摘要
研究背景
肺部炎症反应过程中,构成微循环交换血管最内层的肺微血管内皮细胞(pulmonary microvascular endothelial cells, PMVECs)是炎症因子首先攻击的效应细胞,其病理生理改变之一是细胞通透性增加,严重时可导致肺水肿,这是急性肺损伤/急性呼吸窘迫综合征(acute lung injury/acute respiratory distress syndrome, ALI/ARDS)的主要发病环节之一。多种信号转导通路参与PMVECs通透性调控,其中蛋白激酶C(protein kinase C, PKC)信号转导通路被广泛研究。已知活化的PKC磷酸化底物蛋白、促进PMVECs骨架重构以及细胞间黏附连接物解聚,从而增加PMVECs通透性,但细胞内PKC下游信号转导错综复杂,所以PKC底物及其在PMVECs通透性调控中的作用机制值得深入研究。此外,在人体研究PMVECs通透性变化十分困难,目前关于PMVECs通透性的研究结果主要来自体外细胞实验,所以PMVECs单层的融合状态决定实验结果精确性。
src抑制的蛋白激酶C底物(src-suppressed C kinase substrate, SSeCKS)是一新发现的PKC底物,可被活化的PKC磷酸化。多种细胞株研究发现SSeCKS蛋白参与肌动蛋白骨架和血脑屏障的动态调控,脂多糖(lipopolysaccharide, LPS)诱导大鼠PMVECs高表达SSeCKS蛋白,这些研究提示SSeCKS可能是PMVECs通透性调控的重要效应分子,但其在炎症因子如白介素(interleukin, IL)-1β和肿瘤坏死因子(tumor necrosin factor, TNF)-α诱导PMVECs通透性增加中的具体作用仍未阐明。除LPS外,其他在肺部炎症反应过程中发挥重要作用的炎症介质是否参与PMVECs中SSeCKS的诱导表达也未见报道。
IL-17F是一种新的炎症因子,大量研究表明在支气管上皮细胞和静脉内皮细胞中,IL-17F通过诱导IL-6、IL-8和细胞间黏附分子-1表达触发一系列炎症反应;肺炎克雷伯杆菌、支原体或白色念珠菌感染后,肺部增加的IL-17F诱发中性粒细胞聚集和变态反应,这些研究提示IL-17F是参与肺部炎症反应的重要细胞因子。目前关于肺部IL-17F作用机制的研究主要集中在支气管上皮细胞,并且对其细胞内信号转导通路知之甚少,PMVECs是肺部炎症反应时的主要效应细胞,但关于IL-17F对PMVECs的刺激效应以及细胞内信号转导通路的研究却未见报道。
目的
观察PMVECs单层融合前后的生物学特征。融合PMVECs单层用于实验,观察IL-17F对PMVECs通透性的影响以及SSeCKS信号通路在IL-1β、TNF-α和IL-17F诱导PMVECs通透性变化中的作用,为认识ALI/ARDS发生机制提供实验依据。
方法
体外培养大鼠PMVECs;在Transwell和硝酸醋酸混合纤维膜上构建PMVECs单层;倒置显微镜、苏木素-伊红染色、跨内皮细胞电阻(transendothelial electrical resistance, TER)、异硫氰酸荧光素标记葡聚糖法(Pd)以及Hank’s平衡液法(LP)观察细胞单层生物学特征;逆转录聚合酶链反应分析SSeCKS的mRNA变化;免疫印迹技术分析PKC活性以及SSeCKS蛋白变化;应用异硫氰酸荧光素标记的鬼笔环肽观察PMVECs肌动蛋白骨架改变;此外,PMVECs还被特异性的SSeCKS小分子干扰RNA(small interfering RNA, siRNA)转染。
结果
1)1×105/cm2的PMVECs接种Transwell后,TER随接种时间延长稳定升高,接种后第四天达最高,尔后维持,第十天TER开始下降,而倒置显微镜观察到细胞接种后第三天已融合成单层;2×105/cm2的PMVECs接种Transwell后,TER达高峰时间提前到接种后第二天,第九天TER开始下降;不同密度接种组的TER最高值间比较无显著性差异(P > 0.05)。静息状态下融合PMVECs单层TER为48.07±2.84 ?·cm2、Pd为6.19±0.43×10-6 cm/s、LP为6.80±0.62×10-7 cm/s/cm H2O。
2)10mg/L LPS分别刺激融合PMVECs单层0.5h和2h后,与未处理对照组比较,TER显著下降、LP和Pd均显著增加。
3)5ng/ml IL-1β和10ng/ml TNF-α分别激活PMVECs的PKC信号通路和诱导SSeCKS的mRNA和蛋白高表达,IL-1β+TNF-α诱导SSeCKS表达的效应更显著;BIM (1μmol/L,PKC抑制剂)显著下调PMVECs的PKC活性和抑制IL-1β+TNF-α诱导SSeCKS的mRNA和蛋白高表达,而蛋白激酶A(protein kinase A, PKA)抑制剂(H89,10μmol/L)和蛋白酪氨酸激酶(protein tyrosine kinase, PTK)抑制剂(金雀异黄素,50μg/ml)不影响IL-1β+TNF-α诱导SSeCKS的mRNA和蛋白高表达。
4)与未处理组比较,50nmol/L SSeCKS-siRNA转染PMVECs 12h后,SSeCKS mRNA表达显著下降、24h达最低值、持续到转染后96h,SSeCKS-siRNA抑制SSeCKS蛋白表达的最佳时间在转染48h后,抑制率接近50%,抑制效应持续到转染后120h;SSeCKS-siRNA转染还以浓度依赖方式(10、20、50 nmol/L)沉默SSeCKS mRNA和蛋白表达。无论是脂质体空转染、普通阴性对照siRNA转染还是SSeCKS-siRNA转染均使融合PMVECs的静息状态TER轻微下降、静息状态Pd略升高,但与未处理组比较无显著性差异(P > 0.05)。
5 ) IL-1β+TNF-α刺激显著下调PMVECs的TER和增加PMVECs的Pd ,SSeCKS-siRNA转染可显著下调IL-1β+TNF-α诱导细胞单层通透性的增加,但改善效果与BIM的改善效果比较存在差异(P < 0.05)。
6)不同浓度IL-17F(0.1、1、10、100ng/ml)刺激PMVECs 3h后,下降的TER和增加的Pd与未处理组比较差异显著;100ng/ml IL-17F刺激细胞0.5h后,Pd与未处理组比较增加了15%、3~6h后Pd达高峰、显著增加的Pd持续到刺激后24h;同Pd变化,IL-17F刺激PMVECs单层时,TER呈时间依赖性下降;1、10μmol/L BIM均能改善IL-17F诱导增加的Pd和下降的TER,但都未能使其恢复到正常值。
7)IL-17F刺激3h后,PMVECs的丝状肌动蛋白重组:从细胞周边向细胞中心转移并成平行束状堆积、增厚,同时细胞旁缝隙显著增大;流式细胞技术检测发现IL-17F刺激后,PMVECs中丝状肌动蛋白的相对荧光强度曲线向右位移、其平均荧光强度与未处理组比较显著增加,而1、10μmol/L BIM均能有效改善IL-17F诱导PMVECs肌动蛋白骨架的重构。
8)不同浓度(0.1、1、10、100ng/ml)IL-17F刺激PMVECs后,SSeCKS的mRNA和蛋白表达均显著增加;100ng/ml IL-17F刺激PMVECs时,SSeCKS基因表达在刺激后3h达高峰,而蛋白表达在刺激后6h达高峰;IL-17F刺激PMVECs 5min后,SSeCKS蛋白的丝氨酸被磷酸化、30min后显著、3~6h后达高峰、后维持在较高的磷酸化水平至刺激后24h,而1μmol/L BIM预孵育后,IL-17F诱导增加的蛋白磷酸化水平显著下降;此外,IL-17F刺激PMVECs 30min后,不溶于Triton-X-100的SSeCKS蛋白显著增加,3~6h后,不溶于Triton-X-100的SSeCKS蛋白达高峰;最后,静息状态时,SSeCKS蛋白大部分集中在细胞质,IL-17F刺激30min后,细胞膜部SSeCKS蛋白的量与静息状态比较差异显著,刺激90min后,SSeCKS大部分集中在细胞膜。
结论
1)TER联合倒置显微镜较倒置显微镜单独应用能更准确判定PMVECs单层融合状态;2)TER、Pd、LP均能有效检测PMVECs单层通透性变化,TER联合Pd更多地反映PMVECs通透性变化信息;3)SSeCKS表达增加上调PMVECs通透性在ALI/ARDS复杂机制中占重要地位,IL-1β、TNF-α和IL-17F是PMVECs高表达SSeCKS基因和蛋白的强有力诱导剂,PKC而非PKA、PTK信号通路参与其表达调控;4)IL-17F通过激活PKC-SSeCKS信号转导通路以及诱导PKC依赖的F-actin骨架重构来增加PMVECs通透性,从而促进肺部炎症反应。
