Vascular Barrier Disruption Research Articles

Key Regulatory Effect of Activated HIF-1α/VEGFA Signaling Pathway in Systemic Capillary Leak Syndrome Confirmed by Bioinformatics Analysis

Systemic capillary leak syndrome (SCLS) is a rare disorder characterized by capillary leakage of plasma fluids throughout the endothelium. The mechanism of SCLS is still unknown. Vascular endothelial growth factor (VEGF), an inducer or barrier disruption, is markedly upregulated in SCLS. This study was to investigate the molecular mechanisms involving SCLS-related inflammation and neuron damage in SCLS remain unclear. Data files of GSE97287 dataset were extracted and processed for identification of differentially expressed genes (DEGs), including upregulated adrenomedullin (ADM) gene, hypoxia-inducible factor-1α (HIF-1α) and VEGFA; and downregulated aldehyde dehydrogenase 1A1 (ALDH1A1) gene and interleukin (IL)-2 receptor β (IL-2RB) gene. Weighted gene coexpression network analysis (WGCNA) was performed for DEGs and four significant modules were identified and were enriched Gene Ontology (GO) biological processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to immune response, angiogenesis, neuroblast proliferation, HIF-1 signaling pathway, and Parkinson's disease. The activated HIF-1α/VEGFA signaling in SCLS patients might well be responsible for the impaired inflammatory, nervous, and immune systems.

Microbiota-driven gut vascular barrier disruption is a prerequisite for non-alcoholic steatohepatitis development

Fatty liver disease, including non-alcoholic fatty liver (NAFLD) and steatohepatitis (NASH), has been associated with increased intestinal barrier permeability and translocation of bacteria or bacterial products into the blood circulation. In this study, we aimed to unravel the role of both intestinal barrier integrity and microbiota in NAFLD/NASH development. C57BL/6J mice were fed with high-fat diet (HFD) or methionine-choline-deficient diet for 1 week or longer to recapitulate aspects of NASH (steatosis, inflammation, insulin resistance). Genetic and pharmacological strategies were then used to modulate intestinal barrier integrity. We show that disruption of the intestinal epithelial barrier and gut vascular barrier (GVB) are early events in NASH pathogenesis. Mice fed HFD for only 1 week undergo a diet-induced dysbiosis that drives GVB damage and bacterial translocation into the liver. Fecal microbiota transplantation from HFD-fed mice into specific pathogen-free recipients induces GVB damage and epididymal adipose tissue enlargement. GVB disruption depends on interference with the WNT/β-catenin signaling pathway, as shown by genetic intervention driving β-catenin activation only in endothelial cells, preventing GVB disruption and NASH development. The bile acid analogue and farnesoid X receptor agonist obeticholic acid (OCA) drives β-catenin activation in endothelial cells. Accordingly, pharmacologic intervention with OCA protects against GVB disruption, both as a preventive and therapeutic agent. Importantly, we found upregulation of the GVB leakage marker in the colon of patients with NASH. We have identified a new player in NASH development, the GVB, whose damage leads to bacteria or bacterial product translocation into the blood circulation. Treatment aimed at restoring β-catenin activation in endothelial cells, such as administration of OCA, protects against GVB damage and NASH development. The incidence of fatty liver disease is reaching epidemic levels in the USA, with more than 30% of adults having NAFLD (non-alcoholic fatty liver disease), which can progress to more severe non-alcoholic steatohepatitis (NASH). Herein, we show that disruption of the intestinal epithelial barrier and gut vascular barrier are early events in the development of NASH. We show that the drug obeticholic acid protects against barrier disruption and thereby prevents the development of NASH, providing further evidence for its use in the prevention or treatment of NASH.

