Wheatley E M, McKeown-Longo P J, Vincent P A, Saba T M
Department of Physiology and Cell Biology, Albany Medical College, New York 12208.
Am J Physiol. 1993 Aug;265(2 Pt 1):L148-57. doi: 10.1152/ajplung.1993.265.2.L148.
Plasma fibronectin, a dimeric adhesive protein in blood, incorporates into the subendothelial and interstitial matrix in the lung especially during vascular injury. Fibronectin in the matrix is believed to influence cell-cell interaction and endothelial cell adhesion to the collagen-rich extracellular matrix. We previously observed that addition of purified soluble human plasma fibronectin (hFn) to cultured pulmonary endothelial monolayers attenuates the increase in protein permeability of such monolayers exposed to tumor necrosis factor-alpha (TNF-alpha). In the current study, we determined the specificity of this permeability response to fibronectin by comparing hFn to two other purified adhesive proteins in human plasma, i.e., vitronectin (Vn) and fibrinogen (Fg). We also determined whether matrix incorporation was essential for this hFn-mediated protective response by comparing normal intact hFn to either hFn alkylated with N-ethylmaleimide (NEM) or to purified 160/180-kDa hFn fragments, since these alternate forms of fibronectin are believed to exhibit limited ability to incorporate into matrix. Calf pulmonary artery endothelial (CPAE) monolayers (3-4 days postseeding) were exposed to human recombinant TNF-alpha for 18 h at a medium concentration of 200 U/ml followed by assessment of protein permeability using transendothelial 125I-labeled albumin clearance. Dimeric hFn (600 micrograms/ml) significantly (P < 0.05) reduced the TNF-induced increase in endothelial monolayer permeability. Vn or Fg, added at equal molar concentrations to the hFn, were unable to attenuate endothelial permeability. Immunofluorescent analysis utilizing antibodies specific to either hFn, human Vn, or human Fg revealed incorporation of the exogenous hFn into the extracellular matrix, but no matrix incorporation of Vn or Fg. Both NEM-treated dimeric hFn as well as purified 160/180-kDa fragments of hFn, which cannot incorporate into the matrix, were also unable to prevent the TNF-induced increase in protein permeability. Thus the ability for soluble hFn to reduce the TNF-induced increase in lung endothelial monolayer permeability was specific and dependent on its incorporation into the extracellular matrix.
血浆纤连蛋白是血液中的一种二聚体黏附蛋白,尤其在血管损伤时会整合到肺的内皮下和间质基质中。基质中的纤连蛋白被认为会影响细胞间相互作用以及内皮细胞与富含胶原蛋白的细胞外基质的黏附。我们之前观察到,向培养的肺内皮单层细胞中添加纯化的可溶性人血浆纤连蛋白(hFn)可减弱此类单层细胞在暴露于肿瘤坏死因子-α(TNF-α)时蛋白质通透性的增加。在当前研究中,我们通过将hFn与人类血浆中的另外两种纯化黏附蛋白即玻连蛋白(Vn)和纤维蛋白原(Fg)进行比较,确定了这种对纤连蛋白通透性反应的特异性。我们还通过将正常完整的hFn与用N-乙基马来酰亚胺(NEM)烷基化的hFn或纯化的160/180-kDa hFn片段进行比较,确定了基质整合对于这种hFn介导的保护反应是否必不可少,因为据信这些纤连蛋白的替代形式整合到基质中的能力有限。将小牛肺动脉内皮(CPAE)单层细胞(接种后3 - 4天)以200 U/ml的中等浓度暴露于人类重组TNF-α 18小时,然后使用跨内皮125I标记白蛋白清除率评估蛋白质通透性。二聚体hFn(600微克/毫升)显著(P < 0.05)降低了TNF诱导的内皮单层通透性增加。以与hFn等摩尔浓度添加的Vn或Fg无法减弱内皮通透性。利用针对hFn、人类Vn或人类Fg的特异性抗体进行的免疫荧光分析显示,外源性hFn整合到了细胞外基质中,但Vn或Fg未整合到基质中。NEM处理的二聚体hFn以及无法整合到基质中的纯化160/180-kDa hFn片段也无法阻止TNF诱导的蛋白质通透性增加。因此,可溶性hFn降低TNF诱导的肺内皮单层通透性增加的能力是特异性的,并且依赖于其整合到细胞外基质中。