the AT1 receptor antagonist, TCV-116, showed further reductions of tumor growth, tumor-associated angiogenesis, and VEGFa expression. In vitro study, the expression of VEGFa mRNA and the production of VEGFa protein in LLC cells were significantly increased by AII, which were cancelled by AT1 receptor antagonist, CV-11974. Although the expression of other angiogenic factors, such as angiopoietin-1, angiopoietin-2, epidermal growth factor, and VEGF receptor 2 mRNA, was also investigated in tumor tissues, the expression of VEGFa was most correlated with tumor size among those other angiogenic factors. VEGFa induction by AT1 receptor-signaling in both host and tumor tissues is one of key regulators of tumor growth and tumor-associated angiogenesis. In conclusion, tumor tissue RAS as well as host tissue RAS were found to have an important role in tumor growth. AT1 receptor-signaling blockade may be a novel and effective target in the treatment of cancer. The renin-angiotensin system (RAS) plays important roles in the regulation of cardiovascular homeostasis and blood pressure. 1 Many pathophysiological activities of angiotensin II (AII) are known to be mediated by the seven transmembrane receptors. Two major subtypes of AII receptors, namely AT1 receptor and AT2 receptor, have been identified, with the former having receptor subtypes, AT1a and AT1b. 2 Most of AII functions in the cardiovascular system are mediated through the AT1 receptor, and in rodents they are mediated through the AT1a receptor. [3][4][5][6] Recently, many reports have suggested that AII is involved in other functions, such as apoptosis, vascular remodeling, and inflammation. 7-9 As regards vascular remodeling, several studies have shown that AII promotes proliferation, migration, and growth factor synthesis in several types of vascular cells, including smooth muscle cells and pericytes. [10][11][12][13] Other studies have also investigated the angiogenic effects of exogenous AII in vivo angiogenesis models. 14-17 Furthermore, recent studies have revealed local expression of several components of the RAS in various cancer cells and tissues. 18 A large-scale clinica...

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Abstract-We have recently cloned a novel molecule that interacts with the angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP).In this study, we tested the hypothesis that ATRAP modulates angiotensin II-induced responses in vascular smooth muscle cells. The results of immunoprecipitation and bioluminescence resonance energy transfer assay demonstrated a direct interaction between ATRAP and AT1R at baseline and showed that angiotensin II enhanced the interaction of these proteins Ͼ2-fold. The results of immunofluorescence analysis also demonstrated that Ͼ65% of ATRAP constitutively colocalized with an endosome marker. Although only 36% of ATRAP colocalized with AT1R at baseline, angiotensin II enhanced the colocalization of these molecules and made 92% of ATRAP colocalize with AT1R on a quantitative fluorescence analysis. Overexpression of ATRAP by adenoviral transfer decreased the cell surface AT1R number from 4.33 to 2.13 fmol/10 6 cells at baseline and from 3.04 to 1.26 fmol/10 6 cells even after removal of angiotensin II. ATRAP also suppressed angiotensin II-mediated increases in c-fos gene transcription and transforming growth factor-␤ production. Furthermore, this suppression was accompanied by inhibition of angiotensin II-induced activation of 5-bromodeoxyuridine incorporation. Finally, ATRAP knockdown by small-interference RNA activated angiotensin II-induced c-fos gene expression, which was effectively inhibited by valsartan, an AT1R-specific antagonist. These results indicate that ATRAP promotes internalization of AT1R and attenuates the angiotensin II-mediated c-fos-transforming growth factor-␤ pathway and proliferative response in vascular smooth muscle cells, suggesting a novel strategy to inhibit vascular fibrosis and remodeling through a novel and specific blockade of AT1R signaling. (AT1R) is a member of the G protein-coupled receptor superfamily and activates G proteins through the third intracellular loop and the intracellular carboxyl-terminal (C-terminal) tail of the receptor. 1,2 The C-terminal cytoplasmic end of AT1R is involved in the control of AT1R internalization independent of G protein coupling, and it plays an important role in linking receptormediated signal transduction to the specific biological response to angiotensin II (Ang II), such as cardiovascular fibrosis and remodeling. 3,4 Using a yeast 2-hybrid screening system, we recently cloned a novel AT1R-associated protein (ATRAP) that specifically interacts with the C-terminal cytoplasmic domain of AT1R. [5][6][7] We showed that ATRAP is expressed in a variety of tissues and suppresses Ang IImediated hypertrophic responses in cardiac myocytes. 8,9 ARAP1 is another protein that was found recently to interact with the C-terminal domain of AT1R. 10 Characterization of ARAP1 has revealed that ARAP1 binds and promotes recycling of AT1R to the plasma membrane, indicating its role in the receptor-recycling pathway. In this study, we examined the function of ATRAP in Ang II-induced fibrotic and proliferative responses of rat ...