Tura Aysegül, Grisanti Salvatore, Petermeier Katrin, Henke-Fahle Sigrid
University Eye Hospital of the Center of Ophthalmology at the Eberhard-Karls University of Tübingen, Tübingen, Germany.
Invest Ophthalmol Vis Sci. 2007 May;48(5):2152-61. doi: 10.1167/iovs.06-1271.
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.
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.
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.
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.
通过在体外使用强效Rho激酶抑制剂H - 1152P，分析Rho激酶信号传导在人眼Tenon囊成纤维细胞伤口愈合活性中的作用。
通过MTT试验确定H - 1152P的最佳浓度。通过BrdU掺入和Ki - 67免疫染色测量细胞增殖，而通过乙锭同二聚体-1染料排除法研究细胞活力。通过α-平滑肌肌动蛋白（SMA）免疫染色和Alexa 488 - 鬼笔环肽染色展示肌动蛋白细胞骨架组织。在受限胶原凝胶上并通过划痕伤口试验研究细胞迁移，随后进行Ki - 67和纤连蛋白免疫染色。在接种有成纤维细胞的漂浮胶原凝胶中分析H - 1152P对收缩的影响，随后对其进行纤连蛋白免疫染色处理。通过免疫印迹分析检测内收蛋白水平以及该蛋白的蛋白激酶A（PKA）依赖性磷酸化，以排除对PKA的干扰。
与10μM该抑制剂孵育4天的细胞中，BrdU掺入和Ki - 67上调降低了80%至90%（P < 0.01）。H - 1152P以剂量依赖性方式导致应力纤维解体，且不产生毒性作用，对PKA途径也无明显干扰。H - 1152P还显著抑制细胞迁移3至3.5倍，并使胶原晶格收缩五倍，同时对纤连蛋白网络组装产生剂量依赖性损害。
这些发现表明Rho激酶在人眼Tenon囊成纤维细胞伤口愈合活性中发挥作用，并显示出H - 1152P作为抑制这些事件的安全且特异性手段的潜力。
