Search

Acute respiratory distress syndrome (ARDS) is a severe lung condition without targeted therapy that is characterized by the disruption of epithelial and endothelial barriers. The role of the tight junction protein occludin in the pathogenesis of this disease is unknown, although it has previously been deemed redundant in some tissues. The aim of the present study is to determine whether occludin is required for lung function by controlling alveolar barrier integrity in mouse models. Immunofluorescence staining of lungs from ARDS patients revealed a significant decrease in occludin expression compared to controls. Gene delivery of shRNA against occludin in the mouse lung reduced occludin levels and induced lung injury, as assessed by wet-to-dry-ratio, histology, and cellularity and protein content of bronchial alveolar lavage fluid. Conversely, gene delivery of an occludin-expressing plasmid increased occludin expression and dampened endotoxin-induced lung injury. In primary rat alveolar epithelial cells, occludin levels were positively correlated with barrier integrity, as well as membrane localization of claudin-18, another tight junction protein. Collectively, our data demonstrate that occludin plays a significant role in alveolar barrier function and that targeting occludin may provide a new therapeutic approach for ARDS. [ABSTRACT FROM AUTHOR]

The procoagulant thrombin promotes polymorphonuclear leukocyte (PMN) adhesion to endothelial cells by a mechanism involving expression of intercellular adhesion molecule-1 (ICAM-1) via an NF-[kappa]B-dependent pathway. We now provide evidence that activation of c-Src is crucial in signaling thrombin-induced ICAM-1 expression via tyrosine phosphorylation of RelA/p65. Stimulation of human umbilical vein endothelial cells with thrombin resulted in a time-dependent activation of c-Src, with maximal activation occurring at 30 min after thrombin challenge. Inhibition of c-Src by pharmacological and genetic approaches impaired thrombin-induced NF-[kappa]B-dependent reporter activity and ICAM-1 expression. Analysis of the NF-[kappa]B pathway revealed that the effect of c-Src inhibition occurred independently of I[kappa]B[alpha] degradation and NF-[kappa]B DNA binding function and was not associated with exchange of NF-[kappa]B dimers. Phosphorylation of RelA/p65 at [Ser.sup.536], an event mediating the transcriptional activity of DNA-bound RelA/p65, was also insensitive to c-Src inhibition. Interestingly, thrombin induced association of c-Src with RelA/p65, and inhibition of c-Src prevented this response, indicating that this interaction is contingent on activation of c-Src. We also observed that thrombin induced tyrosine phosphorylation of RelA/p65, and this phosphorylation was lost upon inhibition of c-Src, consistent with the requirement of activated c-Src for interaction with RelA/p65. These data implicate an important role of c-Src in phosphorylating RelA/p65 to promote the transcriptional activity of NF-[kappa]B and thereby ICAM-1 expression in endothelial cells. tyrosine kinases; NF-[kappa]B; adhesion molecules

Lung inflammatory disease is characterized by increased polymorphonuclear leukocyte (PMN) infiltration and vascular permeability. PMN infiltration into tissue involves signaling between endothelial cells and migrating PMNs, which leads to alterations in the organization of adherens junctions (AJs). We addressed the possible role of the protein constituents of AJs, endothelium-specific vascular-endothelial (VE)-cadherin, in the migration of PMNs. Studies were made using VE-cadherin mutant constructs lacking the extracellular domain ([DELTA]EXD) or, additionally, lacking the COOH-terminus [beta]-catenin-binding domain ([DELTA]EXD[DELTA][beta]). Either construct was transduced in pulmonary microvessel endothelia of mice using cationic liposome-encapuslated cDNA constructs injected intravenously. Optimal expression of constructs was seen by Western blot analysis within 24 h. Vessel wall liquid permeability measured as the lung microvessel capillary filtration coefficient increased threefold in [DELTA]EXD-transduced lungs, indicating patency of interendothelial junctions, whereas the control [DELTA]EXD[DELTA][beta] construct was ineffective. To study lung tissue PMN recruitment, we challenged mice intraperitoneally with LPS (3 mg/kg) for 6 h and measured PMN numbers by bronchoalveolar lavage and their accumulation morphometrically in lung tissue. [DELTA]EXD expression markedly reduced the PMN sequestration and migration seen in nontransfected (control wild type) or [DELTA]EXD[DELTA] [beta]-transfected (negative control) mice challenged with LPS. In addition, [DELTA]EXD transfection suppressed LPS-induced activation of NF-[kappa]B and consequent ICAM-1 expression. These results suggest that disassembly of VE-cadherin junctions serves as a negative signal for limiting transendothelial PMN migration secondary to decreased ICAM-1 expression in the mouse model of LPS-induced sepsis. vascular endothelial cadherin; intercellular adhesion molecule-1; polymorphonuclear leukocyte adhesion; vascular endothelial-cadherin mutants

