[Show abstract][Hide abstract] ABSTRACT: Growth arrest DNA damage inducible alpha (GADD45a) is a stress-induced gene we have shown to participate in the pathophysiology of ventilator-induced lung injury (VILI) via regulation of mechanical stress-induced Akt ubiquitination and phosphorylation. The regulation of GADD45a expression by mechanical stress and its relationship with acute lung injury (ALI) susceptibility and severity, however, remains unknown.
PLoS ONE 01/2014; 9(6):e100169. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Increased lung vascular permeability and alveolar edema are cardinal features of inflammatory conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). We previously demonstrated that PBEF/NAMPT, the pro-inflammatory cytokine encoded by NAMPT (known as pre-B cell colony enhancing factor (PBEF), participates in ARDS and VILI inflammatory syndromes. The present study evaluated post-transcriptional regulation of PBEF/NAMPT gene expression in human lung endothelium via 3'UTR miRNA binding. In silico analysis identified hsa-miR-374a and hsa-miR-568 as potential miRNA candidates. Increased PBEF/NAMPT transcription (RT-PCR) and expression (western blotting) induced by 18% cyclic stretch (CS) (2 hrs-3.4±0.06 mRNA fold increase, 10 hrs-1.5±0.06 protein FI) and by LPS (4 hrs- 3.8±0.2 mRNA FI, 48 hrs-2.6±0.2 protein FI) was significantly attenuated by transfection with mimics of hsa-miR-374a or hsa-miR-568 (40-60% reductions each). LPS and 18% CS each increased the activity of a PBEF/NAMPT 3'UTR luciferase reporter (2.4-3.25 FI) with induction reduced by mimics of each miRNA (44-60% reduction). Specific miRNA inhibitors (antagomirs) for each PBEF/NAMPT miRNA significantly increased the endogenous PBEF/NAMPT mRNA (1.4-3.4±0.1 FI) and protein levels (1.2-1.4±0.1 FI) and 3'UTR luciferase activity (1.4-1.7±0.1 FI) compared with negative antagomir controls. Collectively, these data demonstrate that increased PBEF/NAMPT expression induced by bioactive agonists (excessive mechanical stress, LPS) involves epigenetic regulation with hsa-miR-374a and hsa-miR-568 representing novel therapeutic strategies to reduce inflammatory lung injury.
American Journal of Respiratory Cell and Molecular Biology 09/2013; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The lipid membrane not only provides a rich interface with an array of receptor signaling complexes with which a cell communicates, but it also serves as a source of lipid derived bioactive molecules. In pathologic conditions of acute lung injury (ALI) associated with activation of oxidative stress, unsaturated phosphatidyl cholines overlooking a luminal space undergo oxidation leading to generation of fragmented phospholipids such as 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPC), or 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) full length oxygenation products (oxPAPC). Using Langmuir monolayers as models of the lipid bilayer, we evaluated the propensity of these phospholipids to solubilize from the cell membrane. The results suggest that lysoPC is rapidly released as it is produced, while oxPAPC has a longer membrane bound lifetime. After being released from cell membranes, these oxidized phospholipids exhibit potent agonist-like effects on neighboring cells. Therefore, we correlate the presence of the two phospholipid groups with the onset and resolution of increased vascular leakiness associated with ALI through testing their effect on vascular endothelial barrier integrity. Our work shows that cells respond differently to these two groups of products of phosphatidyl choline oxidation. LysoPC disrupts cell-cell junctions and increases endothelial permeability while oxPAPC enhances endothelial barrier. These data suggest a model whereby rapid release of lysoPC results in onset of ALI associated vascular leak, and the release of a reserve of oxPAPC as oxidative stress subsides restores the vascular barrier properties.
