LPS-induced Endothelial Barrier Dysfunction Research Articles

Alleviation of LPS-induced Endothelial Injury due to GHRH Antagonist Treatment.

GHRH is produced in the hypothalamus and affects various tissues beyond the pituitary, including the lungs. GHRH antagonists exert anti-inflammatory properties in several experimental models of disease, but their role inprotecting the endothelial barrier during inflammation is less understood. This study investigates the effects ofGHRHAnt on LPS-induced endothelial dysfunction. BPAEC and HMVEC-L cells were exposed to LPS to induce endothelial injury. GHRHAnt was administered eitherpre- or post-LPS treatment. Western blot analysis was used to evaluate protein expression levels. Paracellularpermeability was assessed utilizing FITC-dextran assay to evaluate endothelial barrier function. GHRHAnt post-treatment significantly reduced LPS-induced MLC2 phosphorylation and cofilin activation inBPAECs. Furthermore, pretreatment with GHRHAnt enhanced barrier function and ameliorated LPS-inducedhyperpermeability in both human and bovine endothelial cells. GHRHAnt treatment mitigates LPS-induced endothelial barrier dysfunction. These findings suggest that GHRHAntcould serve as potential therapeutic agents towards endothelial dysfunction-related illness (e.g. sepsis).

MiR-98-3p alleviates lipopolysaccharide-induced pulmonary microvascular endothelial barrier dysfunction by targeting DKK3 in sepsis-induced acute lung injury.

Endothelial barrier dysfunction is critical for the pathogenesis of sepsis-induced acute lung injury (ALI). Lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cells (HPMECs) are widely used as the cell model of sepsis-associated ALI for exploration of endothelial barrier dysfunction. Dickkopf (DKK) family proteins were reported to mediate endothelial functions in various diseases. The present study explored the effect of Dickkopf-3 (DKK3) on endothelial barrier permeability, angiogenesis, and tight junctions in LPS-stimulated HPMECs. RT-qPCR was required for detecting DKK3 and miR-98-3p expression. The angiogenesis of HPMECs was evaluated by tube formation assays. Monolayer permeability of HPMECs was examined by Transwell rhodamine assays. The protein expression of DKK3 and tight junctions in HPMECs was measured via western blotting. Luciferase reporter assay was used to verify the interaction between miR-98-3p and DKK3. LPS treatment inhibited angiogenetic ability while increasing the permeability of HPMECs. DKK3 expression was upregulated while miR-98-3p level was reduced in LPS-treated HPMECs. DKK3 knockdown alleviated HPMEC injury triggered by LPS stimulation. MiR-98-3p targeted DKK3 in HPMECs. Overexpression of miR-98-3p protects HPMECs from the LPS-induced endothelial barrier dysfunction, and the protective effect was reversed by DKK3 overexpression. MiR-98-3p ameliorates LPS-evoked pulmonary microvascular endothelial barrier dysfunction in sepsis-associated ALI by targeting DKK3.

Activating transcription factor 6 protects against endothelial barrier dysfunction

BackgroundEndothelial hyperpermeability is associated with sepsis and acute respiratory distress syndrome (ARDS). The identification of molecular pathways involved in barrier dysfunction; may reveal promising therapeutic targets to combat ARDS. Unfolded protein response (UPR) is a highly conserved molecular pathway, which ameliorates endoplasmic reticulum stress. The present work focuses on the effects of ATF6, which is a UPR sensor, in lipopolysaccharides (LPS)-induced endothelial hyperpermeability. MethodsThe in vitro effects of AA147 and Ceapin-A7 in LPS-induced endothelial barrier dysfunction were investigated in bovine pulmonary artery endothelial cells (BPAEC). Small interfering (si) RNA was utilized to “silence” ATF6, and electric cell-substrate impedance sensing (ECIS) measured transendothelial resistance. Fluorescein isothiocyanate (FITC)-dextran assay was utilized to assess paracellular permeability. Protein expression levels were evaluated with Western blotting, and cell viability with MTT assay. ResultsWe demonstrated that AA147 prevents LPS-induced barrier disruption by counteracting Cofilin and myosin light chain 2 (MLC2) activation, as well as VE-Cadherin phosphorylation. Moreover, this ATF6 inducer opposed LPS-triggered decrease in transendothelial resistance (TEER), as well as LPS-induced paracellular hyperpermeability. On the other hand, ATF6 suppression due to Ceapin-A7 or small interfering RNA exerted the opposite effects, and potentiated LPS-induced endothelial barrier disruption. Moderate concentrations of both ATF6 modulators did not affect cell viability. ConclusionsATF6 activation protects against endothelial barrier function, suggesting that this UPR sensor may serve as a therapeutic target for sepsis and ARDS.

