WST11 PDT of New Vessels Induces Apoptosis of Endothelial Cells Through a Non–Caspase Dependent Mechanism and Peroxynitrite Production

Abstract

Abstract: : Purpose:To evaluate the mechanism of action of photodynamic treatment (PDT) using a new hydrosoluble photosensitizer (WST11) on rat corneal angiogenesis. Methods: To induce a strong angiogenesis, Elvax 40 implants sequestering 75µg of LPS are prepared and implanted in the mid stroma of rat corneas. PDT of the new vessels (NV) was carried out with 10mg/Kg WST11 infused IV for 3 minutes, a 3.5 mm laser (753nm) beam, a fluence of 140 J/cm2 (600mW/cm2) and a DLI of 1min. NV occlusion was evaluated clinically by the extent of bleaching of the irradiated area during and immediately after PDT. 6 and 24hours, 2 and day 8 after treatment the eyes enucleated and snap frozen in OCT. 8 microns cryo sections of the treated corneal region are prepared. Immunohistochemistry for NOSII, nitrotyrosine, activated caspase–3, ED1 and OX42, LEI (leucocyte elastase inhibitor) and TUNEL assay are performed to detect caspase–dependent and caspase independent events of apoptosis. Results:Occlusion of the treated NV is observed during the laser delivery. As early as 6 hours after PDT, but more markedly after 24 hours, typical images of apoptotic nuclei were observed in NV endothelial cells (EC). The image of apoptotic nuclei is associated with an increased production of NOSII. The treated NV EC remain negative for TUNEL reaction at all time points after PDT. Nitrotyrosine production is observed only in NV EC within the PDT area and is strictly confined to the NV lumen. Leukocyte elastase inhibitor (LEI), a caspase independent marker of apoptosis, is expressed in the EC cytoplasm 6 hours after PDT treatment. The LEI translocated from the cytoplasm to the nucleus 24 hours and 8 days after PDT. These observations demonstrate that a transformation of LEI to L–DNase II activity occurs driving the apoptotic events in WST11 PDT. No evidence for EC activation of caspase dependent pathways is detected after WST11 PDT. Conclusions: WST11 PDT induces NO and peroxynitrite production within the NV EC, leading to their apoptosis through a non–caspase dependent mechanism. These apoptotic events are associated with the caspase independent LEI–L–DNase II pathway.

Keywords: neovascularization • apoptosis/cell death • photodynamic therapy