Chemistry and physics of lipids 07/2013; · 2.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelial cell (EC) barrier disruption induced by inflammatory agonists such as thrombin leads to potentially lethal physiological dysfunction such as alveolar flooding, hypoxemia and pulmonary edema. Thrombin stimulates paracellular gap and F-actin stress fiber formation, triggers actomyosin contraction and alters EC permeability through multiple mechanisms that include protein kinase C (PKC) activation. We previously have shown that the ezrin, radixin, and moesin (ERM) actin-binding proteins differentially participate in S1P-induced EC barrier enhancement. Phosphorylation of a conserved threonine residue in the C terminus of ERM proteins causes conformational changes in ERM to unmask binding sites and is considered a hallmark of ERM activation. In the present study we test the hypothesis that ERM proteins are phosphorylated on this critical threonine residue by thrombin-induced signaling events and explore the role of the ERM family in modulating thrombin-induced cytoskeletal rearrangement and EC barrier function. Thrombin promotes ERM phosphorylation at this threonine residue (Ezrin-567, Radixin-564, Moesin-558) in a PKC-dependent fashion and induces translocation of phosphorylated ERM to the EC periphery. Thrombin-induced ERM threonine phosphorylation is likely synergistically mediated by protease-activated receptors PAR1 and PAR2. Using the siRNA approach, depletion of either moesin alone, or of all three ERM proteins, significantly attenuates thrombin-induced increase in EC barrier permeability (TER), cytoskeletal rearrangements, paracellular gap formation and accumulation of di-phospho-MLC. In contrast, radixin depletion exerts opposing effects on these indices. These data suggest that ERM proteins play important differential roles in the thrombin-induced modulation of EC permeability, with moesin promoting barrier dysfunction and radixin opposing it.
[Show abstract][Hide abstract] ABSTRACT: Increased lung vascular permeability, the consequence of endothelial cell (EC) barrier dysfunction, is a cardinal feature of inflammatory conditions such as acute lung injury and sepsis and leads to lethal physiological dysfunction characterized by alveolar flooding, hypoxemia and pulmonary edema. We previously demonstrated that the non-muscle myosin light chain kinase isoform (nmMLCK) plays a key role in agonist-induced pulmonary EC barrier regulation. The present study evaluated post-transcriptional regulation of MYLK expression, the gene encoding nmMLCK, via 3'UTR binding by microRNAs (miRNAs) with in silico analysis identifying hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p and hsa-miR-1290 as miRNA candidates. We identified increased MYLK gene transcription (RT-PCR) induced by TNF-α (24 hrs, 4.7±0.45 fold increase), LPS (4 hrs, 2.85±0.15 FI) and 18% cyclic stretch (24 hrs, 4.6±0.24 FI) that was attenuated by transfection of human lung EC with mimics of hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p or hsa-miR-1290 (20-80% reductions by each miRNA). TNF-α, LPS and 18% CS each increased the activity of a MYLK 3'UTR luciferase reporter (2.5-7.0 FI) with induction reduced by mimics of each miRNA (30%-60% reduction). MiRNA inhibitors (antagomirs) for each MYLK miRNA significantly increased 3'UTR luciferase activity (1.2-2.3 FI) and rescued the decreased MLCK-3'UTR reporter activity produced by miRNA mimics (70-110% increases for each miRNA, p<0.05). These data demonstrate that increased human lung EC expression of MYLK by bioactive agonists (excessive mechanical stress, LPS, TNF-α is regulated (in part) by specific miRNAs (hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, hsa-miR-1290) representing a novel therapeutic strategy for reducing inflammatory lung injury.
American Journal of Respiratory Cell and Molecular Biology 03/2013; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Endothelial cell (EC) barrier dysfunction induced by inflammatory agonists is a frequent pathophysiologic event in multiple diseases. The platelet-derived phospholipid sphingosine-1 phosphate (S1P) reverses this dysfunction by potently enhancing the EC barrier through a process involving Rac GTPase-dependent cortical actin rearrangement as an integral step. In this study we explored the role of the ezrin, radixin, and moesin (ERM) family of actin-binding linker protein in modulating S1P-induced human pulmonary EC barrier enhancement. S1P induces ERM translocation to the EC periphery and promotes ERM phosphorylation on a critical threonine residue (Ezrin-567, Radixin-564, Moesin-558). This phosphorylation is dependent on