Retina Investigation of the Regulation of Roundabout4 by Hypoxia-Inducible Factor-1a in Microvascular Endothelial Cells Rui Tian,1 Zaoxia Liu,1 Hui Zhang,1 Xuexun Fang,2 Chenguang Wang,1 Shounan Qi,1 Yan Cheng,1 and Guanfang Su1 1Department of Ophthalmology, Second Hospital of Jilin University, Changchun, Jilin, China 2School of Life Science, Jilin University, Changchun, China Correspondence: Guanfang Su, De- PURPOSE. We determined if hypoxia-inducible factor-1a (HIF-1a) and Roundabout4 (Robo4) partment of Ophthalmology, Second colocalized in fibrovascular membranes (FVM) from patients with proliferative diabetic Hospital of Jilin University, 218 retinopathy (PDR), and investigated the regulation of HIF-1a on Robo4 in microvascular Ziqiang Street, Changchun, Jilin, endothelial cells under normoxic and hypoxic conditions in vitro. 130021, China; [email protected]. METHODS. Immunofluorescence and confocal laser scanning microscopy were done to analyze Submitted: March 21, 2014 the colocalization of HIF-1a and Robo4 in the FVM. Expression of HIF-1a was knocked down Accepted: February 27, 2015 by small interfering RNA (siRNA) technology to study its effects on Robo4 expression of human retinal endothelial cells (HREC) and human dermal microvascular endothelial cells Citation: Tian R, Liu Z, Zhang H, et al. (HDMEC) under normoxic and/or hypoxic conditions. Full-length human a gene was Investigation of the regulation of HIF-1 Roundabout4 by hypoxia-inducible transfected into HREC and HDMEC using GFP lentivirus vectors to overexpress HIF-1a under factor-1a in microvascular endothelial normoxic conditions. The HIF-1a and Robo4 mRNA and protein expressions were quantified cells. Invest Ophthalmol Vis Sci. by real-time PCR and Western blot. A cell proliferation, migration assay, and flow cytometry 2015;56:2586–2594. DOI:10.1167/ were used to analyze the effect of HIF-1a regulation on Robo4 in HREC under hypoxic iovs.14-14409 conditions. RESULTS. Colocalization of HIF-1a and Robo4 in vessels of FVM was confirmed by immunofluorescence staining. Knockdown of HIF-1a expression by siRNA in the HREC and HDMEC inhibited Robo4 expression in mRNA and protein level, while overexpressed HIF-1a increased Robo4 mRNA and protein expression. Silencing HIF-1a in endothelial cells under hypoxic conditions inhibited cell invasion and proliferation, which showed that HIF-1a and Robo4 overexpression due to hypoxic conditions correlated with HREC migration and proliferation. CONCLUSIONS. Both HIF-1a and Robo4 may have a vital role during the formation of FVM. The increased or decreased expression of Robo4 by stimulation or knockdown of HIF-1a suggesting that Robo4 is positively regulated by HIF-1a under normoxic and hypoxic conditions in microvascular endothelial cells in vitro. The HIF-1a gene promotes HREC invasion and proliferation by transcriptionally upregulating Robo4 under hypoxic conditions. Keywords: Robo4, hypoxia-inducible factor-1a, microvascular endothelial cells, fibrovascular membranes, proliferative diabetic retinopathy iabetic retinopathy is a severe microvascular complication In the conditions of low oxygen, hundreds of proteins D associated with diabetes mellitus, and a hallmark of this related to angiogenesis, cell proliferation, cell survival and complication is the formation of a fibrovascular membrane metabolism were activated through HIF-1 pathway.6 To (FVM) that can greatly threaten patients’ visual function. An stimulate the expression of hypoxia induced-protein, for increasing number of reports demonstrate that chronic retinal example VEGF, erythropoietin, and angiopoietins, HIF-1a has hypoxia and ischemia have important roles in FVM develop- to translocate to the nucleus, dimerizes with HIF-1b, and binds ment. Due to its essential role in systemic responses to hypoxia, to hypoxia response elements within the promoters of several hypoxia-inducible factor 1a (HIF-1a), an oxygen sensitive genes.7–10 transcription factor, has been associated with angiogenesis Roundabout4 (Robo4), the fourth member of the Robo gene and FVM development.1–3 Particularly, an increased expression family, receives the most attention among its gene family, of HIF-1a is found in the vitreous humor and in the because it is expressed specifically in the vasculature and is fibrovascular tissues of eyes of proliferative diabetic retinopathy upregulated at sites of angiogenesis.11–13 It is reported that (PDR) patients. Experimentally and clinically, HIF-1a has a either knockdown or overexpression of Robo4 on zebrafish mediating or contributing role in PDR, and, therefore, it can impairs vessel formation, suggesting Robo4 has a key role in serve as a potential target for therapeutic intervention of retinal embryonic angiogenesis.14 Moreover, in Robo4-knockout mice neovascularization.4,5 vascular leakage was found more severe than normal mice, Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783 2586 Downloaded from jov.arvojournals.org on 09/25/2021 Regulation of Robo4 by HIF-1a IOVS j April 2015 j Vol. 56 j No. 4 j 2587 indicating Robo4 is essential in stabilizing