Background
Pulmonary microvascular endothelial cells (PMVECs) lined at the critical interface between the blood and microvessel are primary targets of inflammatory cytokines during lung inflammation. A pathophysiologic response of PMVECs to inflammatory cytokines is permeability increase that contributes to high-protein pulmonary edema, which is a characteristic of acute lung injury and acute respiratory distress syndrome (ALI/ARDS). It is well known that the protein kinase C (PKC) signaling pathway significantly contributes to the breakdown of PMVECs barrier though phosphorylation of proteins which can promote cytoskeletal reorganization or dissolution of the adherens junctions. Given the complex interplay between PKC signaling pathway and the possible contribution of other signaling pathways, further studies should be required to delineate downstream targets of PKC and the mechanisms that regulate endothelial barrier function in PMVECs. In addition, it is very difficult in vivo to study changes of PMVECs permeability, and considerable achievements in this research area come from in vitro experiments of cultured cells, therefore, the integrality of study can be determined by the confluent state of PMVECs.
Src-suppressed C kinase substrate (SSeCKS) is a PKC substrate that is identified to be phosphorylated by PKC. Of particular interest, evidences that SSeCKS is involved in a dramatic rearrangement of the actin cytoskeleton and the modulation of the blood-brain barrier permeability have been presented. Moreover, in rat PMVECs (RPMVECs), increased levels of SSeCKS have been found after challenge of lipopolysaccharide (LPS). These implicate that SSeCKS is possibly one of the critical proteins in the pathogenesis of endothelial hyperpermeability after exposure of PMVECs to various inflammatory factors, such as interleukin (IL)-1βand tumor necrosin factor (TNF)-α. Nevertheless, the exact role of SSeCKS in the regulation of PMVECs permeability is not well understood. Additionally, besides LPS, other potential inducers of SSeCKS in RPMVECs remain ill-defined.
Interleukin-17F is a novel proinflammatory cytokine. Accumulating studies have showed that IL-17F triggers a variety of inflammatory responses such as inducing inflammatory factors, including IL-6, IL-8, and intracellular adhesion molecule-1, in airway epithelial cells and vein endothelial cells. Of note, in lung, increased expression of IL-17F has been found in animal models after Klebsiella pneumoniae, Mycoplasma pneumoniae and Candida albicans infection, and to induce pulmonary neutrophilia and an additive effect on antigen-induced allergic inflammatory responses. These findings have suggested that IL-17F is one of the key cytokines regulating lung inflammation. However, up to date, studies about mechanisms of IL-17F in lung focus on bronchial epithelium, and signaling pathways activated by IL-17F in cells have not been elucidated. It is well known that PMVECs are involved in lung inflammatory disease, yet, little information exists in the role of IL-17F and its signaling pathway in PMVECs.
Objective
The aim of this study is firstly to observe some biological characteristics of PMVECs monolayers before and after confluence, and then, we sought to investigate the effects of IL-17F on confluent PMVECs monolayers and assess the role of PKC in this process. The role of SSeCKS in the modulation of PMVECs permeability elicited by IL-1β, TNF-αand IL-17F is also investigated. These may be helpful for elucidating the pathogenesis of ALI/ARDS.
