The role of VASP in regulation of cAMP- and Rac 1-mediated endothelial barrier stabilization

Regulation of actin dynamics is critical for endothelial barrier functions. We provide evidence that the actin-binding protein vasodilator-stimulated phosphoprotein (VASP) is required for endothelial barrier maintenance. Baseline permeability was significantly increased in VASP-deficient (VASP(-/-)) microvascular myocardial endothelial cells (MyEnd) in the absence of discernible alterations of immunostaining for adherens and tight junctions. We tested whether VASP is involved in the endothelium-stabilizing effects of cAMP or Rac 1. Forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 (CNF-1) to activate Rac 1 were equally efficient to stabilize barrier functions in VASP(-/-) and wild-type (wt) cells. In wt cells, VASP was phosphorylated in response to F/R but did not localize to intercellular junctions. In contrast, CNF-1 and expression of constitutively active Rac 1 induced translocation of VASP to cell borders in wt cells, where it colocalized with active Rac 1. In VASP(-/-) cells, Rac 1 activity was reduced to 0.4 of wt levels in controls and increased approximately 20-fold in response to CNF-1 compared with 7-fold activation in wt cells. Moreover, inactivation of Rac 1 by lethal toxin led to a greater increase of permeability compared with wt cells. All these data suggest that VASP is involved in the regulation of Rac 1 activity. Taking these findings together, our study indicates that VASP at least in part stabilizes endothelial barrier functions by control of Rho-family GTPases.     

Increased spreading,Rac/p21-activated kinase (PAK) activity,and compromised cell motility in cells deficient in vasodilator-stimulated phosphoprotein (VASP)

Ena/VASP (Drosophila Enabled/vasodilator-stimulated phosphoprotein) proteins are key regulators that promote or inhibit actin-based motility, cell adhesion, and various aspects of axon guidance. However, a conclusive concept of Ena/VASP functions remains elusive. Here, we report that VASP-deficient fibroblasts, despite normal mammalian Enabled (Mena) and Ena-VASP-like (Evl) expression levels, are highly spread. VASP(-/-) cells cover about twice the substrate surface area as wild type cells, while cell volumes are unchanged. In accordance with these observations, activation of the Rac/p21-activated kinase (PAK) pathway, a crucial element in the regulation of cell spreading, is markedly enhanced in VASP(-/-) cells. Thus, in the absence of VASP Rac activation is dramatically prolonged, and PAK activity is elevated after stimulation with platelet-derived growth factor or serum, respectively. Moreover, VASP-deficient cells show compromised migration and reorientation in a wound healing assay. Collectively, our results reveal a VASP-dependent modulation of the Rac/PAK pathway and Rac/PAK-regulated processes, like cell motility and polarization.     

Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alphaII-spectrin-VASP complexes

Directed cortical actin assembly is the driving force for intercellular adhesion. Regulated by phosphorylation, vasodilator-stimulated phosphoprotein (VASP) participates in actin fiber formation. We screened for endothelial proteins, which bind to VASP, dependent on its phosphorylation status. Differential proteomics identified αII-spectrin as such a VASP-interacting protein. αII-Spectrin binds to the VASP triple GP₅-motif via its SH3 domain. cAMP-dependent protein kinase–mediated VASP phosphorylation at Ser157 inhibits αII-spectrin–VASP binding. VASP is dephosphorylated upon formation of cell–cell contacts and in confluent, but not in sparse cells, αII-spectrin colocalizes with nonphosphorylated VASP at cell–cell junctions. Ectopic expression of the αII-spectrin SH3 domain at cell–cell contacts translocates VASP, initiates cortical actin cytoskeleton formation, stabilizes cell–cell contacts, and decreases endothelial permeability. Conversely, the permeability of VASP-deficient endothelial cells (ECs) and microvessels of VASP-null mice increases. Reconstitution of VASP-deficient ECs rescues barrier function, whereas αII-spectrin binding-deficient VASP mutants fail to restore elevated permeability. We propose that αII-spectrin–VASP complexes regulate cortical actin cytoskeleton assembly with implications for vascular permeability.     

