Abstract
Abstract: :
Purpose: Focal adhesion kinase (FAK), a tyrosine kinase localized to cellular focal adhesions, becomes phosphorylated upon cellular adhesion. FAK is involved in processes integral to angiogenesis such as cell growth, survival, and migration. Intense FAK immunoreactivity is associated with developing vasculature. Angiogenic growth factors are coupled to FAK activation. We asked if human retinal endothelial cells (HREC) exposed to the angiogenic factor insulin like growth factor-1 (IGF-1) could modulate FAK and examined whether overexpression of either FAK or FAK-related non-kinase (FRNK), an inhibitor of FAK, could influence HREC migration and in vivo angiogenesis. Methods: HREC were exposed to IGF-1 then FAK protein levels and phosphorylation were examined. Other HREC cultures were transiently transfected with empty plasmid (CDM8), or plasmid expressing either FAK or FRNK. FAK/FRNK expression was confirmed by Western. Transfected HREC were used to evaluate migration in response to IGF-1 using modified Boyden chambers. We used the neonatal murine model of hyperoxia-induced retinal neovascularization to test the effects of local FAK and FRNK expression on angiogenesis. Mice were injected intravitreally on post-natal day 1 (P1) with the plasmids as above. At P17 mice were perfused with fluorescein-dextran, eyes enucleated, retinas dissected and examined by fluorescence microscopy. Results: HREC adhesion leads to phosphorylation of FAK on tyrosine 397. IGF-1 did not have a consistent effect on FAK phosphorylation. Transfection of HREC with FAK resulted in a 44% increase (p<0.001) in cell migration in response to IGF-1, whereas transfection with FRNK resulted in a 20% decrease (p<0.05) in cell migration compared to control plasmid-transfected cells. Local FAK expression in mouse eyes led to formation of numerous large vascular tufts resembling glomeruli, whereas FRNK resulted in decreased neovascularization compared to control plasmid-injected animals. Conclusions: HREC attachment induces FAK phosphorylation; the effect of IGF-1 on FAK phosphorylation is variable and may be due to confounding effects of IGF binding proteins expressed by HREC. Enhanced expression of FAK augmented cell migration in response to IGF-1, whereas FRNK overexpression reduced the ability of HREC to respond to this growth factor. These studies are the first to demonstration that FAK stimulates, whereas FRNK decreases, retinal angiogenesis in vivo. Modulating FAK/FRNK system may provide a novel approach to modulating pathological retinal angiogenesis.
Keywords: neovascularization • extracellular matrix • cell adhesions/cell junctions