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Aims

Hypoxia-inducible factor 1 (HIF-1) is a heterodimer composed of HIF-1α and HIF-1β subunits. HIF-1 is known to promote tissue vascularization by activating the transcription of genes encoding angiogenic factors, which bind to receptors on endothelial cells (ECs) and bone marrow-derived angiogenic cells (BMDACs). In this study, we analysed whether HIF-1 activity in the responding ECs and BMDACs is also required for cutaneous vascularization during burn wound healing.

Methods and results

We generated mice with floxed alleles at the Hif1a or Arnt locus encoding HIF-1α and HIF-1β, respectively. Expression of Cre recombinase was driven by the Tie2 gene promoter, which is expressed in ECs and bone marrow cells. Tie2Cre+ and Tie2Cre− mice were subjected to burn wounds of reproducible diameter and depth. Deficiency of HIF-1α or HIF-1β in Tie2-lineage cells resulted in delayed wound closure, reduced vascularization, decreased cutaneous blood flow, impaired BMDAC mobilization, and decreased BMDAC homing to burn wounds.

Conclusion

HIF-1 activity in Tie2-lineage cells is required for the mobilization and homing of BMDACs to cutaneous burn wounds and for the vascularization of burn wound tissue.

The mechanisms that regulate angiogenesis in hypoxia or hypoxic microenvironment are modulated by several pro- and antiangiogenic factors. Hypoxia-inducible factors (HIFs) have been established as the basic and major inducers of angiogenesis, but understanding the role of interacting proteins is becoming increasingly important to elucidate the angiogenic processes of a hypoxic response. In particular, with regard to wound healing and the novel therapies for vascular disorders such as ischemic brain and heart attack, it is essential to gain insights in the formation and regulation of HIF transcriptional machineries related to angiogenesis. Further, identification of alternative ways of inhibiting tumor growth by disrupting the growth-triggering mechanisms of increasing vascular supply via angiogenesis depends on the knowledge of how tumor cells develop their own vasculature. Here, we review our findings on the interactions of basic HIFs, HIF-1α and HIF-2α, with their regulatory binding proteins, histone deacetylase 7 (HDAC7) and translation initiation factor 6 (Int6), respectively. The present results and discussion revealed new regulatory interactions of HIF-related mechanisms.

The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important molecular target for anticancer drug discovery. In a T47D cell-based reporter assay, the Caulerpa spp. algal pigment caulerpin (1) inhibited hypoxia-induced as well as 1,10-phenanthroline-induced HIF-1 activation. The angiogenic factor vascular endothelial growth factor (VEGF) is regulated by HIF-1. Caulerpin (10 μM) suppressed hypoxic induction of secreted VEGF protein and the ability of hypoxic T47D cell-conditioned media to promote tumor angiogenesis in vitro. Under hypoxic conditions, 1 (10 μM) blocked the induction of HIF-1α protein, the oxygen-regulated subunit that controls HIF-1 activity. Reactive oxygen species produced by mitochondrial complex III are believed to act as a signal of cellular hypoxia that leads to HIF-1α protein induction and activation. Further mechanistic studies revealed that 1 inhibits mitochondrial respiration at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Under hypoxic conditions, it is proposed that 1 may disrupt mitochondrial ROS-regulated HIF-1 activation and HIF-1 downstream target gene expression by inhibiting the transport or delivery of electrons to complex III.

Impaired wound healing in the elderly represents a major clinical problem. Delineating the cellular and molecular mechanisms by which aging impairs wound healing may lead to the development of improved treatment strategies for elderly patients with non-healing wounds. Neovascularization is an essential step in wound healing, and bone marrow-derived angiogenic cells (BMDACs) play an important role in vascularization. Using a mouse full-thickness burn wound model, we demonstrate that perfusion and vascularization of burn wounds were impaired by aging and were associated with dramatically reduced mobilization of BMDACs bearing the cell surface molecules CXCR4 and Sca1. Expression of stromal-derived factor 1 (SDF-1), the cytokine ligand for CXCR4, was significantly decreased in peripheral blood and burn wounds of old mice. Expression of hypoxia-inducible factor (HIF)-1α was detected in burn wounds from young (2-month-old), but not old (2-year-old), mice. When BMDACs from young donor mice were injected intravenously, homing to burn wound tissue was impaired in old recipient mice, whereas the age of the BMDAC donor mice had no effect on homing. Our results indicate that aging impairs burn wound vascularization by impairing the mobilization of BMDACs and their homing to burn wound tissue as a result of impaired HIF-1 induction and SDF-1 signaling.