Macrophage migration inhibitory factor is induced by thrombin and factor Xa in endothelial cells

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, has been shown to play a role in wound-healing processes. In this study, we investigated whether protease-activated receptor (PAR)-1 and PAR-2 mediated MIF expression in human endothelial cells. Thrombin, factor Xa (FXa), and trypsin induced MIF expression in human dermal microvascular endothelial cells and human umbilical vein endothelial cells, but other proteases, including kallikrein and urokinase, failed to do so. Thrombin-induced MIF mRNA expression was significantly reduced by the thrombin-specific inhibitor hirudin. Thrombin receptor activation peptide-6, a synthetic PAR-1 peptide, induced MIF mRNA expression, suggesting that PAR-1 mediates MIF expression in response to thrombin. The effects of FXa were blocked by antithrombin III, but not by hirudin, indicating that FXa might enhance MIF production directly rather than via thrombin stimulation. The synthetic PAR-2 peptide SLIGRL-NH(2) induced MIF mRNA expression, showing that PAR-2 mediated MIF expression in response to FXa. Concerning the signal transduction, a mitogen-activated protein kinase kinase inhibitor (PD98089) and a nuclear factor (NF)-kappaB inhibitor (SN50) suppressed the up-regulation of MIF mRNA in response to thrombin, FXa, and PAR-2 agonist stimulation, whereas a p38 inhibitor (SB203580) had little effect. These facts indicate that up-regulation of MIF by thrombin or FXa is regulated by p44/p42 mitogen-activated protein kinase-dependent pathways and NF-kappaB-dependent pathways. Moreover, we found that PAR-1 and PAR-2 mRNA expression in endothelial cells was enhanced by MIF. Furthermore, we examined the inflammatory response induced by PAR-1 and PAR-2 agonists injected into the mouse footpad. As shown by footpad thickness, an indicator of inflammation, MIF-deficient mice (C57BL/6) were much less sensitive to either PAR-1 or PAR-2 agonists than wild-type mice. Taken together, these results suggest that MIF contributes to the inflammatory phase of the wound healing process in concert with thrombin and FXa via PAR-1 and PAR-2.     

Modulation of Rac1 activity by ADMA/DDAH regulates pulmonary endothelial barrier function

Endogenously produced nitric oxide synthase inhibitor, asymmetric methylarginine (ADMA) is associated with vascular dysfunction and endothelial leakage. We studied the role of ADMA, and the enzymes metabolizing it, dimethylarginine dimethylaminohydrolases (DDAH) in the regulation of endothelial barrier function in pulmonary macrovascular and microvascular cells in vitro and in lungs of genetically modified heterozygous DDAHI knockout mice in vivo. We show that ADMA increases pulmonary endothelial permeability in vitro and in in vivo and that this effect is mediated by nitric oxide (NO) acting via protein kinase G (PKG) and independent of reactive oxygen species formation. ADMA-induced remodeling of actin cytoskeleton and intercellular adherens junctions results from a decrease in PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and a subsequent down-regulation of Rac1 activity. The effects of ADMA on endothelial permeability, Rac1 activation and VASP phosphorylation are prevented by overexpression of active DDAHI and DDAHII, whereas inactive DDAH mutants have no effect. These findings demonstrate for the first time that ADMA metabolism critically determines pulmonary endothelial barrier function by modulating Rac1-mediated remodeling of the actin cytoskeleton and intercellular junctions.     

Impaired integrin‐mediated adhesion contributes to reduced barrier properties in VASP‐deficient microvascular endothelium

