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

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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 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.

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Authors and Affiliations

  1. Department of Ophthalmology, Ludwig-Maximilians-University, Mathildenstrasse 8, 80336, Munich, Germany

    Jakob Siedlecki, Ben Asani, Christian Wertheimer, Anna Hillenmayer, Andreas Ohlmann, Claudia Priglinger, Siegfried Priglinger, Armin Wolf & Kirsten Eibl-Lindner

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  1. Jakob Siedlecki
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  2. Ben Asani
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  3. Christian Wertheimer
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  4. Anna Hillenmayer
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  5. Andreas Ohlmann
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  7. Siegfried Priglinger
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Correspondence to Jakob Siedlecki.

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Siedlecki, J., Asani, B., Wertheimer, C. et al. Combined VEGF/PDGF inhibition using axitinib induces αSMA expression and a pro-fibrotic phenotype in human pericytes. Graefes Arch Clin Exp Ophthalmol 256, 1141–1149 (2018). https://doi.org/10.1007/s00417-018-3987-8

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  • DOI: https://doi.org/10.1007/s00417-018-3987-8

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