The role of different VEGF isoforms in scar formation after glaucoma filtration surgery

Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. Our previous studies showed a differential role of VEGF isoforms in retinal physiological angiogenesis. We also demonstrated that non-selective inhibition of VEGF by bevacizumab had a beneficial effect on surgical outcome after glaucoma filtration surgery by reducing angiogenesis as well as fibrosis. However, the function of the VEGF isoforms in pathological angiogenesis and wound healing in the eye still remains unidentified. This study was designed to elucidate the differential roles of VEGF isoforms in scar formation after trabeculectomy. Furthermore, we also investigated whether pegaptanib (Macugen™, Pfizer), an aptamer which specifically blocks VEGF165, could improve surgical outcome by reducing postoperative scarring. VEGF-R2 and neuropilin-1 (NRP-1) expression was analyzed in vitro by RT-PCR, and were found to be expressed at higher levels in human umbilical vein endothelial cells (HUVEC) as compared to Tenon fibroblasts (TF). The effect of the different VEGF isoforms (VEGF121, VEGF165 and VEGF189) and pegaptanib on cell proliferation was determined via WST-1 assay. Endothelial cell proliferation was stimulated after addition of VEGF121 and VEGF165, whereas VEGF121 and VEGF189 increased fibroblast growth. These effects on proliferation were associated with an activation of the ERK pathway, as revealed using the TransAM c-Myc assay. Inhibition of the ERK pathway, by PD98059 administration, significantly reduced VEGF isoform induced cell growth. A dose-dependent reduction of endothelial cell proliferation was observed after pegaptanib administration, while only the highest dose was able to inhibit fibroblast growth. Next, the in vivo effect of pegaptanib was investigated in a rabbit model of trabeculectomy. The surgical outcome was evaluated by performing clinical investigations (IOP, bleb area, height and survival), as well as histomorphometric analyses of angiogenesis (CD31), inflammation (CD45) and fibrosis (Sirius Red). A single postoperative application of pegaptanib had a beneficial impact on surgical outcome, mainly by reducing angiogenesis, but not inflammation or collagen deposition. Repeated injections slightly improved surgical outcome, but again solely by reducing angiogenesis. In summary, our results revealed that the VEGF isoforms play a differential role in ocular wound healing: VEGF165 and VEGF121 predominantly affect blood vessel growth, whereas VEGF189 is rather involved in fibrosis, an important process in wound healing.

► VEGF isoforms play a different role in scar formation after glaucoma surgery.
► Endothelial cell proliferation is stimulated after addition of VEGF121 and VEGF165.
► VEGF121 and VEGF189 increase Tenon fibroblast growth.
► Pegaptanib administration slightly improves surgical outcome after glaucoma surgery.
► Pegaptanib reduces postoperative angiogenesis but has no effect on fibrosis.