Recent studies point to a significant role of vasodilator‐stimulated phosphoprotein (VASP) in the maintenance of endothelial barrier functions in vivo and in vitro. Moreover, it has been reported that VASP is required for activation of the small GTPase Rac 1. However, little is known whether VASP is involved in the regulation of cell adhesion molecules that are critical for maintenance of the endothelial barrier. Here we demonstrate that impaired barrier properties in VASP‐deficient (VASP?/?) microvascular myocardial endothelial cells (MyEnd) correlated with both impaired integrin‐mediated adhesion as revealed by laser tweezer trapping and reduced integrin‐dependent cell migration. This was paralleled by reduction of focal adhesions at the cell periphery as well as of β₁‐integrin and VE‐cadherin cytoskeletal anchorage. Incubation of MyEnd VASP wt with RGD peptide to block interaction of integrins with extracellular matrix (ECM) reduced barrier properties and Rac 1 activity in wt endothelial monolayers mimicking the situation in VASP (?/?) cells under resting conditions. Moreover, cAMP‐mediated Rac 1 activation was reduced under conditions of impaired integrin‐mediated adhesion in wt cells and cAMP‐induced increase in VE‐cadherin cytoskeletal anchorage was abolished in VASP (?/?) endothelium. In summary, these data indicate that VASP is required for integrin‐mediated adhesion which stabilizes endothelial barrier properties at least in part by facilitating Rac 1 activation. J. Cell. Physiol. 220: 357–366, 2009. © 2009 Wiley‐Liss, Inc.     

Involvement of Local Lamellipodia in Endothelial Barrier Function

Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.     

Thrombin enhances the barrier function of rat microvascular endothelium in a PAR-1-dependent manner

Thrombin is a multifunctional coagulation protease with pro- and anti-inflammatory vascular effects. We questioned whether thrombin may have segmentally differentiated effects on pulmonary endothelium. In cultured rat endothelial cells, rat thrombin (10 U/ml) recapitulated the previously reported decrease in transmonolayer electrical resistance (TER), F-actin stress fiber formation, paracellular gap formation, and increased permeability. In contrast, in rat pulmonary microvascular endothelial cells (PMVEC), isolated on the basis of Griffonia simplicifolia lectin recognition, thrombin increased TER, induced fewer stress fibers, and decreased permeability. To assess for differential proteinase-activated receptor (PAR) expression as a basis for the different responses, PAR family expression was analyzed. Both pulmonary artery endothelial cells and PMVEC expressed PAR-1 and PAR-2; however, only PMVEC expressed PAR-3, as shown by both RT-PCR and Western analysis. PAR-1 activating peptides (PAR-APs: SFLLRN-NH(2) and TFLLRN-NH(2)) were used to confirm a role for the PAR-1 receptor. PAR-APs (25-250 muM) also increased TER, formed fewer stress fibers, and did not induce paracellular gaps in PMVEC in contrast to that shown in pulmonary artery endothelial cells. These results were confirmed in isolated perfused rat lung preparations. PAR-APs (100 mug/ml) induced a 60% increase in the filtration coefficient over baseline. However, by transmission electron microscopy, perivascular fluid cuffs were seen only along conduit veins and arteries without evidence of intra-alveolar edema. We conclude that thrombin exerts a segmentally differentiated effect on endothelial barrier function in vitro, which corresponds to a pattern of predominant perivascular fluid cuff formation in situ. This may indicate a distinct role for thrombin in the microcirculation.     

Inhibitory effect of thrombin on the expression of secretory group IIA phospholipase A₂

It is well known that the expression level of secretory group IIA phospholipase A(2) (sPLA(2)-IIA) is elevated in inflammatory diseases and lipopolysaccharide (LPS) up-regulates the expression of sPLA(2)-IIA in human umbilical vein endothelial cells (HUVECs). Recently, lower concentration thrombin could elicit anti-inflammatory responses in HUVECs. Here, the effects of lower concentration thrombin on the expression of sPLA(2)-IIA in LPS-stimulated HUVECs were investigated. Prior treatment of cells with thrombin (25-75 pM) inhibited LPS-induced sPLA(2)-IIA expression by activating its receptor, protease-activated receptor-1 (PAR-1). And pretreatment of cells with either PI3-kinase inhibitor (LY294002) or cholesterol depleting agent (methyl-β-cyclodextrin, MβCD) abolished the inhibitory activity of thrombin against sPLA(2)-IIA expression. Therefore, these results suggest that PAR-1 activation by lower concentration thrombin inhibited LPS mediated expression of sPLA(2)-IIA by PAR-1 and PI3-kinase-dependent manner in lipid raft on the HUVECs.     

Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger

Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na⁺/Ca²⁺ exchanger (NCX) operating in the Ca²⁺-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca²⁺ influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na⁺-K⁺-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans'' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.     

