Caveolin-1在氧化应激致肺血管通透性增加中的作用和调控机制
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摘要
研究目的：血管通透性(vascular permeability)增加及其引起的组织水肿是多种炎症性疾病如急性肺损伤(acute lung injury)等共同的病理特点。其包括跨细胞通透性(transcellular permeability)和细胞旁通透性(paracellular permeability)两个方面。在生理条件下,内皮细胞间连接严格限制血液中生物大分子如白蛋白的通过,血管内皮对白蛋白的基础通透性主要取决于caveolae介导的的跨细胞转运(transcytosis)。病理情况下,活性氧(reactive oxygen species, ROS)包括超氧阴离子(·O2-)、过氧化氢(H2O2)等产生增加,形成氧化应激状态,是诱导血管通透性增加的一个重要因素,但其作用机制尚不完全清楚。有研究表明,氧化剂主要通过破坏细胞间连接从而增加细胞旁通透性。
Caveolin-1(Cav-1)为分子量为21-24kDa的整合膜蛋白,是caveolae的主要结构和调节蛋白,在内皮细胞中表达丰富。我们前期的研究表明,caveolae介导的白蛋白跨细胞转运,不仅在生理状态下维持正常的组织胶体渗透压起决定性作用,而且在炎症(如活化的中性粒细胞)导致的肺血管通透性增加中也起重要作用,且此过程依赖于Cav-1的磷酸化。此外,近期多项研究也指出,Cav-1也参与了对细胞间粘附和细胞旁通透性的调节。但是,Cav-1对氧化应激致肺血管通透性增加的两条途径的调节作用及其调控机制目前尚无报道。
研究方法：本研究用H2O2作为外源性ROS的来源,诱导氧化应激。体外实验利用培养的大鼠肺微血管内皮细胞(Rat lung microvascular endothelial cells,RLMVECs),或用野生型Cav-1 (WT-Cav-1) cDNA和不能被磷酸化的突变型Cav-1(Y14F-Cav-1) cDNA转染RLMVECs,构建两种稳定转染细胞系,其中内源性Cav-1的表达水平未改变,但是外源性Y14F-Cav-1具有显性负效应(dominant-negative effect)可同时抑制内源性Cav-1的磷酸化,研究H2O2诱导的Cav-1磷酸化对白蛋白跨细胞通透性和细胞旁通透性的调节作用和调控机制。免疫沉淀和免疫印迹法检测蛋白Cav-1、Src激酶和c-Ab1激酶的磷酸化水平。利用小干扰RNA (Small interfering RNA, siRNA)介导的基因沉默技术,抑制Cav-1和c-Ab1激酶的表达。以甲基环糊精(methyl-β-cyclodextrin,MβCD)和Src激酶抑制剂PP2分别破环caveolae结构和抑制Src激酶活性。利用放射性同位素碘125(125Ⅰ)标记的白蛋白检测白蛋白的内吞和跨细胞转运。免疫荧光和共聚焦显微镜技术检测白蛋白内吞,Cav-1和β-catenin的细胞定位和共定位并且观察细胞间连接的完整性。测定跨内皮电阻(transendothelial electrical resistance, TER),反映内皮屏障功能(细胞旁通透性)的变化。免疫共沉淀技术测定Cav-1和β-catenin,β-catenin和VE-cadherin之间的相互作用及其变化。利用细胞膜和细胞质分离技术检测蛋白的转位。
体内实验用野生型(Cav-1+/+)和Cav-1基因敲除(Cav-1-/-)小鼠,采用新鲜制备的脂质体介导的基因转染法,将WT-Cav-1和Y14F-Cav-1 cDNA转染到Cav-1-/-小鼠体内,使肺血管内皮细胞重新表达Cav-1蛋白(WT-Cav-1或者Y14F-Cav-1),然后利用小鼠的原位离体肺灌流模型,研究H2O2诱导的Cav-1磷酸化在肺血管通透性增加和肺水肿形成中的作用。免疫印迹法检测Cav-1和磷酸化Cav-1的蛋白表达。测定125Ⅰ标记白蛋白的血管外漏出量,计算肺白蛋白通透性和表面积的乘积(Permeability-surface area [PS] product),定量评价肺微血管通透性。测量肺的湿干比,判断肺水肿程度。
结果：体外实验结果(1) H2O2 (0.05-0.8mmol/L)可增加Cav-1氨基端的第14位酪氨酸磷酸化,并呈浓度依赖性。从5min开始升高,30min达最高峰,60min恢复到基础水平。同时,Src激酶的第418位酪氨酸磷酸化与Cav-1的磷酸化同步增加；c-Abl激酶的磷酸化也呈浓度依赖性的增高。低浓度H2O2 (0.2mmol/L)引起的Cav-1磷酸化可被PP2完全抑制,而c-Abl siRNA对其无抑制作用。高浓度的H2O2(0.6mmol／L)诱导的磷酸化,只能部分被PP2抑制,并且c-Ab1 siRNA对其也有部分抑制作用。另外发现,PP2可明显抑制c-Ab1激酶的磷酸化。(2)H2O2 (0.05-0.2mmol/L)不损伤细胞间连接,不破环内皮屏障功能,但是可以浓度依赖性的增加白蛋白的细胞内吞和跨细胞转运,从而增加跨细胞通透性。并且发现,H2O2诱导的白蛋白内吞和跨细胞转运,可以被MβCD、Cav-1 siRNA以及PP2阻断,但是不受c-Ab1激酶活性的影响,揭示磷酸化的Cav-1发挥重要作用。进一步实验显示,过表达Y14F-Cav-1与过表达WT-Cav-1的细胞相比,明显减弱了H2O2介导的白蛋白内吞和跨细胞转运。(3)H2O2(0.4-0.8mmol/L)可降低TER,诱导内皮细胞间隙形成,其中H2O2 (0.4-0.6mmol/L)诱导的TER的降低可在5h内完全恢复,所以选择0.6mmol/LH2O2作为破坏正常细胞间连接,增加细胞旁通透性的药物浓度。结果显示,与正常细胞相比,0.6mmol/L H2O2可使过表达WT-Cav-1细胞的TER降低进一步加剧,而过表达Y14F-Cav-1的细胞、PP2和c-Abl siRNA均可使TER降低的幅度减弱,且c-Abl siRNA与PP2相比,可使TER更快恢复。更值得注意的是,0.2mmol/L H2O2就足以使过表达WT-Cav-1的细胞TER明显降低,而此浓度对于正常细胞和过表达Y14F-Cav-1的细胞无效；(4)免疫荧光结果显示,在正常情况下,存在明显的Cav-1与β-catenin共定位于细胞-细胞邻接处。免疫共沉淀也表明,Cav-1与β-catenin间存在相互作用。().6mmol/L H2O2可降低Cav-1与β-catenine的相互作用,使β-catenin由细胞膜转移到细胞质,破坏β-catenin和VE-cadherin的复合体,并观察到细胞间隙。而对于过表达WT-Cav-1的细胞,0.2mmol/L H2O2即可引起上述变化,但对于过表达Y14F-Cav-1的细胞无影响。
体内实验结果表明,O.5mmol/L H2O2处理Cav-1+/+小鼠,可增加PS product和肺湿干比。而处理Cav-1-/-小鼠, PS product和肺湿干比未见增加。重新表达WT-Cav-1的Cav-1-/-小鼠,可以恢复与Cav-1+/+小鼠相同的对H2O2的敏感性,而重新表达Y14F-Cav-1的Cav-1-/-小鼠仍然未发生肺通透性增加和肺水肿。
结论：(1)H2O2可诱导Cav-1的酪氨酸磷酸化。其中低浓度H2O2诱导的Cav-1磷酸化主要依赖于Src激酶,而高浓度H2O2诱导的Cav-1磷酸化同时依赖于Src和c-Ab1激酶。(2)Cav-1的磷酸化介导了低浓度的H2O2诱导的白蛋白跨细胞通透性增加。(3)Cav-1的磷酸化介导了高浓度H2O2诱导的白蛋白细胞旁通透性增加。其机制主要是通过阻碍Cav-1与细胞连接蛋白β-catenin的结合,使β-catenin由细胞膜转移到细胞质,从而减少β-catenin和VE-cadherin的复合体,破环细胞连接的稳定性。(4)Cav-1的磷酸化介导了H2O2诱导的肺血管通透性增加和肺水肿。
意义：本研究首次证明了磷酸化的Cav-1不仅参与了氧化剂诱导的血管内皮跨细胞通透性增加,更重要的是其可以调节细胞间连接,在细胞旁通透性增加中也起关键作用。首次将导致通透性增加的两条原本认为孤立的途径用一个蛋白即磷酸化的Cav-1联系起来。同时也揭示了,炎症状态下跨细胞通透性和细胞旁通透性之间可能存在协同关系。磷酸化的Cav-1可能是抑制氧化剂诱导的肺血管通透性增加,阻止肺水肿的重要的治疗靶点,因此对进一步认识急性肺损伤的发病机制及寻求更有效的治疗方法有重大意义。
