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Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated alpha subunit and a constitutively expressed beta subunit. Although HIF-1 is regulated mainly by oxygen tension through the oxygen-dependent degradation of its alpha subunit, in vitro it can also be modulated by cytokines, hormones and genetic alterations. To investigate HIF-1 activation in vivo, we determined the spatial and temporal distribution of HIF-1 in healthy mice subjected to varying fractions of inspiratory oxygen. Immunohistochemical examination of brain, kidney, liver, heart, and skeletal muscle revealed that HIF-1alpha is present in mice kept under normoxic conditions and is further increased in response to systemic hypoxia. Moreover, immunoblot analysis showed that the kinetics of HIF-1alpha expression varies among different organs. In liver and kidney, HIF-1alpha reaches maximal levels after 1 h and gradually decreases to baseline levels after 4 h of continuous hypoxia. In the brain, however, HIF-1alpha is maximally expressed after 5 h and declines to basal levels by 12 h. Whereas HIF-1beta is constitutively expressed in brain and kidney nuclear extracts, its hepatic expression increases concomitantly with HIF-1alpha. Overall, HIF-1alpha expression in normoxic mice suggests that HIF-1 has an important role in tissue homeostasis.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a master transcriptional regulator of oxygen homeostasis and a possible target for gene therapy of ischemic disease. Although the role of oxygen concentration in HIF-1a protein stabilization is well established, it is less clear whether and how oxygen-regulated mechanisms contribute to HIF-1a protein modifications, nuclear translocation, heterodimerization with the b-subunit, recruitment of cofactors, and gene trans-activation. Because the HIF-1a protein is proteolytically degraded under normoxic conditions, we established two HeLa Tet-Off cell lines (HT42 and HT43), which inducibly overexpress high levels of HIF-1a under normoxic conditions, allowing to distinguish hypoxia-dependent from hypoxia-independent activation mechanisms. Using these cells, we found that normoxically induced HIF-1a is localized to the nucleus, binds DNA, and trans-activates reporter and endogenous target genes. The levels of p53 expression remained unaffected. The MAP kinase inhibitor PD98059 attenuated HIF-1a protein modifications and trans-activation ability but not protein stabilization and DNA-binding activity. Because overexpressed HIF-1a is fully localized to the nucleus but displays only partial DNA-binding and trans-activation activity, mitogen-activated protein kinase-dependent phosphorylation might be required for full HIF-1 activation. HIF-1a protein was also overexpressed in vivo, following the transplantation of HT42 cells into nude mice, demonstrating the feasibility of HIF-1a gene transfer.

Little is known about the factors influencing the hemoglobin switch in vertebrates during development. Inasmuch as the mammalian conceptus is exposed to changing oxygen tensions in utero, we examined the effect of different oxygen concentrations on beta-globin switching. We used an in vitro model of mouse embryogenesis based on the differentiation of blastocyst-derived embryonic stem cells to embryoid bodies (EBs). Cultivation of EBs at increasing oxygen concentrations (starting at 1% O2) did not influence the temporal expression pattern of embryonic (betaH1) globin compared to the normoxic controls (20% O2). In contrast, when compared to normoxically grown EBs, expression of fetal/adult (betamaj) globin in EBs cultured at varying oxygen concentrations was delayed by about 2 days and persisted throughout differentiation. Quantitation of hemoglobin in EBs using a 2,7-diaminofluorene-based colorimetric assay revealed the appearence of hemoglobin in two waves, an early and a late one. This observation was verified by spectrophotometric analysis of hemoglobin within single EBs. These two waves might reflect the switch of erythropoiesis from yolk sac to fetal liver. Reduced oxygenation is known to activate the hypoxia-inducible factor-1 (HIF-1), which in turn specifically induces expression of a variety of genes among them erythropoietin (EPO). Although EBs increased EPO expression upon hypoxic exposure, the altered beta-globin appearance was not related to EPO levels as determined in EBs overexpressing EPO. Since mRNA from both mouse HIF-1alpha isoforms was detected in all EBs tested at different differentiation stages, we propose that HIF-1 modulates beta-globin expression during development.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a master regulator of oxygen homeostasis. Protein stability and transactivation function of the alpha subunit are controlled by iron- and oxygen-dependent hydroxylation of proline and asparagine residues. The anti-mycotic ciclopirox olamine (CPX) is a lipophilic bidentate iron chelator that stabilizes HIF-1alpha under normoxic conditions at lower concentrations than other iron chelators, probably by inhibiting HIF-1alpha hydroxylation. As shown by the inhibition of iron-dependent quenching of FITC-labeled deferoxamine (DFX) fluorescence, CPX appears to have an even higher affinity for iron than DFX. Initial observations that treatment with 1% CPX, but not with placebo, occasionally caused reddening of wound margins in a mouse skin wound model prompted us to investigate the capability of CPX to induce angiogenesis. CPX-induced HIF-1-mediated reporter gene activity and endogenous HIF-1 target gene expression, including elevation of transcription, mRNA, and protein levels of the vascular endothelial growth factor (VEGF). In the chick chorioallantoic membrane assay, inert polymer disks containing CPX but not the solvent alone induced angiogenesis. In summary, these results suggest that CPX induces angiogenesis in vivo via HIF-1 and VEGF induction. Therefore, CPX might serve as an alternative to recombinant VEGF treatment or to VEGF gene therapy for therapeutic angiogenesis.

In the first-trimester mammalian fetus, skin wounds heal with perfect reconstitution of the dermal architecture without scar formation. Understanding environmental molecular regulation in fetal wound healing may reveal scar-limiting therapeutical strategies for the prevention of postnatal scarring wound repair. Therefore, we performed studies on fetal skin oxygenation and skin and wound expression of hypoxia-inducible factor 1alpha (HIF-1alpha) in the sheep model in vivo and performed studies on the potential relevance of HIF-1alpha during wound healing in vitro. Skin oxygen partial pressure levels were hypoxic throughout normal development. In nonscarring fetal skin at gestation day (GD)60, HIF-1alpha could be detected neither in healthy nor in wounded tissue. At GD100, in wounds with minimal scar formation, HIF-1alpha was expressed in fibroblasts and was markedly up-regulated at the wound edge. In scarring fetal wounds at GD120, HIF-1alpha was predominantly expressed in inflammatory cells. Expression of transforming growth factor beta3 (TGF-beta3), a potent antiscarring cytokine, overlapped with HIF-1a expression at GD100. HIF-1alpha-deficient mouse embryonic fibroblasts showed impaired migratory capabilities and demonstrated that TGF-beta3, but not proscarring TGF-beta1, manifests hypoxia- and HIF-1alpha-dependent regulation. In conclusion, HIF-1alpha-dependent regulation of a potent antiscarring cytokine may provide new strategies for antiscarring manipulation of wound healing.

SPECIFIC AIMSDespite the pivotal role the hypoxia-inducible factor 1α (HIF-1α) plays in physiological and pathological processes, little is known regarding the time frame and mechanisms involved in...