threatening fibrosis in the eye, and that an equilibrium shift in the balance between VEGF and CTGF levels in vitreous regulates the switch from the angiogenic phase to fibrosis in conditions such as PDR. Therefore, we investigated vitreous VEGF and CTGF levels in correlation with degree of fibrosis and neovascularization in a series of patients with PDR and other vitreoretinal disorders, and studied the effect of anti-VEGF treatment on the course of PDR in human patients. Methods Patients We investigated 68 vitreous samples of patients with PDR (N = 32), macular hole (N = 13) and idiopathic macular pucker (N = 23), who were operated by pars plana vitrectomy. The study was conducted according to the Declaration of Helsinki and approved by the institutional review board of the Academic Medical Center at the University of Amsterdam. Informed written consent was obtained from each patient. Clinical data, which allowed grading of fibrosis, activity of neovascularization, degree of haemorrhage, and presence and type of diabetes, were obtained from the pre-operative ophthalmic and ultrasound examinations, the patient files and from per-operative observations, using a standardized form. Fibrosis was graded as 0 when there was no fibrosis, as 1 when there were a few pre-retinal membranes (as limited as in macular pucker), as 2 when white pre- retinal fibrotic membranes with limited extension into the vitreous were present, and as 3 when abundant white membranes reaching into the vitreous body were observed. Neovascularization was graded as 0 when absent, as 1 (quiescent) when only non-perfused vessels were present, and as 2 (active) when there were perfused preretinal capillaries [26]. Degree of haemorrhage was graded as 0 when all media were clear and all fundus details were visible, as 1 when media were a little clouded but the fundus could still be examined, as 2 when the disk was obscured by haemorrhage, and as 3 when fundus details could not be obtained. In addition, we investigated retrospectively, after informed consent, the clinical records of 4 patients with PDR and one patient with proliferative retinopathy as a complication of branch retinal vein occlusion, who were treated with either pan-retinal photocoagulation (2 cases of PDR and the case of branch retinal vein occlusion) or with intravitreal injections with bevacizumab, an anti-VEGF agent, followed by pan-retinal laser treatment (2 cases of PDR). Sample collection Undiluted vitreous samples (0.5–1 ml) were obtained by using a vitrectome at the start of a classic three-port pars plana vitrectomy with the infusion line in position but not opened. The vitreous was transferred to sterile Eppendorf tubes and immediately frozen in dry ice. The samples were kept at 280uC until assayed. Measurement of CTGF and VEGF levels by enzyme-linked immunosorbent assays (ELISA) After thawing, vitreous samples were centrifuged at 14.000 rpm for 15 minutes at 4uC, and supernatant was collected. Concen- trations of CTGF were measured in samples by means of sandwich ELISAs, using two distinct monoclonal antibodies specifically recognizing the N-terminal part of the CTGF protein (FibroGen, San Francisco CA, USA) as described previously [10]. Purified recombinant human CTGF (FibroGen) was used as standard. Concentrations of VEGF165 were determined by means of sandwich ELISA according to the manufacturer’s protocol (R&D Systems, Minneapolis MS, USA). All ELISAs were carried out in duplicate. Angio-Fibrotic Switch Statistical analysis Both CTGF and VEGF levels were tested for normal distribution using the Shapiro-Wilk test (W.0.90). VEGF levels in vitreous showed a right skewed distribution and were log10 transformed to obtain a normal distribution. Associations between gender, age, diabetes type, degree of neovascularization, degree of haemorrhage and degree of fibrosis were assessed by ANOVA with correction for age and gender (included as covariates) where appropriate, and for unequal variances due to inter-assay variability. Least square means were estimated based on the model and, in case of an overall significant difference, compared using a post-hoc test. The correlation between CTGF and log10(VEGF) is expressed as Spearman correlation coefficient. In addition, univariate and multiple ordinal logistic regression analyses were performed with degree of fibrosis or neovascular- ization activity as dependent (outcome) variables. These effects were expressed as odds ratios with a 95% confidence interval. A two-tailed p-value ,0.05 was considered statistically significant. All analyses were carried out using SAS (Version 9) software (SAS Institute, Cary NC, USA). Results Analysis of all patients The research variables and characteristics of the 68 patients studied are shown in Table 1. There was a significant difference in age between the patients diagnosed with a macular hole or macular pucker and patients with PDR (66.4 versus 54.2, respectively; p = 0.001). Mean vitreous levels of CTGF and log10(VEGF) were significantly higher in patients with PDR than in patients diagnosed with a macular hole or macular pucker (p,0.001 for both growth factors, corrected for age (covariate)). Univariate analysis of all 68 patients showed that CTGF and log10(VEGF) levels in vitreous correlated strongly (p,0.001). A scatter plot of the correlation between CTGF levels and log10(VEGF) levels is shown (Figure 1). Univariate analysis of all 68 patients also showed that levels of both growth factors were associated with diabetes-associated variables (i.e. the presence of diabetes, degree of neovascularization, fibrosis and haemorrhage; all p,0.001). CTGF and log10(VEGF) levels did not differ between genders. Log10(VEGF) levels were not associated with age, whereas CTGF levels were (p = 0.03). Multivariate analysis showed that mean CTGF levels were only associated with degree of fibrosis (p,0.001), whereas log10(VEGF) levels were only significantly associated with degree of neovascularization (p,0.001). Analysis of patients with PDR Most parameters which correlated with VEGF and CTGF levels were associated with diabetes (i.e. neovascularization, haemorrhage and fibrosis) in the group of 68 patients. However, the 13 macular hole patients and 23 macular pucker patients did not suffer from diabetes (Table 1). Therefore, to exclude this confounder, we repeated the statistical analysis on the data of the 32 patients with PDR who all had diabetes. Similar to the entire patient population, CTGF levels in the vitreous of patients with PDR were significantly associated with degree of fibrosis (p = 0.042), and not significantly with degree of neovascularization (p = 0.072), whereas log10(VEGF) levels were associated with degree of neovascularization (p = 0.011), and not with degree of fibrosis (p = 0.665) (Figure 1). We also evaluated the association between the ratio CTGF/log10(VEGF) and degree of fibrosis or degree of neovascularization, respectively. The ratio CTGF/ log10(VEGF) was associated with degree of fibrosis (p = 0.009), but not with degree of neovascularization (p = 0.635) (Figure 2). PLoS ONE | www.plosone.org 2 July 2008 | Volume 3 | Issue 7 | e2675