Hypoxia-Induced Apelin Expression Regulates Endothelial Cell Proliferation and Regenerative Angiogenesis

Abstract

Abstract-Apelin has been identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ. This peptide exerts a variety of cardiovascular effects and particularly acts as an activator of angiogenesis. Importantly, hypoxia has been reported to regulate apelin expression but the molecular mechanism underlying hypoxia-induced apelin expression and the relationship with the physiological response of the apelin/APJ system are still not established. Here, we demonstrate that apelin expression is induced by hypoxia in cultured endothelial and vascular smooth muscle cells as well as in lung from mice exposed to acute hypoxia. Transient transfection experiments show that hypoxia-inducible transcriptional activation of apelin requires an intact hypoxia-responsive element (ϩ813/ϩ826) located within the first intron of the human apelin gene. Chromatin immunoprecipitation assay reveals that hypoxia-inducible factor-1␣ binds to the endogenous hypoxia-responsive element site of the apelin gene. Moreover, overexpression of hypoxia-inducible factor-1␣ increases the transcriptional activity of a reporter construct containing this hypoxia-responsive element, whereas small interfering RNA-mediated hypoxia-inducible factor-1␣ knockdown abolishes hypoxia-induced apelin expression. Finally, microinterfering RNA-mediated apelin or APJ receptor knockdown inhibits both hypoxia-induced endothelial cell proliferation in vitro and hypoxia-induced vessel regeneration in the caudal fin regeneration of Fli-1 transgenic zebrafish. The hypoxia-induced apelin expression may, thus, provide a new mechanism involved in adaptive physiological and pathophysiological response of vascular cells to low oxygen level. Key Words: hypoxia Ⅲ transcriptional regulation Ⅲ endothelial cell growth-APJ Ⅲ zebrafish model T he apelin peptide, originally isolated from bovine stomach tissue extracts, has been identified as the endogenous ligand of the human orphan G protein-coupled receptor APJ, a receptor related to the angiotensin II receptor type 1. Apelin and APJ are distributed in various tissues where it exerts a broad range of physiological actions, including heart contractility, appetite, and drinking behavior or the hypothalamicpituitary-adrenal axis. 1 The role of apelin in the cardiovascular system is currently the most documented. Apelin and APJ were reported to be expressed in cardiomyocytes, endothelial, and vascular smooth muscle cells. 2,3 Apelin-deficient mice are viable, but they present impaired cardiac contractility with aging and pressure overload, indicating that apelin/ APJ pathway is important for maintaining cardiac function. 4 Apelin was reported to positively regulate angiogenesis both in vitro and in vivo. Apelin stimulates endothelial cell proliferation, migration, and tube formation in vitro. 5,6 In the frog embryo, apelin is crucial for the normal development of the embryonic vasculature. 7,8 Apelin expression is upregulated during tumor angiogenesis, and its overexpression was reported to increase the in vivo ...

The apelin receptor APJ: journey from an orphan to a multifaceted regulator of homeostasis

“…The pathophysiological effects of apelin in angiogenesis have also been reported for the liver, where the apelinergic system is a factor in portosystemic collaterisation and splanchnic neovascularisation in portal hypotensive rats (Tiani et al 2009) as well as in neovascularisation during liver cirrhosis (Principe et al 2008). However, apelin may have therapeutic effects in ischaemia recovery due to vessel regeneration and endothelial proliferation (Eyries et al 2008) and blood vessel diameter regulation (Kidoya et al 2010). These findings indicate that apelin is a crucial factor for angiogenesis and that there may be therapeutic potential in both the disruption of its signalling (e.g.…”

“…In pluripotent embryonic stem cells, apelin induces the phosphorylation (but not full activation) of p70S6K, via the upstream activation of ERK1/2 (D'Aniello et al 2009). Although in one study of hypoxia apelin has been shown to induce the phosphorylation of p70S6K via mTOR in mouse embryonic endothelial cells (Eyries et al 2008), the protective action of apelin in the rat ischaemic heart does not appear to be mediated by p70S6K (Kleinz & Baxter 2008).…”

“…Putative hypoxia response elements (HREs) are present in the apelin gene promoter and intron sequence of various species in silico (Cox et al 2006), and hypoxia has subsequently been found to up-regulate the expression of apelin in cardiac myocytes (Ronkainen et al 2007), and hypoxia-inducible factor 1a (HIF1a) has been reported to induce the expression of apelin in adipocytes (Glassford et al 2007). Additional studies have determined that hypoxia-inducible apelin up-regulation is mediated by HIF1a at a HRE within the APLN intron (conserved in rat and mouse apelin genes) between C813 and C826 bp (Eyries et al 2008), indicating that apelin may play a role in the homeostatic response to low oxygen levels. Insulin has also been shown to increase the expression of apelin in human and mouse adipocytes, via PI3K, protein kinase C (PKC), mitogen-activated and ERK kinase (MAPK) 1 (Boucher et al 2005) and HIF1a (Glassford et al 2007), while aldosterone has been shown to decrease the expression of apelin in 3T3-L1 adipocytes via the p38 MAPK pathway (Jiang et al 2013).…”

The apelin/APJ system induces maturation of the tumor vasculature and improves the efficiency of immune therapy

“…Apelin and APJ mediate a wide range of physiological actions, including angiogenesis (Cox et al, 2006;Eyries et al, 2008;Kasai et al, 2008;Kidoya et al, 2008), heart contractility, blood pressure regulation (Dai et al, 2006) and other effects (Sorhede Winzell et al, 2005;Lambrecht et al, 2006;Lago et al, 2007). Apelin and APJ are expressed on ECs of newly developing blood vessels during angiogenesis (Kidoya et al, 2008), and it has been reported that apelin expression is induced by hypoxia in ECs (Eyries et al, 2008).…”

Role of three adipokines in metabolic syndrome

“…6 Furthermore, a role in the control of food intake and stimulation of gastric, endothelial, and vascular smooth muscle cell proliferation have been reported. [7][8][9] An increase in plasma apelin concentrations in human and animal models has been observed in patients with metabolic syndrome. 10 In this syndrome, apelin has a beneficial effect on the improvement of insulin sensitivity associated with an intervention both in glucose and lipid metabolism.…”