Expression of Vascular Endothelial Growth Factor (VEGF) and Effects of Anti–VEGF Treatment in the Rat Chorioretina After Photodynamic Therapy With Verteporfin

Abstract

Abstract: : Purpose: Therapies targeting VEGF are being developed to treat choroidal neovascularization (CNV) due to AMD and initial studies have demonstrated activity in this disease. VEGF has been reported to be up–regulated in human retina after PDT with verteporfin (Visudyne^®, Novartis AG); however, the role that VEGF plays in the posterior segment in response to PDT is still unknown. Blocking VEGF may prolong CNV closure after PDT, but an anti–VEGF therapy may also extend the choriocapillaris hypoperfusion noticed after PDT, which may lead to potential damage to the retina. We are interested in the combination of PDT and therapies targeting VEGF, and specifically, how to optimally and safely apply both therapies. We investigated the role of VEGF in the biological responses of the chorioretina to PDT using an anti–VEGF small interfering RNA (siRNA) in the rat eye. Here we report on the expression of VEGF in the chorioretina exposed to PDT and the impact of an anti–VEGF siRNA on the choriocapillaris after PDT. Methods: PDT was performed in Long Evans rats using an intravenous bolus injection of 9 mg/m² verteporfin followed by 25 J/cm² light delivered to the retina at a fluence rate of 150 mW/cm². A siRNA that suppressed VEGF expression in rat C6 glioma cells in vitro or, as a control, a siRNA against luciferase was intravitreally injected before or after PDT. VEGF mRNA extracted from retinas was quantified by real–time RT–PCR. The expression of VEGF and VEGF receptors 1 and 2 was also evaluated by immunohistochemistry. Choriocapillaris perfusion was assessed by fluorescein angiography and histology. Results: The choriocapillaris exposed to PDT was non–perfused for 7–14 days under the tested regimen. Rapid up–regulation of VEGF, VEGFR1 and VEGFR2, and phosphorylation of tyrosine residues on both VEGFRs were detected by immunohistology in the retina 24 h post–PDT. An increased level of VEGF mRNA was also confirmed in the PDT–treated retinas by real–time RT–PCR. Results of experiments on the effects of siRNA on PDT–induced VEGF expression and choriocapillaris closure will be shown. Conclusions: PDT induced a temporary choriocapillaris closure which was accompanied by an up–regulation of VEGF and its receptors in the chorioretina. The impact of the anti–VEGF siRNA on choriocapillaris hypoperfusion after PDT is being evaluated and may provide guidance on the clinical evaluation of the combined use of verteporfin PDT with anti–VEGF therapies for the treatment of CNV due to AMD.

Keywords: choroid: neovascularization • photodynamic therapy • age-related macular degeneration