The Pyruvate Dehydrogenase Complex Mitigates LPS-Induced Endothelial Barrier Dysfunction by Metabolic Regulation.

Sepsis is a fatal health issue induced by an aberrant host response to infection, and it correlates with organ damage and a high mortality rate. Endothelial barrier dysfunction and subsequent capillary leakage play major roles in sepsis-induced multiorgan dysfunction. Anaerobic glycolysis is the primary metabolic mode in sepsis and the pyruvate dehydrogenase complex (PDHC) serves as a critical hub in energy regulation. Therefore, it is important to understand the role of PDHC in metabolic regulation during the development of sepsis-induced endothelial barrier dysfunction.In present study, human umbilical vein endothelial cells (HUVECs) and C57 BL/6 mice were treated with lipopolysaccharide (LPS) as models of endotoxemia. LPS increased basal glycolysis, compensatory glycolysis, and lactate secretion, indicating increased glycolysis level in endothelial cells (ECs). Activation of PDHC with dichloroacetate (DCA) reversed LPS-induced glycolysis, allowing PDHC to remain in the active dephosphorylated state, thereby preventing lactic acid production and HUVECs monolayers barrier dysfunction, as assessed by transendothelial electrical resistance and Fluorescein Isothiocyanate-labeled dextran. The in vivo study also showed that the lactate level and vascular permeability were increased in LPS-treated mice, but pretreatment with DCA attenuated these increases. The LPS-treated HUVEC model showed that DCA reversed LPS-induced phosphorylation of pyruvate dehydrogenase E1α Ser293 and Ser300 to restore PDHC activity. Immunoprecipitation results showed that LPS treatment increased the acetylation level of PDH E1α in HUVECs.Our study suggested that activation of PDHC may represent a therapeutic target for treatment of LPS-induced endothelial barrier dysfunction.

Inhibition of microRNA-92a ameliorates lipopolysaccharide-induced endothelial barrier dysfunction by targeting ITGA5 through the PI3K/Akt signaling pathway in human pulmonary microvascular endothelial cells

Overwhelming inflammation and extensive alveolar-endothelial injury are characteristic pathological features of acute respiratory distress syndrome (ARDS)). MicroRNAs are involved in the regulation of a variety of cellular processes including endothelial damage and inflammatory responses. However, little is known about their function and the molecules regulating lung microvascular endothelial injury. Here, we determined the levels of microRNA-92a (miR-92a) in lipopolysaccharide (LPS)-induced human pulmonary microvascular endothelial cells (HPMECs). We found that miR-92a expression was greater in HPMECs treated with LPS than in control cells. Inhibition of miR-92a through transfection with a miR-92a inhibitor significantly increased HPMECs migration, enhanced tube formation, and improved endothelial cell barrier dysfunction. Inhibition of miR-92a ameliorated the inflammatory response by decreasing the release of the proinflammatory factors IL-6 and TNF-α. In addition, integrin α5 (ITGA5) was found to be a target gene of miR-92a in LPS-induced endothelial barrier dysfunction. Western blot analysis showed that inhibition of miR-92a may ameliorate endothelial barrier dysfunction by activating the PI3K/Akt signaling pathway. Together, these results reveal an important role of miR-92a in LPS-induced endothelial barrier dysfunction, and suggest that miR-92a may have potential as a prognostic indicator and a future target for the treatment of acute lung injury (ALI)/ARDS.