activation of PKC isoforms and Rac1. The majority of ERM phosphorylation on these critical threonine residues after S1P occurs in moesin and ezrin. Baseline radixin phosphorylation is higher than in the other two ERM proteins but does not increase after S1P. S1P-induced moesin and ezrin threonine phosphorylation is not mediated by the barrier enhancing receptor S1PR1 because siRNA downregulation of S1PR1 fails to inhibit these phosphorylation events, while stimulation of EC with the S1PR1-specific agonist SEW2871 fails to induce these phosphorylation events. Silencing of either all ERM proteins or radixin alone (but not moesin alone) reduced S1P-induced Rac1 activation and phosphorylation of the downstream Rac1 effector PAK1. Radixin siRNA alone, or combined siRNA for all three ERM proteins, dramatically attenuates S1P-induced EC barrier enhancement (measured by transendothelial electrical resistance (TER), peripheral accumulation of di-phospho-MLC, and cortical cytoskeletal rearrangement. In contrast, moesin depletion has the opposite effects on these parameters. Ezrin silencing partially attenuates S1P-induced EC barrier enhancement and cytoskeletal changes. Thus, despite structural similarities and reported functional redundancy, the ERM proteins differentially modulate S1P-induced alterations in lung EC cytoskeleton and permeability. These results suggest that ERM activation is an important regulatory event in EC barrier responses to S1P.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Applied Physiology 11/2010; 110(1):213-24. · 3.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Elevation in intracellular cAMP level has been associated with increased endothelial barrier integrity and linked to the activation of protein kinase A (PKA). Recent studies have shown a novel mechanism of cAMP-mediated endothelial barrier regulation via cAMP-dependent nucleotide exchange factor Epac1 and Rap1 GTPase. This study examined a contribution of PKA-dependent and PKA-independent pathways in the human pulmonary endothelial (EC) barrier protection by cAMP. Synthetic cAMP analog, 8-bromoadenosine-3',5'-cyclic monophosphate (Br-cAMP), induced dose-dependent increase in EC transendothelial electrical resistance which was associated with activation of PKA, Epac/Rap1, and Tiam/Vav/Rac cascades and significantly attenuated thrombin-induced EC barrier disruption. Both specific Epac/Rap1 activator 8CPT-2Me-cAMP (8CPT) and specific PKA activator N(6)-benzoyl-adenosine-3',5'-cyclic monophosphate (6Bnz) enhanced EC barrier, suppressed thrombin-induced EC permeability, and independently activated small GTPase Rac. SiRNA-induced Rac knockdown suppressed barrier protective effects of both PKA and Epac signaling in pulmonary EC. Intravenous administration of either 6Bnz, or 8CPT, significantly reduced lung vascular leak in the murine model of lung injury induced by high tidal volume mechanical ventilation (HTV, 30 ml/kg, 4 h), whereas combined treatment with 6Bnz and 8CPT showed no further additive effects. This study dissected for the first time PKA and Epac pathways of lung EC barrier protection caused by cAMP elevation and identified Rac GTPase as a hub for PKA and Epac signaling leading to enhancement of lung vascular barrier.
Microvascular Research 12/2009; 79(2):128-38. · 2.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Actomyosin contraction directly regulates endothelial cell (EC) permeability, but intracellular redistribution of cytoskeletal tension associated with EC permeability is poorly understood. We used atomic force microscopy (AFM), EC permeability assays, and fluorescence microscopy to link barrier regulation, cell remodeling, and cytoskeletal mechanical properties in EC treated with barrier-protective as well as barrier-disruptive agonists. Thrombin, vascular endothelial growth factor, and hydrogen peroxide increased EC permeability, disrupted cell junctions, and induced stress fiber formation. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, hepatocyte growth factor, and iloprost tightened EC barriers, enhanced peripheral actin cytoskeleton and adherens junctions, and abolished thrombin-induced permeability and EC remodeling. AFM force mapping and imaging showed differential distribution of cell stiffness: barrier-disruptive agonists increased stiffness in the central region, and barrier-protective agents decreased stiffness in the center and increased it at the periphery. Attenuation of thrombin-induced permeability correlates well with stiffness changes from the cell center to periphery. These results directly link for the first time the patterns of cell stiffness with specific EC permeability responses.
