Focal Adhesion Kinase Signaling Pathway Regulate The Formation Of Choroidal Neovascularization

Background Choroidal neovascularization (CNV) is now known the leading cause of vision loss in age related macular degeneration (AMD), while its pathogenesis is still poorly understood. Retinal pigment epithelium (RPE) cells are known to quickly respond and adapt to environmental stresses such as ischemia and metabolic changes by expressing a number of various genes. In the early stages of CNV development, RPE produce cytokines and growth factors promoting CNV development. Subsequently, proliferating choroidal vessels surmount Bruch's membrane and spread in the subpigment epithelial and subretinal space to form a CNV membrane.The underlying mechanism of CNV is multifactorial and complex. Growth factors and cell adhesion molecules have been implicated in CNV. RPE cells participating in angiogenesis get signals from several sources, such as soluble stimulators and extracellular matrix (ECM). Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, plays a crucial role in linking signals initiated by the integrins or growth factor receptors to intracellular cytoskeletal and signaling proteins, thus controlling essential cellular processes such as growth, survival, migration and differentiation. Extensive evidence has shown that FAK is activated in response to both the ECM and soluble signaling factors. Recent work from several laboratories points out the importance of FAK in influencing distinct steps of the angiogenic response and suggests a critical role of FAK in pathological retinal angiogenesis. But to our knowledge, no study investigating the effect of FAK on CNV formation has been reported previously.Purpose It would be helpful for our understanding of CNV to study the effect of FAK on CNV formation. In this study, we investigate the expression of FAK in the experimental CNV models and evaluate the effect of several CNV risk factors on the expression of FAK in cultured RPE cells. Then we used siRNA specific blockade of FAK in RPE cells, seek to evaluate the role of FAK in key steps involved in CNV.Methods⑴The BN rats underwent laser rupture of Bruch's membrane to induce CNV and were killed at 1, 3, 7, and 14 days after laser injury. Immunofluorescenc and Western blot were processed to detect FAK protein.⑵Cultured human RPE cells were exposed to hypoxia, H2O2, ECM and out segment of photoreceptors (POS), differently. The expression of FAK and pFAK in cultured RPE cells were examined by Western blot.⑶RPE cells were cultured under hypoxia and RNA interference (RNAi) technique was used to knock down the FAK gene in RPE cells. Expression of hypoxia inducible factor-1α(HIF-1α) and vascular endothelial growth factor (VEGF) in RPE cells were investigated by RT-PCR and Western blot.⑷Two kinds of coculture models were used to observe the effects of specific blockade of FAK in RPE cells on the proliferation and migration of choroidal endothelial cells (CEC), respectively.Results⑴FAK was highly expressed in the rat RPE-choroid tissue after photocoagulation. Immunocytochemistry evaluations suggest that the strong up-regulation of FAK expression was located in RPE cells participating in choroidal neovascularization.⑵In vitro experiment shows hypoxia, H2O2, ECM and POS, which were known initiate CNV response, can induce FAK expression in cultured RPE cells.⑶FAK siRNA-treated RPE cells exhibited an inhibition in the expression of HIF-1 and VEGF in response to hypoxia, which indicated that FAK involved in hypoxia signaling in RPE cells.⑷The absence of FAK in RPE cells show inability in reducing the proliferation and migration of CEC under hypoxia condition.Conclusions In summary, we indicate the first time that FAK pathway activation plays a role in the development of laser-induced CNV in rat. In vitro experiment shows FAK pathway involves in several CNV initiated signaling in cultured RPE cells. The absence of FAK effectively reduces gene expression of hypoxia-induced HIF-1αand VEGF in RPE cells in vitro. Furthermore, our data provide strong evidence that specific blockade of FAK in RPE cells resultes in the inhibition of proliferation and migration of CEC. Combining the observation above, this is the first demonstration that FAK modulates the formation of CNV. It is reasonable to propose that FAK siRNA potentially provides a means to attenuate the strong stimuli for neovascularization in CNV-dependent disorder, which could represent a therapeutically relevant strategy for the inhibition of CNV.