Identification of HIF-2Α-Regulated Genes That Play a Role in Human
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Angiogenesis DOI 10.1007/s10456-016-9527-4 ORIGINAL PAPER Identification of HIF-2a-regulated genes that play a role in human microvascular endothelial sprouting during prolonged hypoxia in vitro 1,2 1,2 3,4 Tessa D. Nauta • Marloes van den Broek • Sue Gibbs • 5 6 Tineke C. T. M. van der Pouw-Kraan • Cees B. Oudejans • 1 1 Victor W. M. van Hinsbergh • Pieter Koolwijk Received: 16 June 2016 / Accepted: 28 September 2016 Ó The Author(s) 2016. This article is published with open access at Springerlink.com Abstract During prolonged hypoxic conditions, endothe- differentially regulated genes in hypoxia were compared to lial cells change their gene expression to adjust to the low the genes that were differentially regulated upon silencing oxygen environment. This process is mainly regulated by of HIF-2a in hypoxia. Surprisingly, KEGG pathway anal- the hypoxia-inducible factors, HIF-1a and HIF-2a. ysis showed that metabolic pathways were enriched within Although endothelial cells do not form sprouts during genes upregulated in response to hypoxia and enriched prolonged hypoxic culturing, silencing of HIF-2a partially within genes downregulated upon HIF-2a silencing. restores sprout formation. The present study identifies Moreover, 51 HIF-2a-regulated genes were screened for novel HIF-2a-target genes that may regulate endothelial their role in endothelial sprouting in hypoxia, of which four sprouting during prolonged hypoxia. The gene expression genes ARRDC3, MME, PPARG and RALGPS2 directly profile of primary human microvascular endothelial cells influenced endothelial sprouting during prolonged hypoxic (hMVECs) that were cultured at 20 % oxygen was com- culturing. The manipulation of specific downstream targets pared to hMVECs that were cultured at 1 % oxygen for of HIF-2a provides a new, but to be further evaluated, 14 days by using genome-wide RNA-sequencing. The perspective for restoring reduced neovascularization in several pathological conditions, such as diabetic ulcers or other chronic wounds, for improvement of vascularization Electronic supplementary material The online version of this article (doi:10.1007/s10456-016-9527-4) contains supplementary of implanted tissue-engineered scaffolds. material, which is available to authorized users. Keywords Angiogenesis Á Hypoxia Á HIF-2a Á Genome- & Pieter Koolwijk [email protected] wide RNA-sequencing 1 Department of Physiology, Institute for Cardiovascular Research, VU University Medical Center, De Boelelaan Introduction 1118, Room 11W53, 1081 HV Amsterdam, The Netherlands 2 A-Skin Nederland BV, De Boelelaan 1117, Angiogenesis, the formation of new blood vessels through 1007 MB Amsterdam, The Netherlands endothelial sprouting, is important for tissue growth, 3 Department of Dermatology, VU University Medical Center, development and proper wound healing. Nevertheless, Amsterdam, The Netherlands angiogenesis is associated with several pathological con- 4 Departments of Oral Cell Biology and Dental Material ditions, such as tissue ischemia, solid tumors and adult Sciences, Academic Center for Dentistry Amsterdam macular degeneration of the eye [1]. Usually, these disor- (ACTA), University of Amsterdam and Vrije Universiteit, Amsterdam, The Netherlands ders are accompanied by loss of adequate blood supply or enhanced metabolic demand, leading to reduced oxygen 5 Department of Molecular Cell Biology and Immunology, Institute for Cardiovascular Research, VU University tension (hypoxia) in the tissue. Not surprisingly, hypoxia is Medical Center, Amsterdam, The Netherlands considered to be one of the most potent initiators of 6 Department of Clinical Chemistry, VU University Medical angiogenesis in vitro and in vivo [2–4] through stabiliza- Center, Amsterdam, The Netherlands tion of the transcription factor hypoxia-inducible factor-1a 123 Angiogenesis (HIF-1a) and subsequent induction of vascular endothelial downstream of HIF-2a in conditions that were previously growth factor (VEGF) [5, 6]. Nonetheless, in chronic shown to be favorable for induction of endothelial tubules, hypoxic tissues often a resistance to induction of neovas- i.e., hMVECs seeded on a 3D-fibrin matrix and stimulated cularization is observed [3, 7–11] and limited expression of by VEGF-A/TNFa [20, 21]. Many hypoxia-responsive HIF-1 has been observed in chronic hypoxic human leg genes have been identified using microarrays in different tissue [12]. Therefore, there is a need of stimulating neo- tumor cell lines [22–28] and human endothelial cells vascularization and temporarily overcoming the endoge- [4, 29]. However, only short-term hypoxia (16–72 h) under nous inhibitory factors that prevent induction of basal conditions was evaluated, but not prolonged