Spleen Tyrosine Kinase phosphorylates VE-cadherin to cause endothelial barrier disruption in acute lung injury.

Increased endothelial cell (EC) permeability is a cardinal feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Tyrosine phosphorylation of VE-cadherin is a key determinant of EC barrier disruption. However, the identity and role of tyrosine kinases in this context are incompletely understood. Here we report that Spleen Tyrosine Kinase (Syk) is a key mediator of EC barrier disruption and lung vascular leak in sepsis. Inhibition of Syk by pharmacological or genetic approaches, each reduced thrombin-induced EC permeability. Mechanistically, Syk associates with and phosphorylates VE-cadherin to cause EC permeability. To study the causal role of endothelial Syk in sepsis-induced ALI, we used a remarkably efficient and cost-effective approach based on gene transfer to generate EC-ablated Syk mice. These mice were protected against sepsis-induced loss of VE-cadherin and inflammatory lung injury. Notably, the administration of Syk inhibitor R788 (fostamatinib); currently in phase II clinical trial for the treatment of COVID-19, mitigated lung injury and mortality in mice with sepsis. These data identify Syk as a novel kinase for VE-cadherin and a druggable target against ALI in sepsis.

Impact of Peptide Sequence on Functional siRNA Delivery and Gene Knockdown with Cyclic Amphipathic Peptide Delivery Agents.

Short-interfering RNA (siRNA) oligonucleotide therapeutics that modify gene expression by accessing RNA-interference (RNAi) pathways have great promise for the treatment of a range of disorders; however, their application in clinical settings has been limited by significant challenges in cellular delivery. Herein, we report a structure-function study using a series of modified cyclic amphipathic cell-penetrating peptides (CAPs) to determine the impact of peptide sequence on (1) siRNA-binding efficiency, (2) cellular delivery and knockdown efficiency, and (3) the endocytic uptake mechanism. Nine cyclic peptides of the general sequence Ac-C[XZ]4CG-NH2 in which X residues are hydrophobic/aromatic (Phe, Tyr, Trp, or Leu) and Z residues are charged/hydrophilic (Arg, Lys, Ser, or Glu) are assessed along with one acyclic peptide, Ac-(WR)4G-NH2. Cyclization is enforced by intramolecular disulfide bond formation between the flanking Cys residues. Binding analyses indicate that strong cationic character and the presence of aromatic residues that are competent to participate in CH-π interactions lead to CAP sequences that most effectively interact with siRNA. CAP-siRNA binding increases in the following order as a function of CAP hydrophobic/aromatic content: His < Phe < Tyr < Trp. Both cationic charge and disulfide-constrained cyclization of CAPs improve uptake of siRNA in vitro. Net neutral CAPs and an acyclic peptide demonstrate less-efficient siRNA translocation compared to the cyclic, cationic CAPs tested. All CAPs tested facilitated efficient siRNA target gene knockdown of at least 50% (as effective as a lipofectamine control), with the best CAPs enabling >80% knockdown. Significantly, gene knockdown efficiency does not strongly correlate with CAP-siRNA internalization efficiency but moderately correlates with CAP-siRNA-binding affinity. Finally, utilization of small-molecule inhibitors and targeted knockdown of essential endocytic pathway proteins indicate that most CAP-siRNA nanoparticles facilitate siRNA delivery through clathrin- and caveolin-mediated endocytosis. These results provide insight into the design principles for CAPs to facilitate siRNA delivery and the mechanisms by which these peptides translocate siRNA into cells. These studies also demonstrate the nature of the relationships between peptide-siRNA binding, cellular delivery of siRNA cargo, and functional gene knockdown. Strong correlations between these properties are not always observed, which illustrates the complexity in the design of optimal next-generation materials for oligonucleotide delivery.

Phosphodiesterase 10A Is a Key Mediator of Lung Inflammation.