cAMP targeting of p38 MAP kinase inhibits thrombin-induced NF-[kappa]B activation and ICAM-1 expression in endothelial cells. Am J Physiol Lung Cell Mol Physiol 287: L1017-L1024, 2004. First published July 9, 2004; doi:10.1152/ajplung.00072.2004.--We investigated the mechanisms by which elevated intracellular cAMP concentration inhibits the thrombin-induced ICAM-1 expression in endothelial cells. Exposure of human umbilical vein endothelial cells to forskolin or dibutyryl cAMP, which increase intracellular cAMP by separate mechanisms, inhibited the thrombin-induced ICAM-1 expression. This effect of cAMP was secondary to inhibition of NF-[kappa]B activity, the key regulator of thrombin-induced ICAM-1 expression in endothelial cells. The action of cAMP occurred downstream of I[kappa]B[alpha] degradation and was independent of NF-[kappa]B binding to the ICAM-1 promoter. We observed that cAMP interfered with thrombin-induced phosphorylation of NF-[kappa]B p65 (Re1A) subunit, a crucial event promoting the activation of the DNA-bound NF-[kappa]B. Because p38 MAPK can induce transcriptional activity of Re1A/p65 without altering the DNA binding function of NF-[kappa]B, we addressed the possibility that cAMP antagonizes thrombin-induced NF-[kappa]B activity and ICAM-1 expression by preventing the activation of p38 MAPK. We observed that treating cells with forskolin blocked the activation of p38 MAPK, and inhibition of p38 MAPK interfered with phosphorylation of Re1A/p65 induced by thrombin. Our data demonstrate that increased intracellular cAMP concentration in endothelial cells prevents thrombin-induced ICAM-1 expression by inhibiting p38 MAPK activation, which in turn prevents phosphorylation of Re1A/p65 and transcriptional activity of the bound NF-[kappa]B. endothelial adhesivity; polymorphonuclear leukocytes; I[kappa]B[alpha] degradation; Re1A/p65 phosphorylation; mitogen-activated protein kinase; nuclear factor-[kappa]B; intercellular adhesion molecule-1; cyclic adenosine 5'-monophosphate

We examined the relationship between neutrophil [polymorphonuclear leukocyte (PMN)] influx and lung vascular injury in response to Escherichia coli pneumonia. We assessed lung tissue PMN uptake by measuring myeloperoxidase and transvascular PMN migration by determining PMN counts in lung interstitium and bronchoalveolar lavage fluid (BALF) in mice challenged intratracheally with E. coli. Lung vascular injury was quantified by determining microvessel filtration coefficient ([K.sub.f,c]), a measure of vascular permeability. We addressed the role of CD18 integrin in the mechanism of PMN migration and lung vascular injury by inducing the expression of neutrophil inhibitory factor, a CD11/CD18 antagonist. In control animals, we observed a time-dependent sixfold increase in PMN uptake, a fivefold increase in airway PMN migration, and a 20-fold increase in interstitial PMN uptake at 6 h after challenge. Interestingly, [K.sub.f,c] increased minimally during this period of PMN extravasation. CD11/CD18 blockade reduced lung tissue PMN uptake consistent with the role of CD18 in mediating PMN adhesion to the endothelium but failed to alter PMN migration in the tissue. Moreover, CD11/CD18 blockade did not affect [K.sub.f,c]. Analysis of BALF leukocytes demonstrated diminished oxidative burst compared with leukocytes from bacteremic mice, suggesting a basis for lack of vascular injury. The massive CD11/CD18-independent airway PMN influx occurring in the absence of lung vascular injury is indicative of an efficient host-defense response elicited by E. coli pneumonia. lung injury; bacteremia; polymorphonuclear leukocyte sequestration; adhesion molecules