Methods
After primary RPMVECs were successfully cultured in vitro, the cells were seeded on Transwell polyester membranes or nitric-acid/acetic-acid membranes to construct in vitro models of PMVECs monolayers. The biological characteristics of cells monolayers before and after confluence were observed by the inverted microscope, staining with hematoxylin-eosin, transendothelial electrical resistance (TER) and changes of permeability as measured by means of fluorescein isothiocyanate (FITC)-dextran (Pd) and Hank’s solution (LP) across monolayers. Additional monolayers were stained using FITC-phalloin for observing the filamentous actin (F-actin) cytoskeletal changes. The gene expression of SSeCKS was analyzed by the reverse transcription-polymerase chains. Immunoblotting was used to determine the PKC activity, the levels of SSeCKS protein expression and the biological alterations of SSeCKS protein in PMVECs. In addition, SSeCKS-specific small interfering RNA (siRNA) was transfected into PMVECs.
Results
1) In group one, after a cell suspension (1×105 cells /cm2) was cultured on Transwell, the normalized TER increased steadily in a time-dependent manner and reached the summit on the fourth day post-seeding, then was sustained. Subsequently, on the tenth day post-seeding, the increased TER began to decrease. However, the monolayer under inversed microscope reached the state of confluence on the third day post-seeding. In group two, a cell suspension (2×105 cells /cm2) was seeded, changes of the normalized TER were similar to that of the group one, reaching the summit on the second day post-seeding and beginning to decrease on the ninth day post-seeding. Of note, no significant difference in the maximum TER was found between both groups. The TER, Pd and LP of confluent PMVECs monolayers under quiescent state were 48.07±2.84 ?·cm2, 6.19±0.43×10-6 cm/s and 6.80±0.62×10-7 cm/s/cm H2O, respectively.
2) After confluent PMVECs monolayers were stimulated with 10 mg/L LPS for both 0.5 h and 2 h, there were significant decreases in TER and increases in Pd and LP when respectively compared with the untreated group.
3) Interleukin-1β(5 ng/ml) and TNF-α(10 ng/ml) activated PKC signaling pathway in PMVECs, and up-regulated the gene and protein expression of SSeCKS. Meanwhile, we found that the expression of SSeCKS was the greatest after stimulation with the cytokine combination of 5 ng/ml IL-1βand 10 ng/ml TNF-α. PKC inhibitor, Bisindolylmaleimide I (BIM, 1μmol/L) significantly decreased both the increased PKC activity and SSeCKS expression induced by the cytokines in PMVECs. However, both the selective protein kinase A (PKA) inhibitor H89 (10μmol/L) and the broad-spectrum protein tyrosine kinase (PTK) inhibitor genistein (50μg/ml) showed no significant inhibitory effects on cytokine-induced SSeCKS gene expression and protein production in PMVECs.
4) Transfection with special SSeCKS-siRNA resulted in a decrease in the expression of SSeCKS gene and protein in a dose-dependent manner (10, 20, 50 nmol/L) and in a time-dependent manner. When compared with the untreated group, significantly inhibitory effects of SSeCKS-siRNA (50 nmol/L) in the expression of SSeCKS gene and protein began at 12 h and 24 h, reached a peak at 24 h and 48 h and were sustained to 96 h and 120 h after PMVECs were transfected, respectively. At 48 h post-transfection, the expression of SSeCKS in PMVECs was inhibited by 50 %. Furthermore, SSeCKS depletion, the mock trasfection and the non-silencing control siRNA transfection induced a slight, but not significant (P > 0.05), decrease in quiescent TER and increase in quiescent Pd across confluent PMVECs monolayers.
5) When compared with the untreated group, application of the cytokine combination (IL-1βand TNF-α) to PMVECs monolayers resulted in a significant increase of Pd and a significant decrease of TER, and then, prior transfection with 50 nmol/L SSeCKS-siRNA significantly attenuated the cytokine-induced effects. Notably, it was found that the effect of BIM in blocking cytokine-induced endothelial barrier dysfunction is more significant than that of SSeCKS depletion.
6) Interleukin-17F induced, in a dose-dependent manner (0.1, 1, 10, 100ng/ml), significant increases of Pd and decreases of TER after PMVECs monolayers were exposed to stimulations for 3 h. The effects of IL-17F were also time dependent. Pd increased ~15 % as compared with quiescent levels after monolayers were treated by 100ng/ml IL-17F for 0.5 h, peaked at 3h and prolonged lag time of 24 h. Similarly, the progressive decrease in TER produced by IL-17F was time-dependent when compared with untreated monolayer. After PMVECs monolayers were pretreated with BIM (1 or 10μmol/L, respectively), the increased Pd and decreased TER induced by IL-17F can be significantly blocked, but did not return to normal levels.
7) In PMVECs, IL-17F stimulation for 3 h produced numerous intercellular gaps and a marked reorganization of F-actin from a web-like peripheral distribution to centrally located parallel stress fibers. The latter became thicker. Meanwhile, after IL-17F stimulation, the flow cytometric profile was shifted to the right and the mean fluorescence intensity of F-actin was conspicuously increased when compared with untreated monolayers. IL-17F-induced cytoskeletal changes indicated above can be significantly amended after PMVECs monolayers were pretreated by 1 and 10μmol/L BIM.
8) After PMVECs were treated with increasing concentrations of IL-17F (0.1, 1, 10, 100 ng/ml) and 100 ng/ml IL-17F for various times, the expression of SSeCKS gene and protein were remarkably up-regulated in a dose-dependent manner, occurred maximally at 3 h and 6 h post-stimulation, respectively. The serine phosphorylation of SSeCKS was weakly detected at 5-minutes point, remarkably occurred at 30-minutes point, reached maximal level at 3 ~ 6 h, and stayed at a slightly high level until 24 h after PMVECs were exposed to100 ng/ml IL-17F. BIM (1μmol/L) can significantly abrogate this phosphorylation induced by IL-17F. In addition, our results demonstrated that treatment with IL-17F significantly increased the amount of SSeCKS in the insoluble fraction in a time-dependent manner, beginning at 30 min and peaking at 3 ~ 6 h post-stimulation. Finally, under quiescent conditions, SSeCKS existed for the most part in the cytosolic fraction of PMVECs, after 30 min treatment, IL-17F resulted in significant translocation of SSeCKS from the cytosol to the membrane. At 90-minutes point, SSeCKS predominantly existed in the membrane fraction.
Conclusions
1) The combination of TER and an inverted microscope is better than the solo application of an inverted microscope to assess the confluent state of PMVECs monolayers.
2) Three methods, TER, Pd and LP, can be availably used to determine PMVECs permeability. Combination of Pd and TER maybe greatly reflect the information of changes in permeability across the in vitro PMVECs monolayer model.
