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Vishal Shinde, James W Foster, Yassine Jamil Daoud, Albert Jun, Shukti Chakravarti; Role of FOXO1 in the progression of Keratoconus.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2923.
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© ARVO (1962-2015); The Authors (2016-present)
Keratoconus (KC) is a progressive, vision debilitating, eye disease where the cornea, with reduced connective tissue extracellular matrix (ECM) is weaker and thinner. RNA sequencing of KC corneas by our laboratory shows decreased expression of the gene encoding Forkhead box protein O1 (FOXO1). It is a transcription factor that regulates apoptosis, cell cycle progression, oxidative stress resistance and ECM genes. The phosphorylation (p) of FOXO1 results in export of FOXO1 from the nucleus to the cytoplasm, thereby inhibiting its transcriptional activity. GWAS studies have shown association of FOXO1 with central corneal thickness as well as keratoconus. Here we investigated a) levels of inactive pFOXO1 in donor (DN) and keratoconus (KC) corneal cells in culture and b) tested the effects of FOXO1 inhibition on collagen production to elucidate FOXO1 involvement in KC pathogenesis.
Stromal cells were isolated from 6 DN and 6 KC corneas and cultured as fibroblasts or keratocytes in serum-free medium. FOXO1, pFOXO1 and ECM proteins were determined in fibroblasts and keratocytes by western blots (WB). Nuclear and cytoplasmic FOXO1 was assessed in serum-starved DN and KC cells by confocal microscopy. Physiological concentration of FOXO1 inhibitor AS1842856 was determined by cell viability assay. DN and KC cells were treated with the AS1842856 and levels of ECM proteins, collagen 1, fibronectin, lumican and decorin were determined by WB.
FOXO1 protein expression was unchanged in KC stromal fibroblasts/keratocytes as compared to DN fibroblasts/keratocytes. However, pFOXO1 was significantly higher in both KC fibroblasts and keratocytes as compared to DN. Levels of pFOXO1 protein were also significantly higher in the KC corneas as compared to DN. Confocal microscopy suggested increased cytoplasmic (inactive) FOXO1 in KC cells. Treatment of DN fibroblasts with AS1842856 led to reductions in Collagen 1 and fibronectin 6-and 7-fold respectively, indicating their regulation by FOXO1 signaling. Levels of Lumican and Decorin were unchanged after similar treatment with the inhibitor.
Altered FOXO1 regulation in KC corneas could be potentially responsible for the observed decreases in collagen 1 and fibronectin in keratoconus.
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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