Lung development occurs under relative hypoxia and the most important oxygen-sensitive response pathway is driven by Hypoxia Inducible Factors (HIF). HIFs are heterodimeric transcription factors of an oxygen-sensitive subunit, HIFα, and a constitutively expressed subunit, HIF1β. HIF1α and HIF2α, encoded by two separate genes, contribute to the activation of hypoxia inducible genes. A third HIFα gene, HIF3α, is subject to alternative promoter usage and splicing, leading to three major isoforms, HIF3α, NEPAS and IPAS. HIF3α gene products add to the complexity of the hypoxia response as they function as dominant negative inhibitors (IPAS) or weak transcriptional activators (HIF3α/NEPAS). Previously, we and others have shown the importance of the Hif1α and Hif2α factors in lung development, and here we investigated the role of Hif3α during pulmonary development. Therefore, HIF3α was conditionally expressed in airway epithelial cells during gestation and although HIF3α transgenic mice were born alive and appeared normal, their lungs showed clear abnormalities, including a post-pseudoglandular branching defect and a decreased number of alveoli. The HIF3α expressing lungs displayed reduced numbers of Clara cells, alveolar epithelial type I and type II cells. As a result of HIF3α expression, the level of Hif2α was reduced, but that of Hif1α was not affected. Two regulatory genes, Rarβ, involved in alveologenesis, and Foxp2, a transcriptional repressor of the Clara cell specific Ccsp gene, were significantly upregulated in the HIF3α expressing lungs. In addition, aberrant basal cells were observed distally as determined by the expression of Sox2 and p63. We show that Hif3α binds a conserved HRE site in the Sox2 promoter and weakly transactivated a reporter construct containing the Sox2 promoter region. Moreover, Hif3α affected the expression of genes not typically involved in the hypoxia response, providing evidence for a novel function of Hif3α beyond the hypoxia response.

Hypoxia-inducible factor-1 (HIF-1) is a key mediator of oxygen homeostasis that was first identified as a transcription factor that is induced and activated by decreased oxygen tension. Upon activation, HIF-1 upregulates the transcription of genes that promote adaptation and survival under hypoxic conditions. HIF-1 is a heterodimer composed of an oxygen-regulated subunit known as HIF-1α and a constitutively expressed HIF-1β subunit. In general, the availability and activity of the HIF-1α subunit determines the activity of HIF-1. Subsequent studies have revealed that HIF-1 is also activated by environmental and physiological stimuli that range from iron chelators to hormones. Preclinical studies suggest that HIF-1 activation may be a valuable therapeutic approach to treat tissue ischemia and other ischemia/hypoxia-related disorders.

The focus of this review is natural product-derived small molecule HIF-1 activators. Natural products, relatively low molecular weight organic compounds produced by plants, animals, and microbes, have been and continue to be a major source of new drugs and molecular probes. The majority of known natural product-derived HIF-1 activators were discovered through pharmacological evaluation of specifically selected individual compounds. The combination of natural products chemistry with appropriate high-throughput screening bioassays could provide an alternative approach to discover novel natural product-derived HIF-1 activators. Potent natural product-derived HIF-1 activators that exhibit a low level of toxicity and side effects hold promise as new treatment options for diseases such as myocardial and peripheral ischemia, and as chemopreventative agents that could be used to reduce the level of ischemia/reperfusion injury following heart attack and stroke.