Nanomedicine: nanotechnology, biology, and medicine 10/2008; 5(1):30-41. · 6.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mechanical ventilation at high tidal volumes compromises the blood-gas barrier and increases lung vascular permeability, which may lead to ventilator-induced lung injury and pulmonary edema. Using pulmonary endothelial cell (ECs) exposed to physiologically [5% cyclic stretch (CS)] and pathologically (18% CS) relevant magnitudes of CS, we evaluated the potential protective effects of hepatocyte growth factor (HGF) on EC barrier dysfunction induced by CS and vascular endothelial growth factor (VEGF). In static culture, HGF enhanced EC barrier function in a Rac-dependent manner and attenuated VEGF-induced EC permeability and paracellular gap formation. The protective effects of HGF were associated with the suppression of Rho-dependent signaling triggered by VEGF. Five percent CS promoted HGF-induced enhancement of the cortical F-actin rim and activation of Rac-dependent signaling, suggesting synergistic barrier-protective effects of physiological CS and HGF. In contrast, 18% CS further enhanced VEGF-induced EC permeability, activation of Rho signaling, and formation of actin stress fibers and paracellular gaps. These effects were attenuated by HGF pretreatment. EC preconditioning at 5% CS before HGF and VEGF further promoted EC barrier maintenance. Our data suggest synergistic effects of HGF and physiological CS in the Rac-mediated mechanisms of EC barrier protection. In turn, HGF reduced the barrier-disruptive effects of VEGF and pathological CS via downregulation of the Rho pathway. These results support the importance of HGF-VEGF balance in control of acute lung injury/acute respiratory distress syndrome severity via small GTPase-dependent regulation of lung endothelial permeability.
[Show abstract][Hide abstract] ABSTRACT: Circulating levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) are increased during acute lung injury; however, combined effects of HGF and VEGF on pulmonary endothelial cell (EC) permeability remain to be elucidated. We have previously shown differential remodeling of focal adhesions (FA) caused by barrier-protective and barrier-disruptive mechanical and chemical stimuli. This study examined a role of FA protein paxillin in the pulmonary EC barrier responses induced by HGF and VEGF. VEGF increased, but HGF decreased, pulmonary EC permeability. These effects were accompanied by differential patterns of site-specific phosphorylation of focal adhesion kinase (FAK) and paxillin and FA redistribution. HGF antagonized random FA formation caused by VEGF challenge and promoted FA accumulation at the cell periphery. HGF attenuated VEGF-induced paxillin redistribution, FA remodeling, and endothelial permeability. SiRNA-based paxillin knockdown attenuated VEGF-induced EC permeability, myosin light chain phosphorylation, and stress fiber and paracellular gap formation. Paxillin knockdown also decreased HGF-induced EC barrier enhancement and suppressed activation of Rac and its effector PAK1. Expression of paxillin-S(273) deficient on PAK1 phosphorylation site prevented HGF-induced cytoskeletal remodeling. These data show a dual role of paxillin in the HGF- and VEGF-mediated endothelial barrier regulation and suggest essential paxillin role in the modulation of Rac-Rho crosstalk. Our results also support a model of pulmonary EC barrier recovery during resolution of ALI via switch from VEGF to HGF signaling.
American Journal of Respiratory Cell and Molecular Biology 08/2008; 40(1):99-107. · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 2-Methoxyestradiol (2ME), a promising anti-tumor agent, is currently tested in phase I/II clinical trial to assess drug tolerance and clinical effects. 2ME is known to affect microtubule (MT) polymerization rather than act through estrogen receptors. We hypothesized that 2ME, similar to other MT inhibitors, disrupts endothelial barrier properties. We show that 2ME decreases transendothelial electrical resistance and increases FITC-dextran leakage across human pulmonary artery endothelial monolayer, which correlates with 2ME-induced MT depolymerization. Pretreatment of endothelium with MT stabilizer taxol significantly attenuates the decrease in transendothelial resistance. 2ME treatment results in the induction of F-actin stress fibers, accompanied by the increase in myosin light chain (MLC) phosphorylation. The experiments with Rho kinase (ROCK) and MLC kinase inhibitors and ROCK small interfering RNA (siRNA) revealed that increase in MLC phosphorylation is attributed to the ROCK activation rather than MLC kinase activation. 2ME induces significant ERK1/2, p38, and JNK phosphorylation and activation; however, only p38 activation is relevant to the 2ME-induced endothelial hyperpermeability. p38 activation is accompanied by a marked increase in MAPKAP2 and 27-kDa heat shock protein (HSP27) phosphorylation level. Taxol significantly decreases p38 phosphorylation and activation in response to 2ME stimulation. Vice versa, p38 inhibitor SB203580 attenuates MT rearrangement in 2ME-challenged cells. Together, these results indicate that 2ME-induced barrier disruption is governed by MT depolymerization and p38- and ROCK-dependent mechanisms. The fact that certain concentrations of 2ME induce endothelial hyperpermeability suggests that the issue of the maximum-tolerated dose of 2ME for cancer treatment should be addressed with caution.