Objective:Vascular endothelial hyperpermeability and protein-rich tissue edema is a key hallmark of inflammatory diseases such as acute lung injury (ALI). Transport of the most abundant plasma protein, albumin, occurs by means of transcellular and paracellular pathways. Under physiological condition, because of restrictive endothelial cell-cell contacts, transcellular transport of albumin from the endothelial lumen to the abluminal perivascular interstitium via caveolae is a primary determinant of basal endothelial permeability. The paracellular permeability induced during inflammation is formed by opening of inter-endothelial cell-cell junctions and disruption of endothelial cell-matrix contacts within the vasculature. It is well known that reactive oxygen species including superoxide and hydrogen peroxide generated by endothelial cells and neutrophils play an important role in the regulation of endothelial hyperpermeability. Although the precise mechanisms have not been completely elucidated, studies have implicated an increase in paracellular permeability via opening the interendothelial junctions by oxidant-dependent signaling mechanism.
Caveolin-1 (Cav-1), the 22-kDa integral membrane protein, is the principal structural and regulatory component of caveolae membranes. We previously showed that pulmonary vascular hyperpermeability induced by activation of neutrophils adherent to the vessel wall is dependent on signaling via Cav-1 phosphorylation and subsequently increased caveolae-mediated transcytosis. Moreover, recent evidence also points to the potential role of Cav-1 in cell-cell adhesion and thus paracellular permeability regulation. However, whether and how Cav-1 participates in the regulation of albumin transcytosis and endothelial barrier disruption induced by oxidant signaling remains important and unknown.
Methods:Rat lung microvascular endothelial cells (RLMVECs) and Cav-1 null mice (cav-1-/-) were used in our studies. RLMVEC lines stably expressing Myc-tagged wild-type Cav-1 (WT-Cav-1) or non-phosphorylatable Cav-1 mutant (Y14F-Cav-1) were generated. Western blot analysis and immunoprecipitation (IP) were performed to determine the protein levels of phosphorylation of Cav-1, Src and c-Abl kinase. Depletion of Cav-1 and c-Abl in RLMVECs was conducted with a specific siRNA. In some experiments, confluent RLMVEC monolayers were incubated with the caveolae disrupting agent methyl-β-cyclodextrin (MpCD) or Src inhibitor PP2. Uptake (endocytosis) and transcytosis of 125I-labled albumin were determined by measuring the radioactivity of samples. Fluorescently-tagged albumin endocytosis, colocalization of Cav-1 andβ-catenin, immunostaining and gap formation were determined by confocal microscopy. Endothelial barrier function was assessed by transendothelial electrical resistance (TER). The associations of Cav-1 andβ-catenin,β-catenin and vascular endothelial (VE)-cadherin were evaluated by Co-IP. Subcellular fractionation was used to isolate membrane and cytosol.
Rescue studies were made in mouse lungs from Cav-1-/- mice by liposome-mediated plasmid cDNA transfection. Myc-tagged WT-Cav-1 and Y14F-Cav-1 cDNA in pcDNA6 plasmid vector were used for Cav-1 repletion studies. Exogenous protein expression and Cav-1 and phospho-Cav-1 levels following H2O2 infusion were assessed by Western blot. In situ isolated lung was perfused with Krebs solution at a constant flow.125I-albumin permeability-surface area (PS) product, which is an index of vascular albumin permeability, was measured. Lung edema was analyzed by wet/dry lung weight ratio.