The FIH hydroxylase is a cellular peroxide sensor that modulates HIF transcriptional activity

HIF asparaginyl hydroxylase (FIH) is shown to be strikingly more sensitive to peroxide than the HIF prolyl hydroxylases, indicating that hypoxia and oxidative stress are distinct regulators of the HIF response.

Hypoxic and oxidant stresses can coexist in biological systems, and oxidant stress has been proposed to activate hypoxia pathways through the inactivation of the ‘oxygen-sensing' hypoxia-inducible factor (HIF) prolyl and asparaginyl hydroxylases. Here, we show that despite reduced sensitivity to cellular hypoxia, the HIF asparaginyl hydroxylase—known as FIH, factor inhibiting HIF—is strikingly more sensitive to peroxide than the HIF prolyl hydroxylases. These contrasting sensitivities indicate that oxidant stress is unlikely to signal hypoxia directly to the HIF system, but that hypoxia and oxidant stress can interact functionally as distinct regulators of HIF transcriptional output.

Hypoxia-inducible factor 1α (HIF-1α) controls the cellular responses to hypoxia, activating transcription of a range of genes involved in adaptive processes such as increasing glycolysis and promoting angiogenesis. However, paradoxically, HIF-1α also participates in hypoxic cell death. Several gene products, such as BNip3, RTP801, and Noxa, were identified as HIF-1α-responsive proapoptotic proteins, but the complicated hypoxic cell death pathways could not be completely explained by the few known genes. Moreover, molecules linking the proapoptotic signals of HIF-1α directly to mitochondrial permeability transition are missing. In this work, we report the identification of an HIF-1α-responsive proapoptotic molecule, HGTD-P. Its expression was directly regulated by HIF-1α through a hypoxia-responsive element on the HGTD-P promoter region. When overexpressed, HGTD-P was localized to mitochondria and facilitated apoptotic cell death via typical mitochondrial apoptotic cascades, including permeability transition, cytochrome c release, and caspase 9 activation. In the process of permeability transition induction, the death-inducing domain of HGTD-P physically interacted with the voltage-dependent anion channel. In addition, suppression of HGTD-P expression by small interfering RNA or antisense oligonucleotides protected against hypoxic cell death. Taken together, our data indicate that HGTD-P is a new HIF-1α-responsive proapoptotic molecule that activates mitochondrial apoptotic cascades.

Hypoxia-inducible factor-1α (HIF-1α) is known as a transactivator for VEGF gene promoter. It can be induced by hypoxia. However, no study has been done so far to dissect HIF-1α-mediated effects from hypoxia or VEGF-mediated effects. By using a HIF-1α knockout (HIF-1α KO) cell system in mouse embryonic fibroblast (MEF) cells, this study analyzes cell migration and HIF-1α, hypoxia and VEGF activation. A hypoxia-mediated HIF-1α induction and VEGF transactivation were observed: both HIF-1α WT lines had significantly increased VEGF transactivation, as an indicator for HIF-1α induction, in hypoxia compared to normoxia; in contrast, HIF-1α KO line had no increased VEGF transactivation under hypoxia. HIF-1α promotes cell migration: HIF-1α-KO cells had a significantly reduced migration compared to that of the HIF-1α WT cells under both normoxia and hypoxia. The significantly reduced cell migration in HIF-1α KO cells can be partially rescued by the restoration of WT HIF-1α expression mediated by adenoviral-mediated gene transfer. Interestingly, hypoxia has no effect on cell migration: the cells had a similar cell migration rate under hypoxic and normoxic conditions for both HIF-1α WT and HIF-1α KO lines, respectively. Collectively, these data suggest that HIF-1α plays a role in MEF cell migration that is independent from hypoxia-mediated effects.

