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: 13.22). 03/2012; 122(4):1427-43. DOI: 10.1172/JCI57322
Source: PubMed


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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    • "In the previous studies, re-vascularization in a murine model occurred within 48 h, oxygen saturation returned to nearly normal levels in control mice 7 days after transplantation and it took up to 19– 21 days for the development of an activated follicle to reach the pre-ovulatory stage (Pedersen, 1970; Skuli et al., 2012). Based on these previous studies, we chose the 2, 7 and 21-day time points for the evaluation of ovarian follicle development and function. "
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    ABSTRACT: STUDY QUESTION Does the preoperative administration of simvastatin and methylprednisolone enhance mouse ovarian quality after auto-transplantation of vitrified-warmed ovarian tissue (OT)? SUMMARY ANSWER Treatment with combined simvastatin and methylprednisolone enhances the quality of transplanted mouse OTs. WHAT IS KNOWN ALREADY The prevention of ischemic injury after transplantation of OT is critical for preserving the ovarian follicles. Preoperative administration of simvastatin (a cholesterol-lowering drug) has beneficial effects on various organ transplantations. Moreover, donor treatment with simvastatin and methylprednisolone (main effects are on immune response) prevents ischemia-reperfusion injury and has a beneficial effect on allograft survival in rat cardiac allografts. STUDY DESIGN, SIZE, DURATION A total of 232 6-week-old B6D2F1 mice were randomly distributed into fresh control, vitrified-warmed control and experimental groups (n = 10-17 per group). The experimental groups were as follows: sham control, simvastatin, methylprednisolone and co-treatment groups. In the experimental groups, the mice were administered simvastatin (5 mg/kg, orally), methylprednisolone (15 mg/kg, i.v.) or a combination of simvastatin and methylprednisolone 2 h before ovariectomy, whereas the sham control mice received normal saline. PARTICIPANTS/MATERIALS, SETTING, METHODS Whole ovaries were removed from the mice and vitrified by two-step vitrification procedures. The vitrified ovaries were warmed 1 week later and auto-transplanted under the bilateral kidney capsules. The ovaries and blood samples were collected 2, 7 and 21 days (D) after transplantation for histological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, immunohistochemistry for CD31 and serum anti-Mullerian hormone (AMH) level estimation. Embryonic development was evaluated after IVF of oocytes obtained from the transplanted ovary. MAIN RESULTS AND THE ROLE OF CHANCE The group that received simvastatin and methylprednisolone showed a significantly improved intact (Grade 1) follicle ratio (D2: P < 0.001, D7: P < 0.05 and D21: P < 0.001), apoptotic follicle ratio (D21: P < 0.05), CD31-positive area (D7: P < 0.05 and D21: P < 0.05) and serum AMH level (D7: P < 0.001) after transplantation when compared with the sham control. However, no difference was noted in the fertilization and blastocyst formation rates, number of total and apoptotic blastomeres per blastocyst and inner cell mass/trophectoderm ratio among the four transplantation groups. LIMITATIONS, REASONS FOR CAUTION Although we evaluated the beneficial effects of simvastatin and methylprednisolone in the present study, we did not unravel the corresponding protective mechanisms. WIDER IMPLICATIONS OF THE FINDINGS Our results suggest that a combination of simvastatin and methylprednisolone has beneficial effects on the quality and functioning of transplanted OT. This combined treatment can potentially be applied clinically to humans and domestic animals subject to further studies. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0055). There are no conflicts of interest to declare. © 2015 The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected] /* */
    Full-text · Article · Sep 2015 · Human Reproduction
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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.
    Full-text · Article · Jun 2014 · Frontiers in Immunology
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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.
    Full-text · Article · May 2014 · Scientific Reports
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