Serine Protease Inhibitor A3K Protects Rabbit Corneal Endothelium From Barrier Function Disruption Induced by TNF-α
Serine Protease Inhibitor A3K Protects Rabbit Corneal Endothelium From Barrier Function Disruption Induced by TNF-α
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DOI：
10.1167/iovs.12-10145
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发表时间：
2013-08-01
影响因子：
4.4
通讯作者：
Liu, Zuguo
中科院分区：
文献类型：
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作者：
Hu, Jiaoyue;Zhang, Zhenhao;Liu, Zuguo
目的.确定丝氨酸蛋白酶抑制剂A3 K（SA 3 K）是否减少TNF-α诱导的兔角膜内皮连接屏障完整性下降。在存在或不存在不同浓度的SA 3 K的情况下，将新西兰兔角膜在含有10%FBS的Dulbecco改良Eagle培养基（DMEM）中离体孵育24小时，所述培养基含有或不含有TNF-α。基于FITC-葡聚糖组织蓄积的测量来确定角膜内皮屏障功能渗透性。通过免疫荧光染色、蛋白质印迹分析和/或RT-PCR方法评价了闭合小带-1（ZO-1）、血管内皮（VE）-钙粘蛋白、丝状肌动蛋白（F-actin）和相关微管以及肌球蛋白轻链（MLC）的顶端连接复合物（AJC）完整性。与未处理的对照组相比，TNF-α（20 ng/mL）使角膜内皮FITC-葡聚糖渗透性增加1.8倍。SA 3 K（100-200 nM）剂量依赖性地抑制TNF-α诱导的渗透性增加。SA 3 K几乎完全逆转了TNF-α诱导的紧密连接ZO-1和关节下粘附连接VE-钙粘蛋白完整性的破坏。有趣的是，SA 3 K通过恢复F-actin双带结构逆转了TNF-α诱导的AJC与细胞骨架F-actin阵列连接的破坏。SA 3 K还减弱TNF-α诱导的微管解体。此外，SA 3 K阻断了由TNF-α引起的MLC磷酸化状态的增加。SA 3 K暴露通过维持AJC的完整性显著降低TNF-α诱导的兔角膜内皮屏障结构和功能的破坏。这些保护作用是由于抑制MLC激活。在体内，SA 3 K可能具有抵消TNF-α诱导的内皮屏障结构完整性和功能下降的治疗潜力。
PURPOSE. To determine if a serine protease inhibitor A3K (SA3K) reduces TNF-alpha-induced declines in rabbit corneal endothelial junctional barrier integrity.METHODS. New Zealand rabbit corneas were incubated ex vivo for 24 hours in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS with or without TNF-alpha, in the presence or absence of SA3K at different concentrations. Corneal endothelial barrier function permeability was determined based on measurements of FITC-dextran tissue accumulation. Apical junctional complex (AJC) integrity was evaluated of zonula occludens-1 (ZO-1), vascular endothelial (VE)-cadherin, and filamentous actin (F-actin) and associated microtubules, as well as myosin light chain (MLC) by immunofluorescent staining, Western blot analysis, and/or RT-PCR.RESULTS. TNF-alpha (20 ng/mL) increased corneal endothelial FITC-dextran permeability by 1.8-fold compared with the untreated control. SA3K (100-200 nM) dose dependently suppressed TNF-alpha-induced increases in permeability. SA3K nearly completely reversed TNF-alpha-induced disruptions of tight junctional ZO-1 and subjacent adherens junctions VE-cadherin integrity. Interestingly, SA3K reversed TNF-alpha-induced disruption of AJC linkage to the cytoskeletal F-actin array by restoring F-actin double-band structures. SA3K also attenuated TNF-alpha-induced microtubule disassembly. Furthermore, SA3K blocked increases in MLC phosphorylation status elicited by TNF-alpha.CONCLUSIONS. SA3K exposure markedly reduced TNF-alpha-induced disruption of barrier structure and function in the rabbit corneal endothelium by maintaining AJC integrity. These protective effects are due to suppression of MLC activation. SA3K may have, in vivo, a therapeutic potential to offset TNF-alpha-induced declines in endothelial barrier structural integrity and function.