Results:1) H2O2 Induced Cav-1 Phosphorylation via Src and c-Abl Kinases. In RLMVECs, H2O2 (0.05-0.8 mmol/L) increased Cav-1 Tyr14 phosphorylation in a concentration-dependent manner. This phosphorylation increased within 5 min, peaked at 30 min, and then returned to basal levels 60 min after H2O2 treatment. Coincident with the increase in Cav-1 phosphorylation levels, we also observed phosphorylation (activation) of c-Src and c-Abl kinase. Cav-1 phosphorylation following exposure to lower concentration of H2O2 (0.2 mmol/L) was completely blocked by pretreatment with PP2 but not c-Abl siRNA. However, the effect of higher concentrations of H2O2 (0.6 mmol/L) which was only partially blocked by PP2 was also inhibited by c-Abl siRNA. Moreover, PP2 significantly blocked H2O2-induced activation of c-Abl kinase.2) Tyrosine Phosphorylation of Cav-1 Signals H2O2-Induced Transcellular Albumin Hyper-permeability. H2O2 (0.05-0.2 mmol/L) caused a concentration-dependent increase in albumin endocytosis and transendothelial transport in endothelial cells, but did not disrupt the endothelial barrier. Pretreated with the MβCD, Cav-1 siRNA and PP2 prevented H2O2-induced increase in transcellular permeability, whereas c-Abl siRNA had no effect. Furthermore, H2O2-induced increase in endocytosis and transcytosis of 125I-albumin was significantly attenuated by Y14F-Cav-1 expression compared to WT-Cav-1-expressing cells.3) Tyrosine Phosphorylation of Cav-1 Signals H2O2-Induced Paracellular Hyperpermeability. H2O2 (0.4-0.8 mmol/L) induced interendothelial cell gap formation, detected as a reduction in TER. The effect of 0.4 and 0.6 mmol/L H2O2 was reversible in that TER returned to basal levels within 5 hours, whereas endothelial cells treated with 0.8 mmol/L H2O2 did not fully recover. Accordingly,0.6 mmol/L H2O2 was chosen for subsequent experiments to explore the mechanism of H2O2-induced loss of monolayer integrity (increase in paracellular permeability).0.2 mmol/L H2O2 did not alter TER in native endothelial cells (non-transfected), but remarkably decreased TER in cells overexpressing WT-Cav-1. Importantly,0.2 mmol/L H2O2 had no effect on TER in cells expressing the same level of mutant Y14F-Cav-1. The decrease in TER induced by 0.6 mmol/L H2O2 in endothelial cells expressing WT-Cav-1 was significantly greater than the response observed in native cells. PP2 or c-Abl siRNA similarly reduced the magnitude of the H2O2-induced decrease in TER and resembled the response observed in endothelial cells expressing Y14F-Cav-1. 4) Cav-1 Phosphorylation Mediates H2O2-Induced Dissociation of VE-cadherin and P-catenin. Confocal images showed significant colocalization of Cav-1 andβ-catenin at cell-cell borders and Co-IP studies also demonstrated an association between Cav-1 with P-catenin at baseline. However, compared to untreated endothelial cells, the association between Cav-1 andβ-catenin was reduced following exposure to 0.6 mmol/L H2O2, which led to an increase inβ-catenin translocation from the membrane to the cytosolic compartments, the disruption of VE-cadherin/β-catenin complexes and wide-spread gap formation. Moreover, such changes were also observed after stimulation with only 0.2 mmol/L H2O2 in WT-Cav-1-expressing cells but not in Y14F-Cav-1-expressing cells.5) H2O2-Induced Vascular Albumin Hyperpermeability and Lung Edema Formation Requires Cav-1 Phosphorylation in Mouse Lungs. H2O2 (0.5 mmol/L) induced a robust increase in Cav-1 phosphorylation coupled to an increase in PS and wet/dry ratio in WT isolated lungs. In contrast, in cav-1-/- mouse lungs, which exhibit reduced basal albumin PS, H2O2 did not induce an increase in albumin and fluid permeability. Furthermore, the vascular hyperpermeability response to H2O2 was completely rescued by expression of WT-Cav-1 in cav-1-/- mouse lung micro vessels but was not restored by the Y14F-Cav-1 mutant.
Conclusions:H2O2 stimulated caveolae-mediated transcellular transport and the opening of interendothelial junctions in a manner dependent on Cav-1 phosphorylation. Our findings implicate Cav-1 phosphorylation as a critical mechanism mediating oxidant-induced pulmonary vascular hyperpermeability. Therefore, therapeutic inhibition of Cav-1 phosphorylation may be an effective means of limiting lung vascular injury by preventing increased transcellular albumin permeability and stabilizing the endothelial junctional barrier.
Significance:Our data show, for the first time, that oxidant-induced increase in caveolae-mediated albumin transport and decrease in endothelial barrier integrity were both dependent on tyrosine phosphorylation of Cav-1, suggesting Cav-1 phosphorylation is a common signal regulating transcellular and paracellular permeability pathways in lung micro vessels. This study provides strong evidence indicating Cav-1 phosphorylation in endothelial cells plays a fundamental role in the mechanism of oxidant-induced pulmonary vascular hyperpermeability. Although these studies focused on oxidative stress as a means of increasing endothelial permeability and inducing acute lung injury, it is possible that our findings have broader applicability in understanding the mechanisms and development of inflammatory pulmonary vascular hyperpermeability. Thus, caveolin-1 phosphorylation may be an important therapeutic target for limiting oxidant-mediated vascular hyperpermeability, protein-rich edema formation, and acute lung injury.
