The Angio-Fibrotic Switch of VEGF and CTGF in Proliferative Diabetic Retinopathy

A shift in the balance between CTGF and VEGF is associated with the switch from angiogenesis to fibrosis in proliferative retinopathy.

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.

Degree of fibrosis

0	No fibrosis	Macular hole, RD without PVR
1	Light fibrosis	Macular pucker
2	Moderate fibrosis	PVR grade a/b  PDR with pre-retinal membranes
3	Severe Fibrosis	PVR grade c/d  PDR with abundant  vitreous membranes

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 2). 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).

Clinical course of proliferative retinopathy after laser or anti-VEGF treatment

In each of the 4 cases of PDR, we observed a transition from pre-retinal angiogenesis to a temporary phase of progressing pre-retinal fibrosis after pan-retinal laser treatment, or after anti-VEGF treatment followed by pan-retinal laser (Figure 3).

Figure 3. Fundus photographs of a patient with proliferative diabetic retinopathy and new vessels (nv) along the lower vascular arcade, before (A) and 8 months after (B) an injection with bevacizumab followed by pan-retinal photocoagulation. Note the increase in fibrosis (f) after anti-VEGF and laser treatment (B).

In the case of branch retinal vein occlusion complicated by local pre-retinal angiogenesis with leaky vessels due to ischemia, the new vessels stopped growing and did not leak anymore after laser treatment, whereas fibrosis did not follow (Figure 4).

Figure 4. Fundus photographs (A, D) and fluorescence imaging of FITC (B, C) of a patient with branch retinal vein occlusion before (A-C) and after (D) treatment with pan-retinal photocoagulation. Note the leaky vessels (A, C) consistent with angiogenesis and the quiet aspect of the vessels after treatment without formation of fibrosis (D). n, normal; nv, neovascularization.

In a non-systematic survey, we studied the clinical files of a small series of four PDR patients with active neovascularization treated with intravitreal bevacizumab, an anti-VEGF antibody, and/or pan-retinal laser. Pan-retinal photocoagulation is a therapy which destroys large areas of retina, and which is known to markedly reduce intra-ocular VEGF levels. In these four patients, we did indeed observe regression of neovascularization and the predicted temporary increase in fibrosis. In another patient with branch retinal vein occlusion complicated by focal pre-retinal neovascularization, fibrosis did not follow laser treatment. This suggests that for the angio-fibrotic switch to occur, CTGF levels need to reach a threshold value and a sufficient number of target cells must be able to elicit the fibrotic response. Therefore, intra-ocular fibrosis appears to be a tightly regulated on-off response like intra-ocular angiogenesis.

Read more:

Kuiper EJ, Van Nieuwenhoven FA, de Smet MD, van Meurs JC, Tanck MW, Oliver N, Klaassen I, Van Noorden CJ, Goldschmeding R, Schlingemann RO.The angio-fibrotic switch of VEGF and CTGF in proliferative diabetic retinopathy. PLoS ONE. 2008 Jul 16;3(7):e2675