Staphylococcus aureus is a major etiological agent of sepsis and induces endothelial cell (EC) barrier dysfunction and inflammation, two major hallmarks of acute lung injury. However, the molecular mechanisms of bacterial pathogen–induced EC barrier disruption are incompletely understood. Here, we investigated the role of microtubules (MT) in the mechanisms of EC barrier compromise caused by heat-killed S. aureus (HKSA). Using a customized monolayer permeability assay in human pulmonary EC and MT fractionation, we observed that HKSA-induced barrier disruption is accompanied by MT destabilization and increased histone deacetylase-6 (HDAC6) activity resulting from elevated reactive oxygen species (ROS) production. Molecular or pharmacological HDAC6 inhibition rescued barrier function in HKSA-challenged vascular endothelium. The HKSA-induced EC permeability was associated with impaired MT-mediated delivery of cytoplasmic linker–associated protein 2 (CLASP2) to the cell periphery, limiting its interaction with adherens junction proteins. HKSA-induced EC barrier dysfunction was also associated with increased Rho GTPase activity via activation of MT-bound Rho-specific guanine nucleotide exchange factor-H1 (GEF-H1) and was abolished by HDAC6 down-regulation. HKSA activated the NF-κB proinflammatory pathway and increased the expression of intercellular and vascular cell adhesion molecules in EC, an effect that was also HDAC6-dependent and mediated, at least in part, by a GEF-H1/Rho-dependent mechanism. Of note, HDAC6 knockout mice or HDAC6 inhibitor–treated WT mice were partially protected from vascular leakage and inflammation caused by both HKSA or methicillin-resistant S. aureus (MRSA). Our results indicate that S. aureus–induced, ROS-dependent up-regulation of HDAC6 activity destabilizes MT and thereby activates the GEF-H1/Rho pathway, increasing both EC permeability and inflammation.

The role of prostaglandin A2 (PGA2) in modulation of vascular endothelial function is unknown. We investigated effects of PGA2 on pulmonary endothelial cell (EC) permeability and inflammatory activation and identified a receptor mediating these effects. PGA2 enhanced the EC barrier and protected against barrier dysfunction caused by vasoactive peptide thrombin and proinflammatory bacterial wall lipopolysaccharide (LPS). Receptor screening using pharmacological and molecular inhibitory approaches identified EP4 as a novel PGA2 receptor. EP4 mediated barrier-protective effects of PGA2 by activating Rap1/Rac1 GTPase and protein kinase A targets at cell adhesions and cytoskeleton: VE-cadherin, p120-catenin, ZO-1, cortactin, and VASP. PGA2 also suppressed LPS-induced inflammatory signaling by inhibiting the NFκB pathway and expression of EC adhesion molecules ICAM1 and VCAM1. These effects were abolished by pharmacological or molecular inhibition of EP4. In vivo, PGA2 was protective in two distinct models of acute lung injury (ALI): LPS-induced inflammatory injury and two-hit ALI caused by suboptimal mechanical ventilation and injection of thrombin receptor–activating peptide. These protective effects were abolished in mice with endothelial-specific EP4 knockout. The results suggest a novel role for the PGA2–EP4 axis in vascular EC protection that is critical for improvement of pathological states associated with increased vascular leakage and inflammation.