3) The increased expression of SSeCKS is involved in the regulation of PMVECs hyperpermeability that maybe play an important role in the process of ALI/ARDS. The expression of SSeCKS gene and protein can be potently induced by IL-1β, TNF-αand IL-17F in PMVECs. It is PKC signaling pathway, neither PKA signaling pathway nor PTK signaling pathway, which is involved in the regulation of SSeCKS expression.
4) IL-17F promotes the lung inflammatory response though the activated PKC-SSeCKS signaling pathway and the PKC-dependent reorganization of actin cytoskeleton that all contribute to PMVECs hyperpermeabilty induced by IL-17F.
引文
1. Kaneda K, Miyamoto K, Nomura S, et al. Intercellular localization of occludins and ZO-1 as a solute transport barrier of the mesothelial monolayer. J Artif Organs, 2006; 9(4):241-50.
2. Siflinger-Birnboim A, Johnson A. Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury. Am J Physiol Lung Cell Mol Physiol, 2003; 284(3):L435-51.
3. Kuebler WM. Inflammatory pathways and microvascular responses in the lung. Pharmacol Rep, 2005; 57 Suppl:196-205.
4. Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability. Physiol Rev, 2006; 86(1):279-367.
5. Tsushima K, King LS, Aggarwal NR, et al. Acute lung injury review. Intern Med, 2009; 48(9):621-30.
6. Hermanns MI, Fuchs S, Bock M, et al. Primary human coculture model of alveolo-capillary unit to study mechanisms of injury to peripheral lung. Cell Tissue Res, 2009; 336(1):91-105.
7. Ofori-Acquah SF, King J, Voelkel N, et al. Heterogeneity of barrier function in the lung reflects diversity in endothelial cell junctions. Microvasc Res, 2008; 75(3):391-402.
8. Lim MJ, Chiang ET, Hechtman HB, et al. Inflammation-induced subcellular redistribution of VE-cadherin, actin, and gamma-catenin in cultured human lung microvessel endothelial cells. Microvasc Res, 2001; 62(3):366-82.
9. Gong P, Angelini DJ, Yang S, et al. TLR4 signaling is coupled to SRC family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia. J Biol Chem, 2008; 283(19):13437-49.
10. Wojciak-Stothard B, Potempa S, Eichholtz T, et al. Rho and Rac but not Cdc42 regulate endothelial cell permeability. J Cell Sci, 2001; 114(Pt 7):1343-55.
11. Schmidt A, Utepbergenov DI, Krause G, et al. Use of surface plasmon resonance for real-time analysis of the interaction of ZO-1 and occludin. Biochem Biophys Res Commun, 2001; 288(5):1194-9.
12. Gillrie MR, Krishnegowda G, Lee K, et al. Src-family kinase dependent disruption of endothelial barrier function by Plasmodium falciparum merozoite proteins. Blood, 2007; 110(9):3426-35.
13. Parthasarathi K, Ichimura H, Monma E, et al. Connexin 43 mediates spread of Ca2+-dependent proinflammatory responses in lung capillaries. J Clin Invest, 2006; 116(8):2193-200.
14. Ying X, Minamiya Y, Fu C, et al. Ca2+ waves in lung capillary endothelium. CircRes, 1996; 79(4):898-908.
15. Usatyuk PV, Parinandi NL, Natarajan V. Redox regulation of
4-hydroxy-2-nonenal-mediated endothelial barrier dysfunction by focal adhesion, adherens, and tight junction proteins. J Biol Chem, 2006; 281(46):35554-66.
16. Quadri SK, Bhattacharya J. Resealing of endothelial junctions by focal adhesion kinase. Am J Physiol Lung Cell Mol Physiol, 2007; 292(1):L334-42.
17. Birukova AA, Adyshev D, Gorshkov B, et al. ALK5 and Smad4 are involved in TGF-beta1-induced pulmonary endothelial permeability. FEBS Lett, 2005; 579(18):4031-7.
18. Clements RT, Minnear FL, Singer HA, et al. RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability. Am J Physiol Lung Cell Mol Physiol, 2005; 288(2):L294-306.
19. Petrache I, Verin AD, Crow MT, et al. Differential effect of MLC kinase in TNF-alpha-induced endothelial cell apoptosis and barrier dysfunction. Am J Physiol Lung Cell Mol Physiol, 2001; 280(6):L1168-78.
20. Koss M, Pfeiffer GR, 2nd, Wang Y, et al. Ezrin/radixin/moesin proteins are phosphorylated by TNF-alpha and modulate permeability increases in human pulmonary microvascular endothelial cells. J Immunol, 2006; 176(2):1218-27.
21. Tinsley JH, Breslin JW, Teasdale NR, et al. PKC-dependent, burn-induced adherens junction reorganization and barrier dysfunction in pulmonary microvascular endothelial cells. Am J Physiol Lung Cell Mol Physiol, 2005; 289(2):L217-23.
22. Tinsley JH, Teasdale NR, Yuan SY. Involvement of PKCdelta and PKD in pulmonary microvascular endothelial cell hyperpermeability. Am J Physiol Cell Physiol, 2004; 286(1):C105-11.
23. Yang S, Yip R, Polena S, et al. Reactive oxygen species increased focal adhesion kinase production in pulmonary microvascular endothelial cells. Proc WestPharmacol Soc, 2004; 47:54-6.
24. Liu T, Guevara OE, Warburton RR, et al. Modulation of HSP27 alters hypoxia-induced endothelial permeability and related signaling pathways. J Cell Physiol, 2009; 220(3):600-10.
25. Angelini DJ, Hyun SW, Grigoryev DN, et al. TNF-alpha increases tyrosine phosphorylation of vascular endothelial cadherin and opens the paracellular pathway through fyn activation in human lung endothelia. Am J Physiol Lung Cell Mol Physiol, 2006; 291(6):L1232-45.
26. An SS, Pennella CM, Gonnabathula A, et al. Hypoxia alters biophysical properties of endothelial cells via p38 MAPK- and Rho kinase-dependent pathways. Am J Physiol Cell Physiol, 2005; 289(3):C521-30.
27. Tiruppathi C, Ahmmed GU, Vogel SM, et al. Ca2+ signaling, TRP channels, and endothelial permeability. Microcirculation, 2006; 13(8):693-708.