Cyclic nucleotides cAMP and cGMP are important regulators of immune cell functions. Phosphodiesterases (PDEs) hydrolyze cAMP and/or cGMP and, thus, play crucial roles in cyclic nucleotide homeostasis. Abnormal alterations of PDE expression have been implicated in several diseases. To understand the function of PDEs in macrophages, we screened for all PDE genes in both peritoneal and alveolar macrophages from C57BL/6J mice and found that PDE4B and PDE10A are highly induced by LPS. A number of PDE4 inhibitors have been used clinically for the treatment of inflammatory lung diseases. However, the role of PDE10A in inflammation is still poorly understood. We therefore investigated the role of PDE10A in macrophage inflammatory response in vitro and acute lung inflammation in vivo. We found that LPS induces a sustained PDE10A expression in macrophages, which is different from a transient induction by PDE4B. PDE10A inhibition blocked LPS-induced MCP-1 expression, but not TNF-α, whereas PDE4B inhibition blocked LPS-induced TNF-α expression, but not MCP-1. In addition, PDE10A inhibition or deficiency decreased LPS-induced HIF-1α protein expression and subsequently suppressed MCP-1 expression. In vivo, PDE10A expression was also elevated in lung tissue after LPS exposure. Global PDE10A knockout or systemic administration of the PDE10A inhibitor TP-10 in mice significantly suppressed inflammatory molecule levels in the lung tissue and bronchoalveolar lavage fluid as well as inflammatory cell infiltration. These findings show that PDE10A plays a critical role in lung inflammation by promoting the activation of resident macrophages and infiltration of neutrophils.

SENCR stabilizes vascular endothelial cell adherens junctions through interaction with CKAP4.

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.

The novel coronary artery disease risk gene JCAD/KIAA1462 promotes endothelial dysfunction and atherosclerosis.

AIMS: Recent genome-wide association studies (GWAS) have identified that the JCAD locus is associated with risk of coronary artery disease (CAD) and myocardial infarction (MI). However, the mechanisms whereby candidate gene JCAD confers disease risk remain unclear. We addressed whether and how JCAD affects the development of atherosclerosis, the common cause of CAD. METHODS AND RESULTS: By mining data in the Genotype-Tissue Expression (GTEx) database, we found that CAD-associated risk variants at the JCAD locus are linked to increased JCAD gene expression in human arteries, implicating JCAD as a candidate causal CAD gene. We therefore generated global and endothelial cell (EC) specific-JCAD knockout mice, and observed that JCAD deficiency attenuated high fat diet-induced atherosclerosis in ApoE-deficient mice. JCAD-deficiency in mice also improved endothelium-dependent relaxation. Genome-wide transcriptional profiling of JCAD-depleted human coronary artery ECs showed that JCAD depletion inhibited the activation of YAP/TAZ pathway, and the expression of downstream pro-atherogenic genes, including CTGF and Cyr61. As a result, JCAD-deficient ECs attracted fewer monocytes in response to lipopolysaccharide (LPS) stimulation. Moreover, JCAD expression in ECs was decreased under unidirectional laminar flow in vitro and in vivo. Proteomics studies suggest that JCAD regulates YAP/TAZ activation by interacting with actin-binding protein TRIOBP, thereby stabilizing stress fiber formation. Finally, we observed that endothelial JCAD expression was increased in mouse and human atherosclerotic plaques. CONCLUSION: The present study demonstrates that the GWAS-identified CAD risk gene JCAD promotes endothelial dysfunction and atherosclerosis, thus highlighting the possibility of new therapeutic strategies for CAD by targeting JCAD.

Autophagy inhibitor 3-methyladenine protects against endothelial cell barrier dysfunction in acute lung injury.

