Retinal Cell Biology Heparanase and Vascular Endothelial Growth Factor Expression Is Increased in Hypoxia-Induced Retinal Neovascularization Jie Hu,1 Xin Song,1 Yi Qing He,2,3 Craig Freeman,3 Christopher R. Parish,3 Ling Yuan,1 Honghua Yu,1 and Shibo Tang1 PURPOSE. Heparanase and VEGF are related closely to angiogenesis etinopathy of prematurity (ROP) is a leading cause of in cancer. The purpose of our study was to evaluate the expression Rchildhood blindness over the world, characterized by and correlation of heparanase and VEGF in hypoxia-induced retinal retinal hypoxia-ischemia and neovascularization.1,2 VEGF, neovascularization. which can be upregulated by hypoxia and ischemia,3,4 is one of the most significant factors involved in retinal diseases METHODS. C57BL/6 oxygen-induced retinopathy (OIR) mice and 5,6 human retinal microvascular endothelial cells (HRECs) were associated with pathologic angiogenesis, including ROP. Logically, anti-VEGF therapy has been used in ROP patients treated with the hypoxia mimetic agent cobalt chloride (CoCl2), 7,8 and in the presence of the heparanase inhibitor phosphomanno- and shown beneficial short-term effects. However, it has pentaose sulfate (Muparfostat, PI-88). Heparanase activity was been shown that direct inhibition of VEGF cannot completely assayed in HRECs, and the expression of heparanase, VEGF protein prevent the formation of new vessels and anti-VEGF therapy requires long-term, repeated administration to decrease the and mRNA were evaluated by immunofluorescence, ELISA, continuous generated VEGF.9 Therefore, it is necessary to find Western blot, and real-time PCR while retinal flat mounts were a way to inhibit the VEGF protein production for better control used to evaluate the area of neovascularization of mice retina. of neovascularization. RESULTS. HREC heparanase activity was increased by treatment A very important step in the process of neovascularization with CoCl2, but was decreased by PI-88. Immunofluorescence is to change the integrity of the extracellular matrix (ECM) and showed that heparanase and VEGF staining was intense in subendothelial basement membrane (BM). Heparanase is an hypoxia-treated HRECs and OIR mice retina, while VEGF staining endo-b-glucuronidase that specifically cleaves heparan sulfate was faint in the normoxia and PI-88–treated ones. Western blot (HS) side chains of HS proteoglycans (HSPGs), which are and real-time PCR results indicated that the expression of essential components of the cell surface and ECM that provide heparanase and VEGF was increased under hypoxic conditions, structural integrity.10 Thus, heparanase is involved directly in and the increase of VEGF was inhibited by PI-88. Retinal flat angiogenesis by degrading HS chains. Degradation of HS side mounts showed that the area of new vessels in retina of OIR mice chains also releases HS-bound angiogenic growth factors, was increased compared to the normoxic mice, and this effect was including FGF-2 and VEGF, and HS fragments, which promote inhibited by PI-88. angiogenic growth factor activity.11 It is known that cells are exposed to hypoxic environment ONCLUSIONS Heparanase is upregulated and associated with the C . 12 VEGF expression in hypoxia-induced retinal diseases. Heparanase in solid tumors. Heparanase activity is upregulated by is involved in hypoxia-induced neovascularization through pro- hypoxia in many tumor cells and overexpression of heparanase correlates with increased VEGF protein and mRNA levels, and moting VEGF expression and may be a new therapeutic target for 13–15 hypoxia-induced neovascularization retinal diseases. (Invest Oph- increased tumor vascularity. These studies suggested a thalmol Vis Sci. 2012;53:6810–6817) DOI:10.1167/iovs.11-9144 correlation between heparanase and hypoxia-induced neovas- cularization in tumors. Therefore, a similar mechanism was hypothesized in which heparanase regulates VEGF mRNA and protein levels, and promotes neovascularization in ROP. In our study, as an initial step toward understanding the pathophys- From the 1State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China; the iologic conditions of ROP, we examined the regulation of 2Department of Oral and Maxillofacial Surgery, Guanghua School of heparanase and VEGF expression in retina under normoxic, Stomatology, Sun Yat-sen University, Guangzhou, China; and the hypoxic, and hypoxia mimic conditions, and the effect of the 3Department of Immunology, The John Curtin School of Medical heparanase inhibitor, phosphomannopentaose sulfate (Mupar- Research, The Australian National University, Canberra, Australia. fostat, PI-88), on the VEGF expression in vitro and in vivo. Supported by the International Cooperation Project of Guang- dong Province (Grant 2008B050100038), the Fundamental Research Funds of State Key Lab of Ophthalmology, Sun Yat-sen University MATERIALS AND METHODS (Grant 2010C04), and Science and Technology Planning Project of Guangdong Province (Grant 