The Protective Role of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Protein Sigma-1 Receptor in Regulating Endothelial Inflammation and Permeability Associated with Acute Lung Injury.

Earlier studies from our lab identified endoplasmic reticulum (ER) chaperone BiP/GRP78, an important component of MAM, to be a novel determinant of endothelial cell (EC) dysfunction associated with acute lung injury (ALI). Sigma1R (Sig1R) is another unique ER receptor chaperone that has been identified to associate with BiP/GRP78 at the MAM and is known to be a pluripotent modulator of cellular homeostasis. However, it is unclear if Sig1R also plays a role in regulating the EC inflammation and permeability associated with ALI. Our data using human pulmonary artery endothelial cells (HPAECs) showed that siRNA-mediated knockdown of Sig1R potentiated LPS-induced the expression of proinflammatory molecules ICAM-1, VCAM-1 and IL-8. Consistent with this, Sig1R agonist, PRE-084, known to activate Sig1R by inducing its dissociation from BiP/GRP78, blunted the above response. Notably, PRE-084 failed to blunt LPS-induced inflammatory responses in Sig1R-depleted cells, confirming that the effect of PRE-084 is driven by Sig1R. Furthermore, Sig1R antagonist, NE-100, known to inactivate Sig1R by blocking its dissociation from BiP/GRP78, failed to block LPS-induced inflammatory responses, establishing that dissociation from BiP/GRP78 is required for Sig1R to exert its anti-inflammatory action. Unlike Sig1R, the siRNA-mediated knockdown or Subtilase AB-mediated inactivation of BiP/GRP78 protected against LPS-induced EC inflammation. Interestingly, the protective effect of BiP/GRP78 knockdown or inactivation was abolished in cells that were depleted of Sig1R, confirming that BiP/GRP78 knockdown/inactivation-mediated suppression of EC inflammation is mediated via Sig1R. In view of these findings, we determined the in vivo relevance of Sig1R in a mouse model of sepsis-induced ALI. The intraperitoneal injection of PRE-084 mitigated sepsis-induced ALI, as evidenced by a decrease in ICAM-1, IL-6 levels, lung PMN infiltration, and lung vascular leakage. Together, these data evidence a protective role of Sig1R against endothelial dysfunction associated with ALI and identify it as a viable target in terms of controlling ALI in sepsis.

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.

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.

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.

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.

Critical role of autophagy regulator Beclin1 in endothelial cell inflammation and barrier disruption.

Recent studies have implicated autophagy in several inflammatory diseases involving aberrant endothelial cell (EC) responses, such as acute lung injury (ALI). However, the mechanistic basis for a role of autophagy in EC inflammation and permeability remain poorly understood. In this study, we impaired autophagy by silencing the essential Beclin1 autophagy gene in human pulmonary artery EC. This resulted in reduced expression of proinflammatory genes in response to thrombin, a procoagulant and proinflammatory mediator whose concentration is elevated in many diseases including sepsis and ALI. These (Beclin1-depleted) cells also displayed a marked decrease in NF-κB activity secondary to impaired DNA binding of RelA/p65 in the nucleus, but exhibited normal IκBα degradation in the cytosol. Further analysis showed that Beclin1 knockdown was associated with impaired RelA/p65 translocation to the nucleus. Additionally, Beclin1 knockdown attenuated thrombin-induced phosphorylation of RelA/p65 at Ser536, a critical event necessary for the transcriptional activity of RelA/p65. Beclin1 silencing also protected against thrombin-induced EC barrier disruption by preventing the loss of VE-cadherin at adherens junctions. Moreover, Beclin1 knockdown reduced thrombin-induced phosphorylation/inactivation of actin depolymerizing protein Cofilin1 and thereby actin stress fiber formation required for EC permeability as well as RelA/p65 nuclear translocation. Together, these data identify Beclin1 as a novel mechanistic link between autophagy and EC dysfunction (inflammation and permeability).

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.

Importins α and ß signaling mediates endothelial cell inflammation and barrier disruption.

