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Johannes Menzel-Severing, Matthias Zenkel, Naoki Okumura, Noriko Koizumi, Shigeru Kinoshita, Ursula Schlotzer-Schrehardt, Friedrich E Kruse; Transcriptional changes in corneal endothelial cell sheets following ROCK inhibition by Ripasudil/K-115 in vitro.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1470.
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© ARVO (1962-2015); The Authors (2016-present)
Inhibition of Rho-kinase (ROCK) was suggested to influence migration and proliferation of corneal endothelial cells in vitro and in vivo. The underlying molecular processes are not yet fully understood. We present the results of a pilot study aimed to determine the effect of ROCK-inhibitor Ripasudil/K-115 on the transcription of cytoskeleton-related and signaling-pathway-related genes in corneal endothelial cells in vitro.
Donor corneas with appropriate research consent were obtained from SightLife (Seattle, WA). A corneal endothelial cell sheet was isolated from three donor corneas by manually stripping Descemet’s membrane (DM). DM scrolls were dissected into two halves and incubated for 24 hours in CorneaMax storage medium (Eurobio, France) at 37°C with or without 30 µM Ripasudil (Selleck Chemicals, Houston, TX), respectively. After isolation of mRNA (RNeasy Micro Kit, Qiagen, The Netherlands) and mRNA amplification (MessageAmp II aRNA Amplification Kit, Thermo Fisher Scientific, Waltham, MA), expression analysis was performed using RT2 Profiler PCR Arrays (Qiagen) for genes related to regulation of cytoskeleton and signal transduction pathways.
Interestingly, following treatment with Ripasudil, no meaningful changes were detected in the levels of expression of 84 genes assayed by the Cytoskeleton Regulators PCR Array. By use of the Signal Transduction PathwayFinder PCR Array, discrete changes were noted to the levels of expression of a number of components of the NFκB-, Wnt- and Hedgehog-pathways. Notably, consistent upregulation was observed for Wnt5a and for NFκB target-gene CCL5/RANTES, while TGF beta target-gene SERPINE1 was downregulated.
Inhibition of TGF beta was reported to suppress fibroblastic changes to human corneal endothelial cell expansion in vitro (Okumura et al. 2012). Induction of Wnt5a through NFκB was suggested to enhance human corneal endothelial cell migration by inactivation of RhoA/ROCK signaling without inducing endothelial-to-mesenchymal transition (Lee and Heur 2014). Our preliminary results further support the notion that chemical inhibition of ROCK may be a useful tool in regenerative therapies for corneal endothelial cell disease.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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