Autophagy is an evolutionarily conserved cellular process that facilitates the continuous recycling of intracellular components (organelles and proteins) and provides an alternative source of energy when nutrients are scarce. Recent studies have implicated autophagy in many disorders, including pulmonary diseases. However, the role of autophagy in endothelial cell (EC) barrier dysfunction and its relevance in the context of acute lung injury (ALI) remain uncertain. Here, we provide evidence that autophagy is a critical component of EC barrier disruption in ALI. Using an aerosolized bacterial lipopolysaccharide (LPS) inhalation mouse model of ALI, we found that administration of the autophagy inhibitor 3-methyladenine (3-MA), either prophylactically or therapeutically, markedly reduced lung vascular leakage and tissue edema. 3-MA was also effective in reducing the levels of proinflammatory mediators and lung neutrophil sequestration induced by LPS. To test the possibility that autophagy in EC could contribute to lung vascular injury, we addressed its role in the mechanism of EC barrier disruption. Knockdown of ATG5, an essential regulator of autophagy, attenuated thrombin-induced EC barrier disruption, confirming the involvement of autophagy in the response. Similarly, exposure of cells to 3-MA, either before or after thrombin, protected against EC barrier dysfunction by inhibiting the cleavage and loss of vascular endothelial cadherin at adherens junctions, as well as formation of actin stress fibers. 3-MA also reversed LPS-induced EC barrier disruption. Together, these data imply a role of autophagy in lung vascular injury and reveal the protective and therapeutic utility of 3-MA against ALI.

Serpine2 deficiency results in lung lymphocyte accumulation and bronchus-associated lymphoid tissue formation.

Serine proteinase inhibitor, clade E, member 2 (SERPINE2), is a cell- and extracellular matrix-associated inhibitor of thrombin. Although SERPINE2 is a candidate susceptibility gene for chronic obstructive pulmonary disease, the physiologic role of this protease inhibitor in lung development and homeostasis is unknown. We observed spontaneous monocytic-cell infiltration in the lungs of Serpine2-deficient (SE2(-/-)) mice, beginning at or before the time of lung maturity, which resulted in lesions that resembled bronchus-associated lymphoid tissue (BALT). The initiation of lymphocyte accumulation in the lungs of SE2(-/-) mice involved the excessive expression of chemokines, cytokines, and adhesion molecules that are essential for BALT induction, organization, and maintenance. BALT-like lesion formation in the lungs of SE2(-/-) mice was also associated with a significant increase in the activation of thrombin, a recognized target of SE2, and excess stimulation of NF-κB, a major regulator of chemokine expression and inflammation. Finally, systemic delivery of thrombin rapidly stimulated lung chemokine expression in vivo These data uncover a novel mechanism whereby loss of serine protease inhibition leads to lung lymphocyte accumulation.-Solleti, S. K., Srisuma, S., Bhattacharya, S., Rangel-Moreno, J., Bijli, K. M., Randall, T. D., Rahman, A., Mariani, T. J. Serpine2 deficiency results in lung lymphocyte accumulation and bronchus-associated lymphoid tissue formation.

Inhibition of Regulatory-Associated Protein of Mechanistic Target of Rapamycin Prevents Hyperoxia-Induced Lung Injury by Enhancing Autophagy and Reducing Apoptosis in Neonatal Mice.

Administration of supplemental oxygen remains a critical clinical intervention for survival of preterm infants with respiratory failure. However, prolonged exposure to hyperoxia can augment pulmonary damage, resulting in developmental lung diseases embodied as hyperoxia-induced acute lung injury and bronchopulmonary dysplasia (BPD). We sought to investigate the role of autophagy in hyperoxia-induced apoptotic cell death in developing lungs. We identified increased autophagy signaling in hyperoxia-exposed mouse lung epithelial-12 cells, freshly isolated fetal type II alveolar epithelial cells, lungs of newborn wild-type mice, and human newborns with respiratory distress syndrome and evolving and established BPD. We found that hyperoxia exposure induces autophagy in a Trp53-dependent manner in mouse lung epithelial-12 cells and in neonatal mouse lungs. Using pharmacological inhibitors and gene silencing techniques, we found that the activation of autophagy, upon hyperoxia exposure, demonstrated a protective role with an antiapoptotic response. Specifically, inhibiting regulatory-associated protein of mechanistic target of rapamycin (RPTOR) in hyperoxia settings, as evidenced by wild-type mice treated with torin2 or mice administered (Rptor) silencing RNA via intranasal delivery or Rptor+/-, limited lung injury by increased autophagy, decreased apoptosis, improved lung architecture, and increased survival. Furthermore, we identified increased protein expression of phospho-beclin1, light chain-3-II and lysosomal-associated membrane protein 1, suggesting altered autophagic flux in the lungs of human neonates with established BPD. Collectively, our study unveils a novel demonstration of enhancing autophagy and antiapoptotic effects, specifically through the inhibition of RPTOR as a potentially useful therapeutic target for the treatment of hyperoxia-induced acute lung injury and BPD in developing lungs.