Wound healing is impaired in elderly patients with diabetes mellitus. We hypothesized that age-dependent impairment of cutaneous wound healing in db/db diabetic mice: (a) would correlate with reduced expression of the transcription factor hypoxia-inducible factor 1α (HIF-1α) as well as its downstream target genes; and (b) could be overcome by HIF-1α replacement therapy. Wound closure, angiogenesis, and mRNA expression in excisional skin wounds were analyzed and circulating angiogenic cells were quantified in db/db mice that were untreated or received electroporation-facilitated HIF-1α gene therapy. HIF-1α mRNA levels in wound tissue were significantly reduced in older (4–6 months) as compared to younger (1.5–2 months) db/db mice. Expression of mRNAs encoding the angiogenic cytokines vascular endothelial growth factor (VEGF), angiopoietin 1 (ANGPT1), ANGPT2, platelet derived growth factor B (PDGF-B), and placental growth factor (PLGF) was also impaired in wounds of older db/db mice. Intradermal injection of plasmid gWIZ-CA5, which encodes a constitutively active form of HIF-1α, followed by electroporation, induced increased levels of HIF-1α mRNA at the injection site on day 3 and increased levels of VEGF, PLGF, PDGF-B, and ANGPT2 mRNA on day 7. Circulating angiogenic cells in peripheral blood increased 10-fold in mice treated with gWIZ-CA5. Wound closure was significantly accelerated in db/db mice treated with gWIZ-CA5 as compared to mice treated with empty vector. Thus, HIF-1α gene therapy corrects the age-dependent impairment of HIF-1α expression, angiogenic cytokine expression, and circulating angiogenic cells that contribute to the age-dependent impairment of wound healing in db/db mice.

Hypoxia-inducible factors (HIFs) are transcription factors that activate the transcription of target genes involved in crucial aspects of cancer development. This study investigated the expression of HIFs and their contribution to the regulation of target genes related to angiogenesis and glucose metabolism in gastric cancer. The data showed that HIFs were over-expressed in gastric cancer and that activation of the target genes was observed mainly in the early stages. Moreover, the results of the present study revealed that only HIF-1α, but not HIF-2α dimerizes with HIF-1β and then regulates expression of target genes in response to hypoxia. The results of the present study demonstrate that HIF-1α and HIF-1β enhances expression of VEGF and glucose metabolism-related genes in response to hypoxia in gastric cancer. These data offer important information regarding HIF pathways in the development of gastric cancer.

Objective

Production of the alpha subunit of Hypoxia Inducible Factor (HIF-1α) is increased in healing wounds, which stimulates expression of Vascular Endothelial Growth Factor (VEGF) to promote angiogenesis. Therefore, take rate of skin grafts may be closely associated with the presence or absence of HIF-1α production in the recipient bed. We compared the take rates of skin grafts between myeloid selective HIF-1α knock-out (KO) and wild-type (WT) mice.

Design

Percentage of healthy graft areas obtained by planimetry and scores for epithelialization and granulation tissue formation obtained by histopathological analysis were compared in twelve KO and twelve WT mice following skin grafting.

Results

Graft take rate was significantly impaired in the KO group, while epithelialization or granulation tissue formation scores did not reveal any significant differences.

Conclusion

HIF-1α in myeloid cells may be an important molecule for re-vascularization of avascular tissues such as skin grafts, probably due to its stimulating effect on angiogenesis.

A classical cellular response to hypoxia is a cessation of growth. Hypoxia-induced growth arrest differs in different cell types but is likely an essential aspect of the response to wounding and injury. An important component of the hypoxic response is the activation of the hypoxia-inducible factor 1 (HIF-1) transcription factor. Although this transcription factor is essential for adaptation to low oxygen levels, the mechanisms through which it influences cell cycle arrest, including the degree to which it cooperates with the tumor suppressor protein p53, remain poorly understood. To determine broadly relevant aspects of HIF-1 function in primary cell growth arrest, we examined two different primary differentiated cell types which contained a deletable allele of the oxygen-sensitive component of HIF-1, the HIF-1α gene product. The two cell types were murine embryonic fibroblasts and splenic B lymphocytes; to determine how the function of HIF-1α influenced p53, we also created double-knockout (HIF-1α null, p53 null) strains and cells. In both cell types, loss of HIF-1α abolished hypoxia-induced growth arrest and did this in a p53-independent fashion. Surprisingly, in all cases, cells lacking both p53 and HIF-1α genes have completely lost the ability to alter the cell cycle in response to hypoxia. In addition, we have found that the loss of HIF-1α causes an increased progression into S phase during hypoxia, rather than a growth arrest. We show that hypoxia causes a HIF-1α-dependent increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27; we also find that hypophosphorylation of retinoblastoma protein in hypoxia is HIF-1α dependent. These data demonstrate that the transcription factor HIF-1 is a major regulator of cell cycle arrest in primary cells during hypoxia.