28. Huang F, Subbaiah PV, Holian O, et al. Lysophosphatidylcholine increases endothelial permeability: role of PKCalpha and RhoA cross talk. Am J Physiol Lung Cell Mol Physiol, 2005; 289(2):L176-85.
29. Frey RS, Gao X, Javaid K, et al. Phosphatidylinositol 3-kinase gamma signaling through protein kinase Czeta induces NADPH oxidase-mediated oxidant generation and NF-kappaB activation in endothelial cells. J Biol Chem, 2006; 281(23):16128-38.
30. Kolosova IA, Ma SF, Adyshev DM, et al. Role of CPI-17 in the regulation of endothelial cytoskeleton. Am J Physiol Lung Cell Mol Physiol, 2004; 287(5):L970-80.
31. Cheng C, Liu H, Ge H, et al. Lipopolysaccharide induces expression of SSeCKS in rat lung microvascular endothelial cell. Mol Cell Biochem, 2007; 305(1-2):1-8.
32. Starnes T, Robertson MJ, Sledge G, et al. Cutting edge: IL-17F, a novel cytokine selectively expressed in activated T cells and monocytes, regulates angiogenesis andendothelial cell cytokine production. J Immunol, 2001; 167(8):4137-40.
33. Tinsley JH, Hunter FA, Childs EW. PKC and MLCK-Dependent, Cytokine-Induced Rat Coronary Endothelial Dysfunction. J Surg Res, 2009; 152(1):76-83.
34. Dull RO, DeWitt BJ, Dinavahi R, et al. Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction. J Appl Physiol, 2004; 97(5):1930-7.
35. Sedgwick JB, Menon I, Gern JE, et al. Effects of inflammatory cytokines on the permeability of human lung microvascular endothelial cell monolayers and differential eosinophil transmigration. J Allergy Clin Immunol, 2002; 110(5):752-6.
36. Warfel JM, Steele AD, D'Agnillo F. Anthrax lethal toxin induces endothelial barrier dysfunction. Am J Pathol, 2005; 166(6):1871-81.
37. Zhang H, Sun GY. LPS induces permeability injury in lung microvascular endothelium via AT(1) receptor. Arch Biochem Biophys, 2005; 441(1):75-83.
38. Chang YS, Munn LL, Hillsley MV, et al. Effect of vascular endothelial growth factor on cultured endothelial cell monolayer transport properties. Microvasc Res, 2000; 59(2):265-77.
39. Chen SF, Fei X, Li SH. A new simple method for isolation of microvascular endothelial cells avoiding both chemical and mechanical injuries. Microvasc Res, 1995; 50(1):119-28.
40.肖贞良,孙耕耘,钱桂生.脂多糖致肺微血管内皮细胞单层通透性损伤的实验研究.中华结核和呼吸杂志, 1999; 22(7):427.
41. Kelly JJ, Moore TM, Babal P, et al. Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability. Am J Physiol, 1998; 274(5 Pt 1):L810-9.
42. Irwin DC, Tissot van Patot MC, Tucker A, et al. Direct ANP inhibition of hypoxia-induced inflammatory pathways in pulmonary microvascular and macrovascular endothelial monolayers. Am J Physiol Lung Cell Mol Physiol, 2005;288(5):L849-59.
43. Nwariaku FE, Rothenbach P, Liu Z, et al. Rho inhibition decreases TNF-induced endothelial MAPK activation and monolayer permeability. J Appl Physiol, 2003; 95(5):1889-95.
44.尤青海,孙耕耘.肺微血管内皮细胞通透性调控的信号转导机制.生理科学进展, 2008; 39 (4):395-8.
45. Lin X, Gelman IH. Calmodulin and cyclin D anchoring sites on the Src-suppressed C kinase substrate, SSeCKS. Biochem Biophys Res Commun, 2002; 290(5):1368-75.
46. Gelman IH. The role of SSeCKS/gravin/AKAP12 scaffolding proteins in the spaciotemporal control of signaling pathways in oncogenesis and development. Front Biosci, 2002; 7(d1782-97.
47. Gelman IH, Lee K, Tombler E, et al. Control of cytoskeletal architecture by the src-suppressed C kinase substrate, SSeCKS. Cell Motil Cytoskeleton, 1998; 41(1):1-17.
48. Nelson PJ, Moissoglu K, Vargas J, Jr., et al. Involvement of the protein kinase C substrate, SSeCKS, in the actin-based stellate morphology of mesangial cells. J Cell Sci, 1999; 112 ( Pt 3)(361-70.
49. Kitamura H, Okita K, Fujikura D, et al. Induction of Src-suppressed C kinase substrate (SSeCKS) in vascular endothelial cells by bacterial lipopolysaccharide. J Histochem Cytochem, 2002; 50(2):245-55.
50. Chapline C, Cottom J, Tobin H, et al. A major, transformation-sensitive PKC-binding protein is also a PKC substrate involved in cytoskeletal remodeling. J Biol Chem, 1998; 273(31):19482-9.
51. Cheng C, Liu H, Ge H, et al. Essential role of Src suppressed C kinase substrates in endothelial cell adhesion and spreading. Biochem Biophys Res Commun, 2007; 358(1):342-8.
52. Lee SW, Kim WJ, Choi YK, et al. SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier. Nat Med, 2003; 9(7):900-6.
53.李惠,孙耕耘,费黎明, et al.脂多糖诱导肺微血管内皮细胞SSeCKS mRNA表达的研究.中国危重病急救医学, 2008; 20(2):65-8.
54. Farley KS, Wang L, Mehta S. Septic pulmonary microvascular endothelial cell injury: role of alveolar macrophage NADPH oxidase. Am J Physiol Lung Cell Mol Physiol, 2009; 296(3):L480-8.
55.刘茹,赵亚萍,夏黎, et al.高脂饮食大鼠血清抵抗素水平及下丘脑细胞因子信号转导抑制因子-3基因表达的变化.实用儿科临床杂志, 2008; 23(20):1573-75.
56. Yan M, Cheng C, Jiang J, et al. Essential role of SRC suppressed C kinase substrates in Schwann cells adhesion, spreading and migration. Neurochem Res, 2009; 34(5):1002-10.
