The Angio-Fibrotic Switch of VEGF and CTGF in Proliferative Diabetic Retinopathy Esther J. Kuiper1, Frans A. Van Nieuwenhoven 2 , Marc D. de Smet1, Jan C. van Meurs3, Michael W. Tanck4, Noelynn Oliver5, Ingeborg Klaassen 1 , Cornelis J. F. Van Noorden 1 , Roel Goldschmeding 2 , Reinier O. 1 Schlingemann * 1 Ocular Angiogenesis Group, Departments of Ophthalmology and Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 2 Department of Pathology, Academic Medical Centre of Utrecht, Utrecht, The Netherlands, 3 Department of Ophthalmology, Rotterdam Eye Hospital, Rotterdam, The Netherlands, 4 Department of Clinical Epidemiological Statistics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 5 FibroGen Inc, San Francisco, California, United States of America Abstract Background: In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) cause blindness by neovascularization and subsequent fibrosis, but their relative contribution to both processes is unknown. We hypothesize that the balance between levels of pro-angiogenic VEGF and pro-fibrotic CTGF regulates angiogenesis, the angio-fibrotic switch, and the resulting fibrosis and scarring. Methods/Principal Findings: VEGF and CTGF were measured by ELISA in 68 vitreous samples of patients with proliferative DR (PDR, N = 32), macular hole (N = 13) or macular pucker (N = 23) and were related to clinical data, including degree of intra-ocular neovascularization and fibrosis. In addition, clinical cases of PDR (n = 4) were studied before and after pan-retinal photocoagulation and intra-vitreal injections with bevacizumab, an antibody against VEGF. Neovascularization and fibrosis in various degrees occurred almost exclusively in PDR patients. In PDR patients, vitreous CTGF levels were significantly associated with degree of fibrosis and with VEGF levels, but not with neovascularization, whereas VEGF levels were associated only with neovascularization. The ratio of CTGF and VEGF was the strongest predictor of degree of fibrosis. As predicted by these findings, patients with PDR demonstrated a temporary increase in intra-ocular fibrosis after anti-VEGF treatment or laser treatment. Conclusions/Significance: CTGF is primarily a pro-fibrotic factor in the eye, and a shift in the balance between CTGF and VEGF is associated with the switch from angiogenesis to fibrosis in proliferative retinopathy. Citation: Kuiper EJ, Van Nieuwenhoven FA, de Smet MD, van Meurs JC, Tanck MW, et al. (2008) The Angio-Fibrotic Switch of VEGF and CTGF in Proliferative Diabetic Retinopathy. PLoS ONE 3(7): e2675. doi:10.1371/journal.pone.0002675 Editor: Tailoi Chan-Ling, University of Sydney, Australia Received December 6, 2007; Accepted June 11, 2008; Published July 16, 2008 Copyright: 2008 Kuiper et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from the Diabetes Fonds Nederland (grant 2001 042) and by the Edmond and Marianne Blaauw Fonds. Competing Interests: Noelynn Oliver is an employee of Fibrogen Inc, Roel Goldschmeding has received research support grants from Fibrogen Inc. * E-mail: r.schlingemann@amc.uva.nl Introduction Blindness from proliferative diabetic retinopathy (PDR) is caused by angiogenesis and fibrosis in the vitreous cavity of the eye [1–4]. PDR is a wound healing-like response in which neovascularization is accompanied by influx of inflammatory cells and development of myofibroblasts. This progresses to a fibrotic phase with fibrovascular contraction causing haemorrhages, retinal detachment and inevitable blindness. Several growth factors have been shown to play a role in PDR, e.g. vascular endothelial growth factor-A (VEGF), transforming growth factor- ß, hepatocyte growth factor, platelet-derived growth factor, and the pro-fibrotic connective tissue growth factor (CTGF) [5,6]. VEGF is considered to be the primary angiogenesis factor in this sequence of events [7–9]. In contrast, the causal factors of fibrosis and scarring and the regulation of the transition from angiogenesis to the fibrotic phase of PDR, which we propose to call the angio- fibrotic switch, remain largely unknown. We have recently shown that vitreous levels of CTGF correlate strongly with degree of fibrosis in vitreo-retinal disorders [10]. In other organs than the eye, CTGF has a similar role and has been identified as a potential therapeutic target for inhibition of pathological fibrosis [11–15]. Interestingly, CTGF is upregulated by VEGF [16–18] and inhibits VEGF-induced angiogenesis by formation of VEGF- CTGF complexes [19,20], suggesting a negative feedback function of CTGF on VEGF-induced angiogenesis. In contrast, recombi- nant CTGF has been shown to cause angiogenesis in the cornea and in other in vivo models in addition to its pro-fibrotic properties [21,22]. Other findings, such as co-localization of CTGF and VEGF in human subretinal neovascularization [23], and increased CTGF levels in vitreous of PDR patients with active neovascu- larization [24], also suggested that CTGF has a causal role in ocular neovascularization. However, we have recently challenged this concept by showing that in CTGF knock-out mice, angiogenesis is not impaired in several experimental models of neovascularization [25]. Based on these findings we propose a novel concept. We hypothesize that CTGF is a primary causal factor in sight- PLoS ONE | www.plosone.org 1 July 2008 | Volume 3 | Issue 7 | e2675