School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
J Ocul Pharmacol Ther. 2010 Feb;26(1):1-10. doi: 10.1089/jop.2009.0025.
Increased actomyosin contraction of the dense band of actin cytoskeleton at the apical junctional complex (perijunctional actomyosin ring, PAMR) breaks down the barrier integrity of corneal endothelium. This study has investigated the efficacy of statins, which inhibit activation of RhoA, in opposing the thrombin-induced loss of barrier integrity of monolayers of cultured bovine corneal endothelium.
Myosin light chain (MLC) phosphorylation, a biochemical measure of actomyosin contraction, was assayed by urea-glycerol gel electrophoresis, followed by western blot analysis. The locus of MLC phosphorylation and changes in the organization of the PAMR were visualized by immunostaining. Phosphorylation of MYPT1, a regulatory subunit of myosin light-chain phosphatase (MLCP), was assessed by Western blot analysis to determine down-regulation of RhoA. The barrier integrity was assessed in terms of trans-endothelial electrical resistance (TER), and further confirmed by determining permeability to FITC dextran (10 kDa) and distribution of ZO-1, a marker of tight junctional assembly.
Lovastatin, a prototype of lipophilic statins, induced MLC dephosphorylation under basal conditions. It opposed increase in phosphorylation of MLC and MYPT1 in response to thrombin and nocodazole, agents known to activate RhoA in the endothelium. Pretreatment with the statin opposed the thrombin- and nocodazole-induced disruption of the PAMR and the thrombin-induced decline in TER. Lovastatin also opposed the thrombin- and nocodazole-induced increase in permeability to FITC dextran and redistribution of ZO-1. However, upon supplementation with GGPP (geranylgeranyl pyrophosphate), lovastatin failed to oppose the effects of thrombin and nocodazole on the PAMR, ppMLC, and ZO-1 distribution.
Lovastatin attenuates RhoA activation in the corneal endothelium presumably by reducing its isoprenylation. This underlies the suppression of the thrombin-induced loss in barrier integrity of the corneal endothelium.
在顶端连接复合体（连接周肌动球蛋白环，PAMR）处的肌动球蛋白致密带的肌球蛋白收缩增加，破坏了角膜内皮的屏障完整性。本研究调查了他汀类药物（抑制 RhoA 激活）抑制凝血酶诱导的培养牛角膜内皮单层屏障完整性丧失的功效。
通过尿素甘油凝胶电泳和 Western blot 分析，测定肌球蛋白轻链（MLC）磷酸化，这是肌动球蛋白收缩的生化测量。通过免疫染色观察 MLC 磷酸化的位置和 PAMR 组织的变化。通过 Western blot 分析评估肌球蛋白轻链磷酸酶（MLCP）的调节亚基 MYPT1 的磷酸化，以确定 RhoA 的下调。通过跨内皮电阻（TER）评估屏障完整性，并进一步通过测定 FITC 葡聚糖（10 kDa）的通透性和紧密连接组装标志物 ZO-1 的分布来确认。
亲脂性他汀类药物洛伐他汀在基础条件下诱导 MLC 去磷酸化。它反对凝血酶和诺考达唑引起的 MLC 和 MYPT1 磷酸化增加，这两种药物已知在内皮细胞中激活 RhoA。用他汀类药物预处理可防止 PAMR 的破坏，防止凝血酶诱导的 TER 下降。洛伐他汀还反对凝血酶和诺考达唑引起的 FITC 葡聚糖通透性增加和 ZO-1 分布重新分布。然而，在用 GGPP（香叶基香叶基焦磷酸）补充时，洛伐他汀未能阻止凝血酶和诺考达唑对 PAMR、ppMLC 和 ZO-1 分布的影响。
洛伐他汀通过减少其异戊烯化来减轻角膜内皮中的 RhoA 激活。这是抑制凝血酶诱导的角膜内皮屏障完整性丧失的基础。