Caveolin-1(Cav-1)为分子量为21-24kDa的整合膜蛋白,是caveolae的主要结构和调节蛋白,在内皮细胞中表达丰富。我们前期的研究表明,caveolae介导的白蛋白跨细胞转运,不仅在生理状态下维持正常的组织胶体渗透压起决定性作用,而且在炎症(如活化的中性粒细胞)导致的肺血管通透性增加中也起重要作用,且此过程依赖于Cav-1的磷酸化。此外,近期多项研究也指出,Cav-1也参与了对细胞间粘附和细胞旁通透性的调节。但是,Cav-1对氧化应激致肺血管通透性增加的两条途径的调节作用及其调控机制目前尚无报道。
研究方法：本研究用H2O2作为外源性ROS的来源,诱导氧化应激。体外实验利用培养的大鼠肺微血管内皮细胞(Rat lung microvascular endothelial cells,RLMVECs),或用野生型Cav-1 (WT-Cav-1) cDNA和不能被磷酸化的突变型Cav-1(Y14F-Cav-1) cDNA转染RLMVECs,构建两种稳定转染细胞系,其中内源性Cav-1的表达水平未改变,但是外源性Y14F-Cav-1具有显性负效应(dominant-negative effect)可同时抑制内源性Cav-1的磷酸化,研究H2O2诱导的Cav-1磷酸化对白蛋白跨细胞通透性和细胞旁通透性的调节作用和调控机制。免疫沉淀和免疫印迹法检测蛋白Cav-1、Src激酶和c-Ab1激酶的磷酸化水平。利用小干扰RNA (Small interfering RNA, siRNA)介导的基因沉默技术,抑制Cav-1和c-Ab1激酶的表达。以甲基环糊精(methyl-β-cyclodextrin,MβCD)和Src激酶抑制剂PP2分别破环caveolae结构和抑制Src激酶活性。利用放射性同位素碘125(125Ⅰ)标记的白蛋白检测白蛋白的内吞和跨细胞转运。免疫荧光和共聚焦显微镜技术检测白蛋白内吞,Cav-1和β-catenin的细胞定位和共定位并且观察细胞间连接的完整性。测定跨内皮电阻(transendothelial electrical resistance, TER),反映内皮屏障功能(细胞旁通透性)的变化。免疫共沉淀技术测定Cav-1和β-catenin,β-catenin和VE-cadherin之间的相互作用及其变化。利用细胞膜和细胞质分离技术检测蛋白的转位。
体内实验用野生型(Cav-1+/+)和Cav-1基因敲除(Cav-1-/-)小鼠,采用新鲜制备的脂质体介导的基因转染法,将WT-Cav-1和Y14F-Cav-1 cDNA转染到Cav-1-/-小鼠体内,使肺血管内皮细胞重新表达Cav-1蛋白(WT-Cav-1或者Y14F-Cav-1),然后利用小鼠的原位离体肺灌流模型,研究H2O2诱导的Cav-1磷酸化在肺血管通透性增加和肺水肿形成中的作用。免疫印迹法检测Cav-1和磷酸化Cav-1的蛋白表达。测定125Ⅰ标记白蛋白的血管外漏出量,计算肺白蛋白通透性和表面积的乘积(Permeability-surface area [PS] product),定量评价肺微血管通透性。测量肺的湿干比,判断肺水肿程度。
结果：体外实验结果(1) H2O2 (0.05-0.8mmol/L)可增加Cav-1氨基端的第14位酪氨酸磷酸化,并呈浓度依赖性。从5min开始升高,30min达最高峰,60min恢复到基础水平。同时,Src激酶的第418位酪氨酸磷酸化与Cav-1的磷酸化同步增加；c-Abl激酶的磷酸化也呈浓度依赖性的增高。低浓度H2O2 (0.2mmol/L)引起的Cav-1磷酸化可被PP2完全抑制,而c-Abl siRNA对其无抑制作用。高浓度的H2O2(0.6mmol／L)诱导的磷酸化,只能部分被PP2抑制,并且c-Ab1 siRNA对其也有部分抑制作用。另外发现,PP2可明显抑制c-Ab1激酶的磷酸化。(2)H2O2 (0.05-0.2mmol/L)不损伤细胞间连接,不破环内皮屏障功能,但是可以浓度依赖性的增加白蛋白的细胞内吞和跨细胞转运,从而增加跨细胞通透性。并且发现,H2O2诱导的白蛋白内吞和跨细胞转运,可以被MβCD、Cav-1 siRNA以及PP2阻断,但是不受c-Ab1激酶活性的影响,揭示磷酸化的Cav-1发挥重要作用。进一步实验显示,过表达Y14F-Cav-1与过表达WT-Cav-1的细胞相比,明显减弱了H2O2介导的白蛋白内吞和跨细胞转运。(3)H2O2(0.4-0.8mmol/L)可降低TER,诱导内皮细胞间隙形成,其中H2O2 (0.4-0.6mmol/L)诱导的TER的降低可在5h内完全恢复,所以选择0.6mmol/LH2O2作为破坏正常细胞间连接,增加细胞旁通透性的药物浓度。结果显示,与正常细胞相比,0.6mmol/L H2O2可使过表达WT-Cav-1细胞的TER降低进一步加剧,而过表达Y14F-Cav-1的细胞、PP2和c-Abl siRNA均可使TER降低的幅度减弱,且c-Abl siRNA与PP2相比,可使TER更快恢复。更值得注意的是,0.2mmol/L H2O2就足以使过表达WT-Cav-1的细胞TER明显降低,而此浓度对于正常细胞和过表达Y14F-Cav-1的细胞无效；(4)免疫荧光结果显示,在正常情况下,存在明显的Cav-1与β-catenin共定位于细胞-细胞邻接处。免疫共沉淀也表明,Cav-1与β-catenin间存在相互作用。().6mmol/L H2O2可降低Cav-1与β-catenine的相互作用,使β-catenin由细胞膜转移到细胞质,破坏β-catenin和VE-cadherin的复合体,并观察到细胞间隙。而对于过表达WT-Cav-1的细胞,0.2mmol/L H2O2即可引起上述变化,但对于过表达Y14F-Cav-1的细胞无影响。
体内实验结果表明,O.5mmol/L H2O2处理Cav-1+/+小鼠,可增加PS product和肺湿干比。而处理Cav-1-/-小鼠, PS product和肺湿干比未见增加。重新表达WT-Cav-1的Cav-1-/-小鼠,可以恢复与Cav-1+/+小鼠相同的对H2O2的敏感性,而重新表达Y14F-Cav-1的Cav-1-/-小鼠仍然未发生肺通透性增加和肺水肿。
结论：(1)H2O2可诱导Cav-1的酪氨酸磷酸化。其中低浓度H2O2诱导的Cav-1磷酸化主要依赖于Src激酶,而高浓度H2O2诱导的Cav-1磷酸化同时依赖于Src和c-Ab1激酶。(2)Cav-1的磷酸化介导了低浓度的H2O2诱导的白蛋白跨细胞通透性增加。(3)Cav-1的磷酸化介导了高浓度H2O2诱导的白蛋白细胞旁通透性增加。其机制主要是通过阻碍Cav-1与细胞连接蛋白β-catenin的结合,使β-catenin由细胞膜转移到细胞质,从而减少β-catenin和VE-cadherin的复合体,破环细胞连接的稳定性。(4)Cav-1的磷酸化介导了H2O2诱导的肺血管通透性增加和肺水肿。
意义：本研究首次证明了磷酸化的Cav-1不仅参与了氧化剂诱导的血管内皮跨细胞通透性增加,更重要的是其可以调节细胞间连接,在细胞旁通透性增加中也起关键作用。首次将导致通透性增加的两条原本认为孤立的途径用一个蛋白即磷酸化的Cav-1联系起来。同时也揭示了,炎症状态下跨细胞通透性和细胞旁通透性之间可能存在协同关系。磷酸化的Cav-1可能是抑制氧化剂诱导的肺血管通透性增加,阻止肺水肿的重要的治疗靶点,因此对进一步认识急性肺损伤的发病机制及寻求更有效的治疗方法有重大意义。
Objective:Vascular endothelial hyperpermeability and protein-rich tissue edema is a key hallmark of inflammatory diseases such as acute lung injury (ALI). Transport of the most abundant plasma protein, albumin, occurs by means of transcellular and paracellular pathways. Under physiological condition, because of restrictive endothelial cell-cell contacts, transcellular transport of albumin from the endothelial lumen to the abluminal perivascular interstitium via caveolae is a primary determinant of basal endothelial permeability. The paracellular permeability induced during inflammation is formed by opening of inter-endothelial cell-cell junctions and disruption of endothelial cell-matrix contacts within the vasculature. It is well known that reactive oxygen species including superoxide and hydrogen peroxide generated by endothelial cells and neutrophils play an important role in the regulation of endothelial hyperpermeability. Although the precise mechanisms have not been completely elucidated, studies have implicated an increase in paracellular permeability via opening the interendothelial junctions by oxidant-dependent signaling mechanism.
