Background: The pathological angiogenesis in the retina is the major cause of vision loss at all ages. Vascular endothelial growth factor (VEGF) has been reported as the most potent inducer of retinal neovascularization. We previously demonstrated that integrin-linked kinase (ILK) regulates retinal vascular endothelial proliferation, migration and tube formation. However, little is known about the existence of cross-talk between ILK and VEGF signaling in retinal vascular endothelial cells and the probable regulatory role of ILK during VEGF induced retinal endothelial cells migration. The purpose of this work was to investigate the role of ILK in VEGF induced retinal neovascularization.
Methods: Cultured retinal endothelial cells (RF/6A) were knocked down for ILK using a small interfering RNA (siRNA). For this, cellular ILK expression was quantified by real-time quantitative PCR, Western blot analysis and immunocytochemical assay, and cytotoxicity of tansfection was determined by MTT assay. Moreover, ILK siRNA transfected RF/6A cells were induced by VEGF, and cell proliferation were determined by MTT assay, cell migration was measured by cell counting in modified Boyden chambers, cell spreading assay and tube formation assay were preformed. Furthermore, the impact of ILK-specific siRNA on VEGF induced VEGFR-2 and downstream signal pathways were tested by Western blot analysis.
Results: The results showed that both ILK mRNA and protein levels were virtually undetectable after transfection with ILK siRNA and blocking the expression of ILK by siRNA significantly inhibited VEGF induced retinal endothelial cell proliferation, attachment, spreading, migration and tube formation. The knockdown of ILK effectively suppressed VEGF induced p38 MAPK and Akt phosphorylation while with no effort on VEGFR-2, ERK and JNK.
Conclusion: We conclude that knockdown of ILK with siRNA effectively inhibited VEGF induced retinal endothelial cell attachment, spreading, migration and tube formation, and p38 MAPK and Akt as downstream signaling pathways of the ILK that regulated VEGF induced retinal neovascularization. This may be a potential therapeutic usefulness in treating ocular neovascularization. |
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