Abstract

P 236

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 S.
University Eye Hospital of the Center of Ophthalmology at the Eberhard-Karls University of Tübingen

Objective: The wound healing activities of Tenon’s capsule fibroblasts following glaucoma filtration surgery result in the occlusion of the channel created for aqueous humor drainage and account for the failure in lowering intraocular pressure. Here, we analyzed the role of the 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 after 24, 48 and 96 hours. 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 alpha-smooth muscle actin(SMA) immunostaining and Alexa 488-phalloidin staining. Cell migration was studied on restrained collagen gels and in a wound-scratch assay followed by Ki-67 and fibronectin immunostainings. 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 immunoblotting to rule out interference with PKA.
Results: Incorporation of BrdU and upregulation of Ki-67 were reduced by 85% 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 3.5 fold and the contraction of collagen lattices 5-fold with a dose-dependent impairment in the assembly of the fibronectin network.
Conclusions: These findings attribute a role for Rho-kinase in the wound healing activities of human Tenon’s capsule fibroblasts and underline the potential of H-1152P as a safe and specific means to suppress these events.

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