Caveolin-1 (Cav-1), the 22-kDa integral membrane protein, is the principal structural and regulatory component of caveolae membranes. We previously showed that pulmonary vascular hyperpermeability induced by activation of neutrophils adherent to the vessel wall is dependent on signaling via Cav-1 phosphorylation and subsequently increased caveolae-mediated transcytosis. Moreover, recent evidence also points to the potential role of Cav-1 in cell-cell adhesion and thus paracellular permeability regulation. However, whether and how Cav-1 participates in the regulation of albumin transcytosis and endothelial barrier disruption induced by oxidant signaling remains important and unknown.
Methods:Rat lung microvascular endothelial cells (RLMVECs) and Cav-1 null mice (cav-1-/-) were used in our studies. RLMVEC lines stably expressing Myc-tagged wild-type Cav-1 (WT-Cav-1) or non-phosphorylatable Cav-1 mutant (Y14F-Cav-1) were generated. Western blot analysis and immunoprecipitation (IP) were performed to determine the protein levels of phosphorylation of Cav-1, Src and c-Abl kinase. Depletion of Cav-1 and c-Abl in RLMVECs was conducted with a specific siRNA. In some experiments, confluent RLMVEC monolayers were incubated with the caveolae disrupting agent methyl-β-cyclodextrin (MpCD) or Src inhibitor PP2. Uptake (endocytosis) and transcytosis of 125I-labled albumin were determined by measuring the radioactivity of samples. Fluorescently-tagged albumin endocytosis, colocalization of Cav-1 andβ-catenin, immunostaining and gap formation were determined by confocal microscopy. Endothelial barrier function was assessed by transendothelial electrical resistance (TER). The associations of Cav-1 andβ-catenin,β-catenin and vascular endothelial (VE)-cadherin were evaluated by Co-IP. Subcellular fractionation was used to isolate membrane and cytosol.
Rescue studies were made in mouse lungs from Cav-1-/- mice by liposome-mediated plasmid cDNA transfection. Myc-tagged WT-Cav-1 and Y14F-Cav-1 cDNA in pcDNA6 plasmid vector were used for Cav-1 repletion studies. Exogenous protein expression and Cav-1 and phospho-Cav-1 levels following H2O2 infusion were assessed by Western blot. In situ isolated lung was perfused with Krebs solution at a constant flow.125I-albumin permeability-surface area (PS) product, which is an index of vascular albumin permeability, was measured. Lung edema was analyzed by wet/dry lung weight ratio.
Results:1) H2O2 Induced Cav-1 Phosphorylation via Src and c-Abl Kinases. In RLMVECs, H2O2 (0.05-0.8 mmol/L) increased Cav-1 Tyr14 phosphorylation in a concentration-dependent manner. This phosphorylation increased within 5 min, peaked at 30 min, and then returned to basal levels 60 min after H2O2 treatment. Coincident with the increase in Cav-1 phosphorylation levels, we also observed phosphorylation (activation) of c-Src and c-Abl kinase. Cav-1 phosphorylation following exposure to lower concentration of H2O2 (0.2 mmol/L) was completely blocked by pretreatment with PP2 but not c-Abl siRNA. However, the effect of higher concentrations of H2O2 (0.6 mmol/L) which was only partially blocked by PP2 was also inhibited by c-Abl siRNA. Moreover, PP2 significantly blocked H2O2-induced activation of c-Abl kinase.2) Tyrosine Phosphorylation of Cav-1 Signals H2O2-Induced Transcellular Albumin Hyper-permeability. H2O2 (0.05-0.2 mmol/L) caused a concentration-dependent increase in albumin endocytosis and transendothelial transport in endothelial cells, but did not disrupt the endothelial barrier. Pretreated with the MβCD, Cav-1 siRNA and PP2 prevented H2O2-induced increase in transcellular permeability, whereas c-Abl siRNA had no effect. Furthermore, H2O2-induced increase in endocytosis and transcytosis of 125I-albumin was significantly attenuated by Y14F-Cav-1 expression compared to WT-Cav-1-expressing cells.3) Tyrosine Phosphorylation of Cav-1 Signals H2O2-Induced Paracellular Hyperpermeability. H2O2 (0.4-0.8 mmol/L) induced interendothelial cell gap formation, detected as a reduction in TER. The effect of 0.4 and 0.6 mmol/L H2O2 was reversible in that TER returned to basal levels within 5 hours, whereas endothelial cells treated with 0.8 mmol/L H2O2 did not fully recover. Accordingly,0.6 mmol/L H2O2 was chosen for subsequent experiments to explore the mechanism of H2O2-induced loss of monolayer integrity (increase in paracellular permeability).0.2 mmol/L H2O2 did not alter TER in native endothelial cells (non-transfected), but remarkably decreased TER in cells overexpressing WT-Cav-1. Importantly,0.2 mmol/L H2O2 had no effect on TER in cells expressing the same level of mutant Y14F-Cav-1. The decrease in TER induced by 0.6 mmol/L H2O2 in endothelial cells expressing WT-Cav-1 was significantly greater than the response observed in native cells. PP2 or c-Abl siRNA similarly reduced the magnitude of the H2O2-induced decrease in TER and resembled the response observed in endothelial cells expressing Y14F-Cav-1. 