Ursula Schlötzer-Schrehardt1, Matthias Zenkel2, Maria Strunz2, Andreas Gießl2, Hannah Schondorf2, Heather da Silva3, Gregory A Schmidt4, Mark A Greiner5, Naoki Okumura6, Noriko Koizumi6, Shigeru Kinoshita7, Theofilos Tourtas2, Friedrich E Kruse2. 1. Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany. Electronic address: ursula.schloetzer-schrehardt@uk-erlangen.de. 2. Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany. 3. CorneaGen, Seattle, Washington, USA. 4. Iowa Lions Eye Bank, Coralville, Iowa, USA. 5. Iowa Lions Eye Bank, Coralville, Iowa, USA; Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA. 6. Department of Biomedical Engineering, Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan. 7. Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Abstract
PURPOSE: Rho-associated kinase (ROCK) inhibitors have 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 functional mechanisms by which ROCK inhibitors modulate corneal endothelial cell regeneration in FECD patients have, however, not been clarified. Here, we analyzed the effect of the ROCK inhibitor ripasudil on corneal endothelial cells of FECD patients and normal donors using ex vivo tissue and in vitro cellular models. DESIGN: Experimental study: laboratory investigation. METHODS: This institutional study used endothelial cell-Descemet membrane lamellae from FECD patients (n = 450) undergoing Descemet membrane endothelial keratoplasty (FECD ex vivo model), normal research-grade donor corneas (n = 30) after scraping off central endothelial cells (ex vivo wound healing model), normal donor corneas (n = 20) without endothelial injury, and immortalized cell lines (n = 3) generated from FECD patients (FECD in vitro model). Descemet membrane lamellae were dissected into halves and incubated for 24-72 hours in storage medium with or without a single dose of 30 μM ripasudil. The effects of ripasudil on expression of genes and proteins related to endothelial cell proliferation, migration, functionality, and endothelial-to-mesenchymal transition were analyzed and complemented by functional assays on FECD cell lines. RESULTS: A single dose of ripasudil induced significant upregulation of genes and proteins related to cell cycle progression, cell-matrix adhesion and migration, as well as endothelial barrier and pump function up to 72 hours, whereas classical markers of endothelial-to-mesenchymal transition were downregulated in both FECD and normal specimens compared to unstimulated controls ex vivo. In addition to stimulation of proliferation and migration, ripasudil-induced changes in expression of functional signature genes could be also verified in FECD cell lines in vitro. CONCLUSIONS: These data support the concept that inhibition of ROCK signaling represents a potent tool in regenerative therapies in FECD patients through reactivation of cell proliferation and migration as well as restoration of endothelial pump and barrier function without inducing adverse phenotypic changes.
PURPOSE: Rho-associated kinase (ROCK) inhibitors have 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 functional mechanisms by which ROCK inhibitors modulate corneal endothelial cell regeneration in FECDpatients have, however, not been clarified. Here, we analyzed the effect of the ROCK inhibitor ripasudil on corneal endothelial cells of FECDpatients and normal donors using ex vivo tissue and in vitro cellular models. DESIGN: Experimental study: laboratory investigation. METHODS: This institutional study used endothelial cell-Descemet membrane lamellae from FECDpatients (n = 450) undergoing Descemet membrane endothelial keratoplasty (FECD ex vivo model), normal research-grade donor corneas (n = 30) after scraping off central endothelial cells (ex vivo wound healing model), normal donor corneas (n = 20) without endothelial injury, and immortalized cell lines (n = 3) generated from FECDpatients (FECD in vitro model). Descemet membrane lamellae were dissected into halves and incubated for 24-72 hours in storage medium with or without a single dose of 30 μM ripasudil. The effects of ripasudil on expression of genes and proteins related to endothelial cell proliferation, migration, functionality, and endothelial-to-mesenchymal transition were analyzed and complemented by functional assays on FECD cell lines. RESULTS: A single dose of ripasudil induced significant upregulation of genes and proteins related to cell cycle progression, cell-matrix adhesion and migration, as well as endothelial barrier and pump function up to 72 hours, whereas classical markers of endothelial-to-mesenchymal transition were downregulated in both FECD and normal specimens compared to unstimulated controls ex vivo. In addition to stimulation of proliferation and migration, ripasudil-induced changes in expression of functional signature genes could be also verified in FECD cell lines in vitro. CONCLUSIONS: These data support the concept that inhibition of ROCK signaling represents a potent tool in regenerative therapies in FECDpatients through reactivation of cell proliferation and migration as well as restoration of endothelial pump and barrier function without inducing adverse phenotypic changes.