hypoxic angiogenesis in severely hypoxic tissues. conditions (14 days). Therefore, we studied gene expres- Despite the progress in understanding the mechanisms sion after long-term hypoxia and with and without silenc- and factors regulating angiogenesis [13, 14], the effects of ing of HIF-2a. By using genome-wide RNA-sequencing, long-term hypoxia on angiogenesis regulation and in par- 51 genes were identified in VEGF-A/TNFa-stimulated ticular the behavior of endothelial cells are still poorly cells that were regulated in a reversed way in hypoxia or by understood. Hypoxia rapidly induces large amounts of HIF-2a silencing and were screened for their ability to VEGF-A via HIF-1 activation in all tissue cells, thus sup- regulate sprouting of human endothelial cells in a 3D fibrin plying a major angiogenesis-inducing factor in the envi- matrix. ronment. It activates VEGF receptors on the endothelium which induce proliferation and—often stimulated by additional inflammatory factors—cell migration and inva- Materials and methods sion. However, the transcriptional response of endothelial cells is more complicated and involves—together with Cell culture additional factors—the involvement of HIF-1a and HIF- 2a, which display a partly different spectrum of activation. The study was executed in accordance with the Declaration HIF-1a has been studied extensively and induces mainly of Helsinki and was approved by the University Human tortuous and leaky newly formed vascular structures that Subjects Committee of the VU University Medical Center. are not adequately perfused [15, 16], while HIF-2a Written informed consent was obtained from all donors in (EPAS1), which is abundantly expressed in endothelial accordance with the institutional guidelines. Human cells, induces stabilization of endothelial microvessels, microvascular endothelial cells (hMVECs) were isolated and—in lung microvessels—improves endothelial barrier from foreskin, kindly provided by the Department of function [17]. Indeed, endothelial-specific deletion of HIF- Dermatology (VUmc, Amsterdam), cultured and charac- 2a pointed to a role of HIF-2a in the regulation of angio- terized (CD31, vWF, Ulex europaeus lectin-1 binding, VE- genesis in mice [18, 19], which was suggested to occur cadherin) as previously described [30, 31]. hMVECs were through the induction of the Dll4/Notch signaling pathway. cultured on 1 % gelatin-coated culture plates in culture These data suggest that the endothelial cell has its own medium consisting of Medium 199 supplemented with program to respond adequately to hypoxia, i.e., in such a 100 U/mL penicillin and 100 mg/mL streptomycin (p/s), way that finally a mature, sealed and stabilized new 2mML-glutamine (all Lonza, Verviers, Belgium), 5 U/mL microvascular network is formed. Excess of HIF-1a heparin (Leo Pharmaceutical Products, Weesp, The response, as observed in many tumors, leads to abundant Netherlands), endothelial cell growth factor (ECGF, crude tortuous but not or suboptimal functioning microvessels. extract from bovine brain), 10 % heat-inactivated human As the activation of HIF-1a and HIF-2a is in part transient serum (HSi, Life Technologies) and 10 % heat-inactivated and HIF-2a tends to be longer expressed, the balance newborn calf serum (NBCSi, Lonza). Medium was chan- between sprouting and stabilization may shift during pro- ged every 48 h. Confluent cells were washed with 0.5 mM longed hypoxia. This may underlie our previous observa- EDTA (Merck Millipore) in HBSS, trypsinized (0.05 % tion that prolonged hypoxia reduced the ability of trypsin in EDTA/HBSS, both Lonza) and seeded in a 1:3 endothelial cells to form sprouts. This was partly related to density. Cells were cultured at 37 °C in a water-saturated a reduction of urokinase–plasminogen activator generation, atmosphere of 95 % air and 5 % CO2. hMVECs were used which reduces pericellular proteolysis by the uPA/ until passage 10. uPAR/plasmin pathway and—independently—could be partially relieved by HIF-2a si-RNA [20]. However, the si- Hypoxic cell culture HIF-2a-dependent restoration of sprouting in this study occurred independent of Dll4/Notch signaling. Hypoxic cell culture conditions were maintained inside a The current study investigates which genes and path- custom designed hypoxic workstation (T.C.P.S., Rotselaar, ways are differentially regulated in prolonged hypoxia and Belgium), with a CO2 and O2 controlled (via injection of 123 Angiogenesis N2), humidified incubator (Sanyo, Etten-leur, The Nether- RNA isolation and genome-wide RNA-sequencing lands), placed inside a T4 glovebox (Jacomex, Dagneux, France) equipped with an O2X1 oxygen transmitter (GE hMVECs were cultured for 14 days at normoxic or hypoxic Panametrics, Billerica, USA). The oxygen concentration conditions. Upon confluency, cells were starved for 18 h in inside