Phospholipase C-ε signaling mediates endothelial cell inflammation and barrier disruption in acute lung injury.

Phospholipase C-ε (PLC-ε) is a unique PLC isoform that can be regulated by multiple signaling inputs from both Ras family GTPases and heterotrimeric G proteins and has primary sites of expression in the heart and lung. Whereas the role of PLC-ε in cardiac function and pathology has been documented, its relevance in acute lung injury (ALI) is unclear. We used PLC-ε(-/-) mice to address the role of PLC-ε in regulating lung vascular inflammation and injury in an aerosolized bacterial LPS inhalation mouse model of ALI. PLC-ε(-/-) mice showed a marked decrease in LPS-induced proinflammatory mediators (ICAM-1, VCAM-1, TNF-α, IL-1ß, IL-6, macrophage inflammatory protein 2, keratinocyte-derived cytokine, monocyte chemoattractant protein 1, and granulocyte-macrophage colony-stimulating factor), lung neutrophil infiltration and microvascular leakage, and loss of VE-cadherin compared with PLC-ε(+/+) mice. These data identify PLC-ε as a critical determinant of proinflammatory and leaky phenotype of the lung. To test the possibility that PLC-ε activity in endothelial cells (EC) could contribute to ALI, we determined its role in EC inflammation and barrier disruption. RNAi knockdown of PLC-ε inhibited NF-κB activity in response to diverse proinflammatory stimuli, thrombin, LPS, TNF-α, and the nonreceptor agonist phorbol 13-myristate 12-acetate (phorbol esters) in EC. Depletion of PLC-ε also inhibited thrombin-induced expression of NF-κB target gene, VCAM-1. Importantly, PLC-ε knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and formation of actin stress fibers. These data identify PLC-ε as a novel regulator of EC inflammation and permeability and show a hitherto unknown role of PLC-ε in the pathogenesis of ALI.

Airway epithelial cell PPARγ modulates cigarette smoke-induced chemokine expression and emphysema susceptibility in mice.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent, chronic inflammatory lung disease with limited existing therapeutic options. While modulation of peroxisome proliferator-activating receptor (PPAR)-γ activity can modify inflammatory responses in several models of lung injury, the relevance of the PPARG pathway in COPD pathogenesis has not been previously explored. Mice lacking Pparg specifically in airway epithelial cells displayed increased susceptibility to chronic cigarette smoke (CS)-induced emphysema, with excessive macrophage accumulation associated with increased expression of chemokines, Ccl5, Cxcl10, and Cxcl15. Conversely, treatment of mice with a pharmacological PPARγ activator attenuated Cxcl10 and Cxcl15 expression and macrophage accumulation in response to CS. In vitro, CS increased lung epithelial cell chemokine expression in a PPARγ activation-dependent fashion. The ability of PPARγ to regulate CS-induced chemokine expression in vitro was not specifically associated with peroxisome proliferator response element (PPRE)-mediated transactivation activity but was correlated with PPARγ-mediated transrepression of NF-κB activity. Pharmacological or genetic activation of PPARγ activity abrogated CS-dependent induction of NF-κB activity. Regulation of NF-κB activity involved direct PPARγ-NF-κB interaction and PPARγ-mediated effects on IKK activation, IκBα degradation, and nuclear translocation of p65. Our data indicate that PPARG represents a disease-relevant pathophysiological and pharmacological target in COPD. Its activation state likely contributes to NF-κB-dependent, CS-induced chemokine-mediated regulation of inflammatory cell accumulation.

