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

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...

Although vascular smooth muscle cells (VSMCs) are widely used in cardiovascular research, their phenotypic change under various culture conditions is problematic to evaluate the experimental results obtained. The levels of angiotensin (Ang) type 1/2 (AT1/AT2) receptors as well as contractile and structural proteins are degraded through culture passages. The present study demonstrated that heparin recovered Ang receptors and differentiation markers, such as desmin, SM-22 and smooth muscle a-actin in VSMCs at the ninth passage. Heparin also potenciated Ang II-induced activation for ERK1/2 and p38. These results suggest a potential value of heparin-treated VSMCs as the model for analysis of Ang-mediated signal transduction under physiological condition.