Role of Neutrophil Extracellular Traps and Vesicles in Regulating Vascular Endothelial Permeability

The microvascular endothelium serves as the major barrier that controls the transport of blood constituents across the vessel wall. Barrier leakage occurs during infection or sterile inflammation, allowing plasma fluid and cells to extravasate and accumulate in surrounding tissues, an important pathology underlying a variety of infectious diseases and immune disorders. The leak process is triggered and regulated by bidirectional communications between circulating cells and vascular cells at the blood-vessel interface. While the molecular mechanisms underlying this complex process remain incompletely understood, emerging evidence supports the roles of neutrophil-endothelium interaction and neutrophil-derived products, including neutrophil extracellular traps and vesicles, in the pathogenesis of vascular barrier injury. In this review, we summarize the current knowledge on neutrophil-induced changes in endothelial barrier structures, with a detailed presentation of recently characterized molecular pathways involved in the production and effects of neutrophil extracellular traps and extracellular vesicles. Additionally, we discuss the therapeutic implications of altering neutrophil interactions with the endothelial barrier in treating inflammatory diseases

Neutrophil Extracellular Traps and Vascular Barrier Injury

Sepsis is a life-threatening inflammatory condition with high morbidity and mortality rates. Though improvements to diagnosis and management of sepsis have been made, the complexity of the disease process and an incomplete understanding of its endpoint mechanisms have prevented major breakthroughs for early diagnosis and treatment. Representing a common endpoint in a number of inflammatory injuries including sepsis, endothelial barrier dysfunction causes fluid leakage and leukocyte infiltration that leads to tissue damage and multiple organ failure. Therefore, elucidating mechanisms of endothelial barrier regulation is needed to further develop targeted therapies in inflammatory disease. Sepsis is characterized by a hyperinflammatory response to infection that involves activation of many types of immune cells, especially neutrophils. Neutrophils release a number of pro-inflammatory mediators that can have damaging effects on surrounding tissues, including the microvascular endothelium. In this study, we investigated neutrophil-released products, specifically neutrophil extracellular traps (NETs), for their effects on microvascular endothelial barrier function. We utilized a number of in vitro and in vivo approaches to study endothelial barrier function in response to the release of NETs during sepsis. We found that histones, especially citrullinated histone 3 (H3Cit), have barrier-disrupting capabilities. In response to H3Cit, disruption of the endothelial barrier is characterized by opening of adherens junctions and reorganization of the actin cytoskeleton. These mechanisms do not seem to be dependent on the typical hyperpermeability signaling mediated by Rho, MLCK, PKC, Src, or FAK; however, the barrier-enhancing pharmacological agent forskolin can block the barrier dysfunction induced by H3Cit. These results provide evidence that targeting H3Cit may hold therapeutic potential to limit tissue injury during inflammatory conditions like sepsis

Thrombin-cleaved syndecan-3/-4 ectodomain fragments mediate endothelial barrier dysfunction.

ObjectiveThe endothelial glycocalyx constitutes part of the endothelial barrier but its degradation leaves endothelial cells exposed to transmigrating cells and circulating mediators that can damage the barrier or promote intercellular gaps. Syndecan proteins are key components of the endothelial glycocalyx and are shed during disease states where expression and activity of proteases such as thrombin are elevated. We tested the ability of thrombin to cleave the ectodomains of syndecans and whether the products could act directly on endothelial cells to alter barrier function.Approach and resultsUsing transmission electron microscopy, we illustrated the presence of glycocalyx in human lung microvasculature. We confirmed expression of all syndecan subtypes on the endothelial surface of agarose-inflated human lungs. ELISA and western blot analysis suggested that thrombin can cleave syndecan-3/-4 ectodomains to produce fragments. In vivo, syndecan-3 ectodomain fragments increased extravasation of albumin-bound Evans blue in mouse lung, indicative of plasma protein leakage into the surrounding tissue. Syndecan-3/-4 ectodomain fragments decreased transendothelial electrical resistance, a measure of cell-cell adhesive barrier integrity, in a manner sensitive to a Rho kinase inhibitor. These effects were independent of glycosylation and thrombin receptor PAR1. Moreover, these cleavage products caused rapid VE-cadherin-based adherens junction disorganization and increased F-actin stress fibers, supporting their direct effect on endothelial paracellular permeability.ConclusionsWe suggest that thrombin can cleave syndecan-3/4 ectodomain into fragments which interact with endothelial cells causing paracellular hyperpermeability. This may have important implications in the pathogenesis of vascular dysfunction during sepsis or thrombotic disease states where thrombin is activated

Neutrophil-mediated Vascular Barrier Injury: Role of Neutrophil Extracellular Traps

Neutrophils play an essential role in host defense against infection or injury. While neutrophil activation is necessary for pathogen clearance and tissue repair, a hyperactive response can lead to tissue damage and microcirculatory disorders, a process involving complex neutrophil–endothelium cross talk. This review highlights recent research findings about neutrophil-mediated signaling and structural changes, including those induced by neutrophil extracellular traps, which ultimately lead to vascular barrier injury

Focal adhesion kinase and Src mediate microvascular hyperpermeability caused by fibrinogen- γC- terminal fragments.

ObjectivesWe previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvβ3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation.Methods and resultsUsing intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in γC-mediated hyperpermeability. In addition, γC-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition.ConclusionThe FAK-Src pathway contributes to γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation

AKT2 Maintains Brain Endothelial Claudin-5 Expression and Selective Activation of IR/AKT2/FOXO1-Signaling Reverses Barrier Dysfunction

Inflammation-induced blood–brain barrier (BBB) dysfunction and microvascular leakage are associated with a host of neurological disorders. The tight junction protein claudin-5 (CLDN5) is a crucial protein necessary for BBB integrity and maintenance. CLDN5 is negatively regulated by the transcriptional repressor FOXO1, whose activity increases during impaired insulin/AKT signaling. Owing to an incomplete understanding of the mechanisms that regulate CLDN5 expression in BBB maintenance and dysfunction, therapeutic interventions remain underdeveloped. Here, we show a novel isoform-specific function for AKT2 in maintenance of BBB integrity. We identified that AKT2 during homeostasis specifically regulates CLDN5-dependent barrier integrity in brain microvascular endothelial cells (BMVECs) and that intervention with a selective insulin-receptor (IR) agonist, demethylasterriquinone B1 (DMAQ-B1), rescued IL-1β-induced AKT2 inactivation, FOXO1 nuclear accumulation, and loss of CLDN5-dependent barrier integrity. Moreover, DMAQ-B1 attenuated preclinical CLDN5-dependent BBB dysfunction in mice subjected to experimental autoimmune encephalomyelitis. Taken together, the data suggest a regulatory role for IR/AKT2/FOXO1-signaling in CLDN5 expression and BBB integrity during neuroinflammation