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).

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.

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.

Prevalence and pattern of cross-reacting antibodies to HIV in patients with tuberculosis.

In many countries, HIV testing among tuberculosis (TB) patients is recommended so that both infections are appropriately treated. Cross-reacting antibodies to HIV antigens have been reported for several conditions, including TB, leprosy, malaria, and rheumatoid arthritis. To study the pattern and prevalence of cross-reacting antibodies to HIV antigens, we examined sera from 153 HIV-negative TB patients and 40 healthy individuals in Chennai, south India. We also studied the differences in cross-reactivity of various HIV antigens using two different Western blot kits. Of the 153 samples studied, 80 were tested using HIV Western blot and 73 were tested using INNOLIA. Most patients in the study had concordantly negative ELISA and rapid tests, and no subject had a positive Western blot. However, seven TB patients had antibodies that cross-reacted with HIV antigens, giving rise to an indeterminate result. While p51/55 was the most frequently recognized antigen in the Western blot assay, antibodies to sgp120 was most frequently identified in INNOLIA. Sequence similarities between the two organisms could be responsible for eliciting cross-reacting antibodies, since a few related epitopes were identified in HIV and Mycobacterium. These findings could have potential implications for the development of diagnostics and vaccines.