Hypoxia-induced apelin expression regulates endothelial cell proliferation and regenerative angiogenesis.

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 (+813/+826) located within the first intron of the human apelin gene. Chromatin immunoprecipitation assay reveals that hypoxia-inducible factor-1alpha binds to the endogenous hypoxia-responsive element site of the apelin gene. Moreover, overexpression of hypoxia-inducible factor-1alpha increases the transcriptional activity of a reporter construct containing this hypoxia-responsive element, whereas small interfering RNA-mediated hypoxia-inducible factor-1alpha 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.

Atherogenic properties of LDL particles modified by human group X secreted phospholipase A2 on human endothelial cell function.

Increasing evidence suggests that secreted phospholipases A2 (sPLA2s) play an important role in the pathophysiology of atherosclerosis. Among sPLA2s, the human group X (hGX) enzyme has the highest catalytic activity toward phosphatidylcholine, one of the major phospholipid species of cell membranes and low-density lipoprotein (LDL). Our study examined the presence of hGX sPLA2 in human atherosclerotic lesions and investigated the ability of hGX modified LDL to alter human endothelial cell (HUVEC) function. Our results show that hGX sPLA2 is present in human atherosclerotic lesions and that the hydrolysis of LDL by hGX sPLA2 results in a modified particle that induces lipid accumulation in human monocyte-derived macrophages. Acting on endothelial cells, hGX-modified LDL activates the MAP kinase pathway, which leads to increased arachidonic acid release, increased expression of adhesion molecules on the surface of HUVEC, and increased adhesion of monocytes to HUVEC monolayers. Together, our data suggest that LDL modified by hGX, rather than hGX itself may have strong proinflammatory and proatherogenic properties, which could play an important role in the propagation of atherosclerosis.

Phorbol ester induction of angiotensin-converting enzyme transcription is mediated by Egr-1 and AP-1 in human endothelial cells via ERK1/2 pathway.

Angiotensin-converting enzyme (ACE) is an enzyme that plays a major role in vasoactive peptide metabolism, and it has been implicated in various cardiovascular diseases. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, has been shown to increase ACE mRNA at the transcriptional level in human umbilical vein endothelial cells. We have investigated the transcriptional mechanism involved in protein kinase C induction of the ACE gene. Deletion and transfection analyses have revealed that two regions are required for PMA-inducible gene expression. The first is a G+C-rich region located in the proximal ACE promoter bearing overlapping consensus recognition sequences for stimulatory protein-1 (Sp1) and early growth response gene 1 (Egr-1). Electrophoretic mobility shift assay and supershift experiments have shown that Egr-1 is present in the specific nucleoprotein complex induced by PMA in human umbilical vein endothelial cells. The second region is located in the distal ACE promoter. DNase I footprinting analysis restricted this region to a 21-bp element containing a cAMP-responsive element/12-O-tetradecanoylphorbol 13-acetate-responsive element sequence. Electrophoretic mobility shift assays and supershift analyses have revealed that activating protein 1 (AP-1) is the transcription factor binding the cAMP-responsive element/12-O-tetradecanoylphorbol 13-acetate-responsive element located in the ACE promoter after PMA stimulation. Mutations of either Egr-1 or AP-1 binding sites partially abrogate ACE expression induced by PMA, whereas mutation of both sites totally abrogates PMA-induced ACE expression. Treatment of cells with PD98059, a mitogen-activated protein kinase kinase-1-specific inhibitor, inhibited PMA-induced ACE expression. Our results demonstrate that the two transcription factors, Egr-1 and AP-1, are involved in the PMA-induced ACE transcriptional activation in human endothelial cells via the activation of the extracellular signal-regulated kinase 1/2 signaling pathway.

Identification of hypoxia-response element in the human endothelial nitric-oxide synthase gene promoter.

The human endothelial nitric-oxide synthase gene (heNOS) is constitutively expressed in endothelial cells, and its expression is induced under hypoxia. The goal of this study was to search for regulatory elements of the endothelial nitric-oxide synthase (eNOS) gene responsive to hypoxia. Levels of eNOS mRNA, measured by real time reverse transcriptase-PCR analysis, were increased, and heNOS promoter activity was enhanced by hypoxia as compared with normoxia control experiments. Promoter truncation followed by footprint analysis allowed the mapping and identification of the hypoxia-responsive elements at position -5375 to -5366, closely related to hypoxia-inducible factor (HIF)-responsive element (HRE). To test whether known HIF-1 and HIF-2 are involved in hypoxia-induced heNOS promoter activation, HMEC-1 and HUVEC were transiently transfected with HIF-1alpha and HIF-1beta or HIF-2alpha and HIF-1beta expression vectors. Exogenous HIF-2 markedly increased luciferase reporter activity driven by the heNOS promoter in its native location. The induction of luciferase was conserved with the antisense construct and was increased in cotransfection experiments when this fragment was cloned 5' to the proximal 785-bp fragment of the eNOS promoter. Deletion analysis and site-directed mutagenesis demonstrated that the two contiguous HIF consensus binding sites spanning bp -5375 to -5366 relative to the transcription start site were both functional for heNOS promoter activity induction by hypoxia and by HIF-2 overexpression. In conclusion, we demonstrate that heNOS is a hypoxia-inducible gene, whose transcription is stimulated through HIF-2 interaction with two contiguous HRE sites located at -5375 to -5366 of the heNOS promoter.