Hypoxia-inducible factors (HIF1α/HIF2α) are key transcription factors that promote angiogenesis. The overexpression of degradation-resistant HIF mutants is considered a promising pro-angiogenic therapeutic tool. We compared the transcriptional activity of HIF1α/HIF2α mutants that obtained their resistance to oxygen-dependent degradation either by deletion of their entire oxygen-dependent degradation (ODD) domain or by replacement of prolyl residues that are crucial for oxygen-dependent degradation. Although all HIF mutants translocated into the nucleus, HIF1α and HIF2α mutants inclosing the point mutations were significantly more effective in trans-activating the target gene VEGF and in inducing tube formation of endothelial cells than mutants lacking the complete ODD domain.

SUMMARY

Hypoxia Inducible Factors (HIFs) regulate adaptive responses to changes in oxygen (O2) tension during embryogenesis, tissue ischemia, and tumorigenesis. Because HIF deficient embryos exhibit a number of developmental defects, the precise role of HIF in early vascular morphogenesis has been uncertain. Using para-aortic splanchnopleural (P-Sp) explant cultures we show that deletion of the HIF-β subunit (ARNT) results in defective hematopoiesis and the inhibition of both vasculogenesis and angiogenesis. These defects are rescued upon the addition of wild type Sca-1+ hematopoietic cells or recombinant VEGF. Arnt−/− embryos exhibit reduced levels of VEGF protein and increased numbers of apoptotic hematopoietic cells. These results suggest that HIF coordinates early endothelial cell emergence and vessel development by promoting hematopoietic cell survival and paracrine growth factor production.

The vascular network delivers oxygen (O2) and nutrients to all cells within the body. It is therefore not surprising that O2 availability serves as a primary regulator of this complex organ. Most transcriptional responses to low O2 are mediated by hypoxia-inducible factors (HIFs), highly conserved transcription factors that control the expression of numerous angiogenic, metabolic, and cell cycle genes. Accordingly, the HIF pathway is currently viewed as a master regulator of angiogenesis. HIF modulation could provide therapeutic benefit for a wide array of pathologies, including cancer, ischemic heart disease, peripheral artery disease, wound healing, and neovascular eye diseases. Hypoxia promotes vessel growth by upregulating multiple pro-angiogenic pathways that mediate key aspects of endothelial, stromal, and vascular support cell biology. Interestingly, recent studies show that hypoxia influences additional aspects of angiogenesis, including vessel patterning, maturation, and function. Through extensive research, the integral role of hypoxia and HIF signaling in human disease is becoming increasingly clear. Consequently, a thorough understanding of how hypoxia regulates angiogenesis through an ever-expanding number of pathways in multiple cell types will be essential for the identification of new therapeutic targets and modalities.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that is composed of a hypoxia-inducible α subunit (HIF-1α and HIF-2α) and a constitutively expressed β subunit (HIF-1β). HIF mediates the adaptation of cells and tissues to low oxygen concentrations. It also plays an important role in tumorigenesis and constitutes an important therapeutic target in anti-tumor therapy. We have screened a number of reported HIF inhibitors for their effects on HIF-transcriptional activity and found that the DNA damage inducing agents camptothecin and mitomycin C produced the most robust effects. Camptothecin is a reported inhibitor of HIF-1α translation, while mitomycin C has been reported to induce p53-dependent HIF-1α degradation. In this study we demonstrate that the inhibitory effect of mitomycin C on HIF-1α protein expression is not dependent on p53 and protein degradation, but also involves HIF-1α translational regulation. Initiation of a DNA damage response with the small molecule p53 activator NSC-652287 (RITA) has been reported to inhibit HIF-1α protein synthesis by increasing the phosphorylation of eIF2α. However, we show here that even when eIF2α phosphorylation is prevented, the DNA damage inducing drugs mitomycin C, camptothecin and NSC-652287 still inhibit HIF-1α protein synthesis to the same extent. The inhibitory effects of camptothecin on HIF-1α expression but not that of mitomycin C and NSC-652287 were dependent on cyclin-dependent kinase activity. In conclusion, specific types of DNA damage can bring about selective inhibition of HIF-1α protein synthesis. Further characterization of the involved mechanisms may reveal important novel therapeutic targets.

