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.

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.

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 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).