The maintenance of endothelial barrier integrity is absolutely essential to prevent the vascular leak associated with pneumonia, pulmonary edema resulting from inhalation of toxins, acute elevation to high altitude, traumatic and septic lung injury, acute lung injury (ALI), and its life‐threatening complication, acute respiratory distress syndrome (ARDS). In addition to the long‐known edemagenic and inflammatory agonists, emerging evidences suggest that factors of endothelial cell (EC) mechanical microenvironment such as blood flow, mechanical strain of the vessel, or extracellular matrix stiffness also play an essential role in the control of endothelial permeability and inflammation. Recent studies from our group and others have demonstrated that substrate stiffening causes endothelial barrier disruption and renders EC more susceptible to agonist‐induced cytoskeletal rearrangement and inflammation. Further in vivo studies have provided direct evidence that proinflammatory stimuli increase lung microvascular stiffness which in turn exacerbates endothelial permeability and inflammation and perpetuates a vicious circle of lung inflammation. Accumulating evidence suggests a key role for RhoA GTPases signaling in stiffness‐dependent mechanotransduction mechanisms defining EC permeability and inflammatory responses. Vascular stiffening is also known to be a key contributor to other cardiovascular diseases such as arterial pulmonary hypertension (PH), although the precise role of stiffness in the development and progression of PH remains to be elucidated. This review summarizes the current understanding of stiffness‐dependent regulation of pulmonary EC permeability and inflammation, and discusses potential implication of pulmonary vascular stiffness alterations at macro‐ and microscale in development and modulation of ALI and PH.

Extracellular histones released into the circulation following trauma, sepsis, and ARDS may act as potent damage-associated molecular pattern signals leading to multiple organ failure. Endothelial cell (EC) dysfunction caused by extracellular histones has been demonstrated in vitro and in vivo; however, precise mechanistic details of histone-induced EC dysfunction and exacerbation of ongoing inflammation remain poorly understood. This study investigated the role of extracellular histones in exacerbating preexisting endothelial dysfunction and acute lung injury. Histone subunits H3 and H4, but not H1, H2A, or H2B, induced permeability in human pulmonary EC. H3 and H4 at concentrations above 30 µg/mL caused EC inflammation reflected by activation of the NF-κB pathway, transcriptional activation, and release of cytokines and chemokines including IL-6 and IL-8, and increased mRNA and protein expression of EC adhesion molecules VCAM-1 and ICAM-1. Pharmacological inhibitors targeting Toll-like receptor TLR4 but not TLR2/6, blocked histone-induced EC dysfunction. H3 and H4 also strongly augmented EC permeability and inflammation caused by Gram-negative and Gram-positive bacterial particles, endotoxin, and TNFα. Heparin blocked histone-induced augmentation of EC inflammation caused by endotoxin and TNFα. Injection of histone in mouse models of lung injury caused by bacterial wall lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus (HKSA) augmented ALI parameters: increased protein content, cell count, and inflammatory cytokine secretion in bronchoalveolar lavage fluid. Important clinical significance of these findings is in the demonstration that even a modest increase in extracellular histone levels can act as a severe exacerbating factor in conjunction with other EC barrier disruptive or proinflammatory agents.

Synthetic amphipathic helical peptides (SAHPs) designed as apolipoprotein A-I mimetics are known to bind to class B scavenger receptors (SR-Bs), SR-BI, SR-BII, and CD36, receptors that mediate lipid transport and facilitate pathogen recognition. In this study, we evaluated SAHPs, selected for targeting human CD36, by their ability to attenuate LPS-induced inflammation, endothelial barrier dysfunction, and acute lung injury (ALI). L37pA, which targets CD36 and SR-BI equally, inhibited LPS-induced IL-8 secretion and barrier dysfunction in cultured endothelial cells while reducing lung neutrophil infiltration by 40% in a mouse model of LPS-induced ALI. A panel of 20 SAHPs was tested in HEK293 cell lines stably transfected with various SR-Bs to identify SAHPs with preferential selectivity toward CD36. Among several SAHPs targeting both SR-BI/BII and CD36 receptors, ELK-B acted predominantly through CD36. Compared with L37pA, 5A, and ELK SAHPs, ELK-B was most effective in reducing the pulmonary barrier dysfunction, neutrophil migration into the lung, and lung inflammation induced by LPS. We conclude that SAHPs with relative selectivity toward CD36 are more potent at inhibiting acute pulmonary inflammation and dysfunction. These data indicate that therapeutic strategies using SAHPs targeting CD36, but not necessarily mimicking all apolipoprotein A-I functions, may be considered a possible new treatment approach for inflammation-induced ALI and pulmonary edema.