4) Cav-1 Phosphorylation Mediates H2O2-Induced Dissociation of VE-cadherin and P-catenin. Confocal images showed significant colocalization of Cav-1 andβ-catenin at cell-cell borders and Co-IP studies also demonstrated an association between Cav-1 with P-catenin at baseline. However, compared to untreated endothelial cells, the association between Cav-1 andβ-catenin was reduced following exposure to 0.6 mmol/L H2O2, which led to an increase inβ-catenin translocation from the membrane to the cytosolic compartments, the disruption of VE-cadherin/β-catenin complexes and wide-spread gap formation. Moreover, such changes were also observed after stimulation with only 0.2 mmol/L H2O2 in WT-Cav-1-expressing cells but not in Y14F-Cav-1-expressing cells.5) H2O2-Induced Vascular Albumin Hyperpermeability and Lung Edema Formation Requires Cav-1 Phosphorylation in Mouse Lungs. H2O2 (0.5 mmol/L) induced a robust increase in Cav-1 phosphorylation coupled to an increase in PS and wet/dry ratio in WT isolated lungs. In contrast, in cav-1-/- mouse lungs, which exhibit reduced basal albumin PS, H2O2 did not induce an increase in albumin and fluid permeability. Furthermore, the vascular hyperpermeability response to H2O2 was completely rescued by expression of WT-Cav-1 in cav-1-/- mouse lung micro vessels but was not restored by the Y14F-Cav-1 mutant.
Conclusions:H2O2 stimulated caveolae-mediated transcellular transport and the opening of interendothelial junctions in a manner dependent on Cav-1 phosphorylation. Our findings implicate Cav-1 phosphorylation as a critical mechanism mediating oxidant-induced pulmonary vascular hyperpermeability. Therefore, therapeutic inhibition of Cav-1 phosphorylation may be an effective means of limiting lung vascular injury by preventing increased transcellular albumin permeability and stabilizing the endothelial junctional barrier.
Significance:Our data show, for the first time, that oxidant-induced increase in caveolae-mediated albumin transport and decrease in endothelial barrier integrity were both dependent on tyrosine phosphorylation of Cav-1, suggesting Cav-1 phosphorylation is a common signal regulating transcellular and paracellular permeability pathways in lung micro vessels. This study provides strong evidence indicating Cav-1 phosphorylation in endothelial cells plays a fundamental role in the mechanism of oxidant-induced pulmonary vascular hyperpermeability. Although these studies focused on oxidative stress as a means of increasing endothelial permeability and inducing acute lung injury, it is possible that our findings have broader applicability in understanding the mechanisms and development of inflammatory pulmonary vascular hyperpermeability. Thus, caveolin-1 phosphorylation may be an important therapeutic target for limiting oxidant-mediated vascular hyperpermeability, protein-rich edema formation, and acute lung injury.
引文
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46. Shajahan AN, Timblin BK, Sandoval R, Tiruppathi C, Malik AB, Minshall RD. Role of Src induced dynamin-2 phosphorylation in caveolae-mediated endocytosis in endothelial cells. J Biol Chem.2004;279:20392-20400.
47. Xu N, Rahman A, Minshall RD, Tiruppathi C, Malik AB. β2-Integrin blockade driven by Eselectinpromoter prevents neutrophil sequestration and lung injury in mice. Circ Res.2000;87:254-260.
48. Hu G, Ye RD, Dinauer MC, Malik AB, Minshall RD. Neutrophil caveolin-1 expression contributes to mechanism of lung inflammation and injury. Am J Physiol. 2008;294:L178-186.
49. Kern DF, Malik AB:Microvascular albumin permeability in isolated perfused lung: effects of EDTA. JAppl Physiol.1985;58:372-375.
50. Tiruppathi C, Song W, Bergenfeldt M, Sass P, Malik AB. Gp60 activation mediates
albumin transcytosis in endothelial cells by a tyrosine kinase-dependent pathway. J Biol Chem.1997;272:25968-25975.
1 Bokoch GM, Knaus UG. NADPH oxidases:Not just for leukocytes anymore! Trends Biochem. Sci,2003,28 (9):502-508.
2 Hu G, Place AT, Minshall RD. Regulation of endothelial permeability by Src kinase signaling:Vascular leakage versus transcellular transport of drugs and macromolecules. Chem Biol Interact,2008,171(2):177-189.
3 Wallez Y, Cand F, Cruzalegui F, et al. Src kinase phosphorylates vascular endothelial-cadherin in response to vascular endothelial growth factor:Identification of tyrosine 685 as the unique target site. Oncogene,2007,26 (7):1067-1077.
4 Baumeister U, Funke R., Ebnet K, et al. Association of Csk to VE-cadherin and inhibition of cell proliferation. Embo. J,2005,24 (9):1686-1695.
5 Gavard J, Gutkind JS. VEGF controls endothelial-cell permeability by promoting
the beta-arrestin-dependent endocytosis of VE-cadherin. Nat. Cell. Biol,2006,8 (11):1223-1234.
6 Usatyuk PV, Natarajan V. Regulation of reactive oxygen species-induced endothelial cell-cell and cell-matrix contacts by focal adhesion kinase and adherens junction proteins. Am. J. Physiol. Lung. Cell. Mol. Physiol,2005,289 (6): L999-L1010.
7 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,2004,47:54-56.
8 Quadri SK, Bhattacharya J. Resealing of endothelial junctions by focal adhesion kinase. Am. J. Physiol. Lung. Cell. Mol. Physiol,2007,292 (1):L334-L342.
9 van Buul JD, Anthony EC, Fernandez-Borja M,et al. Proline-rich tyrosine kinase 2 (Pyk2) mediates vascular endothelial-cadherin-based cell-cell adhesion by regulating beta-catenin tyrosine phosphorylation. J. Biol. Chem,2005,280 (22): 21129-21136.