Thrombin selectively engages LIM kinase 1 and slingshot-1L phosphatase to regulate NF-κB activation and endothelial cell inflammation.

Endothelial cell (EC) inflammation is a central event in the pathogenesis of many pulmonary diseases such as acute lung injury and its more severe form acute respiratory distress syndrome. Alterations in actin cytoskeleton are shown to be crucial for NF-κB regulation and EC inflammation. Previously, we have described a role of actin binding protein cofilin in mediating cytoskeletal alterations essential for NF-κB activation and EC inflammation. The present study describes a dynamic mechanism in which LIM kinase 1 (LIMK1), a cofilin kinase, and slingshot-1Long (SSH-1L), a cofilin phosphatase, are engaged by procoagulant and proinflammatory mediator thrombin to regulate these responses. Our data show that knockdown of LIMK1 destabilizes whereas knockdown of SSH-1L stabilizes the actin filaments through modulation of cofilin phosphorylation; however, in either case thrombin-induced NF-κB activity and expression of its target genes (ICAM-1 and VCAM-1) is inhibited. Further mechanistic analyses reveal that knockdown of LIMK1 or SSH-1L each attenuates nuclear translocation and thereby DNA binding of RelA/p65. In addition, LIMK1 or SSH-1L depletion inhibited RelA/p65 phosphorylation at Ser(536), a critical event conferring transcriptional competency to the bound NF-κB. However, unlike SSH-1L, LIMK1 knockdown also impairs the release of RelA/p65 by blocking IKKß-dependent phosphorylation/degradation of IκBα. Interestingly, LIMK1 or SSH-1L depletion failed to inhibit TNF-α-induced RelA/p65 nuclear translocation and proinflammatory gene expression. Thus this study provides evidence for a novel role of LIMK1 and SSH-1L in selectively regulating EC inflammation associated with intravascular coagulation.

Regulation of Rela/p65 and endothelial cell inflammation by proline-rich tyrosine kinase 2.

We investigated the role of proline-rich tyrosine kinase 2 (Pyk2) in the mechanism of NF-κB activation and endothelial cell (EC) inflammation induced by thrombin, a procoagulant serine protease released in high amounts during sepsis and other inflammatory conditions. Stimulation of ECs with thrombin resulted in a time-dependent activation of Pyk2. RNA interference knockdown of Pyk2 attenuated thrombin-induced activity of NF-κB and expression of its target genes, vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1. Pyk2 knockdown impaired thrombin-induced activation of IκB kinase (IKK) and phosphorylation (Ser32 and Ser36) of IkappaBα, but, surprisingly, failed to prevent IκBα degradation. However, depletion of IKKα or IKKß was effective in inhibiting IκBα phosphorylation/degradation, as expected. Intriguingly, Pyk2 knockdown impaired nuclear translocation and DNA binding of RelA/p65, despite the inability to prevent IκBα degradation. In addition, Pyk2 knockdown was associated with inhibition of RelA/p65 phosphorylation at Ser536, which is important for transcriptional activity of RelA/p65. Depletion of IKKα or IKKß each impaired RelA/p65 phosphorylation. Taken together, these data identify Pyk2 as a critical regulator of EC inflammation by virtue of engaging IKK to promote the release and the transcriptional capacity of RelA/p65, and, additionally, by its ability to facilitate the nuclear translocation of the released RelA/p65. Thus, specific targeting of Pyk2 may be an effective anti-inflammatory strategy in vascular diseases associated with EC inflammation and intravascular coagulation.

