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Abstract #18169 Published in IGR 9-2
The Rho-kinase inhibitor H-1152P suppresses the wound-healing activities of human Tenon's capsule fibroblasts in vitro
Tura A; Grisanti S; Petermeier K; Henke-Fahle SInvestigative Ophthalmology and Visual Science 2007; 48: 2152-2161
PURPOSE: To analyze the role of Rho-kinase signaling in the wound-healing activities of human Tenon's capsule fibroblasts by using H-1152P, a potent inhibitor of this kinase, in vitro. METHODS: The optimal concentration of H-1152P was determined by MTT test. Cell proliferation was measured by BrdU incorporation and Ki-67 immunostaining, whereas cell viability was investigated by ethidium homodimer-1 dye exclusion. The actin cytoskeleton organization was demonstrated by α-smooth muscle actin (SMA) immunostaining and Alexa 488-phalloidin staining. Cell migration was studied on restrained collagen gels and in a scratch-wound assay followed by Ki-67 and fibronectin immunostaining. The effect of H-1152P on contraction was analyzed in floating collagen gels populated with fibroblasts, which were subsequently processed for fibronectin immunostaining. The levels of adducin and the protein kinase A (PKA)-dependent phosphorylation of this protein were detected by immunoblot analysis, to rule out interference with PKA. RESULTS: Incorporation of BrdU and upregulation of Ki-67 were reduced by 80% to 90% in cells incubated with 10 μm of this inhibitor for 4 days (P < 0.01). H-1152P caused the disassembly of stress fibers in a dose-dependent manner without exerting toxic effects and without a considerable interference with the PKA-pathway. H-1152P also significantly suppressed cell migration 3- to 3.5-fold and the contraction of collagen lattices fivefold with a dose-dependent impairment in the assembly of the fibronectin network. CONCLUSIONS: These findings imply a role for Rho-kinase in the wound-healing activities of human Tenon's capsule fibroblasts and show the potential of H-1152P as a safe and specific means to suppress these events.
Dr. A. Tura, University Eye Hospital of the Center of Ophthalmology at the Eberhard-Karls University of Tübingen, Tübingen, Germany. Ayseguel.tura@med.uni-tuebingen.de
Classification:
11.14 Investigational drugs; pharmacological experiments (Part of: 11 Medical treatment)
12.8.10 Woundhealing antifibrosis (Part of: 12 Surgical treatment > 12.8 Filtering surgery)