Rac1/MAPK/ERK 通路介导脂多糖LPS 诱导的人内皮细胞 单层通透性增高机制研究

目的:探讨LPS诱导的人内皮细胞单层通透性改变的分子机制。方法:应用逆转录病毒为载体,感染并筛选稳定表达持续活化型Rac1和主导抑制型Rac1的人HUVEC细胞,应用LPS刺激并观察细胞骨架蛋白F-actin和HUVEC单层通透性的改变。同时应用Western blot方法检测LPS刺激前后细胞中MAPK/ERK信号通路的改变及加入PD98059阻断ERK表达后,细胞内F-actin的改变情况。结果:与正常HUVEC相比较,LPS刺激后,感染活化型Rac1和主导抑制型Rac1的HUVEC中F-actin重构并形成大量应力纤维,细胞单层通透性显著增加。而抑制型Rac1感染后的HUVEC中F-actin无重构现象,同时细胞单层通透性无明显增加。LPS刺激前后,各组细胞中ERK1/2总蛋白均无明显改变。LPS刺激后,感染活化型Rac1的HUVEC中,p-ERK增加。经PD98059阻断后,细胞内p-ERK表达下降同时伴随F-actin解聚发生。结论:LPS诱导的内皮细胞通透性增加是经过Rac1-MAPK/ERK通路介导的。     

Thrombin-induced ATP release from human umbilical vein endothelial cells

ATP and its degradation products play an important role as signaling molecules in the vascular system, and endothelial cells are considered to be an important source of nucleotide release. To investigate the mechanism and physiological significance of endothelial ATP release, we compared different pharmacological stimuli for their ability to evoke ATP release from first passage cultivated human umbilical vein endothelial cells (HUVECs). Agonists known to increase intracellular Ca(2+) levels (A23187, histamine, thrombin) induced a stable, non-lytic ATP release. Since thrombin proved to be the most robust and reproducible stimulus, the molecular mechanism of thrombin-mediated ATP release from HUVECs was further investigated. ATP rapidly increased with thrombin (1 U/ml) and reached a steady-state level after 4 min. Loading the cells with BAPTA-AM to capture intracellular calcium suppressed ATP release. The thrombin-specific, protease-activated receptor 1 (PAR-1)-specific agonist peptide TFLLRN (10 μM) fully mimicked thrombin action on ATP release. To identify the nature of the ATP-permeable pathway, we tested various inhibitors of potential ATP channels for their ability to inhibit the thrombin response. Carbenoxolone, an inhibitor of connexin hemichannels and pannexin channels, as well as Gd(3+) were highly effective in blocking the thrombin-mediated ATP release. Specifically targeting connexin43 (Cx43) and pannexin1 (Panx1) revealed that reducing Panx1 expression significantly reduced ATP release, while downregulating Cx43 was ineffective. Our study demonstrates that thrombin at physiological concentrations is a potent stimulus of endothelial ATP release involving PAR-1 receptor activation and intracellular calcium mobilization. ATP is released by a carbenoxolone- and Gd(3+)- sensitive pathway, most likely involving Panx1 channels.     

Thrombin inhibits HMGB1-mediated proinflammatory signaling responses when endothelial protein C receptor is occupied by its natural ligand

High mobility group box 1 (HMGB1) is involved in the pathogenesis of vascular diseases. Unlike activated protein C (APC), the activation of PAR-1 by thrombin is known to elicit proinflammatory responses. To determine whether the occupancy of EPCR by the Gla-domain of APC is responsible for the PAR-1-dependent antiinflammatory activity of the protease, we pretreated HUVECs with the PC zymogen and then activated PAR-1 with thrombin. It was found that thrombin downregulates the HMGB1-mediated induction of both TNF-α and IL-6 and inhibits the activation of both p38 MAPK and NF-κB in HUVECs pretreated with PC. Furthermore, thrombin inhibited HMGB1-mediated hyperpermeability and leukocyte adhesion/migration by inhibiting the expression of cell adhesion molecules in HUVECs if EPCR was occupied. Collectively, these results suggest the concept that thrombin can initiate proinflammatory responses in vascular endothelial cells through the activation of PAR-1 may not hold true for normal vessels expressing EPCR under in vivo conditions. [BMB Reports 2013; 46(11): 544-549]     

Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked Ras/mitogen-activated protein kinase pathway

We addressed the mechanisms of restoration of cell surface proteinase-activated receptor-1 (PAR-1) by investigating thrombin-activated signaling pathways involved in PAR-1 re-expression in endothelial cells. Exposure of endothelial cells transfected with PAR-1 promoter-luciferase reporter construct to either thrombin or PAR-1 activating peptide increased the steady-state PAR-1 mRNA and reporter activity, respectively. Pretreatment of reporter-transfected endothelial cells with pertussis toxin or co-expression of a minigene encoding 11-amino acid sequence of COOH-terminal Galphai prevented the thrombin-induced increase in reporter activity. Pertussis toxin treatment also prevented thrombin-induced MAPK phosphorylation, indicating a role of Galphai in activating the downstream MAPK pathway. Expression of constitutively active Galphai2 mutant or Gbeta1gamma2 subunits increased reporter activity 3-4-fold in the absence of thrombin stimulation. Co-expression of dominant negative mutants of either Ras or MEK1 with the reporter construct inhibited the thrombin-induced PAR-1 expression, whereas constitutively active forms of either Ras or MEK1 activated PAR-1 expression in the absence of thrombin stimulation. Expression of dominant negative Src kinase or inhibitors of phosphoinositide 3-kinase also prevented the MAPK activation and PAR-1 expression. We conclude that thrombin-induced activation of PAR-1 mediates PAR-1 expression by signaling through Gi1/2 coupled to Src and phosphoinositide 3-kinase, and thereby activating the downstream Ras/MAPK cascade.     

Thrombin Has Biphasic Effects on the Nitric Oxide-cGMP Pathway in Endothelial Cells and Contributes to Experimental Pulmonary Hypertension

Background

A potential role for coagulation factors in pulmonary arterial hypertension has been recently described, but the mechanism of action is currently not known. Here, we investigated the interactions between thrombin and the nitric oxide-cGMP pathway in pulmonary endothelial cells and experimental pulmonary hypertension.

Principal Findings

Chronic treatment with the selective thrombin inhibitor melagatran (0.9 mg/kg daily via implanted minipumps) reduced right ventricular hypertrophy in the rat monocrotaline model of experimental pulmonary hypertension. In vitro, thrombin was found to have biphasic effects on key regulators of the nitric oxide-cGMP pathway in endothelial cells (HUVECs). Acute thrombin stimulation led to increased expression of the cGMP-elevating factors endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) subunits, leading to increased cGMP levels. By contrast, prolonged exposition of pulmonary endothelial cells to thrombin revealed a characteristic pattern of differential expression of the key regulators of the nitric oxide-cGMP pathway, in which specifically the factors contributing to cGMP elevation (eNOS and sGC) were reduced and the cGMP-hydrolyzing PDE5 was elevated (qPCR and Western blot). In line with the differential expression of key regulators of the nitric oxide-cGMP pathway, a reduction of cGMP by prolonged thrombin stimulation was found. The effects of prolonged thrombin exposure were confirmed in endothelial cells of pulmonary origin (HPAECs and HPMECs). Similar effects could be induced by activation of protease-activated receptor-1 (PAR-1).

Conclusion

These findings suggest a link between thrombin generation and cGMP depletion in lung endothelial cells through negative regulation of the nitric oxide-cGMP pathway, possibly mediated via PAR-1, which could be of relevance in pulmonary arterial hypertension.     

Protease-activated receptor-2 (PAR-2) in brain microvascular endothelium and its regulation by plasmin and elastase

