Lipid Signaling, Lipidomics, and Vasculotoxicity Laboratory, Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Dorothy M. Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, Ohio, USA.
Toxicol Mech Methods. 2012 Jun;22(5):383-96. doi: 10.3109/15376516.2012.673089.
Lung vascular alterations and pulmonary hypertension associated with oxidative stress have been reported to be involved in idiopathic lung fibrosis (ILF). Therefore, here, we hypothesize that the widely used lung fibrosis inducer, bleomycin, would cause cytoskeletal rearrangement through thiol-redox alterations in the cultured lung vascular endothelial cell (EC) monolayers. We exposed the monolayers of primary bovine pulmonary artery ECs to bleomycin (10 µg) and studied the cytotoxicity, cytoskeletal rearrangements, and the macromolecule (fluorescein isothiocyanate-dextran, 70,000 mol. wt.) paracellular transport in the absence and presence of two thiol-redox protectants, the classic water-soluble N-acetyl-L-cysteine (NAC) and the novel hydrophobic N,N'-bis-2-mercaptoethyl isophthalamide (NBMI). Our results revealed that bleomycin induced cytotoxicity (lactate dehydrogenase leak), morphological alterations (rounding of cells and filipodia formation), and cytoskeletal rearrangement (actin stress fiber formation and alterations of tight junction proteins, ZO-1 and occludin) in a dose-dependent fashion. Furthermore, our study demonstrated the formation of reactive oxygen species, loss of thiols (glutathione, GSH), EC barrier dysfunction (decrease of transendothelial electrical resistance), and enhanced paracellular transport (leak) of macromolecules. The observed bleomycin-induced EC alterations were attenuated by both NAC and NBMI, revealing that the novel hydrophobic thiol-protectant, NBMI, was more effective at µM concentrations as compared to the water-soluble NAC that was effective at mM concentrations in offering protection against the bleomycin-induced EC alterations. Overall, the results of the current study suggested the central role of thiol-redox in vascular EC dysfunction associated with ILF.
肺血管改变和与氧化应激相关的肺动脉高压已被报道与特发性肺纤维化（IPF）有关。因此，在这里，我们假设广泛使用的肺纤维化诱导剂博来霉素会通过巯基氧化还原改变引起培养的肺血管内皮细胞（EC）单层中的细胞骨架重排。我们将原代牛肺动脉 EC 的单层暴露于博来霉素（10μg）中，并在不存在和存在两种巯基氧化还原保护剂的情况下研究细胞毒性、细胞骨架重排以及大分子（异硫氰酸荧光素-葡聚糖，70000mol.wt.）的旁细胞转运。我们的结果表明，博来霉素以剂量依赖性方式诱导细胞毒性（乳酸脱氢酶渗漏）、形态改变（细胞圆化和伪足形成）和细胞骨架重排（肌动蛋白应力纤维形成和紧密连接蛋白 ZO-1 和闭合蛋白的改变）。此外，我们的研究表明形成了活性氧、巯基（谷胱甘肽，GSH）损失、EC 屏障功能障碍（跨内皮电阻降低）和增强的大分子旁细胞转运（渗漏）。博来霉素诱导的 EC 改变被 NAC 和 NBMI 均减弱，表明新型疏水性巯基保护剂 NBMI 在µM 浓度下比在 mM 浓度下有效的水溶性 NAC 更有效，可提供针对博来霉素诱导的 EC 改变的保护。总体而言，本研究的结果表明巯基氧化还原在与特发性肺纤维化相关的血管 EC 功能障碍中起核心作用。
