Integrin–Linked–Kinase (ILK) and Ocular Neovascularization: Evaluation of ILK Inhibitors in Endothelial and RPE Cells

Abstract

Abstract: : Purpose: The integrin–linked kinase (ILK) is a multi–domain serine/threonine kinase implicated in phosphatidylinositol 3–kinase (PI3K)–dependent signaling. The PI3K/ILK signaling pathway, through activated PKB/Akt, regulates cell survival, angiogenesis, and inflammation. We sought to evaluate the role of PI3K in general, and ILK in particular, in retinal and choroidal vasculopathies by using small molecule inhibitors of ILK in endothelial and retinal pigmented epithelial cells. Methods: Human umbilical vein endothelial cells (HUVEC), the microvascular endothelial cell line HMEC–1, and the adult retinal pigmented epithelial cell line ARPE19 were cultured in medium with ILK inhibitors (IC₅₀ ranging from 8 to 241 nM) and, as a control, the PI3K inhibitor LY294002. Cell proliferation and cytotoxicity were measured by ³H–thymidine uptake and MTS assay, respectively. Endothelial cell migration was determined using modified Bowden chambers and serum as chemoattractant. Apoptosis was quantified by measuring caspase–3 activity from cell lysate. Vascular endothelial growth factor (VEGF) release from ARPE19 cells was assessed by ELISA. Results:ILK inhibitors reduced VEGF–induced HUVEC and serum–induced HMEC–1 proliferation (IC₅₀ ranging from 0.9 – to 33.7 µM) without significantly affecting cell viability. Endothelial cell apoptosis was induced only at high doses of the small molecules. Inhibition of ILK also reduced endothelial cell migration. ILK inhibitors reduced the secretion of VEGF from ARPE–19 (IC₅₀ ranging from 9 to 19.1 µM) at doses that did not significantly impact cell viability. Similarly, ILK inhibitors were not cytotoxic on confluent ARPE–19 at concentrations up to 80 µM. Conclusions: ILK may be a potential target for ocular neovascularization, as ILK inhibitors suppressed endothelial cell proliferation and migration as well as VEGF secretion from ARPE–19, while showing a favorable safety profile in vitro. The role of ILK in choroidal neovascularization is currently being evaluated in an animal model.

Keywords: age-related macular degeneration • signal transduction • neovascularization