Gas6 Attenuates Sepsis-Induced Tight Junction Injury and Vascular Endothelial Hyperpermeability via the Axl/NF-κB Signaling Pathway

Vascular endothelial functional dysregulation and barrier disruption are involved the initiation and development of sepsis. Growth arrest-specific protein 6 (Gas6), one of the endogenous ligands of TAM receptors (Tyro3, Axl, and Mertk), is confirmed to have beneficial functions in hemostasis, inflammation, and cancer growth. Here, we demonstrated the protective effects of Gas6 on multi-organ dysfunction syndrome (MODS) in sepsis and the underlying mechanisms. We investigated Gas6-ameliorated MODS by inhibiting vascular endothelial hyperpermeability in a mouse model of sepsis. Additionally, in vitro, under lipopolysaccharide (LPS) stimulation in vascular endothelial cells, Gas6 attenuated vascular endothelial hyperpermeability by reinforcing the tight junction proteins occludin, zonula occludens-1 (ZO-1), and claudin5. Furthermore, Gas6 substantially suppressed NF-κB p65 activation. In addition, blocking the Gas6 receptor, Axl, partially reduced the protective effect of Gas6 on the vascular endothelial barrier and diminished the inhibitive effect of Gas6 on NF-κB p65 activation. Taken together, this study suggests that Gas6 has a protective effect on MODS in sepsis by inhibiting the vascular endothelial hyperpermeability and alteration of tight junction and that the Axl/NF-κB signaling pathway underlies these effects.

Inhibitory effects of collismycin C and pyrisulfoxin A on particulate matter-induced pulmonary injury

BackgroundInhalation of fine particulate matter (PM2.5) is associated with elevated pulmonary injury caused by the loss of vascular barrier integrity. Marine microbial natural products isolated from microbial culture broths were screened for pulmonary protective effects against PM2.5. Two 2,2′-bipyridine compounds isolated from a red alga-associated Streptomyces sp. MC025—collismycin C (2) and pyrisulfoxin A (5)—were found to inhibit PM2.5-mediated vascular barrier disruption. PurposeTo confirm the inhibitory effects of collismycin C and pyrisulfoxin A on PM2.5-induced pulmonary injury Study designIn this study, we investigated the beneficial effects of collismycin C and pyrisulfoxin A on PM-induced lung endothelial cell (EC) barrier disruption and pulmonary inflammation. MethodsPermeability, leukocyte migration, proinflammatory protein activation, reactive oxygen species (ROS) generation, and histology were evaluated in PM2.5-treated ECs and mice. ResultsCollismycin C and pyrisulfoxin A significantly scavenged PM2.5-induced ROS and inhibited the ROS-induced activation of p38 mitogen-activated protein kinase as well as activated Akt, which helped in maintaining endothelial integrity, in purified pulmonary endothelial cells. Furthermore, collismycin C and pyrisulfoxin A reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in the bronchoalveolar lavage fluid of PM-treated mice. ConclusionThese data suggested that collismycin C and pyrisulfoxin A might exert protective effects on PM-induced inflammatory lung injury and vascular hyperpermeability.

Inhibition of interleukin-6 trans-signaling prevents inflammation and endothelial barrier disruption in retinal endothelial cells

Vascular inflammation plays a critical role in the pathogenesis of diabetic retinopathy. Recently, Interleukin-6 (IL-6) trans-signaling via soluble IL-6 receptor (sIL-6R) has emerged as a prominent regulator of inflammation in endothelial cells. This study was designed to test the hypothesis that selective inhibition of the IL-6 trans-signaling pathway will attenuate inflammation and subsequent barrier disruption in retinal endothelial cells. Human retinal endothelial cells (HRECs) were exposed to IL-6 and sIL-6R to induce IL-6 trans-signaling and the commercially available compound sgp130Fc (soluble gp-130 fused chimera) was used to selectively inhibit IL-6 trans-signaling. IL-6 trans-signaling activation caused a significant increase in STAT3 phosphorylation, expression of adhesion molecules, ROS production and apoptosis in HRECs whereas a significant decrease in mitochondrial membrane potential and NO production was observed in IL-6 trans-signaling activated cells. These changes were not observed in cells pre-treated with sgp130Fc. IL-6 trans-signaling activation was sufficient to cause barrier disruption in endothelial monolayers and pre-treatment of HRECs with sgp130Fc, maintained endothelial barrier function similar to that of untreated cells. Thus, in conclusion, these results indicate that IL-6 trans-signaling is an important mediator of inflammation, apoptosis and barrier disruptive effects in the retinal endothelial cells and inhibition of the IL-6 trans-signaling pathway using sgp130-Fc attenuates vascular inflammation and endothelial barrier disruption.