Unfractionated heparin ameliorates pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization in acute lung injury

BackgroundEndothelial barrier dysfunction is central to the pathogenesis of sepsis-associated acute lung injury (ALI). Microtubule (MT) dynamics in vascular endothelium are crucial for the regulation of endothelial barrier function. Unfractionated heparin (UFH) possesses various biological activities, such as anti-inflammatory activity and endothelial barrier protection during sepsis.MethodsHere, we investigated the effects and underlying mechanisms of UFH on lipopolysaccharide (LPS)-induced endothelial barrier dysfunction. C57BL/6 J mice were randomized into vehicle, UFH, LPS and LPS + UFH groups. Intraperitoneal injection of 30 mg/kg LPS was used to induce sepsis. Mice in the LPS + UFH group received intravenous UFH 0.5 h prior to LPS injection. Human pulmonary microvascular endothelial cells (HPMECs) were cultured for analyzing the effects of UFH on LPS-induced and nocodazole-induced hyperpermeability, F-actin remodeling, and LPS-induced p38 MAPK activation.ResultsUFH pretreatment significantly attenuated LPS-induced pulmonary histopathological changes, and increased the lung W/D ratio and Evans blue accumulation in vivo. Both in vivo and in vitro studies showed that UFH pretreatment blocked the LPS-induced increase in guanine nucleotide exchange factor (GEF-H1) expression and myosin phosphatase target subunit 1 (MYPT1) phosphorylation, and microtubule (MT) disassembly in LPS-induced ALI mouse model and human pulmonary microvascular endothelial cells (HPMECs). These results suggested that UFH ameliorated LPS-induced endothelial barrier dysfunction by inhibiting MT disassembly and GEF-H1 expression. In addition, UFH attenuated LPS-induced hyperpermeability of HPMECs and F-actin remodeling. In vitro, UFH pretreatment inhibited LPS-induced increase in monomeric tubulin expression and decrease in tubulin polymerization and acetylation. Meanwhile, UFH ameliorates nocodazole-induced MTs disassembly and endothelial barrier dysfunction.Additionally, UFH decreased p38 phosphorylation and activation, which was similar to the effect of the p38 MAPK inhibitor, SB203580.ConclusionsUFH exert its protective effects on pulmonary microvascular endothelial barrier dysfunction via microtubule stabilization and is associated with the p38 MAPK pathway.

Geniposide ameliorates TNBS-induced experimental colitis in rats via reducing inflammatory cytokine release and restoring impaired intestinal barrier function.

Geniposide is an iridoid glycosides purified from the fruit of Gardenia jasminoides Ellis, which is known to have antiinflammatory, anti-oxidative and anti-tumor activities. The present study aimed to investigate the effects of geniposide on experimental rat colitis and to reveal the related mechanisms. Experimental rat colitis was induced by rectal administration of a TNBS solution. The rats were treated with geniposide (25, 50 mg·kg-1·d-1, ig) or with sulfasalazine (SASP, 100 mg·kg-1·d-1, ig) as positive control for 14 consecutive days. A Caco-2 cell monolayer exposed to lipopolysaccharides (LPS) was used as an epithelial barrier dysfunction model. Transepithelial electrical resistance (TER) was measured to evaluate intestinal barrier function. In rats with TNBS-induced colitis, administration of geniposide or SASP significantly increased the TNBS-decreased body weight and ameliorated TNBS-induced experimental colitis and related symptoms. Geniposide or SASP suppressed inflammatory cytokine (TNF-α, IL-1β, and IL-6) release and neutrophil infiltration (myeloperoxidase activity) in the colon. In Caco-2 cells, geniposide (25-100 μg/mL) ameliorated LPS-induced endothelial barrier dysfunction via dose-dependently increasing transepithelial electrical resistance (TER). The results from both in vivo and in vitro studies revealed that geniposide down-regulated NF-κB, COX-2, iNOS and MLCK protein expression, up-regulated the expression of tight junction proteins (occludin and ZO-1), and facilitated AMPK phosphorylation. Both AMPK siRNA transfection and AMPK overexpression abrogated the geniposide-reduced MLCK protein expression, suggesting that geniposide ameliorated barrier dysfunction via AMPK-mediated inhibition of the MLCK pathway. In conclusion, geniposide ameliorated TNBS-induced experimental rat colitis by both reducing inflammation and modulating the disrupted epithelial barrier function via activating the AMPK signaling pathway.

Urinary trypsin inhibitor attenuates LPS-induced endothelial barrier dysfunction by upregulation of vascular endothelial-cadherin expression.