10 Verin AD. Tyrosine phosphorylation and endothelial cell barrier regulation. American Journal of Pathology,2005,166(4):955-957.
11 Stockton RA, Schaefer E, Schwartz MA. p21-activated kinase regulates endothelial permeability through modulation of contractility. J. Biol. Chem,2004,279 (45): 46621-46630.
12 Martyn KD, Kim MJ, Quinn MT, et al. p21-activated kinase (Pak) regulates N.A. DPH oxidase activation in human neutrophils. Blood,2005,106 (12):3962-3969.
13 Pantano C, Shrivastava P, McElhinney B, et al. Hydrogen peroxide signaling through tumor necrosis factor receptor 1 leads to selective activation of c-Jun N-terminal kinase. J. Biol. Chem,2003,278 (45):44091-44096.
14 Mong PY, Petrulio C, Kaufman H.L, 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),550-558.
15 Yuan SY, Breslin JW, Perrin R, et al. Microvascular permeability in diabetes and
insulin resistance. Microcirculation,2007,14 (4-5):363-373.
16 Waschke J, Golenhofen N, Kurzchalia TV, et al. Protein kinase C-mediated endothelial barrier regulation is caveolin-1-dependent. Histochem. Cell. Biol,2006, 126(1):17-26.
17 Tonks NK. Redox redux:Revisiting PTPs and the control of cell signaling. Cell, 2005,121 (5):667-670.
18 Shasby DM. Cell-cell adhesion in lung endothelium. Am. J. Physiol. Lung. Cell. Mol. Physiol,2007,292 (3):L593-L607.
19 Xu G, Craig AW, Greer P, et al. Continuous association of cadherin with beta-catenin requires the non-receptor tyrosine-kinase Fer. J. Cell. Sci,2004,117 (Pt 15):3207-3219.
20 Sui XF, Kiser TD, Hyun SW, et al. Receptor protein tyrosine phosphatase micro regulates the paracellular pathway in human lung microvascular endothelia. Am. J. Pathol,2005,166 (4):1247-1258.
21 Ahmmed GU, Malik, AB. Functional role of TRPC channels in the regulation of endothelial permeability. Pflugers Arch,2005,451 (1):131-142.
22 Tiruppathi C, Ahmmed GU, Vogel SM, et al. Ca2+signaling, TRP channels, and endothelial permeability. Microcirculation,2006,13 (8):693-708.
23 Kwan HY, Huang Y, Yao X. TRP channels in endothelial function and dysfunction. Biochim. Biophys. Acta,2007,1772 (8):907-914.
24 Singh I, Knezevic N, Ahmmed GU, et al.Galphaq-TRPC6-mediated Ca2+entry induces RhoA activation and resultant endothelial cell shape change in response to thrombin. J Biol Chem,2007,282:7833-7843.
25 Ahmmed GU, Mehta D, Vogel S, et al. Protein kinase Calpha phosphorylates the TRPC1 channel and regulates store-operated Ca2+entry in endothelial cells. J. Biol. Chem,2004,279 (20):20941-20949.
26 Mehta D, Ahmmed GU, Paria BC, et al. RhoA interaction with inositol 1,4,5-trisphosphate receptor and transient receptor potential channel-1 regulates Ca2+entry. Role in signaling increased endothelial permeability. J. Biol. Chem, 2003,278 (35):33492-33500.
27 Alvarez DF, King JA, Weber D, et al. Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier:A novel mechanism of acute lung injury. Circ. Res,2006,99 (9):988-995.
28 Townsley MI, King JA, Alvarez DF. Ca2+channels and pulmonary endothelial permeability:Insights from study of intact lung and chronic pulmonary hypertension. Microcirculation,2006,13 (8):725-739.
29 Hecquet CM, Ahmmed GU, Vogel SM, et al. Role of TRPM2 channel in mediating H2O2-induced Ca2+entry and endothelial hyperpermeability. Circ. Res,2008, 102:347-355.
30 Biswas P, Canosa S, Schoenfeld D, et al. PECAM-1 affects GSK-3beta-mediated beta-catenin phosphorylation and degradation. Am. J. Pathol,2006,169 (1): 314-324.
31 Maas M, Wang R, Paddock C, et al. Reactive oxygen species induce reversible PECAM-1 tyrosine phosphorylation and SHP-2 binding. Am. J. Physiol. Heart Circ. Physiol,2003,285 (6):H2336-H2344.
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26 Miyawaki-Shimizu K, Predescu D, Shimizu J, Broman M, Predescu S, Malik AB. siRNA-induced caveolin-1 knockdown in mice increases lung vascular permeability via the junctional pathway. Am J Physiol.2006;290:L405-L413.
27 Waschke J, Golenhofen N, Kurzchalia TV, Drenckhahn D. Protein kinase C-mediated endothelial barrier regulation is caveolin-1-dependent. Histochem Cell Biol.2006;126:17-26.
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44. John TA, Vogel SM, Tiruppathi C, Malik AB, Minshall RD. Quantitative analysis of albumin uptake and transport in the rat micro vessel endothelial monolayer. Am J Physiol.2003;284:L187-196.
45. Tiruppathi C, Malik AB, Del Vecchio PJ, Keese CR, Giaever I. Electrical method for detection of endothelial cell shape change in real time:assessment of endothelial barrier function. Proc Natl Acad Sci USA.1992;89:7919-7923.
46. Shajahan AN, Timblin BK, Sandoval R, Tiruppathi C, Malik AB, Minshall RD. Role of Src induced dynamin-2 phosphorylation in caveolae-mediated endocytosis in endothelial cells. J Biol Chem.2004;279:20392-20400.
47. Xu N, Rahman A, Minshall RD, Tiruppathi C, Malik AB. β2-Integrin blockade driven by Eselectinpromoter prevents neutrophil sequestration and lung injury in mice. Circ Res.2000;87:254-260.
48. Hu G, Ye RD, Dinauer MC, Malik AB, Minshall RD. Neutrophil caveolin-1 expression contributes to mechanism of lung inflammation and injury. Am J Physiol. 2008;294:L178-186.
49. Kern DF, Malik AB:Microvascular albumin permeability in isolated perfused lung: effects of EDTA. JAppl Physiol.1985;58:372-375.