Schisandrin Attenuates Lipopolysaccharide-Induced Lung Injury by Regulating TLR-4 and Akt/FoxO1 Signaling Pathways.

Objective: Acute lung injury is a severe clinic condition with limited therapeutic approaches. This study evaluated whether schisandrin (Sch), an ingredient of Schisandra chinensis, has preventive effects on endothelium and epithelium injury induced by lipopolysaccharide (LPS) and the underlying mechanisms.Methods: Male Wistar rats were continuously infused with LPS (5 mg/kg/h) via the left jugular vein for 90 min. In some rats, Sch (2.5 mg/kg/h) was administrated through the left jugular vein 30 min before LPS infusion. Leukocyte recruitment, levels of inflammatory cytokines, lung histology and edema, vascular and alveolar barrier disruption and related proteins were evaluated at indicated time point after LPS challenge.Results: LPS infusion for 90 min resulted in an increased leukocyte adhesion to pulmonary venules and overproduction of cytokine and chemokine in both serum and lung homogenate. At 8 h after termination of LPS infusion, obvious Evans blue extravasation and lung edema were observed, along with an increased apoptosis, a decreased expression of tight junction and adherent junction proteins, and a reduction in von Willebrand factor (vWF) and keratin, all of which were attenuated by Sch treatment. Meanwhile, the LPS-elicited activation of TLR-4/NF-κB/MAPK and FoxO1 signaling was inhibited by Sch.Conclusion: The present study revealed that pretreatment with Sch alleviated lung endothelium and epithelium injury after LPS stimulation, which is attributable to inhibition of cell injury and activation of cell regeneration via regulation of TLR-4/NF-κB/MAPK and Akt/FoxO1 signaling pathway.

The E3 Ubiquitin Ligase WWP2 Regulates Endosomal Trafficking of the Purinergic Receptor P2Y1

P2Y1 is a G protein‐coupled receptor (GPCR) for adenosine diphosphate (ADP) that signals to produce pro‐inflammatory effects in blood vessels. P2Y1 signaling contributes to vascular endothelial barrier disruption, leading to cardiovascular diseases such as atherosclerosis. When bound to ADP, P2Y1 internalizes and is either recycled for reuse at the cell surface or sorted to and degraded by lysosomes to attenuate signaling. P2Y1 lysosomal sorting is mediated by binding to the adaptor protein ALIX. Since ALIX activity is regulated by ubiquitination by the WW domain‐containing E3 ubiquitin protein ligase 2 (WWP2), we hypothesized that WWP2 is required for P2Y1 degradation. Here, we show that P2Y1 colocalizes with WWP2 at late endosomes upon ADP agonist stimulation. In addition, we demonstrate that depletion of endogenous WWP2 inhibits P2Y1 sorting into the lumen of late endosomes. We are currently examining how receptor signaling regulates WWP2 translocation to late endosomes and interaction with ALIX. Our evidence suggests that WWP2 is required at endosomes for P2Y1 degradation in response to agonist stimulation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PAR1 and p38 MAPK Regulation of Endothelial Pro‐Inflammatory Responses

