Anti–FasL Neutralizing Antibody Increases the Efficacy of PDT and Reduces the Apoptotic Damage in a Rat Laser Model

Abstract

Abstract: : Photodynamic therapy(PDT) is an effective treatment for choroidal neovascularization(CNV),but may require multiple sessions to limit vision loss. Preclinical studies have demonstrated damage to adjacent retinal structures which may accumulate with multiple treatments.The antiapoptotic properties of a neutralizing anti–FasL antibody may offer neuroprotection and improve the effectiveness of PDT.Purpose:To investigate the efficacy of PDT in combination with the anti–FasL antibody in a laser injury model of CNV in the rat. Materials and Methods:Choroidal neovascular membranes were induced using Argon/Dye laser in Brown–Norway rats treated with continuous administration (via subcutaneous pump) of either FasL neutralizing or isotype–matched control antibody at a total dose of 5 mg/kg. PDT was performed 14 days after CNV induction at a verteporfin dose of 3 mg/m2, irradiance of 600 mW/cm2 and fluence of 25 J/cm2. CNV closure on fluorescein angiograms was assessed 24 hours later in a masked fashion using grading standards. Retinal levels of Bcl–2,Bax, Bcl xL and Bid were measured by immunoblotting whereas cytosolic–mitochondrial levels of cytochrome c were measured by ELISA. Retinal activation of caspase–3 and –6 were measured with an activity assay.Results:Anti–FasL antibody reduced the angiographic leakage from CNV (97.4% of the lesions in the animals treated with the control antibody were stage IIA and IIB vs 82% in the anti–FasL treated group, P<0.001) and increased the efficacy of verteporfin PDT on CNV closure (70% of the lesions in the animals treated with the control antibody and PDT were angiographically not perfused vs 100% of lesions treated with anti–FasL and PDT). PDT decreased the retinal expression of antiapoptotic proteins such as bcl–2 and bcl–xL, increased the expression of pro–apoptotic proteins such as bax and induced cleavage of bid and cytoplasmic release of cytochrome c. PDT also induced activation of caspase 3 and 6 in the retina. Treatment with the anti–FasL antibody attenuated the PDT–induced reduction of bcl–2 and bcl–xL expression, inhibited bid cleavage and cytochrome c cytoplasmic release and reduced the PDT–induced increased bax expression.Conclusions: Anti–FasL administration decreased the angiographic leakage and increased the efficacy of PDT for CNV closure in a rat model, yet reduced retinal apoptotic damage induced by PDT. This suggests that combination of PDT with anti–FasL neutralizing agents may limit damage to normal structures and improve PDT results.

Keywords: cell death/apoptosis • choroid: neovascularization • photodynamic therapy