57. Rao R, Redha R, Macias-Perez I, et al. Prostaglandin E2-EP4 receptor promotes endothelial cell migration via ERK activation and angiogenesis in vivo. J Biol Chem, 2007; 282(23):16959-68.
58.费黎明,孙耕耘,李惠. TNF-α对大鼠肺微血管内皮细胞SSeCKS mRNA表达的影响.临床肺科杂志, 2008; 13(6):686-7.
59. van der Poll T. Immunotherapy of sepsis. Lancet Infect Dis, 2001; 1(3):165-74.
60. Alba-Loureiro TC, Martins EF, Miyasaka CK, et al. Evidence that arachidonic acid derived from neutrophils and prostaglandin E2 are associated with the induction of acute lung inflammation by lipopolysaccharide of Escherichia coli. Inflamm Res, 2004; 53(12):658-63.
61. Lv T, Shen X, Shi Y, et al. TLR4 is essential in acute lung injury induced by unresuscitated hemorrhagic shock. J Trauma, 2009; 66(1):124-31.
62. Wu SH, Liao PY, Dong L, et al. Signal pathway involved in inhibition by lipoxin A(4) of production of interleukins induced in endothelial cells by lipopolysaccharide. Inflamm Res, 2008; 57(9):430-7.
63. Coats SR, Covington JW, Su M, et al. SSeCKS gene expression in vascular smooth muscle cells: regulation by angiotensin II and a potential role in the regulation of PAI-1 gene expression. J Mol Cell Cardiol, 2000; 32(12):2207-19.
64. Lin X, Tombler E, Nelson PJ, et al. A novel src- and ras-suppressed protein kinase C substrate associated with cytoskeletal architecture. J Biol Chem, 1996; 271(45):28430-8.
65.郭葆玉. RNA干扰技术.药物生物技术, 2008; 15(2):137-41.
66. Mountain DJ, Singh M, Menon B, et al. Interleukin-1beta increases expression and activity of matrix metalloproteinase-2 in cardiac microvascular endothelial cells: role of PKCalpha/beta1 and MAPKs. Am J Physiol Cell Physiol, 2007; 292(2):C867-75.
67. Defazio G, Nico B, Trojano M, et al. Inhibition of protein kinase C counteracts TNFalpha-induced intercellular adhesion molecule 1 expression and fluid phase endocytosis on brain microvascular endothelial cells. Brain Res, 2000; 863(1-2):245-8.
68. Weaver CT. Th17: The ascent of a new effector T-cell subset. Preface. Eur J Immunol, 2009; 39(3):634-6.
69. Kawaguchi M, Onuchic LF, Li XD, et al. Identification of a novel cytokine, ML-1, and its expression in subjects with asthma. J Immunol, 2001; 167(8):4430-5.
70. Kuestner RE, Taft DW, Haran A, et al. Identification of the IL-17 receptor related molecule IL-17RC as the receptor for IL-17F. J Immunol, 2007; 179(8):5462-73.
71. Ge D, You Z. Expression of interleukin-17RC protein in normal human tissues. Int Arch Med, 2008; 1(1):19.
72. Li X. Act1 modulates autoimmunity through its dual functions in CD40L/BAFF and IL-17 signaling. Cytokine, 2008; 41(2):105-13.
73. Venkatachalam K, Mummidi S, Cortez DM, et al. Resveratrol inhibits high glucose-induced PI3K/Akt/ERK-dependent interleukin-17 expression in primarymouse cardiac fibroblasts. Am J Physiol Heart Circ Physiol, 2008; 294(5):H2078-87.
74. Wu Q, Martin RJ, Rino JG, et al. IL-23-dependent IL-17 production is essential in neutrophil recruitment and activity in mouse lung defense against respiratory Mycoplasma pneumoniae infection. Microbes Infect, 2007; 9(1):78-86.
75. Tan W, Huang W, Gu X, et al. IL-17F/IL-17R interaction stimulates granulopoiesis in mice. Exp Hematol, 2008; 36(11):1417-27.
76. Happel KI, Dubin PJ, Zheng M, et al. Divergent roles of IL-23 and IL-12 in host defense against Klebsiella pneumoniae. J Exp Med, 2005; 202(6):761-9.
77. Suzuki S, Kokubu F, Kawaguchi M, et al. Expression of interleukin-17F in a mouse model of allergic asthma. Int Arch Allergy Immunol, 2007; 143 Suppl 1(89-94.
78. Hymowitz SG, Filvaroff EH, Yin JP, et al. IL-17s adopt a cystine knot fold: structure and activity of a novel cytokine, IL-17F, and implications for receptor binding. EMBO J, 2001; 20(19):5332-41.
79. Oda N, Canelos PB, Essayan DM, et al. Interleukin-17F induces pulmonary neutrophilia and amplifies antigen-induced allergic response. Am J Respir Crit Care Med, 2005; 171(1):12-8.
80. Numasaki M, Takahashi H, Tomioka Y, et al. Regulatory roles of IL-17 and IL-17F in G-CSF production by lung microvascular endothelial cells stimulated with IL-1beta and/or TNF-alpha. Immunol Lett, 2004; 95(1):97-104.
81. Numasaki M, Tomioka Y, Takahashi H, et al. IL-17 and IL-17F modulate GM-CSF production by lung microvascular endothelial cells stimulated with IL-1beta and/or TNF-alpha. Immunol Lett, 2004; 95(2):175-84.
82. Kawaguchi M, Kokubu F, Matsukura S, et al. Induction of C-X-C chemokines, growth-related oncogene alpha expression, and epithelial cell-derived neutrophil-activating protein-78 by ML-1 (interleukin-17F) involves activation of Raf1-mitogen-activated protein kinase kinase-extracellular signal-regulated kinase1/2 pathway. J Pharmacol Exp Ther, 2003; 307(3):1213-20.
83. Kawaguchi M, Fujita J, Kokubu F, et al. IL-17F-induced IL-11 release in bronchial epithelial cells via MSK1-CREB pathway. Am J Physiol Lung Cell Mol Physiol, 2009; 296(5):L804-10.
84. Kawaguchi M, Kokubu F, Huang SK, et al. The IL-17F signaling pathway is involved in the induction of IFN-gamma-inducible protein 10 in bronchial epithelial cells. J Allergy Clin Immunol, 2007; 119(6):1408-14.