Summary Background Data

Mechanical forces play an important role in tissue neovascularisation and are a constituent part of modern wound therapies. The mechanisms by which Vacuum Assisted Closure (VAC) modulates wound angiogenesis are still largely unknown.

Objective

To investigate how VAC treatment affects wound hypoxia and related profiles of angiogenic factors as well as to identify the anatomical characteristics of the resultant, newly formed vessels.

Methods

Wound neovascularization was evaluated by morphometric analysis of CD31- stained wound cross sections as well as by corrosion casting analysis. Wound hypoxia and mRNA expression of HIF-1α and associated angiogenic factors were evaluated by pimonidazole hydrochloride staining and quantitative RT-PCR, respectively. VEGF protein levels were determined by western blot analysis.

Results

VAC-treated wounds were characterized by the formation of elongated vessels aligned in parallel and consistent with physiologically function, compared to occlusive dressing control wounds that showed formation of tortuous, disoriented vessels. Moreover, VAC-treated wounds displayed a well-oxygenated wound bed, with hypoxia limited to the direct proximity of the VAC-foam interface, where higher VEGF levels were found. By contrast, occlusive dressing control wounds showed generalized hypoxia, with associated accumulation of HIF-1α and related angiogenic factors.

Conclusions

The combination of established gradients of hypoxia and VEGF expression along with mechanical forces exerted by VAC therapy was associated with the formation of more physiological blood vessels compared to occlusive dressing control wounds. These morphological changes are likely a necessary condition for better wound healing.

The pathogenesis of rheumatoid arthritis (RA) and osteoarthritis (OA) remains obscure, although angiogenesis appears to play an important role. We recently confirmed an overexpression of two angiogenic factors, namely vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor (PD-ECGF), by the lining and stromal cells of the synovium in both conditions. Because hypoxia inducible factor (HIF)-1α and HIF-2α are essential in regulating transcription of the VEGF gene, active participation of HIF-α molecules in the pathogenesis of these arthritides is anticipated. We investigated the immunohistochemical expression of HIF-1α and HIF-2α in the synovium of 22 patients with RA, 34 patients with OA and 22 'normal' nonarthritic individuals, in relation to VEGF, VEGF/KDR (kinase insert domain protein receptor) vascular activation, PD-ECGF and bcl-2. A significant cytoplasmic and nuclear overexpression of HIF-1α and HIF-2α was noted in the synovial lining and stromal cells of both diseases relative to normal. Overexpression of HIF-αs was related to high microvessel density, high PD-ECGF expression and high VEGF/KDR receptor activation, suggesting HIF-α-dependent synovial angiogenesis in OA. By contrast, the activation of the angiogenic VEGF/KDR pathway was persistently increased in RA, as indeed was microvessel density and the expression of PD-ECGF, irrespective of the extent of HIF-α expression, indicating a cytokine-dependent angiogenesis. In all cases, the VEGF/KDR vascular activation was significantly lower in OA than in RA, suggesting a relative failure of the HIF-α pathway to effectively produce a viable vasculature for OA, which is consistent with the degenerative nature of the disease. The activation of the HIF-α pathway occurs in both RA and OA, although for unrelated reasons.

