Cholesterol-Dependent Modulation of Stem Cell Biomechanics: Application to Adipogenesis.

Cell mechanics has been shown to regulate stem cell differentiation. We have previously reported that altered cell stiffness of mesenchymal stem cells can delay or facilitate biochemically directed differentiation. One of the factors that can affect the cell stiffness is cholesterol. However, the effect of cholesterol on differentiation of human mesenchymal stem cells remains elusive. In this paper, we demonstrate that cholesterol is involved in the modulation of the cell stiffness and subsequent adipogenic differentiation. Rapid cytoskeletal actin reorganization was evident and correlated with the cell's Young's modulus measured using atomic force microscopy. In addition, the level of membrane-bound cholesterol was found to increase during adipogenic differentiation and inversely varied with the cell stiffness. Furthermore, cholesterol played a key role in the regulation of the cell morphology and biomechanics, suggesting its crucial involvement in mechanotransduction. To better understand the underlying mechanisms, we investigated the effect of cholesterol on the membrane-cytoskeleton linker proteins (ezrin and moesin). Cholesterol depletion was found to upregulate the ezrin expression which promoted cell spreading, increased Young's modulus, and hindered adipogenesis. In contrast, cholesterol enrichment increased the moesin expression, decreased Young's modulus, and induced cell rounding and facilitated adipogenesis. Taken together, cholesterol appears to regulate the stem cell mechanics and adipogenesis through the membrane-associated linker proteins.

Mechanical stress induces pre-B-cell colony-enhancing factor/NAMPT expression via epigenetic regulation by miR-374a and miR-568 in human lung endothelium.

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 pre-B-cell colony-enhancing factor (PBEF)/NAMPT, the proinflammatory cytokine encoded by NAMPT, participates in ARDS and VILI inflammatory syndromes. The present study evaluated posttranscriptional regulation of PBEF/NAMPT gene expression in human lung endothelium via 3'-untranslated region (UTR) microRNA (miRNA) binding. In silico analysis identified hsa-miR-374a and hsa-miR-568 as potential miRNA candidates. Increased PBEF/NAMPT transcription (by RT-PCR) and expression (by Western blotting) induced by 18% cyclic stretch (CS) (2 h: 3.4 ± 0.06 mRNA fold increase (FI); 10 h: 1.5 ± 0.06 protein FI) and by LPS (4 h: 3.8 ± 0.2 mRNA FI; 48 h: 2.6 ± 0.2 protein FI) were significantly attenuated by transfection with mimics of hsa-miR-374a or hsa-miR-568 (40-60% reductions each). LPS and 18% CS 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 (i.e., excessive mechanical stress, LPS) involves epigenetic regulation with hsa-miR-374a and hsa-miR-568, representing novel therapeutic strategies to reduce inflammatory lung injury.

Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function.

MicroRNA regulation of nonmuscle myosin light chain kinase expression in human lung endothelium.

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 nonmuscle myosin light chain kinase isoform (nmMLCK) plays a key role in agonist-induced pulmonary EC barrier regulation. The present study evaluated posttranscriptional regulation of MYLK expression, the gene encoding nmMLCK, via 3' untranslated region (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 induced by TNF-α (24 h; 4.7 ± 0.45 fold increase [FI]), LPS (4 h; 2.85 ± 0.15 [FI]), and 18% cyclic stretch (24 h; 4.6 ± 0.24 FI) that was attenuated by transfection of human lung ECs 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% cyclic stretch 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, and hsa-miR-1290), representing a novel therapeutic strategy for reducing inflammatory lung injury.

Ezrin/radixin/moesin proteins differentially regulate endothelial hyperpermeability after thrombin.

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 sphingosine-1 phosphate-induced EC barrier enhancement. Phosphorylation of a conserved threonine residue in the COOH-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 Thr567, radixin Thr564, moesin Thr558) 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 (transendothelial electrical resistance), cytoskeletal rearrangements, paracellular gap formation, and accumulation of phospho-myosin light chain. In contrast, radixin depletion exerts opposing effects on these indexes. 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.

Putative protein partners for the human CPI-17 protein revealed by bacterial two-hybrid screening.

We have previously demonstrated that PKC-potentiated inhibitory protein of protein phosphatase-1 (CPI-17) is expressed in lung endothelium. CPI-17, a specific inhibitor of myosin light chain phosphatase (MLCP), is involved in the endothelial cytoskeletal and barrier regulation. In this paper, we report the identification of fourteen putative CPI-17 interacting proteins in the lung using BacterioMatch Two-Hybrid System. Five of them: plectin 1 isoform 1, alpha II spectrin, OK/SW-CL.16, gelsolin isoform a, and junction plakoglobin are involved in actin cytoskeleton organization and cell adhesion, suggesting possible significance of these binding partners in CPI-17-mediated cytoskeletal reorganization of endothelial cells. Furthermore, we confirmed the specific interaction between plakoglobin and CPI-17, which is affected by the phosphorylation status of CPI-17 in human lung microvascular endothelial cells.

