Therapeutic potential of VEGF and PlGF inhibition in scar formation after glaucoma filtration surgery

Abstract

Glaucoma is a neurodegenerative disease and the second most important cause of irreversible blindness. This disease is characterized by a raised intraocular pressure (IOP) and by progressive retinal ganglion cell degeneration, resulting in irreversible visual field loss. Filtration surgery (trabeculectomy) remains the most effective therapy to reduce IOP in glaucoma patients. However, in 30% of the cases this surgery fails due to subconjunctival wound healing. Pharmacological enhancement of trabeculectomy using different anti-scarring agents was found to significantly improve rates of surgical success. However, the nonspecific mechanism of action of these agents may result in severe side-effects. Therefore, there is still a need for alternative strategies to prevent filtration failure. Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. VEGF-R2 mediates most biologically relevant VEGF responses, including vascular permeability, cell migration and proliferation. Alternative splicing of a single VEGF-gene results in multiple isoforms, such as VEGF121, VEGF165 and VEGF189. Different anti-VEGF treatments are used in the ophthalmological practice, some of which have more selective properties. Pegaptanib (Macugen , Pfizer) is an aptamer that selectively prevents the binding of VEGF165 to its receptor, whereas bevacizumab (AvastinTM; Genentech) is a humanized, monoclonal full-length antibody against all isoforms. Placental growth factor (PlGF) is a VEGF-homologue which only binds to VEGF-R1. Therefore, this growth factor only acts on pathological angiogenesis and inflammation and is not involved in physiological angiogenic processes. A monoclonal anti-PlGF antibody against mouse PlGF-2 was developed by ThromboGenics NV. In this thesis work, we explored the hypothesis that VEGF and PlGF are playing an important role in scar formation after glaucoma filtration surgery and that they may be targets for improvement of filtration surgery outcome. The exact role of the different VEGF isoforms in the process of wound healing was also studied. The effect of non-selective and selective VEGF inhibitors and inhibition of PlGF on ocular wound healing was investigated in vivo using different animal models of glaucoma filtration surgery. In the first part of this thesis, we demonstrated that VEGF and PlGF are important players in scar formation in vitro. Aqeuous humor of glaucoma patients was collected and aqueous levels of VEGF and PlGF were significantly upregulated compared to control (cataract). Tenon fibroblasts (TF) and endothelial cells, both regarded as key players in wound healing after glaucoma surgery, were cultured and the expression of VEGF, PlGF and their receptors was determined. Quantitative real-time RT-PCR showed that both growth factors and their receptors (VEGF-R1 and VEGF-R2) were indeed expressed on these cells. Moreover, VEGF and PlGF were able to significantly stimulate proliferation of Tenon fibroblasts.  In a second chapter, we showed that the different VEGF isoforms were differentially involved in ocular wound healing. We demonstrated that VEGF121 and VEGF165 significantly stimulated proliferation of endothelial cells, by activating the ERK pathway. PD98059, an inhibitor of the ERK pathway, significantly inhibited VEGF121 and VEGF165-induced endothelial cell proliferation. We also reported the effect of the different isoforms on TF growth. We showed that TF proliferation was mainly induced by VEGF121 and VEGF189, associated with an activation of the ERK pathway. These proliferative effects were significantly reduced by the ERK pathway inhibitor PD98059. Thus, our results indicated that VEGF isoforms play a differential role not only in ocular angiogenesis but also in wound healing. VEGF165 was found to be the most important isoform in inducing endothelial cell proliferation, while VEGF189 was the most prominent in inducing TF proliferation. VEGF121 clearly affected both cell types. In a third section, the therapeutic potential of selective versus non-selective VEGF inhibition (pegaptanib versus bevacizumab) was investigated in vitro and in vivo by analyzing the area and survival of the filtration bleb area and process of wound healing of rabbit eyes following surgery. Proliferation assays on endothelial cells and TF suggested that non-selective VEGF inhibition might be anti-angiogenic and anti-fibrotic, while selective VEGF165 blockage might mainly inhibit angiogenesis. Indeed, these results were confirmed in our rabbit model for glaucoma surgery. A single administration of bevacizumab at the time of trabeculectomy could improve the surgical outcome by reducing postoperative angiogenesis during the initial phase, and collagen deposition at later stages. Pegaptanib injection(s) improved surgery outcome less efficiently by reducing angiogenesis only. Thus, our in vitro and in vivo data indicated that selective VEGF165 inhibition by pegaptanib was less effective than non-selective VEGF inhibition by bevacizumab in reducing scarring after glaucoma surgery, presumably due to retained action of VEGF121 and VEGF189 isoforms, which had a more pronounced effect on the Tenon fibroblasts as compared to VEGF165. Finally, in the last part, the therapeutic potential of PlGF inhibition was investigated. We showed that anti-VEGF therapy prevented post-surgical scarring by inhibiting angiogenesis and collagen deposition, but did not influence inflammation. Therefore, in collaboration with ThromboGenics NV, the known anti-angiogenic and anti-inflammatory, and possibly anti-fibrotic properties of anti-PlGF-antibody were studied in vitro and in vivo in a mouse model of glaucoma surgery. Administration of anti-PlGF antibody could reduce proliferation of TF in vitro and could improve the surgical outcome in vivo. Postoperative inflammation and angiogenesis was reduced during the initial phase and collagen deposition at later time points. Direct comparison showed that inhibition of PlGF possibly seemed to be even more effectively than inhibition of VEGF-R2, which only had an effect on angiogenesis and fibrosis. In conclusion, we showed that VEGF and PLGF are playing an important role in the process of wound healing after glaucoma surgery. Our study also sheds new light on the differential function of the VEGF isoforms in the mechanisms of angiogenesis and wound healing. VEGF165 and VEGF121 predominantly affect blood vessel growth, whereas VEGF189 seems to be more important in fibrosis. These insights may have important therapeutic implications for glaucoma patients. VEGF treatment - particularly non-selective - could improve the surgical outcome in glaucoma patients by reducing angiogenesis and fibrosis. On the other hand, inhibition of PlGF seems effective in improving surgical outcome by reducing inflammation, angiogenesis and collagen deposition. The insights generated within this thesis may open new perspectives not only for glaucoma surgery, but also for modulation of postoperative scarring in general.status: publishe