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Friedrich E Kruse, Theofilos Tourtas, Matthias Zenkel, Shigeru Kinoshita, Ursula Schlotzer-Schrehardt; Potential functional restoration of corneal endothelial cells in Fuchs corneal dystrophy by ROCK inhibitor (Ripasudil). Invest. Ophthalmol. Vis. Sci. 2019;60(9):2172. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Rho-associated kinase (ROCK) inhibitor (Ripasudil) has been successfully used as a rescue strategy in eyes that failed to clear after descemetorhexis without endothelial graft for treatment of Fuchs endothelial corneal dystrophy (FECD). The molecular mechanisms underlying the positive effects of Ripasudil on corneal endothelial cells are not yet fully understood. Here, we analyzed the effect of Ripasudil on the expression of genes related to proliferation, migration, adhesion, endothelial-mesenchymal transformation (EMT) and endothelial function in corneal endothelial cells ex vivo.
Endothelial cell-Descemet’s membrane (EDM) complexes were prepared by manual stripping from normal donor corneas unsuitable for transplantation and from FECD patients during DMEK surgery. Corneal endothelial cells were either left intact or centrally scraped off with a forceps (wound healing model). EDM scrolls were dissected into two halves and incubated up to 48 hours in storage medium with or without 30 µM Ripasudil. Gene expression was analyzed by RT2 Profiler PCR Arrays and specific qRT-PCR primer assays, and protein expression was analyzed by immunohistochemistry.
Ripasudil caused significant upregulation of the levels of genes related to cell cycle progression (e.g. CCND1/2), cell-matrix adhesion (e.g. ITGA1/4/8/M, LAMA4/B1) and endothelial cell function (e.g. ZO-1, CX43, ATPA1, AQP1, SLC4A11, SLC16A7), whereas markers of EMT (e.g. ACTA2, ACTB, ZEB1, VIM, FAK1) were significantly downregulated in both normal wounded and FECD specimens compared to unstimulated controls. Findings were confirmed at the protein level by confocal microscopy. By use of the Signal Transduction PathwayFinder PCR Array, discrete changes were noted in expression levels of a number of components of the Wnt- and TGF-ß/BMP signaling pathways upon treatment with Ripasudil.
Our results support the concept that inhibition of ROCK by Ripasudil may be a useful tool in regenerative therapies for corneal endothelial cell disease through functional restoration of endothelial cells in FECD patients without induction of adverse phenotypic changes.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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