Angiogenesis is a critical component of mammalian brain adaptation to prolonged hypoxia. Hypoxia-induced angiogenesis is mediated by hypoxia inducible factor-1 (HIF-1) dependent transcriptional activation of growth factors, such as vascular endothelial growth factor (VEGF). Microvascular angiogenesis occurs over a three week period in the rodent brain. We have recently reported that HIF-1α accumulation and transcriptional activation of HIF target genes in the aged cortex of 24 month F344 rats is significantly attenuated during acute hypoxic exposure. In the present study, we show that cortical HIF-1α accumulation and HIF-1 activation remains absent during chronic hypoxic exposure in the aged rat brain (24 month F344). Despite this lack of HIF-1 activation, there is no significant difference in baseline or post-hypoxic brain capillary density counts between the young (3 month F344) and old age groups. VEGF mRNA and protein levels are significantly elevated in the aged cortex despite the lack of HIF-1 activation. Other HIF-independent mediators of hypoxia inducible genes could be involved during chronic hypoxia in the aged brain. PPAR-γ coactivator (PGC)-1α, a known regulator of VEGF gene transcription, is elevated in the young and aged cortex during the chronic hypoxic exposure. Overall, our results suggest a compensatory HIF-1 independent preservation of hypoxic-induced microvascular angiogenesis in the aged rat brain.

Localized tissue hypoxia is a consequence of vascular compromise or rapid cellular proliferation and is a potent inducer of compensatory angiogenesis. The oxygen-responsive transcriptional regulator hypoxia-inducible factor 2α (HIF-2α) is highly expressed in vascular ECs and, along with HIF-1α, activates expression of target genes whose products modulate vascular functions and angiogenesis. However, the mechanisms by which HIF-2α regulates EC function and tissue perfusion under physiological and pathological conditions are poorly understood. Using mice in which Hif2a was specifically deleted in ECs, we demonstrate here that HIF-2α expression is required for angiogenic responses during hindlimb ischemia and for the growth of autochthonous skin tumors. EC-specific Hif2a deletion resulted in increased vessel formation in both models; however, these vessels failed to undergo proper arteriogenesis, resulting in poor perfusion. Analysis of cultured HIF-2α–deficient ECs revealed cell-autonomous increases in migration, invasion, and morphogenetic activity, which correlated with HIF-2α–dependent expression of specific angiogenic factors, including delta-like ligand 4 (Dll4), a Notch ligand, and angiopoietin 2. By stimulating Dll4 signaling in cultured ECs or restoring Dll4 expression in ischemic muscle tissue, we rescued most of the HIF-2α–dependent EC phenotypes in vitro and in vivo, emphasizing the critical role of Dll4/Notch signaling as a downstream target of HIF-2α in ECs. These results indicate that HIF-1α and HIF-2α fulfill complementary, but largely nonoverlapping, essential functions in pathophysiological angiogenesis.

HIF-1α is a nuclear factor important in the transcription of genes controlling angiogenesis including vascular endothelial growth factor (VEGF). Both hypoxia and oxidative stress are known mechanisms for the induction of HIF-1α. Oxidative stress and mitochondrial permeability transition (MPT) are mechanistically important in acetaminophen (APAP) toxicity in the mouse. MPT may occur as a result of oxidative stress and leads to a large increase in oxidative stress. We previously reported the induction of HIF-1α in mice with APAP toxicity and have shown that VEGF is important in hepatocyte regeneration following APAP toxicity. The following study was performed to examine the relative contribution of hypoxia versus oxidative stress to the induction of HIF-1α in APAP toxicity in the mouse. Time course studies using the hypoxia marker pimonidazole showed no staining for pimonidazole at 1 or 2 h in B6C3F1 mice treated with APAP. Staining for pimonidazole was present in the midzonal to periportal regions at 4, 8, 24 and 48 h and no staining was observed in centrilobular hepatocytes, the site of the toxicity. Subsequent studies with the MPT inhibitor cyclosporine A showed that cyclosporine A (CYC; 10 mg/kg) reduced HIF-1α induction in APAP treated mice at 1 and 4 h and did not inhibit the metabolism of APAP (depletion of hepatic non-protein sulfhydryls and hepatic protein adduct levels). The data suggest that HIF-1α induction in the early stages of APAP toxicity is secondary to oxidative stress via a mechanism involving MPT. In addition, APAP toxicity is not mediated by a hypoxia mechanism.