the blood vessels.15 immunostaining procedure. In all cases, negative controls Furthermore, the levels of Robo4 mRNA and the presence, showed only weak staining after incubation at 48C overnight. distribution, and role in retinal cells of Robo4 were studied in Each antibody specimen was immunolabeled twice at least, the FVM from PDR patients. These studies suggest that Robo4 and appropriate Ig controls were included for each experi- may have a role in the formation of FVM and have physiological ment. Slides were mounted in 20% glycerol/PBS, coverslipped, functions in the cells of the retina.16 More recently, Keijiro et and sealed with nail varnish. The observation was carried by an al.17 demonstrated that Robo4 gene downregulation is Olympus FV-1000 laser confocal microscope (Olympus, Center associated with retinal hyperoxia in an oxygen-induced Valley, PA, USA) and image software (FV10-ASW1.7). retinopathy model, which may correspond to the marked regression of the superficial network of vessels that in the Cell Culture central retina and delayed development of the deep plexus. The Robo4 gene was found overexpressed only in The HREC and HDMEC cells were kindly provided by Prof Liu endothelial cells when exposed to hypoxia in vitro,18 Xiaoqing of Tongji University and Prof Pei of the Second consistent with the theory that Robo4 is a gene regulated by Hospital of Jilin University, respectively. The HREC cells were hypoxia. However, the regulatory mechanism has not been cultured in Endothelial Cell Medium (Invitrogen Corp.) in an fully explained to date. Based on the positive expression of HIF- incubator maintained at 378C in an atmosphere containing 5% 1a and Robo4 in FVM in vivo,3,4,16 we hypothesized that HIF- CO2 and air, supplemented with 5% fetal bovine serum (FBS; 1a, a transcriptional regulatory factor, may have a regulatory Gibco, Invitrogen Corp.), 100 units/mL penicillin, 100 lg/mL role on Robo4 expression. To confirm this hypothesis, streptomycin (Sigma-Aldrich Corp.), 1% Endothelial Cell colocalization of HIF-1a and Robo4 in the FVM were analyzed Growth Supplement (Beijing Maichen, Bio Co., China). The by immunofluorescence and confocal laser scanning micros- HDMEC cells were cultured in Dulbecco’s modified eagle copy, and Robo4 expression was quantified during HIF-1a medium (DMEM; Invitrogen Corp.) with 10% FBS, 100 units/ downregulation or overexpression in human retinal endothe- mL penicillin, 100 lg/mL streptomycin at 378C in a humidified lial cells (HREC) and human dermal microvascular endothelial atmosphere containing 5% CO2 and air. cells (HDMEC) under normoxic and/or hypoxic conditions in Hypoxic conditions were gained by culturing cells in a vitro. Our results reveal for the first time to our knowledge that sealed, anaerobic workstation (Concept 400; Ruskin Technol- HIF-1a and Robo4 colocalized in the vessels of FVM and that ogies, Pencoed, Wales, UK), in which the hypoxic environment (1% O , 94% N2, and 5% CO ), temperature (37 C), and Robo4 was positively regulated by HIF-1a under normoxic and 2 2 8 humidity (90%) were kept constant. hypoxic conditions in microvascular endothelial cells in vitro. Small Interfering RNA (siRNA) and Transfection MATERIALS AND METHODS Assays Tissue Samples Using a previously described method,19 the HIF-1a–specific siRNAs were synthesized chemically. The sequence of targeted The study protocol was approved by the Ethics Committee of HIF-1a was CTGGACACAGTGTGTTTGA. Human nonsilencing Jilin University, and informed consent was obtained from all siRNA acted as a negative control (NC) and was used to control patients according to the World Medical Association Declara- for any effects of the siRNA and transfection reagent. The tion of Helsinki. A total of 12 type II diabetes mellitus patients control sequence was AGUCUCCACGUGUACGUTT. Cells were with PDR got involved in this research, four of whom were transfected with siRNAs using Lipofectamine 2000 reagent males and eight were females. All patients were aged 47 to 72 (Invitrogen Corp.) according to the manufacturer’s instruc- years. They accepted pars plana vitrectomy with membrane tions. The HIF-1a siRNA was used to transfect the cells at peeling. The FVM specimens surgically obtained were fixed in different concentrations (such as 10, 20, 50, and 100 nM) for 4% paraformaldehyde (PFA), paraffin embedded, and sections 24, 48, and 72 hours. It was determined that transfection with cut at 4 lm. 100 nM HIF-1a siRNA for 48 hours was the most effective and was selected for the next experiments (data not shown). Under Confocal Immunofluorescence normoxic conditions, HREC and HDMEC were randomly divided into control hypoxia group (N), vector group (NC), Sections were dewaxed and rehydrated through an alcohol to and HIF-1a siRNA group (HIF-1a si). The HREC cells were water gradient, rinsed in 0.01 M PBS for 5 minutes and blocked randomly divided into a normoxia group and hypoxia group. with 10% normal goat serum (Sigma-Aldrich Corp., St. Louis, The hypoxia group was randomly divided into control hypoxia MO, USA) for 30 minutes at 378C.