85.张志刚,何权衡,刘新民, et al.白细胞介素-17在急性肺损伤发病机制中的作用.心肺血管病杂志, 2007; 26(1):38-43.
86. Stasek JE, Jr., Patterson CE, Garcia JG. Protein kinase C phosphorylates caldesmon77 and vimentin and enhances albumin permeability across cultured bovine pulmonary artery endothelial cell monolayers. J Cell Physiol, 1992; 153(1):62-75.
87. Jennings LK, Fox JE, Edwards HH, et al. Changes in the cytoskeletal structure of human platelets following thrombin activation. J Biol Chem, 1981; 256(13):6927-32.
88. Yang XO, Chang SH, Park H, et al. Regulation of inflammatory responses by IL-17F. J Exp Med, 2008; 205(5):1063-75.
89. Hizawa N, Kawaguchi M, Huang SK, et al. Role of interleukin-17F in chronic inflammatory and allergic lung disease. Clin Exp Allergy, 2006; 36(9):1109-14.
90. Al-Ramli W, Prefontaine D, Chouiali F, et al. T(H)17-associated cytokines (IL-17A and IL-17F) in severe asthma. J Allergy Clin Immunol, 2009; 123(5):1185-7.
91. Nembrini C, Marsland BJ, Kopf M. IL-17-producing T cells in lung immunity and inflammation. J Allergy Clin Immunol, 2009; 123(5):986-94; quiz 995-6.
92. Kebir H, Kreymborg K, Ifergan I, et al. Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nat Med, 2007; 13(10):1173-5.
93. Rung-ruangkijkrai T, Fujikura D, Kitamura H, et al. The expression of src-suppressed C kinase substrate (SSeCKS) and uptake of exogenous particles in endothelial and reticular cells. Arch Histol Cytol, 2004; 67(2):135-47.
94. Corbit KC, Trakul N, Eves EM, et al. Activation of Raf-1 signaling by protein kinase C through a mechanism involving Raf kinase inhibitory protein. J Biol Chem, 2003; 278(15):13061-8.
95. Kolch W, Heidecker G, Kochs G, et al. Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature, 1993; 364(6434):249-52.
96. Di Stefano A, Caramori G, Gnemmi I, et al. T helper type 17-related cytokine expression is increased in the bronchial mucosa of stable chronic obstructive pulmonary disease patients. Clin Exp Immunol, 2009; 157(2):316-24.
1. Kuebler WM. Inflammatory pathways and microvascular responses in the lung. Pharmacol Rep, 2005; 57 Supp:l196-205.
2. Zhou C, Wu S. T-type calcium channels in pulmonary vascular endothelium. Microcirculation, 2006; 13(8):645-56.
3. Paria BC, Vogel SM, Ahmmed GU, et al. Tumor necrosis factor-alpha-induced TRPC1 expression amplifies store-operated Ca2+ influx and endothelial permeability. Am J Physiol Lung Cell Mol Physiol, 2004; 287(6):L1303-13.
4. Ito S, Suki B, Kume H, et al. Actin Cytoskeleton Regulates Stretch-activated Ca2+ Influx in Human Pulmonary Microvascular Endothelial Cells. Am J Respir Cell Mol Biol, 2009; [Epub ahead of print].
5. Yin J, Hoffmann J, Kaestle SM, et al. Negative-feedback loop attenuates hydrostatic lung edema via a cGMP-dependent regulation of transient receptor potential vanilloid 4. Circ Res, 2008; 102(8):966-74.
6. Sundivakkam PC, Kwiatek AM, Sharma TT, et al. Caveolin-1 scaffold domain interacts with TRPC1 and IP3R3 to regulate Ca2+ store release-induced Ca2+ entry in endothelial cells. Am J Physiol Cell Physiol, 2009; 296(3):C403-13.
7. Kelly JJ, Moore TM, Babal P, et al. Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+ and permeability. Am J Physiol, 1998; 274(5 Pt 1):L810-9.
8. Cai H, Liu D, Garcia JG. CaM Kinase II-dependent pathophysiological signalling in endothelial cells. Cardiovasc Res, 2008; 77(1):30-4.
9. Tinsley JH, Teasdale NR, Yuan SY. Involvement of PKCdelta and PKD in pulmonary microvascular endothelial cell hyperpermeability. Am J Physiol Cell Physiol, 2004; 286(1):C105-11.
10. Cheng C, Liu H, Ge H, et al. Lipopolysaccharide induces expression of SSeCKS in rat lung microvascular endothelial cell. Mol Cell Biochem, 2007; 305(1-2):1-8.
11. Kolosova IA, Ma SF, Adyshev DM, et al. Role of CPI-17 in the regulation of endothelial cytoskeleton. Am J Physiol Lung Cell Mol Physiol, 2004; 287(5):L970-80.
12. Tinsley JH, Breslin JW, Teasdale NR, et al. PKC-dependent, burn-induced adherens junction reorganization and barrier dysfunction in pulmonary microvascular endothelial cells. Am J Physiol Lung Cell Mol Physiol, 2005; 289(2):L217-23.
13. Birukova AA, Birukov KG, Gorshkov B, et al. MAP kinases in lung endothelial permeability induced by microtubule disassembly. Am J Physiol Lung Cell Mol Physiol, 2005; 289(1):L75-84.
14. Kim KS, Rajagopal V, Gonsalves C, et al. A novel role of hypoxia-inducible factor in cobalt chloride- and hypoxia-mediated expression of IL-8 chemokine in human endothelial cells. J Immunol, 2006; 177(10):7211-24.
15. Usatyuk PV, Parinandi NL, Natarajan V. Redox regulation of 4-hydroxy-2-nonenal-mediated endothelial barrier dysfunction by focal adhesion, adherens, and tight junction proteins. J Biol Chem, 2006; 281(46):35554-66.
16. Nwariaku FE, Rothenbach P, Liu Z, et al. Rho inhibition decreases TNF-induced endothelial MAPK activation and monolayer permeability. J Appl Physiol, 2003; 95(5):1889-95.
17. Mong PY, Petrulio C, Kaufman HL, et al. Activation of Rho kinase by TNF-alpha is required for JNK activation in human pulmonary microvascular endothelial cells. J Immunol, 2008; 180(1):550-8.
18. Liu F, Verin AD, Borbiev T, et al. Role of cAMP-dependent protein kinase Aactivity in endothelial cell cytoskeleton rearrangement. Am J Physiol Lung Cell Mol Physiol, 2001; 280(6):L1309-17.
