Pulmonary and Critical Care Medicine, Rush University Medical Center, 1750 W, Harrison Street, 297 Jelke, Chicago, IL 60612, USA.
Respir Res. 2011 Apr 12;12(1):46. doi: 10.1186/1465-9921-12-46.
The response of lung microvascular endothelial cells (ECs) to lipopolysaccharide (LPS) is central to the pathogenesis of lung injury. It is dual in nature, with one facet that is pro-inflammatory and another that is cyto-protective. In previous work, overexpression of the anti-apoptotic Bcl-XL rescued ECs from apoptosis triggered by siRNA knockdown of intersectin-1s (ITSN-1s), a pro-survival protein crucial for ECs function. Here we further characterized the cyto-protective EC response to LPS and pro-inflammatory dysfunction.
Electron microscopy (EM) analyses of LPS-exposed ECs revealed an activated/dysfunctional phenotype, while a biotin assay for caveolae internalization followed by biochemical quantification indicated that LPS causes a 40% inhibition in biotin uptake compared to controls. Quantitative PCR and Western blotting were used to evaluate the mRNA and protein expression, respectively, for several regulatory proteins of intrinsic apoptosis, including ITSN-1s. The decrease in ITSN-1s mRNA and protein expression were countered by Bcl-XL and survivin upregulation, as well as Bim downregulation, events thought to protect ECs from impending apoptosis. Absence of apoptosis was confirmed by TUNEL and lack of cytochrome c (cyt c) efflux from mitochondria. Moreover, LPS exposure caused induction and activation of inducible nitric oxide synthase (iNOS) and a mitochondrial variant (mtNOS), as well as augmented mitochondrial NO production as measured by an oxidation oxyhemoglobin (oxyHb) assay applied on mitochondrial-enriched fractions prepared from LPS-exposed ECs. Interestingly, expression of myc-ITSN-1s rescued caveolae endocytosis and reversed induction of iNOS expression.
Our results suggest that ITSN-1s deficiency is relevant for the pro-inflammatory ECs dysfunction induced by LPS.
肺微血管内皮细胞（ECs）对脂多糖（LPS）的反应是肺损伤发病机制的核心。它具有双重性质，一方面是促炎的，另一方面是细胞保护的。在以前的工作中，抗凋亡蛋白 Bcl-XL 的过表达挽救了由 intersectin-1s（ITSN-1s）siRNA 敲低引发的 EC 细胞凋亡，ITSN-1s 是一种对 EC 功能至关重要的生存蛋白。在这里，我们进一步研究了 LPS 诱导的 EC 细胞保护反应和促炎功能障碍。
电镜（EM）分析显示 LPS 暴露的 EC 具有激活/功能障碍表型，而生物素内吞测定法用于检测 caveolae 内化，随后进行生物化学定量分析表明，与对照组相比，LPS 导致生物素摄取减少 40%。定量 PCR 和 Western blotting 分别用于评估内在凋亡的几种调节蛋白的 mRNA 和蛋白表达，包括 ITSN-1s。Bcl-XL 和 survivin 的上调以及 Bim 的下调逆转了 ITSN-1s mRNA 和蛋白表达的减少，这些事件被认为可以保护 EC 免受即将发生的凋亡。TUNEL 证实没有发生凋亡，线粒体没有细胞色素 c（cyt c）外排。此外，LPS 暴露引起诱导型一氧化氮合酶（iNOS）和线粒体变体（mtNOS）的激活和诱导，以及通过应用于从 LPS 暴露的 EC 制备的富含线粒体的级分的氧化氧合血红蛋白（oxyHb）测定法测量的线粒体 NO 产生增加。有趣的是，myc-ITSN-1s 的表达挽救了 caveolae 内吞作用并逆转了 iNOS 表达的诱导。
我们的结果表明，ITSN-1s 缺乏与 LPS 诱导的促炎 EC 功能障碍有关。
