The Rho-kinase inhibitor H-1152P suppresses the wound-healing activities of human Tenon's capsule fibroblasts in vitro.
OBJECTIVE
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 alpha-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 microM 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.
Tura
Aysegül
A
University Eye Hospital of the Center of Ophthalmology at the Eberhard-Karls University of Tübingen, Tübingen, Germany. ayseguel.tura@med.uni-tuebingen.de
Grisanti
Salvatore
S
Petermeier
Katrin
K
Henke-Fahle
Sigrid
S
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D003239:Connective Tissue Cells; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors* / Q000502:physiology; D019394:Ki-67 Antigen / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology; D014945:Wound Healing / Q000187:drug effects*; D054460:rho-Associated Kinases; D000099291:Adducins
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000199:Actins; D002148:Calmodulin-Binding Proteins; D003094:Collagen; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D005353:Fibronectins; D047908:Intracellular Signaling Peptides and Proteins; D019394:Ki-67 Antigen; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D000099291:Adducins; C459092:2-methyl-1-((4-methyl-5-isoquinolinyl)sulfonyl)homopiperazine