Endothelial cells line the lumen of blood vessels and create a semi‐permeable barrier that is disrupted during vascular inflammation resulting in increased permeability, tissue edema, and subsequent organ failure. Endothelial dysfunction also results in cytokine production and leukocyte recruitment. However, the molecular mechanisms behind this pro‐inflammatory state remains to be elucidated. Protease‐activated receptor‐1 (PAR1) is a G protein‐coupled receptor for the coagulant protease thrombin and mediates hemostasis, thrombosis, and inflammatory response to vascular injury. However, the distinct mechanism of how endothelial PAR1 promotes vascular pro‐inflammatory responses represents a major gap in knowledge. We recently discovered that thrombin activation of PAR1 stimulates p38 MAPK activation through a non‐canonical pathway mediated by TAB1 to promote endothelial permeability. PAR1‐induced p38 activation has been linked to vascular barrier disruption; however, the mechanism as to how p38 may be integrated into major signaling pathways that promote pro‐inflammatory responses is not known. We found that thrombin‐stimulated p38 signals independent of the classic g‐protein pathways of RhoA activation and MLC phosphorylation to induce endothelial barrier permeability. Instead, thrombin‐stimulated p38 appears to be signaling through phosphorylation of the MK2 and HSP27. We further show MK2 activity is PAR1 and p38 dependent and regulates differential phosphorylation of its direct substrate, HSP27. Additionally we show HSP27 activity is p38 and PAR1‐dependent. The p38‐MK2‐HSP27 signaling pathway also appears to modulate HUVEC monolayer permeability. These studies show thrombin‐induced p38 signals through MK2 and HSP27 cascade to regulate endothelial barrier permeability.Support or Funding InformationT32 Pharmocological Sciences Training Grant (NIGMS T32 GM007752) (C. Rada) NIH NIGM R01 GM116597 (J. Trejo)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

LPS-induced Acute Lung Injury Involves NF-κB-mediated Downregulation of SOX18.

One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.

Antiseptic effects of dabrafenib on TGFBIp-induced septic responses

Transforming growth factor-β-induced protein (TGFBIp), an extracellular protein, is expressed on several cell types in response to TGF-β stimulation. Human umbilical vein endothelial cell (HUVEC)-derived TGFBIp functions as a mediator of sepsis. Screening of bioactive compound libraries is an effective approach for repositioning FDA-approved drugs or discovering new treatments for human diseases (drug repositioning). Dabrafenib (DAB), a B-Raf inhibitor, was initially used for treating metastatic melanoma. The present study determined whether DAB modulated TGFBIp-mediated septic responses in HUVECs and in mice. Antiseptic functions of DAB were examined by measuring permeability, leukocyte adhesion and migration, and proinflammatory protein activation in TGFBIp-stimulated HUVECs and mice. In addition, beneficial effects of DAB on survival rate were examined using a mouse model of sepsis. We found that DAB inhibited TGFBIp-induced vascular barrier disruption, cell adhesion molecule (CAM) expression, and neutrophil adhesion/transendothelial migration toward human endothelial cells. DAB also suppressed TGFBIp-induced hyperpermeability and leukocyte migration in vivo. These results suggest that DAB exerts anti-inflammatory effects by inhibiting hyperpermeability, CAM expression, and leukocyte adhesion and migration, indicating its utility for treating vascular inflammatory diseases.

Pharmacologic Protection of Mitochondrial DNA Integrity May Afford a New Strategy for Suppressing Lung Ischemia-Reperfusion Injury.

Lung ischemia-reperfusion (IR) injury contributes to post-transplant complications, including primary graft dysfunction. Decades of reports show that reactive oxygen species generated during lung IR contribute to pulmonary vascular endothelial barrier disruption and edema formation, but the specific target molecule(s) that "sense" injury-inducing oxidant stress to activate signaling pathways culminating in pathophysiologic changes have not been established. This review discusses evidence that mitochondrial DNA (mtDNA) may serve as a molecular sentinel wherein oxidative mtDNA damage functions as an upstream trigger for lung IR injury. First, the mitochondrial genome is considerably more sensitive than nuclear DNA to oxidant stress. Multiple studies suggest that oxidative mtDNA damage could be transduced to physiologic dysfunction by pathways that are either a direct consequence of mtDNA damage per se or involve formation of proinflammatory mtDNA damage-associated molecular patterns. Second, transgenic animals or cells overexpressing components of the base excision DNA repair pathway in mitochondria are resistant to oxidant stress-mediated pathophysiologic effects. Finally, published and preliminary studies show that pharmacologic enhancement of mtDNA repair or mtDNA damage-associated molecular pattern degradation suppresses reactive oxygen species-induced or IR injury in multiple organs, including preclinical models of lung procurement for transplant. Collectively, these findings point to the interesting prospect that pharmacologic enhancement of DNA repair during procurement or ex vivo lung perfusion may increase the availability of lungs for transplant and reduce the IR injury contributing to primary graft dysfunction.