Nucleocytoplasmic shuttling via importins is central to the function of eukaryotic cells and an integral part of the processes that lead to many human diseases. In this study, we addressed the role of α and ß importins in the mechanism of endothelial cell (EC) inflammation and permeability, important pathogenic features of many inflammatory diseases such as acute lung injury and atherosclerosis. RNAi-mediated knockdown of importin α4 or α3 each inhibited NF-κB activation, proinflammatory gene (ICAM-1, VCAM-1, and IL-6) expression, and thereby endothelial adhesivity towards HL-60 cells, upon thrombin challenge. The inhibitory effect of α4 and α3 knockdown was associated with impaired nuclear import and consequently, DNA binding of RelA/p65 subunit of NF-κB and occurred independently of IκBα degradation. Intriguingly, knockdown of importins α4 and α3 also inhibited thrombin-induced RelA/p65 phosphorylation at Ser536, showing a novel role of α importins in regulating transcriptional activity of RelA/p65. Similarly, knockdown of importin ß1, but not ß2, blocked thrombin-induced activation of RelA/p65 and its target genes. In parallel studies, TNFα-mediated inflammatory responses in EC were refractory to knockdown of importins α4, α3 or ß1, indicating a stimulus-specific regulation of RelA/p65 and EC inflammation by these importins. Importantly, α4, α3, or ß1 knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and by regulating actin cytoskeletal rearrangement. These results identify α4, α3 and ß1 as critical mediators of EC inflammation and permeability associated with intravascular coagulation.

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.

Featured Article: Electroporation-mediated gene delivery of surfactant protein B (SP-B) restores expression and improves survival in mouse model of SP-B deficiency.

Surfactant Protein B Deficiency is a rare but lethal monogenetic, congenital lung disease of the neonate that is unresponsive to any treatment except lung transplantation. Based on the potential that gene therapy offers to treat such intractable diseases, our objective was to test whether an electroporation-based gene delivery approach could restore surfactant protein B expression and improve survival in a compound knockout mouse model of surfactant protein B deficiency. Surfactant protein B expression can be shut off in these mice upon withdrawl of doxycycline, resulting in decreased levels of surfactant protein B within four days and death due to lung dysfunction within four to seven days. Control or one of several different human surfactant protein B-expressing plasmids was delivered to the lung by aspiration and electroporation at the time of doxycycline removal or four days later. Plasmids expressing human surfactant protein B from either the UbC or CMV promoter expressed surfactant protein B in these transgenic mice at times when endogenous surfactant protein B expression was silenced. Mean survival was increased 2- to 5-fold following treatment with the UbC or CMV promoter-driven plasmids, respectively. Histology of all surfactant protein B treated groups exhibited fewer neutrophils and less alveolar wall thickening compared to the control groups, and electron microscopy revealed that gene transfer of surfactant protein B resulted in lamellar bodies that were similar in the presence of electron-dense, concentric material to those in surfactant protein B-expressing mice. Taken together, our results show that electroporation-mediated gene delivery of surfactant protein B-expressing plasmids improves survival, lung function, and lung histology in a mouse model of surfactant protein B deficiency and suggest that this may be a useful approach for the treatment of this otherwise deadly disease. Impact statement Surfactant protein B (SP-B) deficiency is a rare but lethal genetic disease of neonates that results in severe respiratory distress with no available treatments other than lung transplantation. The present study describes a novel treatment for this disease by transferring the SP-B gene to the lungs using electric fields in a mouse model. The procedure is safe and results in enough expression of exogenous SP-B to improve lung histology, lamellar body structure, and survival. If extended to humans, this approach could be used to bridge the time between diagnosis and lung transplantation and could greatly increase the likelihood of affected neonates surviving to transplantation and beyond.

The role of high airway pressure and dynamic strain on ventilator-induced lung injury in a heterogeneous acute lung injury model.

BACKGROUND: Acute respiratory distress syndrome causes a heterogeneous lung injury with normal and acutely injured lung tissue in the same lung. Improperly adjusted mechanical ventilation can exacerbate ARDS causing a secondary ventilator-induced lung injury (VILI). We hypothesized that a peak airway pressure of 40 cmH2O (static strain) alone would not cause additional injury in either the normal or acutely injured lung tissue unless combined with high tidal volume (dynamic strain). METHODS: Pigs were anesthetized, and heterogeneous acute lung injury (ALI) was created by Tween instillation via a bronchoscope to both diaphragmatic lung lobes. Tissue in all other lobes was normal. Airway pressure release ventilation was used to precisely regulate time and pressure at both inspiration and expiration. Animals were separated into two groups: (1) over-distension + high dynamic strain (OD + HDS, n = 6) and (2) over-distension + low dynamic strain (OD + LDS, n = 6). OD was caused by setting the inspiratory pressure at 40 cmH2O and dynamic strain was modified by changing the expiratory duration, which varied the tidal volume. Animals were ventilated for 6 h recording hemodynamics, lung function, and inflammatory mediators followed by an extensive necropsy. RESULTS: In normal tissue (NT), OD + LDS caused minimal histologic damage and a significant reduction in BALF total protein (p < 0.05) and MMP-9 activity (p < 0.05), as compared with OD + HDS. In acutely injured tissue (ALIT), OD + LDS resulted in reduced histologic injury and pulmonary edema (p < 0.05), as compared with OD + HDS. CONCLUSIONS: Both NT and ALIT are resistant to VILI caused by OD alone, but when combined with a HDS, significant tissue injury develops.