We determined the time-dependent effects of conditional expression of neutrophil inhibitory factor (NIF), a specific 41-kDa CD18 integrin antagonist, on the time course of NIF expression and lung PMN (polymorphonuclear leukocyte) infiltration and vascular injury in a model of Escherichia coli-induced sepsis in mice. Studies were made in mice transduced with the E-selectin (ES) promoter-NIF construct (using liposomes) in which the NIF cDNA was driven by the inflammation- and endothelial cell-specific ES promoter. We observed time-dependent expression of NIF in pulmonary vascular endothelium that paralleled the ES expression. Expression of both was evident at 1 h after E. coli challenge, peaked at 3-6 h, and returned to basal level within 48 h. We observed that increases in PMN uptake and transalveolar PMN migration induced by E. coli challenge were reversed in a time-dependent manner following NIF expression in mice. NIF expression also prevented the progression of lung vascular injury and edema formation following E. coli challenge. Thus the conditional expression of NIF using the ES promoter can reverse, in a time-dependent manner, lung PMN infiltration and vascular injury induced by gram-negative sepsis. The results support the model that initial engagement of CD18 integrins enables the further recruitment of additional PMN into lung tissues such that PMN continue to sequester and migrate after E. coli challenge. E-selectin promoter; gram-negative sepsis; CD18 blockade; polymorphonuclear leukocyte migration and uptake; lung vascular permeability

The possible functions of tumor necrosis factor-alpha (TNF-alpha)-induced reactive oxygen species (ROS) generation in endothelial cells in signaling E-selectin gene transcription were investigated. Stimulation of the human pulmonary artery endothelial cells with TNF-alpha resulted in ROS production. Findings indicated that TNF-alpha induces necrosis factor-kappa-B activation and E-selectin gene expression by a pathway involving formation of ROS.

SENCR is a human-specific, vascular cell-enriched long-noncoding RNA (lncRNA) that regulates vascular smooth muscle cell and endothelial cell (EC) phenotypes. The underlying mechanisms of action of SENCR in these and other cell types is unknown. Here, levels of SENCR RNA are shown to be elevated in several differentiated human EC lineages subjected to laminar shear stress. Increases in SENCR RNA are also observed in the laminar shear stress region of the adult aorta of humanized SENCR-expressing mice, but not in disturbed shear stress regions. SENCR loss-of-function studies disclose perturbations in EC membrane integrity resulting in increased EC permeability. Biotinylated RNA pull-down and mass spectrometry establish an abundant SENCR-binding protein, cytoskeletal-associated protein 4 (CKAP4); this ribonucleoprotein complex was further confirmed in an RNA immunoprecipitation experiment using an antibody to CKAP4. Structure-function studies demonstrate a noncanonical RNA-binding domain in CKAP4 that binds SENCR. Upon SENCR knockdown, increasing levels of CKAP4 protein are detected in the EC surface fraction. Furthermore, an interaction between CKAP4 and CDH5 is enhanced in SENCR-depleted EC. This heightened association appears to destabilize the CDH5/CTNND1 complex and augment CDH5 internalization, resulting in impaired adherens junctions. These findings support SENCR as a flow-responsive lncRNA that promotes EC adherens junction integrity through physical association with CKAP4, thereby stabilizing cell membrane-bound CDH5. [ABSTRACT FROM AUTHOR]

