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Despite encouraging preclinical data, therapies to reduce ARDS mortality remains a globally unmet need, including during the COVID-19 pandemic. We previously identified extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a novel damage-associated molecular pattern protein (DAMP) via TLR4 ligation which regulates inflammatory cascade activation. eNAMPT is tightly linked to human ARDS by biomarker and genotyping studies in ARDS subjects. We now hypothesize that an eNAMPT-neutralizing mAb will significantly reduce the severity of ARDS lung inflammatory lung injury in diverse preclinical rat and porcine models. Sprague Dawley rats received eNAMPT mAb intravenously following exposure to intratracheal lipopolysaccharide (LPS) or to a traumatic blast (125 kPa) but prior to initiation of ventilator-induced lung injury (VILI) (4 h). Yucatan minipigs received intravenous eNAMPT mAb 2 h after initiation of septic shock and VILI (12 h). Each rat/porcine ARDS/VILI model was strongly associated with evidence of severe inflammatory lung injury with NFkB pathway activation and marked dysregulation of the Akt/mTORC2 signaling pathway. eNAMPT neutralization dramatically reduced inflammatory indices and the severity of lung injury in each rat/porcine ARDS/VILI model (~ 50% reduction) including reduction in serum lactate, and plasma levels of eNAMPT, IL-6, TNFα and Ang-2. The eNAMPT mAb further rectified NFkB pathway activation and preserved the Akt/mTORC2 signaling pathway. These results strongly support targeting the eNAMPT/TLR4 inflammatory pathway as a potential ARDS strategy to reduce inflammatory lung injury and ARDS mortality., (© 2022. The Author(s).)

Sphingolipids, first described in the brain in 1884, are important structural components of biological membranes of all eukaryotic cells. In recent years, several lines of evidence support the critical role of sphingolipids such as sphingosine, sphingosine-1-phosphate (S1P), and ceramide as anti- or pro-inflammatory bioactive lipid mediators in a variety of human pathologies including pulmonary and vascular disorders. Among the sphingolipids, S1P is a naturally occurring agonist that exhibits potent barrier enhancing property in the endothelium by signaling via G protein-coupled S1P1 receptor. S1P, S1P analogs, and other barrier enhancing agents such as HGF, oxidized phospholipids, and statins also utilize the S1P/S1P1 signaling pathway to generate membrane protrusions or lamellipodia, which have been implicated in resealing of endothelial gaps and maintenance of barrier integrity. A better understanding of sphingolipids mediated regulation of lamellipodia formation and barrier enhancement of the endothelium will be critical for the development of sphingolipid-based therapies to alleviate pulmonary disorders such as sepsis-, radiation-, and mechanical ventilation-induced acute lung injury., (© 2018 Elsevier Inc. All rights reserved.)

Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS). Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The pathobiology of VILI and ARDS shares many inflammatory features including increases in lung vascular permeability due to loss of endothelial cell barrier integrity resulting in alveolar flooding. While there have been advances in the understanding of certain elements of VILI and ARDS pathobiology, such as defining the importance of lung inflammatory leukocyte infiltration and highly induced cytokine expression, a deep understanding of the initiating and regulatory pathways involved in these inflammatory responses remains poorly understood. Prevailing evidence indicates that loss of endothelial barrier function plays a primary role in the development of VILI and ARDS. Thus this review will focus on the latest knowledge related to 1 ) the key role of the endothelium in the pathogenesis of VILI; 2 ) the transcription factors that relay the effects of excessive mechanical stress in the endothelium; 3 ) the mechanical stress-induced posttranslational modifications that influence key signaling pathways involved in VILI responses in the endothelium; 4 ) the genetic and epigenetic regulation of key target genes in the endothelium that are involved in VILI responses; and 5 ) the need for novel therapeutic strategies for VILI that can preserve endothelial barrier function., (Copyright © 2017 the American Physiological Society.)

Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.

We previously identified the marked upregulation of integrin beta4 in human lung endothelial cells (EC) treated with simvastatin, an HMG coA-reductase inhibitor with vascular-protective and anti-inflammatory properties in murine models of acute lung injury (ALI). We now investigate the role of integrin beta4 as a novel mediator of vascular inflammatory responses with a focus on mitogen-activated protein kinases (MAPK) signaling and the downstream expression of the inflammatory cytokines (IL-6 and IL-8) essential for the full elaboration of inflammatory lung injury. Silencing of integrin beta4 (siITGB4) in human lung EC resulted in significant increases in both basal and LPS-induced phosphorylation of ERK 1/2, JNK, and p38 MAPK, consistent with robust integrin beta4 regulation of MAPK activation. In addition, siITB4 increased both basal and LPS-induced expression of IL-6 and IL-8 mRNA and protein secretion into the media. We next observed that integrin beta4 silencing increased basal and LPS-induced phosphorylation of SHP-2, a protein tyrosine phosphatase known to modulate MAPK signaling. In contrast, inhibition of SHP-2 enzymatic activity (sodium stibogluconate) abrogated the increased ERK phosphorylation associated with integrin beta4 silencing in LPS-treated EC and attenuated the increases in levels of IL-6 and IL-8 in integrin-beta4-silenced EC. These findings highlight a novel negative regulatory role for integrin beta4 in EC inflammatory responses involving SHP-2-mediated MAPK signaling. Upregulation of integrin beta4 may represent an important element of the anti-inflammatory and vascular-protective properties of statins and provides a novel strategy to limit inflammatory vascular syndromes., ((c) 2010 Wiley-Liss, Inc.)

The beneficial effects of statins, the most widely prescribed class of drugs in the world, are now recognized to extend well beyond their lipid-lowering properties. Through a combination of both distinct and interdependent effects on endothelial cell (EC) Rho GTPase regulation, NAPDH oxidase activity, NO bioavailability, and differential gene expression, statins confer significant protection of the vasculature. Abundant in vitro data, in addition to myriad reports relying on a range of animal models, now firmly support the idea that these drugs may serve as novel and effective therapeutic agents in a variety of disease states characterized by vascular dysfunction.

Endothelial barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell-cell junctions. Increases in cytosolic Ca2+ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically Galphaq and Galphai2), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of Galphaq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca2+, but involve activation of myosin phosphatase via a novel G-protein-coupled mechanism and PKA.