Single-cell analysis reveals IGF-1 potentiation of inhibition of the TGF-β/Smad pathway of fibrosis in human keratocytes in vitro

Tomislav Sarenac; Martin Trapecar; Lidija Gradisnik; Marjan Slak Rupnik; Dusica Pahor

doi:10.1038/srep34373

Single-cell analysis reveals IGF-1 potentiation of inhibition of the TGF-β/Smad pathway of fibrosis in human keratocytes in vitro

Sci Rep. 2016 Sep 30:6:34373. doi: 10.1038/srep34373.

Authors

Tomislav Sarenac^{1

2}, Martin Trapecar^{2

3}, Lidija Gradisnik², Marjan Slak Rupnik^{2

4}, Dusica Pahor^{1

2}

Affiliations

¹ Department of Ophthalmology, University Medical Centre Maribor, Ljubljanska 5, 2000 Maribor, Slovenia.
² Institute of Physiology, Faculty of Medicine, University of Maribor, Taborska ulica 8, 2000 Maribor, Slovenia.
³ Gladstone Institute of Virology and Immunology, University of California, 1650 Owens Street, 94158 San Francisco, CA, USA.
⁴ Institute of Physiology, Centre for Physiology and Pharmacology, Medical University of Vienna, Währingerstrasse 13A, 1090 Vienna, Austria.

Abstract

Corneal wound healing is often affected by TGF-β-mediated fibrosis and scar formation. Guided fibrosis with IGF-1 and antifibrotic substances might maintain corneal transparency. Primary human corneal keratocytes under serum-free conditions were used as a model of corneal stromal wounding, with markers of corneal fibrosis and opacity studied under TGF-β2 stimulation. Single-cell imaging flow cytometry was used to determine nuclearization of Smad3, and intracellular fluorescence intensity of Smad7 and the corneal crystallin aldehyde dehydrogenase 3A1. Extracellular matrix proteoglycans keratocan and biglycan were quantified using ELISAs. On the TGF-β2 background, the keratocytes were treated with IGF-1, and suberoylanilidehydroxamic acid (SAHA) or halofuginone ± IGF-1. IGF-1 alone decreased Smad3 nuclearization and increased aldehyde dehydrogenase 3A1 expression, with favorable extracellular matrix proteoglycan composition. SAHA induced higher Smad7 levels and inhibited translocation of Smad3 to the nucleus, also when combined with IGF-1. Immunofluorescence showed that myofibroblast transdifferentiation is attenuated and appearance of fibroblasts is favored by IGF-1 alone and in combination with the antifibrotic substances. The TGF-β/Smad pathway of fibrosis and opacity was inhibited by IGF-1, and further with SAHA in particular, and with halofuginone. IGF-1 is thus a valid aid to antifibrotic treatment, with potential for effective and transparent corneal wound healing.