Urinary trypsin inhibitor (UTI) decreases inflammatory cytokine levels and mortality in experimental animal models of inflammation. Here, we observed the effect of UTI on lipopolysaccharide (LPS)-induced hyperpermeability in human umbilical vein endothelial cells (HUVECs) and explored the role of vascular endothelial-cadherin (VE-cadherin) in its effect. The effect of UTI on endothelial barrier hyperpermeability was detected by an electrical cell-substrate impedance sensing (ECIS) system and a transwell chamber system. The expression of VE-cadherin in HUVECs was examined by real-time PCR and western blot. We demonstrated that the alleviation of LPS-induced barrier dysfunction could be achieved by pretreatment with 3000U/mL of UTI. VE-cadherin monoclonal antibody (mAb) could inhibit the protective effects. UTI maintained VE-cadherin expression by increasing protein stability at both the transcriptional and post-transcriptional levels. Meanwhile, VE-cadherin expression on the cell surface increased when the cells were pretreated with UTI. Furthermore, pretreatment with UTI decreased the phosphorylation of VE-cadherin at Tyr658 but not Tyr731. Our data show that prophylactic UTI maintains the endothelial barrier function, increases VE-cadherin expression, and inhibits the phosphorylation of VE-cadherin at Tyr658 under inflammatory conditions. It suggests a scientific and potential clinical therapeutic importance of UTI in treatment of inflammatory disorders.

Histone deacetylase inhibitors prevent pulmonary endothelial hyperpermeability and acute lung injury by regulating heat shock protein 90 function.

Transendothelial hyperpermeability caused by numerous agonists is dependent on heat shock protein 90 (Hsp90) and leads to endothelial barrier dysfunction (EBD). Inhibition of Hsp90 protects and restores transendothelial permeability. Hyperacetylation of Hsp90, as by inhibitors of histone deacetylase (HDAC), suppresses its chaperone function and mimics the effects of Hsp90 inhibitors. In this study we assessed the role of HDAC in mediating lipopolysaccharide (LPS)-induced transendothelial hyperpermeability and acute lung injury (ALI). We demonstrate that HDAC inhibition protects against LPS-mediated EBD. Inhibition of multiple HDAC by the general inhibitors panobinostat or trichostatin provided protection against LPS-induced transendothelial hyperpermeability, acetylated and suppressed Hsp90 chaperone function, and attenuated RhoA activity and signaling crucial to endothelial barrier function. Treatment with the HDAC3-selective inhibitor RGFP-966 or the HDAC6-selective inhibitor tubastatin A provided partial protection against LPS-mediated transendothelial hyperpermeability. Similarly, knock down of HDAC3 and HDAC6 by specific small-interfering RNAs provided significant protection against LPS-induced EBD. Furthermore, combined pharmacological inhibition of both HDAC3 and -6 attenuated the inflammation, capillary permeability, and structural abnormalities associated with LPS-induced ALI in mice. Together these data indicate that HDAC mediate increased transendothelial hyperpermeability caused by LPS and that inhibition of HDAC protects against LPS-mediated EBD and ALI by suppressing Hsp90-dependent RhoA activity and signaling.

Rab5-mediated VE-cadherin internalization regulates the barrier function of the lung microvascular endothelium.

The small GTPase Rab5 has been well defined to control the vesicle-mediated plasma membrane protein transport to the endosomal compartment. However, its function in the internalization of vascular endothelial (VE)-cadherin, an important component of adherens junctions, and as a result regulating the endothelial cell polarity and barrier function remain unknown. Here, we demonstrated that lipopolysaccharide (LPS) simulation markedly enhanced the activation and expression of Rab5 in human pulmonary microvascular endothelial cells (HPMECs), which is accompanied by VE-cadherin internalization. In parallel, LPS challenge also induced abnormal cell polarity and dysfunction of the endothelial barrier in HPMECs. LPS stimulation promoted the translocation of VE-cadherin from the plasma membrane to intracellular compartments, and intracellularly expressed VE-cadherin was extensively colocalized with Rab5. Small interfering RNA (siRNA)-mediated depletion of Rab5a expression attenuated the disruption of LPS-induced internalization of VE-cadherin and the disorder of cell polarity. Furthermore, knockdown of Rab5 inhibited the vascular endothelial hyperpermeability and protected endothelial barrier function from LPS injury, both in vitro and in vivo. These results suggest that Rab5 is a critical mediator of LPS-induced endothelial barrier dysfunction, which is likely mediated through regulating VE-cadherin internalization. These findings provide evidence, implicating that Rab5a is a potential therapeutic target for preventing endothelial barrier disruption and vascular inflammation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-015-1973-4) contains supplementary material, which is available to authorized users.

C-Abl mediated tyrosine phosphorylation of paxillin regulates LPS-induced endothelial dysfunction and lung injury.

Paxillin is phosphorylated at multiple residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and acute lung injury (ALI) remains unclear. We used siRNA and site-specific nonphosphorylable mutants of paxillin to abrogate the function of paxillin to determine its role in lung endothelial permeability and ALI. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (HLMVECs) resulted in enhanced tyrosine phosphorylation of paxillin at Y31 and Y118 with no significant change in Y181 and significant barrier dysfunction. Knockdown of paxillin with siRNA attenuated LPS-induced endothelial barrier dysfunction and destabilization of VE-cadherin. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase, but not by Src and focal adhesion kinase. c-Abl siRNA significantly reduced LPS-induced endothelial barrier dysfunction. Transfection of HLMVECs with paxillin Y31F, Y118F, and Y31/118F double mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization. In vivo, the c-Abl inhibitor AG957 attenuated LPS-induced pulmonary permeability in mice. Together, these results suggest that c-Abl mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury.

P53 protects against LPS-induced lung endothelial barrier dysfunction.

New therapies toward heart and blood vessel disorders may emerge from the development of Hsp90 inhibitors. Several independent studies suggest potent anti-inflammatory activities of those agents in human tissues. The molecular mechanisms responsible for their protective effects in the vasculature remain unclear. The present study demonstrates that the transcription factor p53, an Hsp90 client protein, is crucial for the maintenance of vascular integrity, protects again LPS-induced endothelial barrier dysfunction, and is involved in the mediation of the anti-inflammatory activity of Hsp90 inhibitors in lung tissues. p53 silencing by siRNA decreased transendothelial resistance (a measure of endothelial barrier function). A similar effect was induced by the p53 inhibitor pifithrin, which also potentiated the LPS-induced hyperpermeability in human lung microvascular endothelial cells (HLMVEC). On the other hand, p53 induction by nutlin suppressed the LPS-induced vascular barrier dysfunction. LPS decreased p53 expression in lung tissues and that effect was blocked by pretreatment with Hsp90 inhibitors both in vivo and in vitro. Furthermore, the Hsp90 inhibitor 17-allyl-amino-demethoxy-geldanamycin suppressed the LPS-induced overexpression of the p53 negative regulator MDMX as well as p53 and MDM2 (another p53 negative regulator) phosphorylation in HLMVEC. Both negative p53 regulators were downregulated by LPS in vivo. Chemically induced p53 overexpression resulted in the suppression of LPS-induced RhoA activation and MLC2 phosphorylation, whereas p53 suppression caused the opposite effects. These observations reveal new mechanisms for the anti-inflammatory actions of Hsp90 inhibitors, i.e., the induction of the transcription factor p53, which in turn can orchestrate robust vascular anti-inflammatory responses both in vivo and in vitro.

Abstract 20531: C-Abl Mediated Tyrosine Phosphorylation of Paxillin is Essential for LPS-Induced Endothelial Barrier Dysfunction and Acute Lung Injury

Paxillin, a multi-domain scaffold-adapter focal adhesion (FA) protein, plays an important role in facilitating protein networking and efficient signaling transduction. Paxillin is phosphorylated at multiple serine/threonine and tyrosine residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and the acute respiratory distress syndrome (ARDS) remains unclear. In this study, we used paxillin-specific siRNA and site-specific non-phosphorylatable mutants of paxillin to abrogate the function of paxillin, both in vitro and in vivo, to determine its role in the regulation of lung endothelial permeability and ARDS. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (ECs) resulted in paxillin accumulation at focal adhesions, enhanced tyrosine phosphorylation of paxillin at Y31 and Y118, and significant endothelial barrier dysfunction. However no significant changes in Y181 phosphorylation by LPS challenge was observed. Paxillin silencing (siRNA) attenuated LPS-induced endothelial barrier dysfunction and dissociation of VE-cadherin from adherens junctions. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase and not by Src or focal adhesion kinase (FAK) in human lung microvascular ECs. Furthermore, down-regulation of c-Abl (siRNA) significantly reduced LPS-mediated endothelial barrier dysfunction. Transfection of human lung microvascular ECs with paxillin Y31, Y118 and Y31/Y118 mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization at adherens junctions. In vivo, knockdown of paxillin with siRNA in mouse lungs ameliorated LPS-induced pulmonary protein leak and lung inflammation. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury.

MicroRNA-147b regulates vascular endothelial barrier function by targeting ADAM15 expression.

A disintegrin and metalloproteinase15 (ADAM15) has been shown to be upregulated and mediate endothelial hyperpermeability during inflammation and sepsis. This molecule contains multiple functional domains with the ability to modulate diverse cellular processes including cell adhesion, extracellular matrix degradation, and ectodomain shedding of transmembrane proteins. These characteristics make ADAM15 an attractive therapeutic target in various diseases. The lack of pharmacological inhibitors specific to ADAM15 prompted our efforts to identify biological or molecular tools to alter its expression for further studying its function and therapeutic implications. The goal of this study was to determine if ADAM15-targeting microRNAs altered ADAM15-induced endothelial barrier dysfunction during septic challenge by bacterial lipopolysaccharide (LPS). An in silico analysis followed by luciferase reporter assay in human vascular endothelial cells identified miR-147b with the ability to target the 3′ UTR of ADAM15. Transfection with a miR-147b mimic led to decreased total, as well as cell surface expression of ADAM15 in endothelial cells, while miR-147b antagomir produced an opposite effect. Functionally, LPS-induced endothelial barrier dysfunction, evidenced by a reduction in transendothelial electric resistance and increase in albumin flux across endothelial monolayers, was attenuated in cells treated with miR-147b mimics. In contrast, miR-147b antagomir exerted a permeability-increasing effect in vascular endothelial cells similar to that caused by LPS. Taken together, these data suggest the potential role of miR147b in regulating endothelial barrier function by targeting ADAM15 expression.

Doxycycline hyclate protects lipopolysaccharide-induced endothelial barrier dysfunction by inhibiting the activation of p38 mitogen-activated protein kinase.

Doxycycline hyclate (DOX-h) attenuates inflammatory conditions independent of its antimicrobial effect. This study aimed to observe the effects of DOX-h on lipopolysaccharide (LPS)-induced endothelial barrier dysfunction. The endothelial monolayer permeability of human umbilical vein endothelial cells (HUVECs) was monitored by transendothelial electrical resistance (TEER). The phosphorylation of mitogen-activated protein kinases (MAPKs) and the arrangement of F-actin were detected. The results showed that both pretreatment and simultaneous treatment with DOX-h markedly attenuated the LPS-induced reduction in TEER and the disorganization of F-actin on HUVECs in a dose- and time-dependent manner. LPS mediated the phosphorylation of all three MAPKs (p38, extracellular signal-regulated kinase (ERK)1/2, and c-Jun N-terminal kinase (JNK)), but DOX-h was only able to inhibit the LPS-induced phosphorylation of p38 and JNK. The data further suggested that DOX-h alleviated LPS-evoked TEER reduction and F-actin redistribution by inhibiting the phosphorylation of p38 and its downstream target, heat shock protein (HSP)27. Thus, DOX-h attenuates LPS-induced endothelial barrier dysfunction via inhibition of the p38 MAPK-HSP27-F-actin pathway.

Heat Shock Protein 90 Inhibitors Prevent LPS-Induced Endothelial Barrier Dysfunction by Disrupting RhoA Signaling

Permeability of the endothelial monolayer is increased when exposed to the bacterial endotoxin LPS. Our previous studies have shown that heat shock protein (Hsp) 90 inhibitors protect and restore LPS-mediated hyperpermeability in bovine pulmonary arterial endothelial cells. In this study, we assessed the effect of Hsp90 inhibition against LPS-mediated hyperpermeability in cultured human lung microvascular endothelial cells (HLMVECs) and delineated the underlying molecular mechanisms. We demonstrate that Hsp90 inhibition is critical in the early phase, to prevent LPS-mediated hyperpermeability, and also in the later phase, to restore LPS-mediated hyperpermeability in HLMVECs. Because RhoA is a well known mediator of endothelial hyperpermeability, we investigated the effect of Hsp90 inhibition on LPS-mediated RhoA signaling. RhoA nitration and activity were increased by LPS in HLMVECs and suppressed when pretreated with the Hsp90 inhibitor, 17-allylamino-17 demethoxy-geldanamycin (17-AAG). In addition, inhibition of Rho kinase, a downstream effector of RhoA, protected HLMVECs from LPS-mediated hyperpermeability and abolished LPS-induced myosin light chain (MLC) phosphorylation, a target of Rho kinase. In agreement with these findings, 17-AAG or dominant-negative RhoA attenuated LPS-induced MLC phosphorylation. MLC phosphorylation induced by constitutively active RhoA was also suppressed by 17-AAG, suggesting a role for Hsp90 downstream of RhoA. Inhibition of Src family kinases also suppressed RhoA activity and MLC phosphorylation. Together, these data indicate that Hsp90 inhibition prevents and repairs LPS-induced lung endothelial barrier dysfunction by suppressing Src-mediated RhoA activity and signaling.

Genetic disruption of protein kinase Cδ reduces endotoxin-induced lung injury

The pathogenesis of acute lung injury and acute respiratory distress syndrome is characterized by sequestration of leukocytes in lung tissue, disruption of capillary integrity, and pulmonary edema. PKCδ plays a critical role in RhoA-mediated endothelial barrier function and inflammatory responses. We used mice with genetic deletion of PKCδ (PKCδ(-/-)) to assess the role of PKCδ in susceptibility to LPS-induced lung injury and pulmonary edema. Under baseline conditions or in settings of increased capillary hydrostatic pressures, no differences were noted in the filtration coefficients (k(f)) or wet-to-dry weight ratios between PKCδ(+/+) and PKCδ(-/-) mice. However, at 24 h after exposure to LPS, the k(f) values were significantly higher in lungs isolated from PKCδ(+/+) than PKCδ(-/-) mice. In addition, bronchoalveolar lavage fluid obtained from LPS-exposed PKCδ(+/+) mice displayed increased protein and cell content compared with LPS-exposed PKCδ(-/-) mice, but similar changes in inflammatory cytokines were measured. Histology indicated elevated LPS-induced cellularity and inflammation within PKCδ(+/+) mouse lung parenchyma relative to PKCδ(-/-) mouse lungs. Transient overexpression of catalytically inactive PKCδ cDNA in the endothelium significantly attenuated LPS-induced endothelial barrier dysfunction in vitro and increased k(f) lung values in PKCδ(+/+) mice. However, transient overexpression of wild-type PKCδ cDNA in PKCδ(-/-) mouse lung vasculature did not alter the protective effects of PKCδ deficiency against LPS-induced acute lung injury. We conclude that PKCδ plays a role in the pathological progression of endotoxin-induced lung injury, likely mediated through modulation of inflammatory signaling and pulmonary vascular barrier function.

Abstract 209: Inhibition of AMP-Activated Protein Kinase Accentuates Lipopolysaccharides-Induced Lung Endothelial Barrier Dysfunction and Lung Injury in Vivo

AMP-activated protein kinase (AMPK) is reported to have vascular protective effects, but whether AMPK regulates endothelial barrier functions and protects lung function remains unknown. The aim of this study was to determine the roles of AMPK in lipopolysaccharide (LPS)-induced lung endothelial barrier dysfunction ( in vitro and in vivo ) and lung injury ( in vivo ). Cultured human pulmonary artery endothelial cells (HPAECs) and AMPKα subunit-deficient mice and wild-type (WT) control (C57BL/6J) mice were used to study the molecular mechanisms by which LPS induced lung endothelial barrier dysfunction and lung injury. In cultured HPAECs, LPS markedly increased endothelial hyperpermeabilty in parallel with a marked decrease in both AMPK phosphorylation at Thr172 and AMPK activity. Consistent with this latter observation, AMPK activation with AICAR treatment attenuated LPS-induced endothelial hyperpermeabilty in vitro . Intratracheal administration of LPS (1 mg/kg) in WT C57BL6 mice markedly reduced AMPK phosphorylation at Thr172 in lung tissue extracts, but markedly increased protein contents and cell numbers in bronchial aveolar lavage fluid and Evans blue dye infiltration in the lung in vivo . Pretreatment with AICAR, a potent AMPK activator, markedly reduced LPS-enhanced protein contents, cell counts, and inflammatory cell infiltration into alveolae. These lung injury indicators were significantly enhanced in AMPK-knockout mice compared to WT C57BL6 mice, supporting that AMPK inhibition by LPS accentuated lung injury. Mechanistically, we found that AMPK activation with AICAR attenuated LPS-induced endothelial hyperpermeabilty by activating the Rac/Cdc42/PAK pathway with concomitant inhibition of the Rho pathway, and decreased VE-cadherin phosphorylation at Tyr 658 . We conclude that (1) LPS inhibits AMPK; (2) AMPK inhibition accelerates LPS-induced endothelial barrier dysfunction and lung injury; and (3) AMPK activation attenuates LPS-induced pulmonary vascular hyperpermeability and lung injury.