2011B020800005). Human Retinal Microvascular Endothelial Cell Submitted for publication November 23, 2011; revised April 15 and August 12, 2012; accepted August 22, 2012. Culture and Treatment Disclosure: J. Hu,None;X. Song,None;Y.Q. He,None;C. Human eyes were obtained from the Eye Bank of Zhongshan Freeman,None;C.R. Parish,None;L. Yuan,None;H. Yu,None; S. Tang,None Ophthalmic Center of Sun Yat-sen University (Guangzhou, China), Corresponding author: Jie Hu, The State Key Laboratory of within 20 hours postmortem. Donors of all these eyes were healthy Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen Univer- accident victims. All human materials were in compliance with the sity, Guangzhou 510060, China; [email protected] World Medical Association Declaration of Helsinki ethical principles for Investigative Ophthalmology & Visual Science, October 2012, Vol. 53, No. 11 6810 Copyright 2012 The Association for Research in Vision and Ophthalmology, Inc. Downloaded from iovs.arvojournals.org on 09/26/2021 IOVS, October 2012, Vol. 53, No. 11 Heparanase and Retinal Neovascularization 6811 FIGURE 1. Heparanase activity, heparanase and VEGF protein levels in CoCl2-treated HRECs. HRECs were cultured in normal media (control), or treated with CoCl2, CoCl2 þ PI-88, and CoCl2 þ PBS for 24 hours. A heparan sulfate degrading enzyme assay kit was used to determine heparanase activity (A). Western blot analysis was performed to detect heparanase (B) and VEGF (C) protein levels. An ELISA was performed on HRECs treated with CoCl2, or CoCl2 þ PI-88, and CoCl2 þ PBS (D). Data represent the mean 6 SD of three experiments. *P < 0.05 versus control. †P < 0.05 versus CoCl2 or PBS. medical research. The procedures were as described previously.16 sterile PBS, pH 7.4.18 For experimental control, HRECs were grown in Briefly, retinal tissues were isolated from eyes, and digested with 2% normal medium containing PBS. trypsogen and 0.1% collagenase I (Sigma Chemical Co., St. Louis, MO.) for 20 minutes at 378C, then subjected to centrifugation (1000g for 10 Mouse Model of Oxygen-Induced Retinopathy and minutes). The pellet was resuspended in human endothelial serum-free Treatment medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and 5 ng/mL b-endothelial cell growth factor (b- Experiments were performed on C57BL/6 mice (Laboratory Animal ECGF, Sigma), plated into a 21.5 mm2 culture dish precoated with 5 Center of Zhongshan Ophthalmic Center, Sun Yat-sen University) of both sexes at postnatal day (PD) 17. All experiments were in mg/mL fibronectin (Gibco), and then incubated at 378C with 5% CO . 2 compliance with the ARVO Statement for the Use of Animals in Human retinal microvascular endothelial cells (HRECs) were identified Ophthalmic and Vision Research, and were approved by the Animal by positive staining with anti-VIII factor antibody (Biosynthesis Care Committee of Sun Yat-sen University. The reproducible murine Biotechnology Co., Beijing, China). Only cells at passages 3 to 5 were model of oxygen-induced retinopathy (OIR) has been described used for the experiments. previously.19 Briefly, mice pups with their nursing mothers were HRECs were incubated with normal medium (normal group), 100 exposed to room air between PD 1 and PD 17, using as normal lM cobalt chloride (CoCl2 group), or 100 lM CoCl2 combined with 5 controls (Group 1: normal mice). Mice of the same strain and the same lg/mL heparanase inhibitor PI-88 (PI-88 group; Progen Pharmaceuti- age were exposed to 75% 6 2% oxygen between PD 7 and PD 12, and cals Limited, Brisbane, Australia) for 24 hours.17 PI-88 was dissolved in then returned to room air for 5 days (Group 2: OIR mice). OIR mice Downloaded from iovs.arvojournals.org on 09/26/2021 6812 Hu et al. IOVS, October 2012, Vol. 53, No. 11 FIGURE 2. Confocal microscopy analysis of heparanase and VEGF protein levels in CoCl2-treated HRECs. HRECs were cultured in normal media (control; [A–D]), or treated with CoCl2 (E–H), or with CoCl2 + PI-88 (I–L) for 24 hours. Cells were stained with DAPI (blue;[C, G, K]), and rabbit anti-heparanase (green;[A, E, I]) and mouse anti-VEGF (red;[B, F, J]) antibodies. Low levels of heparanase (A) and VEGF (B) were observed in control HRECs and marked increases in CoCl2-treated cells (E, F). The intense VEGF staining was decreased (J) by PI-88. were injected intraperitoneally with PI-88 at a dose of 25 mg/kg/day dish. Cells were washed with PBS 3 times and fixed in fresh 4% for 5 days from PD 12 to PD 16 (Group 3: OIR mice þ PI-88). A vehicle paraformaldehyde (PFA, pH 7–8) for 10 minutes at room temperature. group received sterile PBS alone from PD 12 to PD 16 (Group 4: OIR Next, HRECs were permeabilized in 0.1% Triton X-100 (Sigma) for 5 mice þ PBS).20,21 Animals were anesthetized with intraperitoneal minutes and blocked with 1% BSA (Sigma) in PBS containing 0.1% injection of 10% chloral hydrate at a dose of 3 mL/kg (Zhongshan Tween 20 (blocking solution) for 60 minutes at room temperature.