Rahman, Arshad, Khandaker N. Anwar, and Asrar B. Malik. Protein kinase C-[Zeta] mediates TNF-[Alpha]-induced ICAM-1 gene transcription in endothelial cells. Am J Physiol Cell Physiol 279: C906-C914, 2000.--We addressed the role of protein kinase C (PKC) isozymes in mediating tumor necrosis factor-[Alpha] (TNF-[Alpha])-induced oxidant generation in endothelial cells, a requirement for nuclear factor-[Kappa]B (NF-[Kappa]B) activation and intercellular adhesion molecule-1 (ICAM-1) gene transcription. Depletion of the conventional (c) and novel (n) PKC isozymes following 24 h exposure of human pulmonary artery endothelial (HPAE) cells with the phorbol ester, phorbol 12-myristate 13-acetate (500 nM), failed to prevent TNF-[Alpha]-induced oxidant generation. In contrast, inhibition of PKC-[Zeta] synthesis by the antisense oligonucleotide prevented the oxidant generation following the TNF-[Alpha] stimulation. We observed that PKC-[Zeta] also induced the TNF-[Alpha]-induced NF-[Kappa]B binding to the ICAM-1 promoter and the resultant ICAM-1 gene transcription. We showed that expression of the dominant negative mutant of PKC-[Zeta] prevented the TNF-[Alpha]-induced ICAM-1 promoter activity, whereas overexpression of the wild-type PKC-[Zeta] augmented the response. These data imply a critical role for the PKC-[Zeta] isozyme in regulating TNF-[Alpha]-induced oxidant generation and in signaling the activation of NF-[Kappa]B and ICAM-1 transcription in endothelial cells. protein kinase C isoforms; oxidants; nuclear factor-[Kappa]B; intercellular adhesion molecule-1; endothelium

True, Andrea L., Arshad Rahman, and Asrar B. Malik. Activation of NF-[Kappa]B induced by [H.sub.2][O.sub.2] and TNF-[Alpha] and its effects on ICAM-1 expression in endothelial cells. Am J Physiol Lung Cell Mol Physiol 279: L302-L311, 2000.--Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-[Kappa]B in response to tumor necrosis factor (TNF)-[Alpha] challenge. In the present study, we investigated the effects of [H.sub.2][O.sub.2] and TNF-[Alpha] in mediating activation of NF-[Kappa]B and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis showed that TNF-[Alpha] exposure of human dermal microvascular endothelial cells (HMEC-1) induced marked increases in ICAM-1 mRNA and cell surface protein expression. In contrast, [H.sub.2][O.sub.2] added at subcytolytic concentrations failed to activate ICAM-1 expression. Challenge with [H.sub.2][O.sub.2] also failed to induce NF-[Kappa]B-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-[Alpha] increased the NF-[Kappa]B-driven gene expression ~10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both [H.sub.2][O.sub.2]- and TNF-[Alpha]-induced NF-[Kappa]B complexes bound to the ICAM-1 promoter, with the binding of the p65 subunit being the most prominent. In vivo phosphorylation studies, however, showed that TNF-[Alpha] exposure induced marked phosphorylation of NF-[Kappa]B p65 in HMEC-1 cells, whereas [H.sub.2][O.sub.2] had no effect. These results suggest that reactive oxygen species generation in endothelial cells mediates the binding of NF-[Kappa]B to nuclear DNA, whereas TNF-[Alpha] generates additional signals that induce phosphorylation of the bound NF-[Kappa]B p65 and confer transcriptional competency to NF-[Kappa]B. redox state; intercellular adhesion molecule-1 promoter; tumor necrosis factor-[Alpha]; oxidants; nuclear factor-[Kappa]B; signaling; hydrogen peroxide