19. Ragette R, Fu C, Bhattacharya J. Barrier effects of hyperosmolar signaling in microvascular endothelium of rat lung. J Clin Invest, 1997; 100(3):685-92.
20. Siddiqui SS, Siddiqui ZK, Uddin S, et al. p38 MAPK activation coupled to endocytosis is a determinant of endothelial monolayer integrity. Am J Physiol Lung Cell Mol Physiol, 2007; 292(1):L114-24.
21. Wang Q, Yerukhimovich M, Gaarde WA, et al. MKK3 and -6-dependent activation of p38alpha MAP kinase is required for cytoskeletal changes in pulmonary microvascular endothelial cells induced by ICAM-1 ligation. Am J Physiol Lung Cell Mol Physiol, 2005; 288(2):L359-69.
22. Liu T, Guevara OE, Warburton RR, et al. Modulation of HSP27 alters hypoxia-induced endothelial permeability and related signaling pathways. J Cell Physiol, 2009; 220(3):600-10.
23. Koss M, Pfeiffer GR, 2nd, Wang Y, et al. Ezrin/radixin/moesin proteins are phosphorylated by TNF-alpha and modulate permeability increases in human pulmonary microvascular endothelial cells. J Immunol, 2006; 176(2):1218-27.
24. Yang S, Yip R, Polena S, et al. Reactive oxygen species increased focal adhesion kinase production in pulmonary microvascular endothelial cells. Proc West Pharmacol Soc, 2004; 4754-6.
25. Wu SH, Liao PY, Dong L, et al. Signal pathway involved in inhibition by lipoxin A(4) of production of interleukins induced in endothelial cells by lipopolysaccharide. Inflamm Res, 2008; 57(9):430-7.
26. Nold-Petry CA, Nold MF, Zepp JA, et al. IL-32-dependent effects of IL-1beta on endothelial cell functions. Proc Natl Acad Sci U S A, 2009; 106(10):3883-8.
27. Su X, Ao L, Zou N, et al. Post-transcriptional regulation of TNF-induced expression of ICAM-1 and IL-8 in human lung microvascular endothelial cells: an obligatoryrole for the p38 MAPK-MK2 pathway dissociated with HSP27. Biochim Biophys Acta, 2008; 1783(9):1623-31.
28. Creighton J, Zhu B, Alexeyev M, et al. Spectrin-anchored phosphodiesterase 4D4 restricts cAMP from disrupting microtubules and inducing endothelial cell gap formation. J Cell Sci, 2008; 121(Pt 1):110-9.
29. Fischmeister R. Is cAMP good or bad? Depends on where it's made. Circ Res, 2006; 98(5):582-4.
30.肖贞良,钱桂生,孙耕耘. TNF-α对大鼠肺微血管内皮细胞β-受体及其相关GRKs的影响.第三军医大学学报, 2004; 26(10):859-62.
31. Zhu B, Kelly J, Vemavarapu L, et al. Activation and induction of cyclic AMP phosphodiesterase (PDE4) in rat pulmonary microvascular endothelial cells. Biochem Pharmacol, 2004; 68(3):479-91.
32. Bogatcheva NV, Zemskova MA, Kovalenkov Y, et al. Molecular mechanisms mediating protective effect of cAMP on lipopolysaccharide (LPS)-induced human lung microvascular endothelial cells (HLMVEC) hyperpermeability. J Cell Physiol, 2009; 221(3):750-9.
33. Pfeil U, Aslam M, Paddenberg R, et al. Intermedin/adrenomedullin-2 is a hypoxia-induced endothelial peptide that stabilizes pulmonary microvascular permeability. Am J Physiol Lung Cell Mol Physiol, 2009;297(5):L837-45.
34. Fukuhara S, Sakurai A, Sano H, et al. Cyclic AMP potentiates vascular endothelial cadherin-mediated cell-cell contact to enhance endothelial barrier function through an Epac-Rap1 signaling pathway. Mol Cell Biol, 2005; 25(1):136-46.
35. Puana R, McAllister RK, Hunter FA, et al. Morphine attenuates microvascular hyperpermeability via a protein kinase A-dependent pathway. Anesth Analg, 2008; 106(2):480-5.
36. Sayner SL, Alexeyev M, Dessauer CW, et al. Soluble adenylyl cyclase reveals the significance of cAMP compartmentation on pulmonary microvascular endothelialcell barrier. Circ Res, 2006; 98(5):675-81.
37. Mong PY, Wang Q. Activation of Rho kinase isoforms in lung endothelial cells during inflammation. J Immunol, 2009; 182(4):2385-94.
38. Birukova AA, Rios A, Birukov KG. Long-term cyclic stretch controls pulmonary endothelial permeability at translational and post-translational levels. Exp Cell Res, 2008; 314(19):3466-77.
39. Solodushko V, Parker JC, Fouty B. Pulmonary microvascular endothelial cells form a tighter monolayer when grown in chronic hypoxia. Am J Respir Cell Mol Biol, 2008; 38(4):491-7.
40. An SS, Pennella CM, Gonnabathula A, et al. Hypoxia alters biophysical properties of endothelial cells via p38 MAPK- and Rho kinase-dependent pathways. Am J Physiol Cell Physiol, 2005; 289(3):C521-30.
41. Chatterjee S, Levitan I, Wei Z, et al. KATP channels are an important component of the shear-sensing mechanism in the pulmonary microvasculature. Microcirculation, 2006; 13(8):633-44.
42. Liu A, Mosher DF, Murphy-Ullrich JE, et al. The counteradhesive proteins, thrombospondin 1 and SPARC/osteonectin, open the tyrosine phosphorylation-responsive paracellular pathway in pulmonary vascular endothelia. Microvasc Res, 2009; 77(1):13-20.
43. Gong P, Angelini DJ, Yang S, et al. TLR4 signaling is coupled to SRC family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia. J Biol Chem, 2008; 283(19):13437-49.
44. Angelini DJ, Hyun SW, Grigoryev DN, et al. TNF-alpha increases tyrosine phosphorylation of vascular endothelial cadherin and opens the paracellular pathway through fyn activation in human lung endothelia. Am J Physiol Lung Cell Mol Physiol, 2006; 291(6):L1232-45.