Dull Randal O, Dinavahi Ramani, Schwartz Lawrence, Humphries Donald E, Berry David, Sasisekharan Ram, Garcia Joe G N
Anesthesiology and Critical Care Medicine, Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland 21287, USA.
Am J Physiol Lung Cell Mol Physiol. 2003 Nov;285(5):L986-95. doi: 10.1152/ajplung.00022.2003. Epub 2003 May 16.
The endothelial glycocalyx is believed to play a major role in microvascular permeability. We tested the hypothesis that specific components of the glycocalyx, via cytoskeletal-mediated signaling, actively participate in barrier regulation. With the use of polymers of arginine and lysine as a model of neutrophil-derived inflammatory cationic proteins, we determined size- and dose-dependent responses of cultured bovine lung microvascular endothelial cell permeability as assessed by transendothelial electrical resistance (TER). Polymers of arginine and lysine >11 kDa produced maximal barrier dysfunction as demonstrated by a 70% decrease in TER. Monomers of l-arginine and l-lysine did not alter barrier function, suggesting a cross-linking requirement of cell surface "receptors". To test the hypothesis that glycosaminoglycans (GAGs) are candidate receptors for this response, we used highly selective enzymes to remove specific GAGs before polyarginine (PA) treatment and examined the effect on TER. Heparinase III attenuated PA-induced barrier dysfunction by 50%, whereas heparinase I had no effect. To link changes in barrier function with structural alterations, we examined actin organization and syndecan localization after PA. PA induced actin stress fiber formation and clustering of syndecan-1 and syndecan-4, which were significantly attenuated by heparinase III. PA-induced cytoskeletal rearrangement and barrier function did not involve myosin light chain kinase (MLCK) or p38 MAPK, as ML-7, a specific MLCK inhibitor, or SB-20358, a p38 MAPK inhibitor, did not alter PA-induced barrier dysfunction. In summary, lung endothelial cell heparan sulfate proteoglycans are key participants in inflammatory cationic peptide-induced signaling that links cytoskeletal reorganization with subsequent barrier dysfunction.
内皮糖萼被认为在微血管通透性中起主要作用。我们检验了这样一个假说，即糖萼的特定成分通过细胞骨架介导的信号传导，积极参与屏障调节。使用精氨酸和赖氨酸聚合物作为中性粒细胞衍生的炎性阳离子蛋白的模型，我们通过跨内皮电阻（TER）评估了培养的牛肺微血管内皮细胞通透性的大小和剂量依赖性反应。精氨酸和赖氨酸聚合物>11 kDa产生了最大的屏障功能障碍，TER降低了70%即证明了这一点。L-精氨酸和L-赖氨酸单体未改变屏障功能，这表明细胞表面“受体”需要交联。为了检验糖胺聚糖（GAGs）是这种反应的候选受体这一假说，我们在聚精氨酸（PA）处理前使用高度选择性的酶去除特定的GAGs，并检查对TER的影响。肝素酶III使PA诱导的屏障功能障碍减轻了50%，而肝素酶I没有效果。为了将屏障功能的变化与结构改变联系起来,我们在PA处理后检查了肌动蛋白组织和syndecan定位。PA诱导肌动蛋白应激纤维形成以及syndecan-1和syndecan-4聚集，而肝素酶III可使其显著减弱。PA诱导的细胞骨架重排和屏障功能不涉及肌球蛋白轻链激酶（MLCK）或p38丝裂原活化蛋白激酶（MAPK），因为特异性MLCK抑制剂ML-7或p38 MAPK抑制剂SB-20358未改变PA诱导的屏障功能障碍。总之，肺内皮细胞硫酸乙酰肝素蛋白聚糖是炎性阳离子肽诱导信号传导的关键参与者，该信号传导将细胞骨架重组与随后的屏障功能障碍联系起来。