Aims: Hypoxia inducible factors 1α and 2α (HIF1α and HIF2α) are hypoxia regulated transcriptional factors, which control the expression of a variety of genes responsible for angiogenesis, glycolysis, and the inhibition of apoptosis. Because angiogenesis and tissue regeneration are integral components of the inflammatory process, this study was designed to investigate the role of HIFα molecules in inflammatory bowel disease.

Methods: Surgical specimens from patients with active ulcerative colitis (UC) and Crohn’s disease (CD) were assessed immunohistochemically for HIF1α and HIF2α reactivity, and the expression of these molecules was compared with the expression of the angiogenic factors thymidine phosphorylase (TP), vascular endothelial growth factor (VEGF), and VEGF–KDR activated vasculature. The vascular density of the lesions was also assessed using anti-CD31 immunostaining.

Results: HIF1α was expressed focally (epithelial cells, stromal fibroblasts, and myocytes) in both UC and CD, whereas HIF2α was expressed focally in UC and diffusely in CD. TP expression was uniformly positive in both diseases. VEGF expression was absent in CD, and weakly positive in UC. The VEGF–KDR reactivity of the submucosal vasculature was only slightly increased in UC and CD compared with normal tissue. The inflammatory cells stained with HIF2α and TP in all cases, but the reactivity was generalised in CD and focal in UC. In both diseases, vascular density was significantly higher than that seen in normal tissue.

Conclusions: The discordant expression of HIF2α and VEGF in CD suggests an inherent deficiency of the intestine to respond to various stresses by the induction of VEGF. This finding should be investigated further.

Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation. Inhibition of Wnt pathway by Osx highlights the potential for feedback control mechanisms involved in bone formation. Hypoxia-inducible factor-1α (HIF-1α) is a master regulator of hypoxia. HIF-1α has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that Osx and HIF-1α cooperatively regulate VEGF expression in osteoblasts. Effects of hypoxia/HIF-1α on osteoblast proliferation and related mechanisms are not well understood. In this study, osteoblast growth under hypoxia was examined. We observed that osteoblast growth was inhibited under hypoxia. To explore possible mechanisms for hypoxia/HIF-1α to inhibit osteoblast proliferation, we tested the effect of hypoxia/HIF-1α on Wnt pathway. Quantitative RT-PCR results revealed that Wnt target genes such as cyclin D1 and c-Myc were downregulated under hypoxia while HIF-1α was upregulated. Treatment of desferrioxamine, a HIF-1α activator, led to further downregulation of expressions of cyclin D1 and c-Myc in osteoblasts. On the contrary, the inhibition of HIF-1α by siRNA in osteoblasts led to the expression increase of cyclin D1 and c-Myc. These data suggest that HIF-1α inhibits Wnt pathway in osteoblasts. To examine the effect of HIF-1α on Wnt pathway, HIF-1α was cotransfected with β-catenin along with Topflash reporter in transient transfection assay. Our results showed that HIF-1α inhibited β-catenin-induced Topflash reporter activity. Interestingly, a synergistic interplay was observed between Osx and HIF-1α in the inhibition of β-catenin-induced Topflash expression. Our findings indicate that Osx and HIF-1α cooperatively inhibit Wnt pathway. This study revealed additional new information of the cooperation between HIF-1α and Osx in osteoblasts.

Background

Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis.

Methodology/Principal Findings

By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90β is the main player in this phenomenon.

Conclusions/Significance

We identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the β isoform of molecular chaperone HSP90.