Tissue Kallikrein Suppresses Laser-Induced Choroidal Neovascularization in Mice

Abstract

Purpose: : Tissue kallikrein is a serine protease that contributes to a flow-dependent arterial dilation through activation of bradykinin B2 receptors coupled with endothelial nitric oxide release. Recently, it has been reported that tissue kallikrein also possesses the antiangiogenic effects through the cleavage of vascular endothelial growth factor (VEGF) 165 isoform and thereby reduces the pathological vascular changes in the murine model of oxygen-induced retinopathy. Here, we investigate the impact of tissue kallikrein in laser-induced choroidal neovascularization (CNV).

Methods: : Male C57Bl/6 mice (7-8 weeks old) were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. CNV was induced by laser photocoagulation (200mW, 75µm, 100msec). The animals received daily subcutaneous injections of tissue kallikrein (50μg/kg), or vehicle control (PBS) for 2 days before laser photocoagulation, and the treatment was continued until the end of the study. Seven days after laser injury, choroidal flat mounts were prepared and the size of the CNV lesions was quantified. The RPE-choroid complex was harvested 3 days after laser injury and the levels of inflammation-associated molecules, monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1, were assessed by enzyme-linked immunosorbent assay. Immunoblotting was performed using the RPE-choroid complex with anti-VEGF164 antibody.

Results: : The animals treated with tissue kallikrein showed a significant decrease in CNV size (27168.3±2432.2µm²), compared with vehicle-treated controls (36374.6±3204.1µm², p<0.05). Tissue kallikrein treatment significantly reduced the levels of MCP-1 (p<0.05) and ICAM-1 (p<0.05). Furthermore, immunoblotting showed the approximately 16kDa-band, presumably corresponding to fragmented VEGF164 protein, in the RPE-choroid complex samples obtained from the animals treated with tissue kallikrein.

Conclusions: : The current data indicate an antiangiogenic property of tissue kallikrein through VEGF164 cleavage in CNV.

Keywords: age-related macular degeneration • vascular endothelial growth factor • drug toxicity/drug effects