Therapeutic Targeting of NF-κB in Acute Lung Injury: A Double-Edged Sword.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a devastating disease that can be caused by a variety of conditions including pneumonia, sepsis, trauma, and most recently, COVID-19. Although our understanding of the mechanisms of ALI/ARDS pathogenesis and resolution has considerably increased in recent years, the mortality rate remains unacceptably high (~40%), primarily due to the lack of effective therapies for ALI/ARDS. Dysregulated inflammation, as characterized by massive infiltration of polymorphonuclear leukocytes (PMNs) into the airspace and the associated damage of the capillary-alveolar barrier leading to pulmonary edema and hypoxemia, is a major hallmark of ALI/ARDS. Endothelial cells (ECs), the inner lining of blood vessels, are important cellular orchestrators of PMN infiltration in the lung. Nuclear factor-kappa B (NF-κB) plays an essential role in rendering the endothelium permissive for PMN adhesion and transmigration to reach the inflammatory site. Thus, targeting NF-κB in the endothelium provides an attractive approach to mitigate PMN-mediated vascular injury, not only in ALI/ARDS, but in other inflammatory diseases as well in which EC dysfunction is a major pathogenic mechanism. This review discusses the role and regulation of NF-κB in the context of EC inflammation and evaluates the potential and problems of targeting it as a therapy for ALI/ARDS.

Essential Role of Rho-Associated Kinase in ABO Immune Complex-Mediated Endothelial Barrier Disruption.

ABO immune complexes (ABO-IC) formed by ABO-incompatible antigen-antibody interaction are associated with hemolysis and platelet destruction in patients transfused with ABO-nonidentical blood products. However, the effects of ABO-IC on endothelial cells (EC) are unclear. ABO-IC were formed in vitro from normal donor-derived plasma and serum. Human pulmonary artery EC (HPAEC) were cultured and treated with media, ABO-identical and -non-identical plasma, and ABO-IC. EC barrier integrity was evaluated using transendothelial electrical resistance (TEER), scanning electron microscopy (SEM), vascular endothelial (VE)-cadherin and phalloidin staining, and Rho-associated Kinase (ROCK) inhibitor treatment. TEER revealed significant/irreversible barrier disruption within 1-2 h of exposure to ABO non-identical plasma and ABO-IC; this occurred independently of EC ABO type. Treatment with ABO-IC resulted in decreased VE-cadherin staining and increased phalloidin staining in a time-dependent manner, suggesting that the resultant increased EC barrier permeability is secondary to actin stress fiber formation and loss of cell surface VE-cadherin. Inhibition of ROCK was effective in protecting against IC-induced barrier disruption even two hours after ABO-IC exposure. ABO-IC causes increased EC barrier permeability by decreasing cell surface VE-cadherin and promoting stress fiber formation, which is preventable by inhibiting ROCK activation to protect against EC contraction and gap formation.

Essential role of cofilin-1 in regulating thrombin-induced RelA/p65 nuclear translocation and intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells.

Activation of RhoA/Rho-associated kinase (ROCK) pathway and the associated changes in actin cytoskeleton induced by thrombin are crucial for activation of NF-kappaB and expression of its target gene ICAM-1 in endothelial cells. However, the events acting downstream of RhoA/ROCK to mediate these responses remain unclear. Here, we show a central role of cofilin-1, an actin-binding protein that promotes actin depolymerization, in linking RhoA/ROCK pathway to dynamic alterations in actin cytoskeleton that are necessary for activation of NF-kappaB and thereby expression of ICAM-1 in these cells. Stimulation of human umbilical vein endothelial cells with thrombin resulted in Ser(3) phosphorylation/inactivation of cofilin and formation of actin stress fibers in a ROCK-dependent manner. RNA interference knockdown of cofilin-1 stabilized the actin filaments and inhibited thrombin- and RhoA-induced NF-kappaB activity. Similarly, constitutively inactive mutant of cofilin-1 (Cof1-S3D), known to stabilize the actin cytoskeleton, inhibited NF-kappaB activity by thrombin. Overexpression of wild type cofilin-1 or constitutively active cofilin-1 mutant (Cof1-S3A), known to destabilize the actin cytoskeleton, also impaired thrombin-induced NF-kappaB activity. Additionally, depletion of cofilin-1 was associated with a marked reduction in ICAM-1 expression induced by thrombin. The effect of cofilin-1 depletion on NF-kappaB activity and ICAM-1 expression occurred downstream of IkappaBalpha degradation and was a result of impaired RelA/p65 nuclear translocation and consequently, RelA/p65 binding to DNA. Together, these data show that cofilin-1 occupies a central position in RhoA-actin pathway mediating nuclear translocation of RelA/p65 and expression of ICAM-1 in endothelial cells.

