Abstract-Hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of vascular endothelial growth factor (VEGF) and other hypoxia-responsive genes. Transgenic expression of a constitutively stable HIF-1␣ mutant increases the number of vascular vessels without vascular leakage, tissue edema, or inflammation. This study aimed to investigate the molecular basis by which HIF-1 mediates the angiogenic response to hypoxia. In primary human endothelial cells, hypoxia, desferrioxamine, or infection with Ad2/HIF-1␣/VP16, an adenoviral vector encoding a constitutively stable hybrid form of HIF-1␣, increased the mRNA and protein levels of VEGF, angiopoietin-2 (Ang-2), and angiopoietin-4 (Ang-4). Infection with Ad2/CMVEV (a control vector expressing no transgene) had no effect. Angiopoietin-1 (Ang-1) expression was not detected in human endothelial cells. Ang-4 was also induced by hypoxia or Ad2/HIF-1␣/VP16 in human cardiac cells, whereas Ang-1 expression remained unchanged. Recombinant Ang-4 protein protected endothelial cells against serum starvation-induced apoptosis and increased cultured endothelial cell migration and tube formation. Ad2/HIF-1␣/VP16 stimulated endothelial cell proliferation and tube formation. Hypoxia-or Ad2/HIF-1␣/VP16-induced tube formation was significantly reduced by a Tie-2 inhibitor. These results suggest that HIF-1 mediates the angiogenic response to hypoxia by upregulating the expression of multiple angiogenic factors. Ang-4 can function similarly as Ang-1 and substitute for Ang-1 to participate in hypoxia-induced angiogenesis. Activation of the angiopoietin/Tie-2 system may play a role in the ability of HIF-1 to induce hypervascularity without excessive permeability. (Circ Res. 2003;93:664-673.)Key Words: hypoxia-inducible factor-1 Ⅲ hypoxia Ⅲ angiogenesis Ⅲ angiopoietins Ⅲ vascular endothelial growth factor A dministration of a single growth factor in the form of protein or gene has been shown to promote tissue neovascularization in animal models and patients. 1,2 However, transgenic overexpression of vascular endothelial growth factor (VEGF) alone in mice results in increased numbers of primarily leaky vascular vessels with tissue edema and inflammation, 3,4 suggesting that VEGF needs to work in conjunction with other angiogenic factors to produce a healthy vasculature. 5,6 In a variety of conditions, such as malignant tumors, wound healing, and myocardial ischemia, hypoxia is a fundamental stimulus for angiogenesis. 7,8 Activation of hypoxia-responsive genes including VEGF is mediated by hypoxia-inducible factor-1 (HIF-1), a heterodimeric basic helix-loop-helix-PAS domain transcription factor. 9,10 HIF-1 is composed of two subunits, HIF-1␣ and HIF-1␤ (aryl hydrocarbon nuclear translocator). Whereas the ␤-subunit protein is constitutively present, the stability of the ␣-subunit and its transcriptional activity are precisely controlled by the intracellular oxygen concentration. [11][12][13][14][15] Knocking-out the HIF-1␣ alleles in mice results in embryonic lethality with vascul...

. Expression of constitutively stable hybrid hypoxia-inducible factor 1␣ protects cultured rat cardiomyocytes against simulated ischemia-reperfusion injury.

Ϫ cells with VHL mutations, the protein of hypoxia-inducible factor-1␣ (HIF-1␣) is constitutively stabilized and the mRNA levels of HIF target genes, including vascular endothelial growth factor (VEGF), are elevated. However, the expression of angiopoietins in these cells and their involvement in angiogenesis are not well known. In this study, we compared the mRNA levels of angiopoietins in human kidney proximal tubule epithelial (RPTE) and RCC4 and RCC786 -0 VHL Ϫ cells. In RPTE cells, angiopoietin-4 (Ang-4) expression was selectively induced by hypoxia or by expression of a hybrid form of HIF-1␣. Under normoxic conditions, the mRNA levels of Ang-4 were higher in RCC4 and RCC786 -0 VHL Ϫ than RPTE cells. Angiopoietin-1 expression was detectable in RCC4 and RCC786 -0 VHL Ϫ cells but not RPTE cells. In RCC786 -0 VHL ϩ cells, which were stably transfected with a wild-type copy of VHL, the mRNA levels of VEGF and Ang-4 were suppressed and the hypoxic response was restored. We also demonstrated that stimulation of endothelial tube formation by conditioned medium harvested from RCC4 cells was inhibited by a soluble Tie-2 receptor. These results suggest that the angiopoietin/Tie-2 system may participate in the angiogenic response to hypoxia in renal tissues and in tumor angiogenesis in renal carcinoma.

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