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H. Ma, M. Azuma, T. R. Shearer; Calpain Inhibitor Reverses VEGF-Induced Endothelial Cell Motility and Tube Formation in vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(13):858.
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Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis and microvascular permeability. VEGF-induced cytoskeletal reorganization plays a crucial role in angiogenesis (1). Cytoskeletal organization in endothelial cells is regulated by calpain cysteine proteases (2). Calpains are a family of 14 calcium-regulated intracellular proteases, which modulate cellular function by limited, specific proteolysis. Calpain-1 (µ-calpain) and calpain-2 (m-calpain) are the two major typical calpain isoforms and are responsible for most calpain activity in endothelial cells. The purpose of the present study was to determine if an orally available form of calpain inhibitor, SNJ-1945, reversed angiogenesis induced by VEGF in vitro.
Human retinal microvascular endothelial cells (HREC) were incubated with VEGF (60-100 ng/ml) for 24 h. Total calpain activities were determined by casein zymography. Proteolysis of endogenous calpain substrate α-spectrin in situ and autolysis of calpain were analyzed by immunoblotting. Angiogenesis in vitro was evaluated by measuring cell migration and tube formation into Matrigel.
Incubation of HREC with VEGF resulted in increased activity of mainly m-calpain. VEGF also increased calpain activation as measured by increased proteolysis of α-spectrin and increased amounts of the autolytic 43kDa fragment of calpain. When the retinal soluble proteins from microvascular endothelial cells were incubated with calcium, calpain activation and autolysis occurred. Treatment of endothelial cells with calpain inhibitor SNJ-1945 reversed VEGF-mediated tube formation and cell motility.
Our results suggested that orally available calpain inhibitor SNJ-1945 might be a possible candidate drug for management of pathological angiogenesis occurring in wet AMD and proliferative retinopathy.1. Leung DW, et al. (1989) Science 26, 1306-13092. Kulkarni S, et al. (2002) J. Biol. Chem. 277, 24435-24441
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