Involvement of rho-kinase pathway in contractile activity of rabbit RPE cells in vivo and in vitro.
OBJECTIVE
Increased alpha-smooth muscle actin (alpha-SMA) expression in epiretinal membranes causes tractional retinal detachment (TRD) in proliferative vitreoretinopathy (PVR). The Rho-A/Rho-associated kinase signaling pathway is a principal mediator of contractile force generation in nonmuscle cells. In the current study, the relation between the Rho-kinase pathway and alpha-SMA expression and type I collagen gel contractile activity in retinal pigment epithelial (RPE) cells was investigated, using Y27632, a specific inhibitor of p160ROCK, and the involvement of the Rho-kinase pathway was evaluated in a rabbit PVR model with cultured RPE cells and platelet-rich plasma (PRP).


METHODS
RPE cells were obtained from rabbits and cultured. The number of passages and the effect of Y27632 on alpha-SMA expression were studied by immunohistochemistry and immunoblot analysis. An in vitro type I collagen gel contraction assay and MTT assay evaluated the effect of Y27632 on RPE cell contractile force and proliferation. Cultured sixth-passage rabbit RPE cells were coinjected with PRP intravitreally, followed by 50 micro M of Y27632, injected weekly. The presence of TRD was assessed until 28 days to evaluate the effect of Y27632 in vivo.


RESULTS
Expression of alpha-SMA was increased according to the passages. Y27632 suppressed alpha-SMA expression in cultured RPE cells and impaired contractile force. Y27632 did not affect the proliferative potential. Y27632 significantly (P < 0.01) reduced TRD development.


CONCLUSIONS
Y27632 decreased alpha-SMA expression and the contractile force generated by RPE cells and attenuated PVR, indicating the involvement of the Rho-kinase pathway in cell-dependent collagen contraction in vitro and in vivo. The drug may affect the biological event by inhibiting alpha-SMA expression, and Y27632 could be useful for preventing PVR.
Zheng
Yuping
Y
Department of Ophthalmology, Osaka University Medical School, Osaka, Japan.
Bando
Hajime
H
Ikuno
Yasushi
Y
Oshima
Yusuke
Y
Sawa
Miki
M
Ohji
Masahito
M
Tano
Yasuo
Y
D000199:Actins / Q000378:metabolism*; D000577:Amides / Q000494:pharmacology*; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D015151:Immunoblotting; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000201:enzymology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology*; D011817:Rabbits; D018630:Vitreoretinopathy, Proliferative / Q000201:enzymology / Q000517:prevention & control; D054460:rho-Associated Kinases
D000199:Actins; D000577:Amides; D024042:Collagen Type I; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases