Cigarette smoke causes lung vascular barrier dysfunction via oxidative stress-mediated inhibition of RhoA and focal adhesion kinase

Cigarette smoke (CS) is a major cause of chronic lung and cardiovascular diseases. Recent studies indicate that tobacco use is also a risk factor for acute lung injury (ALI) associated with blunt trauma. Increased endothelial cell (EC) permeability is a hallmark of ALI. CS increases EC permeability in vitro and in vivo; however, the underlying mechanism is not well understood. In this study, we found that only 6 h of exposure to CS impaired endothelial barrier function in vivo, an effect associated with increased oxidative stress in the lungs and attenuated by the antioxidant N-acetylcysteine (NAC). CS also exacerbated lipopolysaccharide (LPS)-induced increase in vascular permeability in vivo. Similar additive effects were also seen in cultured lung EC exposed to cigarette smoke extract (CSE) and LPS. We further demonstrated that CSE caused disruption of focal adhesion complexes (FAC), F-actin fibers, and adherens junctions (AJ) and decreased activities of RhoA and focal adhesion kinase (FAK) in cultured lung EC. CSE-induced inhibition of RhoA and FAK, endothelial barrier dysfunction, and disassembly of FAC, F-actin, and AJ were prevented by NAC. In addition, the deleterious effects of CSE on FAC, F-actin fibers, and AJ were blunted by overexpression of constitutively active RhoA and of FAK. Our data indicate that CS causes endothelial barrier dysfunction via oxidative stress-mediated inhibition of RhoA and FAK.

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

Circulation ◽

10.1161/circ.130.suppl_2.20531 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Panfeng Fu ◽

Anne E Cress ◽

Ting Wang ◽

Joe G Garcia ◽

Viswanathan Natarajan

Keyword(s):

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Human Lung ◽

Adherens Junctions ◽

Endothelial Barrier ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Lps Challenge

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.

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Effect of p18 on Endothelial Barrier Function by Mediating Vascular Endothelial Rab11a-VE-cadherin Recycling

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab172 ◽

2021 ◽

Author(s):

Bo-Wen Xu ◽

Zhi-Qiang Cheng ◽

Xu-Ting Zhi ◽

Xiao-Mei Yang ◽

Zhi-Bo Yan

Keyword(s):

Barrier Function ◽

Adherens Junctions ◽

Cecal Ligation ◽

Underlying Mechanism ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Recycling Endosome

Abstract Endothelial barrier integrity requires recycling of VE-cadherin to adherens junctions. Both p18 and Rab11a play significant roles in VE-cadherin recycling. However, the underlying mechanism and the role of p18 in activating Rab11a have yet to be elucidated. Performing in vitro and in vivo experiments, we showed that p18 protein bound to VE-cadherin before Rab11a through its VE-cadherin-binding domain (aa 1–39). Transendothelial resistance showed that overexpression of p18 promoted the circulation of VE-cadherin to adherens junctions and the recovery of the endothelial barrier. Silencing of p18 caused endothelial barrier dysfunction and prevented Rab11a-positive recycling endosome accumulation in the perinuclear recycling compartments. Furthermore, p18 knockdown in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide and cecal ligation puncture. This study showed that p18 regulated the pulmonary endothelial barrier function in vitro and in vivo by regulating the binding of Rab11a to VE-cadherin and the activation of Rab11a.

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Cigarette Smoke Causes Lung Endothelial Barrier Dysfunction And Apoptosis Through Oxidative Stress-Mediated Loss Of FAK And RhoA Activities

10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a1956 ◽

2011 ◽

Author(s):

Qing Lu ◽

Julie Newton ◽

Pavlo Sakhatskyy ◽

Mazen O. Al-Qadi ◽

Katie L. Grinnell ◽

...

Keyword(s):

Oxidative Stress ◽

Cigarette Smoke ◽

Endothelial Barrier ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction

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Atrial natriuretic peptide protects against Staphylococcus aureus-induced lung injury and endothelial barrier dysfunction

Journal of Applied Physiology ◽

10.1152/japplphysiol.00284.2010 ◽

2011 ◽

Vol 110 (1) ◽

pp. 213-224 ◽

Cited By ~ 24

Author(s):

Junjie Xing ◽

Nurgul Moldobaeva ◽

Anna A. Birukova

Keyword(s):

Staphylococcus Aureus ◽

Lung Inflammation ◽

Endothelial Barrier ◽

Protective Effects ◽

Barrier Dysfunction ◽

Gram Positive ◽

Gram Negative ◽

Endothelial Barrier Dysfunction

Lung inflammation and alterations in endothelial cell (EC) permeability are key events to development of acute lung injury (ALI). Protective effects of atrial natriuretic peptide (ANP) have been shown against inflammatory signaling and endothelial barrier dysfunction induced by gram-negative bacterial wall liposaccharide. We hypothesized that ANP may possess more general protective effects and attenuate lung inflammation and EC barrier dysfunction by suppressing inflammatory cascades and barrier-disruptive mechanisms shared by gram-negative and gram-positive pathogens. C57BL/6J wild-type or ANP knockout mice (Nppa−/−) were treated with gram-positive bacterial cell wall compounds, Staphylococcus aureus-derived peptidoglycan (PepG) and/or lipoteichoic acid (LTA) (intratracheal, 2.5 mg/kg each), with or without ANP (intravenous, 2 μg/kg). In vitro, human pulmonary EC barrier properties were assessed by morphological analysis of gap formation and measurements of transendothelial electrical resistance. LTA and PepG markedly increased pulmonary EC permeability and activated p38 and ERK1/2 MAP kinases, NF-κB, and Rho/Rho kinase signaling. EC barrier dysfunction was further elevated upon combined LTA and PepG treatment, but abolished by ANP pretreatment. In vivo, LTA and PepG-induced accumulation of protein and cells in the bronchoalveolar lavage fluid, tissue neutrophil infiltration, and increased Evans blue extravasation in the lungs was significantly attenuated by intravenous injection of ANP. Accumulation of bronchoalveolar lavage markers of LTA/PepG-induced lung inflammation and barrier dysfunction was further augmented in ANP−/− mice and attenuated by exogenous ANP injection. These results strongly suggest a protective role of ANP in the in vitro and in vivo models of ALI associated with gram-positive infection. Thus ANP may have important implications in therapeutic strategies aimed at the treatment of sepsis and ALI-induced gram-positive bacterial pathogens.

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p53 protects against LPS-induced lung endothelial barrier dysfunction

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00334.2014 ◽

2015 ◽

Vol 308 (8) ◽

pp. L776-L787 ◽

Cited By ~ 34

Author(s):

Nektarios Barabutis ◽

Christiana Dimitropoulou ◽

Charalampos Birmpas ◽

Atul Joshi ◽

Gagan Thangjam ◽

...

Keyword(s):

Transcription Factor ◽

Negative Regulator ◽

Endothelial Barrier ◽

Barrier Dysfunction ◽

Hsp90 Inhibitors ◽

Endothelial Barrier Dysfunction ◽

Anti Inflammatory ◽

Transcription Factor P53

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.

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Endothelial Barrier Dysfunction Induced by Zinc Oxide Nanoparticles In Vivo and In Vitro and Their Mechanism of Crossing the Endothelial Barrier

Journal of Biomedical Nanotechnology ◽

10.1166/jbn.2019.2696 ◽

2019 ◽

Vol 15 (3) ◽

pp. 443-461 ◽

Cited By ~ 4

Author(s):

Junrong Wu ◽

Yanli Zhang ◽

Qian Yin ◽

Guangman Cui ◽

Longquan Shao

Keyword(s):

Zinc Oxide ◽

Zinc Oxide Nanoparticles ◽

Endothelial Barrier ◽

Oxide Nanoparticles ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction

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Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

Toxicology and Applied Pharmacology ◽

10.1016/j.taap.2012.08.013 ◽

2012 ◽

Vol 265 (2) ◽

pp. 253-262 ◽

Cited By ~ 12

Author(s):

Ming-Chung Lin ◽

Chia-Ling Chen ◽

Tsan-Tzu Yang ◽

Pui-Ching Choi ◽

Chung-Hsi Hsing ◽

...

Keyword(s):

Endothelial Barrier ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction

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Developmental differences in focal adhesion kinase expression modulate pulmonary endothelial barrier function in response to inflammation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00363.2017 ◽

2018 ◽

Vol 315 (1) ◽

pp. L66-L77 ◽

Cited By ~ 4

Author(s):

Lihua Ying ◽

Cristina M. Alvira ◽

David N. Cornfield

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Barrier Function ◽

Developmental Differences ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Barrier Disruption ◽

Adult Mice

Compromised pulmonary endothelial cell (PEC) barrier function characterizes acute respiratory distress syndrome (ARDS), a cause of substantial morbidity and mortality. Survival from ARDS is greater in children compared with adults. Whether developmental differences intrinsic to PEC barrier function contribute to this survival advantage remains unknown. To test the hypothesis that PEC barrier function is more well-preserved in neonatal lungs compared with adult lungs in response to inflammation, we induced lung injury in neonatal and adult mice with systemic lipopolysaccharide (LPS). We assessed PEC barrier function in vivo and in vitro, evaluated changes in the expression of focal adhesion kinase 1 (FAK1) and phosphorylation in response to LPS, and determined the effect of FAK silencing and overexpression on PEC barrier function. We found that LPS induced a greater increase in lung permeability and PEC barrier disruption in the adult mice, despite similar degrees of inflammation and apoptosis. Although baseline expression was similar, LPS increased FAK1 expression in neonatal PEC but increased FAK1 phosphorylation and decreased FAK1 expression in adult PEC. Pharmacologic inhibition of FAK1 accentuated LPS-induced barrier disruption most in adult PEC. Finally, in response to LPS, FAK silencing markedly impaired neonatal PEC barrier function, whereas FAK overexpression preserved adult PEC barrier function. Thus, developmental differences in FAK expression during inflammatory injury serve to preserve neonatal pulmonary endothelial barrier function compared with that of adults and suggest that intrinsic differences in the immature versus pulmonary endothelium, especially relative to FAK1 phosphorylation, may contribute to the improved outcomes of children with ARDS.

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00306.2014 ◽

2015 ◽

Vol 308 (10) ◽

pp. L1025-L1038 ◽

Cited By ~ 17

Author(s):

Panfeng Fu ◽

Peter V. Usatyuk ◽

Abhishek Lele ◽

Anantha Harijith ◽

Carol C. Gregorio ◽

...

Keyword(s):

Lung Injury ◽

Tyrosine Phosphorylation ◽

Endothelial Permeability ◽

Endothelial Barrier ◽

Barrier Dysfunction ◽

Pulmonary Vascular Permeability ◽

Endothelial Barrier Dysfunction ◽

Lps Challenge

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.

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