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Siedlecki, Jakob; Asani, Ben; Wertheimer, Christian; Hillenmayer, Anna; Ohlmann, Andreas; Priglinger, Claudia; Priglinger, Siegfried; Wolf, Armin; Eibl-Lindner, Kirsten (2018): Combined VEGF/PDGF inhibition using axitinib induces alpha SMA expression and a pro-fibrotic phenotype in human pericytes. In: Graefes Archive for Clinical and Experimental Ophthalmology, Vol. 256, No. 6: pp. 1141-1149
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Abstract

Purpose Large trials on anti-VEGF/PDGF (vascular endothelial/platelet-derived growth factor) combination therapy have been established to improve management of neovascular activity in age-related macular degeneration. Targeting pericytes, PDGF is thought to induce vessel regression and reduce fibrovascular scarring. The fate of pericytes exposed to anti-VEGF/PDGF combination therapy is not clear. Therefore, this study was designed to study the influence of anti-VEGF/PDGF on pericyte phenotype and cellular behavior. Methods Human pericytes from placenta (hPC-PL) were treated with axitinib, a tyrosine kinase inhibitor targeting VEGFR1-3 and PDGFR. Toxic effects were excluded using live/dead staining. Phenotypic changes were evaluated using phalloidin staining for actin cytoskeleton and the expression of stress fibers. MRNA and protein expression levels of alpha-smooth muscle actin (alpha SMA) as a marker of proto-myofibroblastic transition were evaluated with real-time PCR and Western blotting. Influences of fibrotic cellular mechanisms were evaluated with a scratch wound migration and a collagen gel contraction assay. Results Treatment with 0.5, 1, and 2.5 mu g/ml axitinib strongly induced a proto-myofibroblast-like actin cytoskeleton with a marked increase in stress fibers. Quantitative real-time PCR and Western blotting revealed these changes to be linked to dose-dependent increases in aSMA mRNA and protein expression. However, fibrotic cellular mechanisms were significantly reduced in the presence of axitinib (scratch wound closure: up to -78.4%, collagen gel contraction: up to -37.4%). Conclusions Combined anti-VEGF/PDGF inhibition seems to induce a proto-myofibroblast-like phenotype in human pericytes in vitro, but reduce profibrotic cellular mechanisms due to prolonged anti-PDGF inhibition.