Hypoxia is an essential developmental and physiological stimulus that plays a key role in the pathophysiology of cancer, heart attack, stroke, and other major causes of mortality. Hypoxia-inducible factor 1 (HIF-1) is the only known mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia. We now report that in Hif1a −/− embryonic stem cells that did not express the O 2 -regulated HIF-1␣ subunit, levels of mRNAs encoding glucose transporters and glycolytic enzymes were reduced, and cellular proliferation was impaired. Vascular endothelial growth factor mRNA expression was also markedly decreased in hypoxic Hif1a −/− embryonic stem cells and cystic embryoid bodies. Complete deficiency of HIF-1␣ resulted in developmental arrest and lethality by E11 of Hif1a −/− embryos that manifested neural tube defects, cardiovascular malformations, and marked cell death within the cephalic mesenchyme. In Hif1a +/+ embryos, HIF-1␣ expression increased between E8.5 and E9.5, coincident with the onset of developmental defects and cell death in Hif1a −/− embryos. These results demonstrate that HIF-1␣ is a master regulator of cellular and developmental O 2 homeostasis.

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Hypoxia-inducible factor 1 (HIF-1) is a dimeric transcription factor composed of HIF-1alpha and HIF-1beta subunits that plays an essential role in mammalian O2 homeostasis. In Hif1a-/- knockout mice, complete deficiency of HIF-1alpha resulted in cardiac and vascular malformations and embryonic lethality at E10.5. Between E8. 75 and E9.25 striking vascular regression and abnormal remodeling occurred in the cephalic region concomitant with marked mesenchymal cell death. Similar vascular defects were observed in HIF-1alpha- and VEGF-deficient embryos and VEGF mRNA expression was not induced by hypoxia in Hif1a-/- embryonic stem cells. Surprisingly, Hif1a-/- embryos demonstrated increased VEGF mRNA expression compared to wild-type embryos. In tissue culture cells, VEGF mRNA expression was induced by glucose deprivation independent of HIF-1alpha, providing a mechanism for increased VEGF mRNA expression in Hif1a-/- embryos, in which absence of adequate tissue perfusion resulted in both O2 and glucose deprivation. Rather than being associated with VEGF deficiency, the vascular defects in Hif1a-/- embryos were spatially correlated with cell death, the onset of which preceded vascular regression.

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates cellular and systemic homeostatic responses to reduced O2 availability in mammals, including angiogenesis, erythropoiesis, and glycolysis. HIF-1 activity is controlled by the O 2-regulated expression of the HIF-1␣ subunit. Under nonhypoxic conditions, HIF-1␣ protein is subject to ubiquitination and proteasomal degradation. Here we report that missense mutations and͞or deletions involving several different regions of HIF-1␣ result in constitutive expression and transcriptional activity in nonhypoxic cells. We demonstrate that hypoxia results in decreased ubiquitination of HIF-1␣ and that missense mutations increase HIF-1␣ expression under nonhypoxic conditions by blocking ubiquitination.

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Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and blood vessel remodeling. Its expression is up-regulated in vascular smooth muscle cells by a number of conditions, including hypoxia. Hypoxia increases the transcriptional rate of VEGF via a 28-base pair enhancer located in the 5-upstream region of the gene. The gas molecules nitric oxide (NO) and carbon monoxide (CO) are important vasodilating agents. We report here that these biological molecules can suppress the hypoxia-induced production of VEGF mRNA and protein in smooth muscle cells. In transient expression studies, both NO and CO inhibited the ability of the hypoxic enhancer we have previously identified to activate gene transcription. Furthermore, electrophoretic mobility shift assays indicated decreased binding of hypoxia-inducible factor 1 (HIF-1) to this enhancer by nuclear proteins isolated from COtreated cells, although HIF-1 protein levels were unaffected by CO. Given that both CO and NO activate guanylyl cyclase to produce cGMP and that a cGMP analog (8-Br-cGMP) showed a similar suppressive effect on the hypoxic induction of the VEGF enhancer, we speculate that the suppression of VEGF by these two gas molecules occurs via a cyclic GMP-mediated pathway.Low oxygen tension is a potent regulator of diverse biological processes, including erythropoiesis, angiogenesis, and vascular cell contractility. These effects are mediated by several proteins that are induced under hypoxic environments and modulate cell-cell interactions, cell proliferation, and differentiation. In the vasculature, hypoxia regulates the expression of genes encoding growth factors such as endothelin-1 (ET-1) 1 , platelet-derived growth factor-B (PDGF-B) and vascular endothelial growth factor (VEGF), as well as genes regulating the production of gas molecules such as nitric oxide (NO) and carbon monoxide (CO) (1)(2)(3)(4)(5). Whereas the expression of the endothelial nitric oxide synthase gene is suppressed by hypoxia, the expression of heme oxygenase-1 (HO-1), the enzyme catalyzing the production of CO, is up-regulated by hypoxia (5).Mechanisms by which hypoxia alters gene expression include transcriptional and post-transcriptional regulation (4, 6, 7). Several hypoxia-responsive cis-acting elements have been identified (8,9). We have reported the presence of a 28-bp enhancer located approximately 980 bp upstream of the VEGF transcription start site, which is necessary and sufficient to up-regulate transcription of the VEGF gene in response to hypoxia (10). This hypoxia response element contains a sequence homologous to (and now has been included into) the hypoxia-inducible factor 1 (HIF-1) consensus (11). HIF-1 is a basic helix-loop-helix transcription factor originally identified to mediate the transcriptional activation of the erythropoietin gene (8) leading to enhanced erythropoiesis under hypoxia. It was subsequently shown to regulate the expression of genes encoding glycolytic enzymes (12) and the gene for VEGF (10, 11) implicating it as an i...

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