A cortactin CTTN coding SNP contributes to lung vascular permeability and inflammatory disease severity in African descent subjects.

The cortactin gene (CTTN), encoding an actin-binding protein critically involved in cytoskeletal dynamics and endothelial cell (EC) barrier integrity, contains single nucleotide polymorphisms (SNPs) associated with severe asthma in Black patients. As loss of lung EC integrity is a major driver of mortality in the Acute Respiratory Distress Syndrome (ARDS), sepsis, and the acute chest syndrome (ACS), we speculated CTTN SNPs that alter EC barrier function will associate with clinical outcomes from these types of conditions in Black patients. In case-control studies, evaluation of a nonsynonymous CTTN coding SNP Ser484Asn (rs56162978, G/A) in a severe sepsis cohort (725 Black subjects) revealed significant association with increased risk of sepsis mortality. In a separate cohort of sickle cell disease (SCD) subjects with and without ACS (177 SCD Black subjects), significantly increased risk of ACS and increased ACS severity (need for mechanical ventilation) was observed in carriers of the A allele. Human lung EC expressing the cortactin S484N transgene exhibited: (i) delayed EC barrier recovery following thrombin-induced permeability; (ii) reduced levels of critical Tyr486 cortactin phosphorylation; (iii) inhibited binding to the cytoskeletal regulator, nmMLCK; and (iv) attenuated EC barrier-promoting lamellipodia dynamics and biophysical responses. ARDS-challenged Cttn+/- heterozygous mice exhibited increased lung vascular permeability (compared to wild-type mice) which was significantly attenuated by IV delivery of liposomes encargoed with CTTN WT transgene but not by CTTN S484N transgene. In summary, these studies suggest that the CTTN S484N coding SNP contributes to severity of inflammatory injury in Black patients, potentially via delayed vascular barrier restoration.

Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium.

Vascular barrier regulation is intimately linked to alterations in the distribution and configuration of the endothelial cell (EC) cytoskeleton in response to angiogenic and edemagenic agonists. Critical actin cytoskeletal rearrangement includes spatially directed increases in myosin light chain (MLC) phosphorylation, catalyzed by Ca(2+)/calmodulin-dependent non-muscle myosin light chain kinase variants (nmMLCK1- and -2), as well as association of nmMLCK with the actin-binding protein, cortactin. As these associations have proven difficult to quantify in a spatially specific manner, we now describe the utility of intensity correlation image analysis and the intensity correlation quotient (ICQ) to quantify colocalization in fixed and live cell imaging assays in human pulmonary artery EC. From baseline ICQ values averaging 0.216 reflecting colocalization of cortactin-DsRed with EGFP-nmMLCK fusion proteins in resting EC, thrombin-induced EC contraction significantly reduced cortactin-DsRed-EGFP-nmMLCK colocalization (nmMLCK1: ICQ=0.118; nmMLCK2: ICQ=0.091) whereas the potent EC barrier-protective agonist, sphingosine 1-phosphate (S1P), significantly increased nmMLCK-cortactin colocalization within lamellipodia (nmMLCK1: ICQ=0.275; nmMLCK2: ICQ=0.334). Over-expression of a cortactin-DsRed mutant fusion protein lacking the SH3 domain, known to be essential for cortactin-nmMLCK association, reduced baseline and S1P-mediated live cell colocalization with each nmMLCK variant (nmMLCK1: ICQ=0.160; nmMLCK2: ICQ=0.157). Similarly, expression of a truncated EGFP-nmMLCK2 mutant lacking cortactin- and actin-binding domains, markedly reduced basal localization in lamellipodia and abolished colocalization with cortactin-DsRed in lamellipodia after S1P (ICQ=-0.148). These data provide insights into the molecular basis for vascular barrier-regulatory cytoskeletal responses and support the utility of sophisticated imaging analyses and methodological assessment to quantify the critical nmMLCK and cortactin interaction during vascular barrier regulation.

Interactions between PBEF and oxidative stress proteins--a potential new mechanism underlying PBEF in the pathogenesis of acute lung injury.

Identification of pre-B-cell colony-enhancing factor (PBEF) interacting partners may reveal new molecular mechanisms of PBEF in the pathogenesis of acute lung injury (ALI). The interactions between PBEF and NADH dehydrogenase subunit 1(ND1), ferritin light chain and interferon induced transmembrane 3 (IFITM3) in human pulmonary vascular endothelial cells were identified and validated. ND1, ferritin and IFITM3 are involved in oxidative stress and inflammation. Overexpression of PBEF increased its interactions and intracellular oxidative stress, which can be attenuated by rotenone. The interaction modeling between PBEF and ND1 is consistent with the corresponding experimental finding. These interactions may underlie a novel role of PBEF in the pathogenesis of ALI.