Peripherally derived FGF21 promotes remyelination in the central nervous system.

Demyelination in the central nervous system (CNS) leads to severe neurological deficits that can be partially reversed by spontaneous remyelination. Because the CNS is isolated from the peripheral milieu by the blood-brain barrier, remyelination is thought to be controlled by the CNS microenvironment. However, in this work we found that factors derived from peripheral tissue leak into the CNS after injury and promote remyelination in a murine model of toxin-induced demyelination. Mechanistically, leakage of circulating fibroblast growth factor 21 (FGF21), which is predominantly expressed by the pancreas, drives proliferation of oligodendrocyte precursor cells (OPCs) through interactions with β-klotho, an essential coreceptor of FGF21. We further confirmed that human OPCs expressed β-klotho and proliferated in response to FGF21 in vitro. Vascular barrier disruption is a common feature of many CNS disorders; thus, our findings reveal a potentially important role for the peripheral milieu in promoting CNS regeneration.

Endothelial lipid phosphate phosphatase-3 deficiency that disrupts the endothelial barrier function is a modifier of cardiovascular development.

AimsLipid phosphate phosphatase-3 (LPP3) is expressed at high levels in endothelial cells (ECs). Although LPP3 is known to hydrolyse the phosphate group from lysolipids such as spingosine-1-phosphate and its structural homologues, the function of Lpp3 in ECs is not completely understood. In this study, we investigated how tyrosine-protein kinase receptor (TEK or Tie2) promoter–dependent deletion of Lpp3 alters EC activities.Methods and resultsLpp3fl/fl mice were crossed with the tg.Tie2Cre transgenic line. Vasculogenesis occurred normally in embryos with Tie2Cre-mediated deletion of Lpp3 (called Lpp3ECKO), but embryonic lethality occurred in two waves, the first wave between E8.5 and E10.5, while the second between E11.5 and E13.5. Lethality in Lpp3ECKO embryos after E11.5 was accompanied by vascular leakage and haemorrhage, which likely resulted in insufficient cardiovascular development. Analyses of haematoxylin- and eosin-stained heart sections from E11.5 Lpp3ECKO embryos showed insufficient heart growth associated with decreased trabeculation, reduced growth of the compact wall, and absence of cardiac cushions. Staining followed by microscopic analyses of Lpp3ECKO embryos revealed the presence of apoptotic ECs. Furthermore, Lpp3-deficient ECs showed decreased gene expression and protein levels of Cyclin-D1, VE-cadherin, Fibronectin, Klf2, and Klf4. To determine the underlying mechanisms of vascular leakage and barrier disruption, we performed knockdown and rescue experiments in cultured ECs. LPP3 knockdown decreased transendothelial electrical resistance and increased permeability. Re-expression of β-catenin cDNA in LPP3-knockdown ECs partially restored the effect of the LPP3 loss, whereas re-expression of p120ctn cDNA did not.ConclusionThese findings demonstrate the essential roles of LPP3 in the maturation of EC barrier integrity and normal cardiovascular development.

