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Ping Yang, Grace M Tewkesbury, Kristen L Buehne, Jae Yang, Peter Baciu, Glenn J Jaffe; Effect of Tec Kinases Inhibitor on Complement-mediated Release of Basic Fibroblast Growth Factor from Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2461.
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© ARVO (1962-2015); The Authors (2016-present)
Complement activation has been implicated in the pathogenesis of AMD. Basic fibroblast growth factor (bFGF) regulates the activity of VEGF and is found in choroidal neovascular membranes removed from patients with AMD. We previously reported complement activation induced bFGF release. PI(4,5)P2, Tec kinase, ATP1A1 and the α chain of the Na/K-ATPase have recently been identified to play an important role in FGF2 secretion. Herein, we investigate the effect of Tec kinases inhibitor on complement-mediated bFGF release in human RPE cells.
Cultured human RPE cells were primed with an anti-RPE antibody and then treated with either C1q-depleted human serum (C1q-Dep) in the presence or absence of LFM-A13 (Tec kinases inhibitor) or normal human serum (NHS) to elicit membrane attack complex (MAC). Controls included heat-inactivated C1q-Dep or heat-inactivated NHS. The levels of 38 cytokine and chemokines in the conditioned media were measured by magnetic luminex performance assay (HCYTMAG-60K-PX38). mRNA expression of Tec, Btk, Itk, Txk and Bmx was evaluated by qPCR. Tec protein expression was examined by Western blot. In the presence or absence of LFM-A13, complement-mediated bFGF release, cell viability and MAC deposition were assayed by ELISA, WST-1 assay and flow cytometry, respectively.
Complement activation increased bFGF, CXCL1, IL-6 and IL-8. Other cytokine/chemokines including IL-1RA, a protein basally expressed and secreted in RPE cells with similar molecular weight and higher plasma concentration than that of bFGF were not increased by complement. Tec mRNA was more abundantly expressed in RPE cells when compared to Btk, Itk, Txk and Bmx. Tec protein was produced in RPE cells. In the presence of LFM-A13, bFGF protein levels in conditioned media were significantly decreased, cell viability was significantly increased and MAC deposition was significantly decreased in complement-treated cells when compared to controls without the inhibitor.
bFGF release induced by complement in human RPE cells may depend on Tec kinase activation and may be independent of membrane disruption. This information enhances our understanding of the role that complement activation plays to mediate neovascularization by bFGF in conditions such as AMD, and may elucidate potential therapeutic targets.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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