Signaling pathways involved in adenosine triphosphate-induced endothelial cell barrier enhancement.

Endothelial barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell-cell junctions. Increases in cytosolic Ca2+ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically Galphaq and Galphai2), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of Galphaq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca2+, but involve activation of myosin phosphatase via a novel G-protein-coupled mechanism and PKA.

Endotoxin- and mechanical stress-induced epigenetic changes in the regulation of the nicotinamide phosphoribosyltransferase promoter.

Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.

Novel role of microtubules in thrombin-induced endothelial barrier dysfunction.

Disturbances in endothelial cell (EC) barrier regulation are critically dependent upon rearrangements of EC actin cytoskeleton. However, the role of microtubule (MT) network in the regulation of EC permeability is not well understood. We examined involvement of MT remodeling in thrombin-induced EC permeability and explored MT regulation by heterotrimeric G12/13 proteins and by small GTPase Rho. Thrombin induced phosphorylation of MT regulatory protein tau at Ser409 and Ser262 and peripheral MT disassembly, which was linked to increased EC permeability. MT stabilization by taxol attenuated thrombin-induced permeability, actin remodeling, and paracellular gap formation and diminished thrombin-induced activation of Rho and Rho-kinase. Expression of activated Galpha12/13 subunits involved in thrombin-mediated signaling or their effector p115RhoGEF involved in Rho activation caused MT disassembly, whereas p115RhoGEF-specific negative regulator RGS preserved MT from thrombin-induced disassembly. Consistent with these results, expression of activated RhoA and Rho-kinase induced MT disassembly. Conversely, thrombin-induced disassembly of peripheral MT network was attenuated by expression of dominant negative RhoA and Rho-kinase mutants or by pharmacological inhibition of Rho-kinase. Collectively, our data demonstrate for the first time a critical involvement of MT disassembly in thrombin-induced EC barrier dysfunction and indicate G-protein-dependent mechanisms of thrombin-induced MT alteration.

Unique Toll-Like Receptor 4 Activation by NAMPT/PBEF Induces NFκB Signaling and Inflammatory Lung Injury.

Ventilator-induced inflammatory lung injury (VILI) is mechanistically linked to increased NAMPT transcription and circulating levels of nicotinamide phosphoribosyl-transferase (NAMPT/PBEF). Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown. We now report that NAMPT/PBEF induces lung NFκB transcriptional activities and inflammatory injury via direct ligation of Toll-like receptor 4 (TLR4). Computational analysis demonstrated that NAMPT/PBEF and MD-2, a TLR4-binding protein essential for LPS-induced TLR4 activation, share ~30% sequence identity and exhibit striking structural similarity in loop regions critical for MD-2-TLR4 binding. Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS. The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.

Differential involvement of ezrin/radixin/moesin proteins in sphingosine 1-phosphate-induced human pulmonary endothelial cell barrier enhancement.

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.

TIMAP is a positive regulator of pulmonary endothelial barrier function.

TGF-beta-inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits; however, its function in EC is not clear. In our pull-down experiments, recombinant TIMAP binds preferentially the beta-isoform of the catalytic subunit of PP1 (PP1cbeta) from pulmonary artery EC. As PP1cbeta, but not PP1calpha, binds with MYPT1 into functional complex, these results suggest that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by small interfering RNA (siRNA) technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP) and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed colocalization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP coimmunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/PKA cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On the contrary, in TIMAP-depleted EC, forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.

Role of vasodilator-stimulated phosphoprotein in cGMP-mediated protection of human pulmonary artery endothelial barrier function.