Unfractionated heparin promotes LPS-induced endothelial barrier dysfunction: A preliminary study on the roles of angiopoietin/Tie2 axis

IntroductionHeparins, including unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH), are anticoagulants approved as a treatment for severe sepsis, which can also prevent apoptosis and inflammation. The aim of this study was to investigate whether UFH prevents vascular leakage induced by lipopolysaccharide (LPS) and to define the role of angiopoietin (Ang)/Tie2 signaling pathway since LPS is usually used to mimic the initiation of sepsis. MethodsHuman pulmonary microvascular endothelial cells (HPMECs) were pretreated with UFH (0.1U/ml-10U/ml), 15min prior to stimulation with LPS (10μg/ml). Those samples not receiving LPS or UFH received an equal volume of Phosphate-buffered saline (PBS). Cells were cultured under various experimental conditions for 2h, 6h or 12h for analysis. Results1) Pretreatment with UFH significantly reduced HPMEC permeability compared with LPS-stimulated groups; 2) Pretreatment with UFH decreased the formation of stress fiber and intracellular gaps induced by LPS; 3) UFH significantly up-regulated gene expression of Tie2 and Ang-1 but down-regulated Ang-2 in HPMECs; 4) UFH prevented LPS-induced decrease in the level of ZO-1. ConclusionThis study demonstrates that UFH enhances endothelial barrier function and Ang/Tie2 axis probably represents one of the mechanisms by which UFH exerts its protective effect.

Protection against LPS-Induced Pulmonary Edema through the Attenuation of Protein Tyrosine Phosphatase–1B Oxidation

One hallmark of acute lung injury is the disruption of the pulmonary endothelial barrier. Such disruption correlates with increased endothelial permeability, partly through the disruption of cell-cell contacts. Protein tyrosine phosphatases (PTPs) are known to affect the stability of both cell-extracellular matrix adhesions and intercellular adherens junctions (AJs). However, evidence for the role of select PTPs in regulating endothelial permeability is limited. Our investigations noted that the inhibition of PTP1B in cultured pulmonary endothelial cells (ECs), as well as in the vasculature of intact murine lungs via the transient overexpression of a catalytically inactive PTP1B, decreased the baseline resistance of cultured EC monolayers and increased the formation of edema in murine lungs, respectively. In addition, we observed that the overexpression of wild-type PTP1B enhanced basal barrier function in vitro. Immunohistochemical analyses of pulmonary ECs and the coimmunoprecipitation of murine lung homogenates demonstrated the association of PTP1B with the AJ proteins β-catenin, p120-catenin, and VE-cadherin both in vitro and ex vivo. Using LPS in a model of sepsis-induced acute lung injury, we showed that reactive oxygen species were generated in response to LPS, which correlated with enhanced PTP1B oxidation, inhibited phosphatase activity, and attenuation of the interactions between PTP1B and β-catenin, as well as enhanced β-catenin tyrosine phosphorylation. Finally, the overexpression of a cytosolic PTP1B fragment, shown to be resistant to nicotinamide adenine dinucleotide phosphate-reduced oxidase-4 (Nox4)-mediated oxidation, significantly attenuated LPS-induced endothelial barrier dysfunction and the formation of lung edema, and preserved the associations of PTP1B with AJ protein components, independent of PTP1B phosphatase activity. We conclude that PTP1B plays an important role in maintaining the pulmonary endothelial barrier, and PTP1B oxidation appears to contribute to sepsis-induced pulmonary vascular dysfunction, possibly through the disruption of AJs.