Zheng Yuping, Bando Hajime, Ikuno Yasushi, Oshima Yusuke, Sawa Miki, Ohji Masahito, Tano Yasuo
Department of Ophthalmology, Osaka University Medical School, Osaka, Japan.
Invest Ophthalmol Vis Sci. 2004 Feb;45(2):668-74. doi: 10.1167/iovs.02-0808.
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).
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.
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.
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.
视网膜前膜中α-平滑肌肌动蛋白（α-SMA）表达增加会导致增殖性玻璃体视网膜病变（PVR）中的牵引性视网膜脱离（TRD）。Rho-A/Rho相关激酶信号通路是非肌肉细胞中产生收缩力的主要介质。在本研究中，使用p160ROCK的特异性抑制剂Y27632，研究了Rho激酶通路与视网膜色素上皮（RPE）细胞中α-SMA表达及I型胶原凝胶收缩活性之间的关系，并在含有培养的RPE细胞和富血小板血浆（PRP）的兔PVR模型中评估了Rho激酶通路的参与情况。
从兔获取RPE细胞并进行培养。通过免疫组织化学和免疫印迹分析研究传代次数及Y27632对α-SMA表达的影响。体外I型胶原凝胶收缩试验和MTT试验评估Y27632对RPE细胞收缩力和增殖的影响。将培养的第六代兔RPE细胞与PRP一起玻璃体内注射，随后每周注射50 μM的Y27632。评估直至28天TRD的存在情况，以评价Y27632在体内的作用。
α-SMA的表达随传代次数增加。Y27632抑制培养的RPE细胞中α-SMA的表达并削弱收缩力。Y27632不影响增殖潜能。Y27632显著（P < 0.01）减少TRD的发生。
Y27632降低α-SMA表达和RPE细胞产生的收缩力，并减轻PVR，表明Rho激酶通路在体外和体内细胞依赖性胶原收缩中起作用。该药物可能通过抑制α-SMA表达影响生物学事件，且Y27632可能对预防PVR有用。