Increased pulmonary endothelial cGMP was shown to prevent endothelial barrier dysfunction through activation of protein kinase G (PKG(I)). Vasodilator-stimulated phosphoprotein (VASP) has been hypothesized to mediate PKG(I) barrier protection because VASP is a cytoskeletal phosphorylation target of PKG(I) expressed in cell-cell junctions. Unphosphorylated VASP was proposed to increase paracellular permeability through actin polymerization and stress fiber bundling, a process inhibited by PKG(I)-mediated phosphorylation of Ser(157) and Ser(239). To test this hypothesis, we examined the role of VASP in the transient barrier dysfunction caused by H(2)O(2) in human pulmonary artery endothelial cell (HPAEC) monolayers studied without and with PKG(I) expression introduced by adenoviral infection (Ad.PKG). In the absence of PKG(I) expression, H(2)O(2) (100-250 microM) caused a transient increased permeability and pSer(157)-VASP formation that were both attenuated by protein kinase C inhibition. Potentiation of VASP Ser(157) phosphorylation by either phosphatase 2B inhibition with cyclosporin or protein kinase A activation with forskolin prolonged, rather than inhibited, the increased permeability caused by H(2)O(2). With Ad.PKG infection, inhibition of VASP expression with small interfering RNA exacerbated H(2)O(2)-induced barrier dysfunction but had no effect on cGMP-mediated barrier protection. In addition, expression of a Ser-double phosphomimetic mutant VASP failed to reproduce the protective effects of activated PKG(I). Finally, expression of a Ser-double phosphorylation-resistant VASP failed to interfere with the ability of cGMP/PKG(I) to attenuate H(2)O(2)-induced disruption of VE-cadherin homotypic binding. Our results suggest that VASP phosphorylation does not explain the protective effect of cGMP/PKG(I) on H(2)O(2)-induced endothelial barrier dysfunction in HPAEC.

Involvement of microtubules, p38, and Rho kinases pathway in 2-methoxyestradiol-induced lung vascular barrier dysfunction.

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.

Potential protein partners for the human TIMAP revealed by bacterial two-hybrid screening.

BacterioMatch Two-Hybrid System (Stratagene) was applied in order to identify potential human TIMAP interaction proteins in the lung. TIMAP highly expressed in endothelial cells and may be involved in endothelial cytoskeletal and barrier regulation. Seven TIMAP interacting partner proteins were identified. Four of identified proteins: cystein and glycine-rich protein 1, eukaryotic translation elongation factor 2, U5 snRNP-specific protein 116 kD, and solute carrier family 3 member 2 are involved in actin cytoskeleton organization, cell adhesion or translation and transcriptional regulation.

GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction.

Endothelial cell (EC) permeability is precisely controlled by cytoskeletal elements [actin filaments, microtubules (MT), intermediate filaments] and cell contact protein complexes (focal adhesions, adherens junctions, tight junctions). We have recently shown that the edemagenic agonist thrombin caused partial MT disassembly, which was linked to activation of small GTPase Rho, Rho-mediated actin remodeling, cell contraction, and dysfunction of lung EC barrier. GEF-H1 is an MT-associated Rho-specific guanosine nucleotide (GDP/GTP) exchange factor, which in MT-unbound state stimulates Rho activity. In this study we tested hypothesis that GEF-H1 may be a key molecule involved in Rho activation, myosin light chain phosphorylation, actin remodeling, and EC barrier dysfunction associated with partial MT disassembly. Our results show that depletion of GEF-H1 or expression of dominant negative GEF-H1 mutant significantly attenuated permeability increase, actin stress fiber formation, and increased MLC and MYPT1 phosphorylation induced by thrombin or MT-depolymerizing agent nocodazole. In contrast, expression of wild-type or activated GEF-H1 mutants dramatically enhanced thrombin and nocodazole effects on stress fiber formation and cell retraction. These results show a critical role for the GEF-H1 in the Rho activation caused by MT disassembly and suggest GEF-H1 as a key molecule involved in cross talk between MT and actin cytoskeleton in agonist-induced Rho-dependent EC barrier regulation.

Pre-B-cell-colony-enhancing factor is critically involved in thrombin-induced lung endothelial cell barrier dysregulation.

Prior genomic and genetic studies identified pre-B-cell colony-enhancing factor (PBEF) as a novel candidate gene and biomarker in acute lung injury (ALI). As increased vascular permeability is a cardinal feature of ALI, we assessed the role of PBEF in in vitro vascular barrier regulation using confluent human pulmonary artery endothelial cell (HPAEC) monolayers. Reductions in PBEF protein expression (>70%) by siRNA significantly attenuated EC barrier dysfunction induced by the potent edemagenic agent, thrombin, reflected by reductions in transendothelial electric resistance (TER, approximately 60% reduction). Furthermore, PBEF siRNA blunted thrombin-mediated increases in Ca(2+) entry, polymerized actin formation, and myosin light chain phosphorylation, events critical to the thrombin-mediated permeability response. Finally, PBEF siRNA also significantly inhibited thrombin-stimulated increase of IL-8 secretion in HPAEC, a chemokine known to induce actin fiber formation and intercellular gap formation of endothelial cells. Taken together, these studies demonstrate that PBEF may be required for complete expression of the thrombin-induced inflammatory response and reveal potentially novel role for PBEF in the regulation of EC Ca(2+)-dependent cytoskeletal rearrangement and endothelial barrier dysfunction. Ongoing studies will continue to address the molecular mechanisms by which PBEF contributes to ALI susceptibility.