C-type natriuretic peptide prevents activation of perivascular mast cells and inflammation in the postischemic microvasculature

Background Atrial- and C-type- natriuretic peptides (ANP, CNP) are both vasodilatatory hormones which are mainly secreted from cardiac atria and vascular endothelium, respectively. Both peptides activate cyclic GMP (cGMP) formation by binding to their specific receptors: the particulate guanylyl cyclases A (GC-A) and B (GC-B). Cyclic GMP via cGMP-dependent protein kinase I (cGKI) was shown to inhibit mast cell (MC) degranulation [1]. During postischemic reperfusion, the activation of MC is detrimental [2]. Due to pleiotropic effects, cytokines released from MC cause tissue damage and vascular leakage. It is unclear whether and how ANP and/or CNP regulate the activity of MC and, thereby, influence ischemia-reperfusion-induced disruption of the microvascular barrier. Methods and results Intravital microscopy was used to visualize the microcirculation within the cremaster muscle of anesthetized mice, and Fluorescein isothiocyanate (FITC)-Dextran was injected intravenously to quantify the microvascular leakage [3,4]. Cremaster blood flow was temporally blocked with an arterial clamp to provoke 30 min of tissue ischemia. During the following 30 min of postischemic reperfusion, the vascular leakage was measured by the ratio of the fluorescent (FITC-Dextran) intensity (RFI) between the adjacent interstitial space and the intravascular lumen. Subsequently resident perivascular degranulated MCs were stained by superfusing Ruthenium Red solution and thereafter counted (number per field). In wildtype mice, ischemia-reperfusion (compared to sham operated cremaster muscles) provoked marked MC degranulation and robust vascular leakage of FITCDextran (as indicated by higher RFI). Local CNP superfusion during reperfusion significantly reduced the amount of degranulated MCs and concomitantly vascular leakage. These effects were mimicked by the membrane-permeable cGKI activator 8-Br-cGMP. The protective effects of 8-Br-cGMP were endothelium-independent, since they were preserved in mice with conditional, endothelial deletion of cGKI. Quantitative real-time RT-PCR revealed local CNP-mRNA expression in the m. cremaster, which was not altered by acute ischemia. In contrast to CNP, ANP superfusion did not significantly alter the amount of degranulated MCs, but still effectively reduced FITC-Dextran leakage resulted from ischemia-reperfusion. Accordingly, in vitro, in cultured human MCs (HMC-1.1, originally developed by Dr. Butterfield and a gift from Dr. Drube) and primary cultured murine bone marrow MC, CNP, but not ANP, induced a marked increase in intracellular cGMP levels and enhanced the phosphorylation of the cytoskeleton-associated vasoactive stimulated phosphoprotein (VASP) at Ser237. Conclusions Our study shows divergent effects of NPs in the postischemic microcirculation. CNP inhibited the ischemic activation of resident, perivascular MCs and consequently the associated microvascular leakage, possibly via MC specific cGMP/cGKI signaling. At difference, ANP had no effect on MCs but still attenuated the postischemic vascular barrier disruption via direct effects on endothelial cells. Our future studies aim to dissect the

Prostacyclin Prevents Pericyte Loss and Demyelination Induced by Lysophosphatidylcholine in the Central Nervous System

Pericytes play pivotal roles in physiological and pathophysiological conditions in the central nervous system. As pericytes prevent vascular leakage, they can halt neuronal damage stemming from a compromised blood-brain barrier. Therefore, pericytes may be a good target for the treatment of neurodegenerative disorders, although evidence is lacking. In this study, we show that prostacyclin attenuates lysophosphatidylcholine (LPC)-mediated vascular dysfunction through pericyte protection in the adult mouse spinal cord. LPC decreased the number of pericytes in an in vitro blood-brain barrier model, and this decrease was prevented by iloprost treatment, a prostacyclin analog. Intrathecal administration of iloprost attenuated vascular barrier disruption after LPC injection in the mouse spinal cord. Furthermore, iloprost treatment diminished demyelination and motor function deficits in mice injected with LPC. These results support the notion that prostacyclin acts on pericytes to maintain vascular barrier integrity.