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.

Preconditioning with endoplasmic reticulum stress ameliorates endothelial cell inflammation.

Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability.

Regulation of endothelial cell inflammation and lung polymorphonuclear lymphocyte infiltration by transglutaminase 2.

We addressed the role of transglutaminase 2 (TG2), a calcium-dependent enzyme that catalyzes cross-linking of proteins, in the mechanism of endothelial cell (EC) inflammation and lung polymorphonuclear lymphocyte (PMN) infiltration. Exposure of EC to thrombin, a procoagulant and proinflammatory mediator, resulted in activation of the transcription factor nuclear factor κB (NF-κB) and its target genes, vascular cell adhesion molecule 1, monocyte chemotactic protein 1, and interleukin 6. RNAi knockdown of TG2 inhibited these responses. Analysis of NF-κB activation pathway showed that TG2 knockdown was associated with inhibition of thrombin-induced DNA binding as well as serine phosphorylation of RelA/p65, a crucial event that controls transcriptional capacity of the DNA-bound RelA/p65. These results implicate an important role for TG2 in mediating EC inflammation by promoting DNA-binding and transcriptional activity of RelA/p65. Because thrombin is released in high amounts during sepsis, and its concentration is elevated in plasma and lavage fluids of patients with acute respiratory distress syndrome, we determined the in vivo relevance of TG2 in a mouse model of sepsis-induced lung PMN recruitment. A marked reduction in NF-κB activation, adhesion molecule expression, and lung PMN sequestration was observed in TG2 knockout mice compared with wild-type mice exposed to endotoxemia. Together, these results identify TG2 as an important mediator of EC inflammation and lung PMN sequestration associated with intravascular coagulation and sepsis.

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.

Critical role of non-muscle myosin light chain kinase in thrombin-induced endothelial cell inflammation and lung PMN infiltration.

The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response. In this study, we developed a new mouse model that permits investigation of lung inflammation associated with intravascular coagulation. Using this mouse model and in vitro approaches, we addressed the role of non-muscle myosin light chain kinase (nmMLCK) in thrombin-induced endothelial cell (EC) inflammation and lung neutrophil (PMN) infiltration. Our in vitro experiments revealed a key role of nmMLCK in ICAM-1 expression by its ability to control nuclear translocation and transcriptional capacity of RelA/p65 in EC. When subjected to intraperitoneal thrombin challenge, wild type mice showed a marked increase in lung PMN infiltration via expression of ICAM-1. However, these responses were markedly attenuated in mice deficient in nmMLCK. These results provide mechanistic insight into lung inflammatory response associated with intravascular coagulation and identify nmMLCK as a critical target for modulation of lung inflammation.

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.

Blocking NF-κB: an inflammatory issue.

The nuclear factor (NF)-κB is considered the master regulator of inflammatory responses. Studies in mouse models have established this transcription factor as an important mediator of many inflammatory disease states, including pulmonary diseases such as acute lung injury and acute respiratory distress syndrome. Endothelial cells provide the first barrier for leukocytes migrating to the inflamed sites and hence offer an attractive cellular context for targeting NF-κB for treatment of these diseases. However, recent studies showing that NF-κB also plays an important role in resolution phase of inflammation and in tissue repair and homeostasis have challenged the view of therapeutic inhibition of NF-κB. This article reviews the regulation of NF-κB in the context of endothelial cell signaling and provides a perspective on why "dampening" rather than "abolishing" NF-κB activation may be a safe and effective treatment strategy for inflammation-associated pulmonary and other inflammatory diseases.

GPR56 Regulates VEGF production and angiogenesis during melanoma progression.

Angiogenesis is a critical step during cancer progression. The VEGF is a major stimulator for angiogenesis and is predominantly contributed by cancer cells in tumors. Inhibition of the VEGF signaling pathway has shown promising therapeutic benefits for cancer patients, but adaptive tumor responses are often observed, indicating the need for further understanding of VEGF regulation. We report that a novel G protein-coupled receptor, GPR56, inhibits VEGF production from the melanoma cell lines and impedes melanoma angiogenesis and growth, through the serine threonine proline-rich segment in its N-terminus and a signaling pathway involving protein kinase Cα. We also present evidence that the two fragments of GPR56, which are generated by autocatalyzed cleavage, played distinct roles in regulating VEGF production and melanoma progression. Finally, consistent with its suppressive roles in melanoma progression, the expression levels of GPR56 are inversely correlated with the malignancy of melanomas in human subjects. We propose that components of the GPR56-mediated signaling pathway may serve as new targets for antiangiogenic treatment of melanoma.