50. Tiruppathi C, Song W, Bergenfeldt M, Sass P, Malik AB. Gp60 activation mediates
albumin transcytosis in endothelial cells by a tyrosine kinase-dependent pathway. J Biol Chem.1997;272:25968-25975.
1 Bokoch GM, Knaus UG. NADPH oxidases:Not just for leukocytes anymore! Trends Biochem. Sci,2003,28 (9):502-508.
2 Hu G, Place AT, Minshall RD. Regulation of endothelial permeability by Src kinase signaling:Vascular leakage versus transcellular transport of drugs and macromolecules. Chem Biol Interact,2008,171(2):177-189.
3 Wallez Y, Cand F, Cruzalegui F, et al. Src kinase phosphorylates vascular endothelial-cadherin in response to vascular endothelial growth factor:Identification of tyrosine 685 as the unique target site. Oncogene,2007,26 (7):1067-1077.
4 Baumeister U, Funke R., Ebnet K, et al. Association of Csk to VE-cadherin and inhibition of cell proliferation. Embo. J,2005,24 (9):1686-1695.
5 Gavard J, Gutkind JS. VEGF controls endothelial-cell permeability by promoting
the beta-arrestin-dependent endocytosis of VE-cadherin. Nat. Cell. Biol,2006,8 (11):1223-1234.
6 Usatyuk PV, Natarajan V. Regulation of reactive oxygen species-induced endothelial cell-cell and cell-matrix contacts by focal adhesion kinase and adherens junction proteins. Am. J. Physiol. Lung. Cell. Mol. Physiol,2005,289 (6): L999-L1010.
7 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,2004,47:54-56.
8 Quadri SK, Bhattacharya J. Resealing of endothelial junctions by focal adhesion kinase. Am. J. Physiol. Lung. Cell. Mol. Physiol,2007,292 (1):L334-L342.
9 van Buul JD, Anthony EC, Fernandez-Borja M,et al. Proline-rich tyrosine kinase 2 (Pyk2) mediates vascular endothelial-cadherin-based cell-cell adhesion by regulating beta-catenin tyrosine phosphorylation. J. Biol. Chem,2005,280 (22): 21129-21136.
10 Verin AD. Tyrosine phosphorylation and endothelial cell barrier regulation. American Journal of Pathology,2005,166(4):955-957.
11 Stockton RA, Schaefer E, Schwartz MA. p21-activated kinase regulates endothelial permeability through modulation of contractility. J. Biol. Chem,2004,279 (45): 46621-46630.
12 Martyn KD, Kim MJ, Quinn MT, et al. p21-activated kinase (Pak) regulates N.A. DPH oxidase activation in human neutrophils. Blood,2005,106 (12):3962-3969.
13 Pantano C, Shrivastava P, McElhinney B, et al. Hydrogen peroxide signaling through tumor necrosis factor receptor 1 leads to selective activation of c-Jun N-terminal kinase. J. Biol. Chem,2003,278 (45):44091-44096.
14 Mong PY, Petrulio C, Kaufman H.L, 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),550-558.
15 Yuan SY, Breslin JW, Perrin R, et al. Microvascular permeability in diabetes and
insulin resistance. Microcirculation,2007,14 (4-5):363-373.
16 Waschke J, Golenhofen N, Kurzchalia TV, et al. Protein kinase C-mediated endothelial barrier regulation is caveolin-1-dependent. Histochem. Cell. Biol,2006, 126(1):17-26.
17 Tonks NK. Redox redux:Revisiting PTPs and the control of cell signaling. Cell, 2005,121 (5):667-670.
18 Shasby DM. Cell-cell adhesion in lung endothelium. Am. J. Physiol. Lung. Cell. Mol. Physiol,2007,292 (3):L593-L607.
19 Xu G, Craig AW, Greer P, et al. Continuous association of cadherin with beta-catenin requires the non-receptor tyrosine-kinase Fer. J. Cell. Sci,2004,117 (Pt 15):3207-3219.
20 Sui XF, Kiser TD, Hyun SW, et al. Receptor protein tyrosine phosphatase micro regulates the paracellular pathway in human lung microvascular endothelia. Am. J. Pathol,2005,166 (4):1247-1258.
21 Ahmmed GU, Malik, AB. Functional role of TRPC channels in the regulation of endothelial permeability. Pflugers Arch,2005,451 (1):131-142.
22 Tiruppathi C, Ahmmed GU, Vogel SM, et al. Ca2+signaling, TRP channels, and endothelial permeability. Microcirculation,2006,13 (8):693-708.
23 Kwan HY, Huang Y, Yao X. TRP channels in endothelial function and dysfunction. Biochim. Biophys. Acta,2007,1772 (8):907-914.
24 Singh I, Knezevic N, Ahmmed GU, et al.Galphaq-TRPC6-mediated Ca2+entry induces RhoA activation and resultant endothelial cell shape change in response to thrombin. J Biol Chem,2007,282:7833-7843.
25 Ahmmed GU, Mehta D, Vogel S, et al. Protein kinase Calpha phosphorylates the TRPC1 channel and regulates store-operated Ca2+entry in endothelial cells. J. Biol. Chem,2004,279 (20):20941-20949.
26 Mehta D, Ahmmed GU, Paria BC, et al. RhoA interaction with inositol 1,4,5-trisphosphate receptor and transient receptor potential channel-1 regulates Ca2+entry. Role in signaling increased endothelial permeability. J. Biol. Chem, 2003,278 (35):33492-33500.
27 Alvarez DF, King JA, Weber D, et al. Transient receptor potential vanilloid 4-mediated disruption of the alveolar septal barrier:A novel mechanism of acute lung injury. Circ. Res,2006,99 (9):988-995.
28 Townsley MI, King JA, Alvarez DF. Ca2+channels and pulmonary endothelial permeability:Insights from study of intact lung and chronic pulmonary hypertension. Microcirculation,2006,13 (8):725-739.
29 Hecquet CM, Ahmmed GU, Vogel SM, et al. Role of TRPM2 channel in mediating H2O2-induced Ca2+entry and endothelial hyperpermeability. Circ. Res,2008, 102:347-355.
30 Biswas P, Canosa S, Schoenfeld D, et al. PECAM-1 affects GSK-3beta-mediated beta-catenin phosphorylation and degradation. Am. J. Pathol,2006,169 (1): 314-324.
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