Autophagy protein ATG7 is a critical regulator of endothelial cell inflammation and permeability.

Endothelial cell (EC) inflammation and permeability are critical pathogenic mechanisms in many inflammatory conditions including acute lung injury. In this study, we investigated the role of ATG7, an essential autophagy regulator with no autophagy-unrelated functions, in the mechanism of EC inflammation and permeability. Knockdown of ATG7 using si-RNA significantly attenuated thrombin-induced expression of proinflammatory molecules such as IL-6, MCP-1, ICAM-1 and VCAM-1. Mechanistic study implicated reduced NF-κB activity in the inhibition of EC inflammation in ATG7-silenced cells. Moreover, depletion of ATG7 markedly reduced the binding of RelA/p65 to DNA in the nucleus. Surprisingly, the thrombin-induced degradation of IκBα in the cytosol was not affected in ATG7-depleted cells, suggesting a defect in the translocation of released RelA/p65 to the nucleus in these cells. This is likely due to suppression of thrombin-induced phosphorylation and thereby inactivation of Cofilin1, an actin-depolymerizing protein, in ATG7-depleted cells. Actin stress fiber dynamics are required for thrombin-induced translocation of RelA/p65 to the nucleus, and indeed our results showed that ATG7 silencing inhibited this response via inactivation of Cofilin1. ATG7 silencing also reduced thrombin-mediated EC permeability by inhibiting the disassembly of VE-cadherin at adherens junctions. Together, these data uncover a novel function of ATG7 in mediating EC inflammation and permeability, and provide a mechanistic basis for the linkage between autophagy and EC dysfunction.

Critical Role of Mortalin/GRP75 in Endothelial Cell Dysfunction Associated with Acute Lung Injury.

Mortalin/GRP75 (glucose regulated protein 75), a member of heat shock protein 70 family of chaperones, is involved in several cellular processes including proliferation and signaling, and plays a pivotal role in cancer and neurodegenerative disorders. In this study, we sought to determine the role of mortalin/GRP75 in mediating vascular inflammation and permeability linked to the pathogenesis of acute lung injury (ALI). In an aerosolized bacterial lipopolysaccharide inhalation mouse model of ALI, we found that administration of mortalin/GRP75 inhibitor mean kinetic temperature-077, both prophylactically and therapeutically, protected against polymorphonuclear leukocytes influx into alveolar airspaces, microvascular leakage, and expression of pro-inflammatory mediators such as interleukin-1ß, E-selectin, and tumor necrosis factor TNFα. Consistent with this, thrombin-induced inflammation in cultured human endothelial cells (EC) was also protected upon before and after treatment with mean kinetic temperature-077. Similar to pharmacological inhibition of mortalin/GRP75, siRNA-mediated depletion of mortalin/GRP75 also blocked thrombin-induced expression of proinflammatory mediators such as intercellular adhesion molecule-1 and vascular adhesion molecule-1. Mechanistic analysis in EC revealed that inactivation of mortalin/GRP75 interfered with the binding of the liberated NF-κB to the DNA, thereby leading to inhibition of downstream expression of adhesion molecules, cytokines, and chemokines. Importantly, thrombin-induced Ca signaling and EC permeability were also prevented upon mortalin/GRP75 inactivation/depletion. Thus, this study provides evidence for a novel role of mortalin/GRP75 in mediating EC inflammation and permeability associated with ALI.

