Abstract
Purpose :
Contractile fibroblastic/myofibroblastic cells derived from retinal pigment epithelial (RPE) cells have been implicated in the fibrotic complications of proliferative vitreoretinopathy. We have previously demonstrated that matrix contraction by RPE-derived cells can be inhibited by dasatinib, an FDA-approved cancer medication that inhibits multiple tyrosine kinases. The purpose of this project was to identify molecular targets affected by dasatinib that contribute to matrix contraction.
Methods :
Primary cultured porcine RPE cells were used between passages 3 to 5. Cells were cultured for 3 days in 25% vitreous fluid supplemented DMEM in the presence or absence of tyrosine kinase inhibitors for the final 24 hours. Western blot analyses were used to determine the phosphorylation status of phospho-tyrosine proteins. A type I collagen contraction assay was utilized to examine matrix contraction by cells.
Results :
Dasatinib reduced tyrosine phosphorylation of multiple proteins. Interestingly, while FAK auto-phosphorylation on Tyr397 was unaffected, auto-phosphorylation on Tyr402 of PYK2, also known as FAK2, was significantly reduced. In agreement with the Western blot data, PF431396, a dual inhibitor of PYK2 and FAK, significantly reduced matrix contraction while PF573228, an inhibitor of FAK but not PYK2, was without effect.
Conclusions :
The inhibitory effect of dasatinib on matrix contraction by RPE-derived cells is, at least in part, due to the prevention of PYK2 activation, which plays a role in the contractile process.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.