Endothelial HIF-2alpha regulates murine pathological angiogenesis and revascularization processes. J Clin Invest

Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6160, USA.
The Journal of clinical investigation (Impact Factor: 13.77). 03/2012; 122(4):1427-43. DOI: 10.1172/JCI57322
Source: PubMed

ABSTRACT 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.

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    • "The physiological response to hypoxia is stabilization of hypoxia-inducible factor (HIF)-1α and HIF-2α, which will dimerize with the β subunit and via binding to hypoxia responsive elements adapt the cell to low oxygen levels (7). HIF-1α and HIF-2α upregulate the transcription of a multitude of cytokines and growth factors (8, 9), but the two transcription factors induce expression of different proteins (10). One of the HIF-regulated proteins is the pleiotropic cytokine erythropoietin (EPO) mainly regulated by HIF-2α (11). "
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    ABSTRACT: The pathogenesis of cerebral malaria (CM) includes compromised microvascular perfusion, increased inflammation, cytoadhesion, and endothelial activation.These events cause blood–brain barrier disruption and neuropathology and associations with the vascular endothelial growth factor (VEGF) signaling pathway have been shown. We studied this pathway in mice infected with Plasmodium berghei ANKA causing murine CM with or without the use of erythropoietin (EPO) as adjunct therapy. ELISA andwestern blottingwas used for quantification of VEGF and relevant proteins in brain and plasma. CM increased levels ofVEGF in brain and plasma and decreased plasma levels of solubleVEGF receptor 2. EPO treatment normalizedVEGF receptor 2 levels and reduced brainVEGF levels. Hypoxiainducible factor (HIF)-1a was significantly upregulated whereas cerebral HIF-2a and EPO levels remained unchanged. Furthermore, we noticed increased caspase-3 and calpain activity in terminally ill mice, as measured by protease-specific cleavage of a-spectrin and p35. In conclusion, we detected increased cerebral and systemic VEGF as well as HIF-1a, which in the brain were reduced to normal in EPO-treated mice. Also caspase and calpain activity was reduced markedly in EPO-treated mice.
    Frontiers in Immunology 06/2014; 5. DOI:10.3389/fimmu.2014.00291
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    • "Moreover, we observed two morphologically distinct populations of cells after 24 hours of ischemia. In vivo studies previously demonstrated the temporal and spatial profile of EC activation during ischemia96061. In one study, ANG2 overexpression was only detected in individual ECs in the first 6–24 hrs of ischemia while prolonged ischemia of 3 days resulted in overexpression of ANG2 by all ECs61. "
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    ABSTRACT: Studying human vascular disease in conventional cell cultures and in animal models does not effectively mimic the complex vascular microenvironment and may not accurately predict vascular responses in humans. We utilized a microfluidic device to recapitulate both shear stress and O2 levels in health and disease, establishing a microfluidic vascular model (μVM). Maintaining human endothelial cells (ECs) in healthy-mimicking conditions resulted in conversion to a physiological phenotype namely cell elongation, reduced proliferation, lowered angiogenic gene expression and formation of actin cortical rim and continuous barrier. We next examined the responses of the healthy μVM to a vasotoxic cancer drug, 5-Fluorouracil (5-FU), in comparison with an in vivo mouse model. We found that 5-FU does not induce apoptosis rather vascular hyperpermeability, which can be alleviated by Resveratrol treatment. This effect was confirmed by in vivo findings identifying a vasoprotecting strategy by the adjunct therapy of 5-FU with Resveratrol. The μVM of ischemic disease demonstrated the transition of ECs from a quiescent to an activated state, with higher proliferation rate, upregulation of angiogenic genes, and impaired barrier integrity. The μVM offers opportunities to study and predict human ECs with physiologically relevant phenotypes in healthy, pathological and drug-treated environments.
    Scientific Reports 05/2014; 4:4951. DOI:10.1038/srep04951 · 5.58 Impact Factor
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    • "A recent report revealed that Int6 suppresses HIF-2α, even during normoxia, and that knock-down of the int6 gene leads to upregulated expression HIF-2α, thereby promoting blood vessel function.16 In addition, a single injection of int6 siRNA promoted angiogenesis through upregulation of HIF-2α-related angiogenic factors and restored function of the ischemic hindlimb in a rat model of peripheral artery disease.20 Another report suggested that endothelial HIF-2α regulates vascular function and tumor angiogenesis in a mouse model.19,21 Therefore, this present study analyzed the function of HIF-2α in ECs through the investigation of transgenic mice overexpressing HIF-2α. "
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    ABSTRACT: Background: The local skin flap procedure is very useful for reconstruction. However, flap necrosis caused by circulatory failure can occur at its distal portion. Hypoxia-inducible factors (HIFs) in endothelial cells (ECs) help to maintain ECs and promote vascularization, and HIF-2α is abundantly expressed in ECs. However, the mechanisms of action of HIF-2α in ECs are not yet fully understood. The aim of this study was to evaluate the in vivo effects of overexpression of HIF-2α in ECs on skin flap survival. Methods: A random pattern skin flap (1.0 × 3.0 cm) was elevated on the dorsum of transgenic mice (Tg mice) with EC-specific HIF-2α conditional overexpression and wild-type littermate control mice (n = 6). Flap survival was evaluated on postoperative day 7. Tissue samples from the skin flaps were harvested and analyzed using Western blotting, quantitative reverse transcriptase-polymerase chain reaction, and immunohistochemistry. Results: The HIF-2α mRNA and protein levels were significantly increased in the Tg mice when compared with control mice. Tg mice had significantly increased skin flap survival areas (72.0% ± 2.7%) when compared with wild-type mice (45.7% ± 1.1%). Moreover, histological examination revealed an increase in the subcutaneous blood vessel counts in the Tg mice. Conclusions: Specific overexpression of HIF-2α in ECs promoted vascularization and enhanced skin flap survival in vivo in a mouse model.
    04/2014; 2(4):e132. DOI:10.1097/GOX.0000000000000083
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