VEGFR-2 inhibition augments cigarette smoke-induced oxidative stress and inflammatory responses leading to endothelial dysfunction.

Vascular endothelial growth factor (VEGF) induces phosphorylation of VEGF receptor-2 (VEGFR-2) and activates the downstream signaling pathway resulting in endothelial cell migration, proliferation, and survival. Cigarette smoking is associated with abnormal vascular and endothelial function, leading to airspace enlargement. Herein, we investigated the mechanism of cigarette smoke (CS) -induced endothelial dysfunction by studying the VEGF-VEGFR-2 signaling in mouse lung and human endothelial cells. CS exposure caused oxidative stress, as shown by increased levels of 4-hydroxy-2-nonenal-adducts in mouse lung and reactive oxygen species generation in human lung microvascular endothelial cells (HMVEC-Ls). Inhibition of VEGFR-2 by a specific kinase inhibitor (NVP-AAD777) enhanced the CS-induced oxidative stress, causing augmented inflammatory cell influx and proinflammatory mediators release in mouse lung. The levels of endothelial nitric oxide synthase (eNOS) and phosphorylated (p) -eNOS in the lungs of mice exposed to CS and/or treated with VEGFR-2 inhibitor were decreased. CS down-regulated VEGFR-2 expression, eNOS levels, and VEGF-induced VEGFR-2 phosphorylation in HMVEC-Ls, resulting in impaired VEGF-induced endothelial cell migration and angiogenesis. Overall, these data show that inhibition of VEGFR-2 augmented CS-induced oxidative stress and inflammatory responses leading to endothelial dysfunction. This explains the mechanism of endothelial dysfunction in smokers and has implications in understanding the pathogenesis of pulmonary and cardiovascular diseases.

RNAi therapeutic strategies for acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS), replacing the clinical term acute lung injury, involves serious pathophysiological lung changes that arise from a variety of pulmonary and nonpulmonary injuries and currently has no pharmacological therapeutics. RNA interference (RNAi) has the potential to generate therapeutic effects that would increase patient survival rates from this condition. It is the purpose of this review to discuss potential targets in treating ARDS with RNAi strategies, as well as to outline the challenges of oligonucleotide delivery to the lung and tactics to circumvent these delivery barriers.

Selective Inactivation of Intracellular BiP/GRP78 Attenuates Endothelial Inflammation and Permeability in Acute Lung Injury.

The role of Endoplasmic Reticulum Chaperone and Signaling Regulator BiP/GRP78 in acute inflammatory injury, particularly in the context of lung endothelium, is poorly defined. In his study, we monitored the effect of SubAB, a holoenzyme that cleaves and specifically inactivates BiP/GRP78 and its inactive mutant SubAA272B on lung inflammatory injury in an aerosolized LPS inhalation mouse model of acute lung injury (ALI). Analysis of lung homogenates and bronchoalveolar lavage (BAL) fluid showed that LPS-induced lung inflammation and injury were significantly inhibited in SubAB- but not in SubAA272B-treated mice. SubAB-treated mice were also protected from LPS-induced decrease in lung compliance. Gene transfer of dominant negative mutant of BiP in the lung endothelium protected against LPS-induced lung inflammatory responses. Consistent with this, stimulation of endothelial cells (EC) with thrombin caused an increase in BiP/GRP78 levels and inhibition of ER stress with 4-phenylbutyric acid (4-PBA) prevented this response as well as increase in VCAM-1, ICAM-1, IL-6, and IL-8 levels. Importantly, thrombin-induced Ca2+ signaling and EC permeability were also prevented upon BiP/GRP78 inactivation. The above EC responses are mediated by intracellular BiP/GRP78 and not by cell surface BiP/GRP78. Together, these data identify intracellular BiP/GRP78 as a novel regulator of endothelial dysfunction associated with ALI.