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. We have reported earlier that primary cultures of rat brain capillary endothelial (RBCE) cells express at least two receptors for thrombin: PAR-1 and PAR-3. In the present study we show that PAR-2 activation by trypsin or by the PAR-2 agonist peptide (SLIGRL) evokes [Ca(2+) ](i) signal in RBCE cells. Taking advantage of RBCE cells expressing PAR-1 and PAR-2, we show that trypsin activates both receptors. The relative agonist activity of trypsin and thrombin on PARs of RBCE cells compared with that of SLIGRL were 112% and 48%, respectively, whereas the potency of trypsin was 10(5) -fold higher than that of SLIGRL. Because under pathological conditions other proteases such as plasmin or leukocyte elastase may reach the cells of the blood-brain barrier, we investigated the effect of these proteases on RBCE cells. Elastase evoked a small increase in [Ca(2+) ](i) but preincubation of cells with elastase dose-dependently reduced the trypsin-induced [Ca(2+) ](i) signal. Plasmin had a 30% inhibitory effect on the trypsin-induced response, and reduced the SLIGRL signal by 20%. It is concluded that PAR-2 is functional in brain capillary endothelium, and that the main fibrinolytic proteases, plasmin and elastase, may regulate PAR-2 signalling under pathological conditions.     

Extracellular β‐nicotinamide adenine dinucleotide (β‐NAD) promotes the endothelial cell barrier integrity via PKA‐ and EPAC1/Rac1‐dependent actin cytoskeleton rearrangement

Extracellular β‐NAD is known to elevate intracellular levels of calcium ions, inositol 1,4,5‐trisphate and cAMP. Recently, β‐NAD was identified as an agonist for P2Y1 and P2Y11 purinergic receptors. Since β‐NAD can be released extracellularly from endothelial cells (EC), we have proposed its involvement in the regulation of EC permeability. Here we show, for the first time, that endothelial integrity can be enhanced in EC endogenously expressing β‐NAD‐activated purinergic receptors upon β‐NAD stimulation. Our data demonstrate that extracellular β‐NAD increases the transendothelial electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration‐dependent manner indicating endothelial barrier enhancement. Importantly, β‐NAD significantly attenuated thrombin‐induced EC permeability as well as the barrier‐compromising effects of Gram‐negative and Gram‐positive bacterial toxins representing the barrier‐protective function of β‐NAD. Immunofluorescence microscopy reveals more pronounced staining of cell–cell junctional protein VE‐cadherin at the cellular periphery signifying increased tightness of the cell‐cell contacts after β‐NAD stimulation. Interestingly, inhibitory analysis (pharmacological antagonists and receptor sequence specific siRNAs) indicates the participation of both P2Y1 and P2Y11 receptors in β‐NAD‐induced TER increase. β‐NAD‐treatment attenuates the lipopolysaccharide (LPS)‐induced phosphorylation of myosin light chain (MLC) indicating its involvement in barrier protection. Our studies also show the involvement of cAMP‐dependent protein kinase A and EPAC1 pathways as well as small GTPase Rac1 in β‐NAD‐induced EC barrier enhancement. With these results, we conclude that β‐NAD regulates the pulmonary EC barrier integrity via small GTPase Rac1‐ and MLCP‐ dependent signaling pathways. J. Cell. Physiol. 223: 215–223, 2010. © 2010 Wiley‐Liss, Inc.     

Plasmin-mediated activation of platelets occurs by cleavage of protease-activated receptor 4

The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmin-induced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a protease-resistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4.     

胰蛋白酶诱导人脐静脉内皮细胞分泌白细胞介素-8

目的：研究胰蛋白酶对IL-8释放的影响。方法：分离、培养人脐静脉内皮细胞（human umbilical vein endothelialcells,HU-VECs）、倒置显微镜观察形态变化,流式细胞术检测内皮细胞标志和蛋白酶活化受体．2（proteinase．activatedreceptor．2,PAR-2）表达,ELISA检测HUVECs培养上清中IL-8水平。结果：HUVECs表达内皮细胞标志和PAR-2。刺激16h,1g／ml胰蛋白酶和100MPAR-2激活肽组HUVECs单层均匀性降低。胰蛋白酶能够显著刺激HUVECs释放IL-8,PAR-2激活肽也诱导IL-8水平升高。蛋白酶抑制剂和PAR-2抑制肽均能够显著抑制胰蛋白酶诱导的IL-8释放。PAR-2激活肽和胰蛋白酶诱导升高的IL-8水平之间成正相关性。结论：胰蛋白酶很可能通过PAR-2激活促进血管内皮细胞释放IL-8。     

Hypoxia/Aglycemia-Induced Endothelial Barrier Dysfunction and Tight Junction Protein Downregulation Can Be Ameliorated by Citicoline

This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs.