Figure 2. Mean levels of CTFG (A, D), geometric mean levels of VEGF (B, E), and mean ratio CTGF/log10(VEGF) (C, F) in relation with degree of neovascularization (A–C) and degree of fibrosis (D–F) in the vitreous of 32 PDR patients. Vertical bars represent 95% confidence intervals. Significant differences between groups are indicated. doi:10.1371/journal.pone.0002675.g002 vessels stopped growing and did not leak anymore after laser treatment, whereas fibrosis did not follow (Figure 4). Discussion The observations presented in this study, set against the background of existing literature on VEGF and CTGF, prompt us to propose a novel concept of the regulation of angiogenesis and fibrosis in ocular disease, and in wound healing in general. In this concept, angiogenesis in the vitreous is driven by VEGF, which amongst other factors upregulates the pro-fibrotic factor CTGF in various cell types in the newly formed neovascular membranes. Increasing levels of CTGF inactivate VEGF by reduction of production and complex formation, and when the equilibrium between these two factors shifts to a certain threshold ratio, the angio-fibrotic switch occurs and fibrosis driven by excess CTGF leads to scarring and blindness. This concept identifies CTGF as a major potential therapeutic target in the treatment of ocular fibrosis, in particular in combination with anti-VEGF agents. What is the evidence that this concept may be true? We have previously shown that vitreous CTGF levels strongly correlate with degree of fibrosis in vitreo-retinal conditions [10], an observation which is in line with the established role of CTGF in pathological fibrosis in other organs [11–13,27–31]. However, exogenous CTGF Angio-Fibrotic Switch has been reported to have angiogenic effects in ocular experimental models [21,22,24,32]. The present study shows that, in human PDR, the CTGF levels and the ratio of CTGF over VEGF are strongly associated with the presence and degree of fibrosis, whereas CTGF levels, in contrast to VEGF levels, do not have an association with neovascularization. Moreover, multivariate analysis revealed that degree of fibrosis was best predicted by the ratio of CTGF over VEGF. VEGF levels do correlate significantly with active neovascularization, in agreement with the widely accepted role of VEGF as a major ocular angiogenic factor [7,8,26,33–35]. We have previously shown that CTGF expression is induced by AGEs in diabetic rat retinas [36], particularly in pericytes [37]. We also have shown in various animal models using CTGF+/2 heterozygous and CTGF2/2 homozygous CTGF-deficient mice that diminished levels or even complete absence of CTGF did not affect neovascularization significantly [25]. These findings support our concept as outlined above. Our findings question the applicability of the concept of CTGF-driven angiogenesis to in vivo ocular disease. Rather, the accumulated evidence indicates that CTGF effects are highly context dependent and, in the specific condition of PDR, cause fibrosis rather than angiogenesis. Observations by others in PDR patients after VEGF inhibition by intravitreal injections with bevacizumab [38–40] may sustain this assumption. In these patients, a remarkable inhibition of angiogenesis is observed. We like to suggest that elevated CTGF levels, which remain in the vitreous after VEGF inhibition, are not able to maintain the angiogenic response, providing further evidence that CTGF has no pro-angiogenic role in PDR. PDR and other proliferative retinopathies, which represent important causes of blindness, are caused by widespread retinal ischemia. In these conditions, pre-retinal neovascularization into the vitreous is the first sign that a fibrovascular wound healing reaction will develop in later stages. VEGF produced by the ischemic retina is considered to be the major causal growth factor in the neovascularization process [8]. In later stages of PDR, scarring mechanisms within the fibrovascular membranes become more important, and fibrosis and vitreoretinal traction cause retinal detachment and blindness [1,3,4,9]. The mechanisms causing this transition, the angio-fibrotic switch, have not been elucidated. Further analysis of human vitreous samples in our study demonstrated a significant correlation between CTGF and log10 (VEGF) levels. This may relate to the fact that VEGF and CTGF are both direct targets of factors such as glucose and AGEs, inherent to the diabetic condition. It may also reflect that VEGF induces CTGF expression in the eye, as has been demonstrated in retinal endothelial cells in vitro [16,17] and in the rat retina in vivo [18,36]. The consequent rise of the ratio of CTGF over VEGF may contribute to the angio-fibrotic shift occurring during progression of PDR: it has been reported that CTGF can influence VEGF availability on different levels, including direct protein-protein binding [19,20,41] and degradation of HIF-1a, which is a transcription factor for VEGF [42]. Angiogenesis may thus decline due to inactivation of VEGF by CTGF. Subsequent fibrosis may be induced by excess of unbound CTGF as was found in kidney [15,43] and skin [30]. An earlier study by Kita et al. [44] did not show a significant correlation between CTGF and VEGF in the vitreous of patients with PDR and proliferative vitreoretinopathy. The reason for the difference with the current results is unknown, but may lie in the fact that we analyzed vitreous of PDR patients only. Our observations that vitreous VEGF levels were significantly higher in active neovascularization than in quiescent neovascu- larization, and were negatively associated to degree of fibrosis, support this concept. It may, however, not be ruled out that other factors such as fibronectin, integrins and proteoglycans have an PLoS ONE | www.plosone.org 4 July 2008 | Volume 3 | Issue 7 | e2675