Quinic acid derivatives from Salicornia herbacea alleviate HMGB1-mediated endothelial dysfunction

The high mobility group box 1 protein (HMGB1) has been targeted in the discovery of dietary supplements or medicinal resources to treat vascular inflammatory diseases. Salicornia herbacea has been utilized as a seasoned vegetable, salad, and traditional medicinal resource as well. Two new (1 and 2) and four known (3–6) caffeoylated quinic acids (CQAs) were isolated from the crude extract of S. herbacea exhibiting anti-HMGB1 activity. The isolated CQAs were further evaluated for their potential to ameliorate HMGB1-mediated vascular barrier disruption. Compounds 1, 3, 5, and 6 exerted vascular protective activity against HMGB1-induced inflammatory responses in both cellular and animal models and their mechanisms were also addressed. The active CQAs increased survival rates of cecal ligation and puncture-induced severe septic models to 20–40%. This study may serve the groundwork for commercializing CQAs as functional food components for prevention and treatment of pathogenic conditions related to endothelial hyperpermeability.

P38/MAPK contributes to endothelial barrier dysfunction via MAP4 phosphorylation-dependent microtubule disassembly in inflammation-induced acute lung injury.

Excessive activation of inflammation and the accompanying lung vascular endothelial barrier disruption are primary pathogenic features of acute lung injury (ALI). Microtubule-associated protein 4 (MAP4), a tubulin assembly-promoting protein, is important for maintaining the microtubule (MT) cytoskeleton and cell-cell junctional structures. However, both the involvement and exact mechanism of MAP4 in the development of endothelial barrier disruption in ALI remains unknown. In this study, lipopolysaccharide (LPS) and tumour necrosis factor-α (TNF-α) were applied to human pulmonary microvascular endothelial cells (HPMECs) to mimic the endothelial damage during inflammation in vitro. We demonstrated that the MAP4 (Ser696 and Ser787) phosphorylation increased concomitantly with the p38/MAPK pathway activation by the LPS and TNF-α stimulation of HPMECs, which induced MT disassembly followed by hyperpermeability. Moreover, the application of taxol, the overexpression of a MAP4 (Ala) mutant, or the application of the p38/MAPK inhibitor SB203580 inhibited the MT disruption and the intracellular junction dysfunction. In contrast, MKK6 (Glu), which constitutively activated p38/MAPK, resulted in microtubule depolymerisation and, subsequently, hyperpermeability. Our findings reveal a novel role of MAP4 in endothelial barrier dysfunction.

Sphingosine-1-phosphate receptor 2 protects against anaphylactic shock through suppression of endothelial nitric oxide synthase in mice

Sphingosine-1-phosphate receptor 2 (S1P(2)) is expressed in vascular endothelial cells (ECs). However, the role of S1P(2) in vascular barrier integrity and anaphylaxis is not well understood. Endothelial nitric oxide synthase (eNOS) generates nitric oxide to mediate vascular leakage, compromising survival in patients with anaphylaxis. We recently observed that endothelial S1P(2) inhibits Akt, an activating kinase of eNOS. We tested the hypothesis that endothelial S1P(2) might suppress eNOS, exerting a protective effect against endothelial barrier disruption and anaphylaxis. Mice deficient in S1P(2) and eNOS underwent antigen challenge or platelet-activating factor (PAF) injection. Analyses were performed to examine vascular permeability and the underlying mechanisms. S1pr2 deletion augmented vascular leakage and lethality after either antigen challenge or PAF injection. PAF injection induced activation of Akt and eNOS in the aortas and lungs of S1pr2-null mice, which were augmented compared with values seen in wild-type mice. Consistently, PAF-induced increase in cyclic guanosine monophosphate levels in the aorta was enhanced in S1pr-null mice. Genetic Nos3 deletion or pharmacologic eNOS blockade protected S1pr2-null mice from aggravation of barrier disruption after antigen challenge and PAF injection. ECs isolated from S1pr2-null mice exhibited greater stimulation of Akt and eNOS, with enhanced nitric oxide production in response to sphingosine-1-phosphate or PAF, compared with that seen in wild-type ECs. Moreover, S1pr2-deficient ECs showed more severe disassembly of adherens junctions with augmented S-nitrosylation of β-catenin in response to PAF, which was restored by pharmacologic eNOS blockade. S1P(2) diminishes harmful robust eNOS stimulation and thereby attenuates vascular barrier disruption, suggesting potential usefulness of S1P(2) agonists as novel therapeutic agents for anaphylaxis.

