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The Rho-Kinase Inhibitor H-1152P Suppresses the Wound-Healing Activities of Human Tenon’s Capsule Fibroblasts In Vitro

From the University Eye Hospital of the Center of Ophthalmology at the Eberhard-Karls University of Tübingen, Tübingen, Germany.

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 -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.