Photodynamic Therapy Induces Caspase–Dependent Apoptosis in Rat CNV Model

Abstract

Purpose: : We have reported that photodynamic therapy (PDT) causes apoptosis in vitro and in vivo, and induces rapid caspase–dependent apoptosis in bovine retinal capillary endothelial cells but details of the signaling mechanisms following PDT of choroidal neovascularization (CNV) remain unclear. In this study we investigated the Akt signaling in caspase–dependent apoptosis in CNV following PDT in the rat CNV model.

Methods: : CNV was induced in the eyes of Brown–Norway rats using Argon laser (100 µm spot size, 0.10 s duration, 150 mW). Verteporfin was injected intravenously at doses of 3, 6, 12 mg/m² 15 minutes before PDT. PDT was performed using laser at the wavelength of 689 nm, irradiance of 600 mW/cm² and fluence of 100 J/cm². Apoptotic cells in CNV were detected by TUNEL assay at various time points (1, 3, 6, 15, 24 and 48 hours) after PDT. Activation of caspase–3 and 9 was determined by immunohistochemical (IHC) analysis with a cleaved caspase specific antibody. Akt activity was determined by Western blot and IHC with a phosphorylated–Akt specific antibody. To access the role of Akt in PDT–induced apoptosis, 5 µg of insulin–like growth factor (IGF)–1, an activator of Akt, with or without 12.5 µM wortomannin, an inhibitor of PI3K–Akt pathway, was injected into the vitreous before PDT.

Results: : The number of TUNEL–positive cells in CNV started to increase at 3 hours after PDT, peaked at 6 hours and then decreased by 24 hours, showing a dose dependence with respect to verteporfin dose. Dephosphorylation of Akt in CNV occurred within one hour of PDT, with subsequent caspase activation. Cleaved caspase–3 and 9 immunoreactivity co–localized with TUNEL–positive cells in CNV. IGF–1 led to significantly increased phosphorylation of Akt and suppressed the number of TUNEL–positive cells in CNV. The effect of IGF–1 was diminished by wortomannin.

Conclusions: : Caspase–dependent apoptosis was induced in CNV following PDT in the rat. Our results suggest that PDT leads to dephosphorylation of Akt and subsequent activation of the caspase–dependent pathway. Understanding the intracellular signaling mechanisms of apoptosis in PDT may lead to a more selective and effective treatment of CNV secondary to AMD.

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