Combined VEGF/PDGF inhibition using axitinib induces αSMA expression and a pro-fibrotic phenotype in human pericytes

• Published in: Graefe's archive for clinical and experimental ophthalmology 2018-06, Vol.256 (6), p.1141-1149
• Main Authors: Siedlecki, Jakob, Asani, Ben, Wertheimer, Christian, Hillenmayer, Anna, Ohlmann, Andreas, Priglinger, Claudia, Priglinger, Siegfried, Wolf, Armin, Eibl-Lindner, Kirsten
• Format: Article
• Language: English
• Subjects: Actin; Age; Basic Science; Collagen; Contraction; Cytoskeleton; Enzyme inhibitors; Gene expression; Genotype & phenotype; Inhibitor drugs; Macular degeneration; Medicine; Medicine & Public Health; mRNA; Ophthalmology; Pericytes; Phalloidin; Phenotypes; Placenta; Platelet-derived growth factor; Protein expression; Protein-tyrosine kinase; Smooth muscle; Vascular endothelial growth factor; Western blotting; Wounds
• ISSN: 0721-832X; 1435-702X
• DOI: 10.1007/s00417-018-3987-8

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 α-smooth muscle actin (αSMA) 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 μg/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 αSMA 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.