It has been shown that oxygen deprivation results in apoptotic cell death, and that hypoxia inducible factor 1 (HIF1) and the tumor suppressor p53 play key roles in this process. However, the molecular mechanism through which hypoxia and HIF1 induce apoptosis is not clear. Here we show that the expression of pro-apoptotic gene BNIP3 is dramatically induced by hypoxia in various cell types, including primary rat neonatal cardiomyocytes. Overexpression of HIF1a, but not p53, induces the expression of BNIP3. Overexpression of BNIP3 leads to a rather unusual type of apoptosis, as no cytochrome c leakage from mitochondria was detected and inhibitors of caspases were unable to prevent cell death. Taken together, these data suggest that HIF1-dependent induction of BNIP3 may play a significant role during hypoxiainduced cell death. Cell Death and Differentiation (2001) 8, 367 ± 376.

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Hypoxia-inducible transcription factors (HIF) mediate complex adaptations to reduced oxygen supply, including neoangiogenesis. Regulation of HIF occurs mainly through oxygen-dependent destruction of its alpha subunit. In the presence of oxygen, two HIFalpha prolyl residues undergo enzymatic hydroxylation, which is required for its proteasomal degradation. We therefore tested whether pharmacological activation of HIFalpha by hydroxylase inhibitors may provide a novel therapeutic strategy for the treatment of ischemic diseases. Three distinct prolyl 4-hydroxylase inhibitors-l-mimosine (L-Mim), ethyl 3,4-dihydroxybenzoate (3,4-DHB), and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamid (S956711)-demonstrated similar effects to hypoxia (0.5% O2) by inducing HIFalpha protein in human and rodent cells. L-Mim, S956711, and, less effectively, 3,4-DHB also induced HIF target genes in cultured cells, including glucose transporter 1 and vascular endothelial growth factor, as well as HIF-dependent reporter gene expression. Systemic administration of L-Mim and S956711 in rats led to HIFalpha induction in the kidney. In a sponge model for angiogenesis, repeated local injection of the inhibitors strongly increased invasion of highly vascularized tissue into the sponge centers. In conclusion, structurally distinct inhibitors of prolyl hydroxylation are capable of inducing HIFalpha and HIF target genes in vitro and in vivo and induce adaptive responses to hypoxia, including angiogenesis.

Blood vessel recruitment is an important feature of normal tissue growth. Here, we examined the role of Akt signaling in coordinating angiogenesis with skeletal muscle hypertrophy. Hypertrophy of C2C12 myotubes in response to insulin-like growth factor 1 or insulin and dexamethasone resulted in a marked increase in the secretion of vascular endothelial growth factor (VEGF). Myofiber hypertrophy and hypertrophy-associated VEGF synthesis were specifically inhibited by the transduction of a dominant-negative mutant of the Akt1 serine-threonine protein kinase. Conversely, transduction of constitutively active Akt1 increased myofiber size and led to a robust induction of VEGF protein production. Akt-mediated control of VEGF expression occurred at the level of transcription, and the hypoxia-inducible factor 1 regulatory element was dispensable for this regulation. The activation of Akt1 signaling in normal mouse gastrocnemius muscle was sufficient to promote myofiber hypertrophy, which was accompanied by an increase in circulating and tissue-resident VEGF levels and high capillary vessel densities at focal regions of high Akt transgene expression. In a rabbit hind limb model of vascular insufficiency, intramuscular activation of Akt1 signaling promoted collateral and capillary vessel formation and an accompanying increase in limb perfusion. These data suggest that myogenic Akt signaling controls both fiber hypertrophy and angiogenic growth factor synthesis, illustrating a mechanism through which blood vessel recruitment can be coupled to normal tissue growth.

Basic fibroblast growth factor (FGF) stimulates the proliferation, differentiation, and motility of multiple cell types. Signal transduction by FGF is mediated by high affinity FGF receptors that have autophosphorylating tyrosine kinase activity and also elicit the release of low molecular weight signaling molecules, including inositol 1,4,5-trisphosphate, diacylglycerol, and arachidonate. We have shown previously that basic FGF-stimulated, phospholipase A2 (PLA2)-mediated arachidonate release regulates endothelial cell (EC) motility (Sa, G., and Fox, P.L. (1994) J. Biol. Chem. 269, 3219-3225). Here we identify the phospholipase responsible for basic FGF-mediated arachidonate release as cytosolic PLA2 (cPLA2) by demonstrating in EC lysates a requirement for micromolar Ca2+, dithiothreitol insensitivity, and inactivation by anti-cPLA2 antiserum. The role of cPLA2 is also indicated by the observed mechanisms of activation which show a requirement for p42 mitogen-activated protein kinase activity, cPLA2 phosphorylation, and cPLA2 translocation from cytosol to membranes. Phosphorylation of cPLA2, arachidonate release from prelabeled intact cells, and cell motility all have similar concentration dependencies on basic FGF. Since arachidonate release is required for basic FGF-stimulated motility of EC, our results show that p42 mitogen-activated protein kinase activation of cPLA2 may be a regulatory event in stimulation of cellular release of this important eicosanoid precursor during cellular responses to basic FGF.