Guanine nucleotide exchange factor-H1 signaling is involved in lipopolysaccharide-induced endothelial barrier dysfunction

Gram-negative bacterial lipopolysaccharide (LPS) leads to the pathologic increase of vascular leakage under septic conditions. However, the mechanisms behind LPS-induced vascular hyperpermeability remain incompletely understood. In this study, we tested hypothesis that guanine nucleotide exchange factor-H1 (GEF-H1) signaling might be a key pathway involved in endothelial cells (ECs) barrier dysfunction. The roles of GEF-H1 signaling pathway in LPS-induced ECs barrier dysfunction were accessed by Evans blue dye-labeled albumin (EB-albumin) leak across the human umbilical vein EC (HUVEC) monolayers and Western blot assays. Furthermore, the effect of GEF-H1 signaling on LPS-induced alteration of cytoskeletal proteins and disruption of cell-cell junctions were analyzed by immunofluorescent analysis and Western blot assays, respectively. We found that LPS could rapidly activated GEF-H1/RhoA/Rho-associated protein kinase (ROCK) signaling pathway in ECs. The LPS-mediated increase in EB-albumin flux across human HUVECs monolayers could be prevented by GEF-H1 depletion or ROCK inactivation. ECs permeability is controlled by actin filaments and cell-cell contact protein complexes. Actin stress fiber formation and/or cell-cell contact proteins loss cause vascular barrier disruption. Here, GEF-H1 knockdown or ROCK inactivation both not only significantly inhibited LPS-induced actin stress fiber formation, phosphorylation of myosin light chain, and myosin-associated phosphatase type 1, but also suppressed LPS-induced loss of occludin, claudin-1, and vascular endothelial (VE)-cadherin in ECs, which suggested that LPS-induced stress fiber formation and cell-cell junctions disruption were closely associated with GEF-H1/RhoA/ROCK signaling activation. Our findings indicate that GEF-H1/RhoA/ROCK pathway in ECs plays an important role in LPS-mediated alteration of cell morphology and disruption of cell-cell junctions, consequently regulate LPS-induced vascular permeability dysfunction.

TLR4 activation of TRPC6-dependent calcium signaling mediates endotoxin-induced lung vascular permeability and inflammation

Lung vascular endothelial barrier disruption and the accompanying inflammation are primary pathogenic features of acute lung injury (ALI); however, the basis for the development of both remains unclear. Studies have shown that activation of transient receptor potential canonical (TRPC) channels induces Ca(2+) entry, which is essential for increased endothelial permeability. Here, we addressed the role of Toll-like receptor 4 (TLR4) intersection with TRPC6-dependent Ca(2+) signaling in endothelial cells (ECs) in mediating lung vascular leakage and inflammation. We find that the endotoxin (lipopolysaccharide; LPS) induces Ca(2+) entry in ECs in a TLR4-dependent manner. Moreover, deletion of TRPC6 renders mice resistant to endotoxin-induced barrier dysfunction and inflammation, and protects against sepsis-induced lethality. TRPC6 induces Ca(2+) entry in ECs, which is secondary to the generation of diacylglycerol (DAG) induced by LPS. Ca(2+) entry mediated by TRPC6, in turn, activates the nonmuscle myosin light chain kinase (MYLK), which not only increases lung vascular permeability but also serves as a scaffold to promote the interaction of myeloid differentiation factor 88 and IL-1R-associated kinase 4, which are required for NF-κB activation and lung inflammation. Our findings suggest that TRPC6-dependent Ca(2+) entry into ECs, secondary to TLR4-induced DAG generation, participates in mediating both lung vascular barrier disruption and inflammation induced by endotoxin.