The endothelium: gatekeeper to lung ischemia-reperfusion injury.

The success of lung transplantation is limited by the high rate of primary graft dysfunction due to ischemia-reperfusion injury (IRI). Lung IRI is characterized by a robust inflammatory response, lung dysfunction, endothelial barrier disruption, oxidative stress, vascular permeability, edema, and neutrophil infiltration. These events are dependent on the health of the endothelium, which is a primary target of IRI that results in pulmonary endothelial barrier dysfunction. Over the past 10 years, research has focused more on the endothelium, which is beginning to unravel the multi-factorial pathogenesis and immunologic mechanisms underlying IRI. Many important proteins, receptors, and signaling pathways that are involved in the pathogenesis of endothelial dysfunction after IR are starting to be identified and targeted as prospective therapies for lung IRI. In this review, we highlight the more significant mediators of IRI-induced endothelial dysfunction discovered over the past decade including the extracellular glycocalyx, endothelial ion channels, purinergic receptors, kinases, and integrins. While there are no definitive clinical therapies currently available to prevent lung IRI, we will discuss potential clinical strategies for targeting the endothelium for the treatment or prevention of IRI. The accruing evidence on the essential role the endothelium plays in lung IRI suggests that promising endothelial-directed treatments may be approaching the clinic soon. The application of therapies targeting the pulmonary endothelium may help to halt this rapid and potentially fatal injury.

Identification of common sequence motifs shared exclusively among selectively packed exosomal pathogenic microRNAs during rickettsial infections.

We previously reported that microRNA (miR)23a and miR30b are selectively sorted into exosomes derived from rickettsia-infected endothelial cells (R-ECExos). Yet, the mechanism remains unknown. Cases of spotted fever rickettsioses have been increasing, and infections with these bacteria cause life-threatening diseases by targeting brain and lung tissues. Therefore, the goal of the present study is to further dissect the molecular mechanism underlying R-ECExos-induced barrier dysfunction of normal recipient microvascular endothelial cells (MECs), depending on their exosomal RNA cargos. Infected ticks transmit the rickettsiae to human hosts following a bite and injections of the bacteria into the skin. In the present study, we demonstrate that treatment with R-ECExos, which were derived from spotted fever group R parkeri infected human dermal MECs, induced disruptions of the paracellular adherens junctional protein VE-cadherin, and breached the paracellular barrier function in recipient pulmonary MECs (PMECs) in an exosomal RNA-dependent manner. We did not detect different levels of miRs in parent dermal MECs following rickettsial infections. However, we demonstrated that the microvasculopathy-relevant miR23a-27a-24 cluster and miR30b are selectively enriched in R-ECExos. Bioinformatic analysis revealed that common sequence motifs are shared exclusively among the exosomal, selectively-enriched miR23a cluster and miR30b at different levels. Taken together, these data warrant further functional identification and characterization of a monopartition, bipartition, or tripartition among ACA, UCA, and CAG motifs that guide recognition of microvasculopathy-relevant miR23a-27a-24 and miR30b, and subsequently results in their selective enrichments in R-ECExos.

Role of interleukin 6 and its soluble receptor on the diffusion barrier dysfunction of alveolar tissue.

During respiratory infection, barrier dysfunction in alveolar tissue can result from "cytokine storm" caused by overly reactive immune response. Particularly, interleukin 6 (IL-6) is implicated as a key biomarker of cytokine storm responsible for and further progression to pulmonary edema. In this study, alveolar-like tissue was reconstructed in a microfluidic device with: (1) human microvascular lung endothelial cells (HULEC-5a) cultured under flow-induced shear stress and (2) human epithelial cells (Calu-3) cultured at air-liquid interface. The effects of IL-6 and the soluble form of its receptor (sIL-6R) on the permeability, electrical resistance, and morphology of the endothelial and epithelial layers were evaluated. The diffusion barrier properties of both the endothelial and epithelial layers were significantly degraded only when IL-6 treatment was combined with sIL-6R. As suggested by recent review and clinical studies, our results provide unequivocal evidence that the barrier dysfunction occurs through trans-signaling in which IL-6 and sIL-6R form a complex and then bind to the surface of endothelial and epithelial cells, but not by classical signaling in which IL-6 binds to membrane-expressed IL-6 receptor. This finding suggests that the role of both IL-6 and sIL-6R should be considered as important biomarkers in developing strategies for treating cytokine storm.

Efficacy and safety of intravenous imatinib in COVID-19 ARDS: a randomized, double-blind, placebo-controlled clinical trial.

PURPOSE: A hallmark of acute respiratory distress syndrome (ARDS) is hypoxaemic respiratory failure due to pulmonary vascular hyperpermeability. The tyrosine kinase inhibitor imatinib reversed pulmonary capillary leak in preclinical studies and improved clinical outcomes in hospitalized COVID-19 patients. We investigated the effect of intravenous (IV) imatinib on pulmonary edema in COVID-19 ARDS. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled trial. Invasively ventilated patients with moderate-to-severe COVID-19 ARDS were randomized to 200 mg IV imatinib or placebo twice daily for a maximum of seven days. The primary outcome was the change in extravascular lung water index (∆EVLWi) between days 1 and 4. Secondary outcomes included safety, duration of invasive ventilation, ventilator-free days (VFD) and 28-day mortality. Posthoc analyses were performed in previously identified biological subphenotypes. RESULTS: 66 patients were randomized to imatinib (n = 33) or placebo (n = 33). There was no difference in ∆EVLWi between the groups (0.19 ml/kg, 95% CI - 3.16 to 2.77, p = 0.89). Imatinib treatment did not affect duration of invasive ventilation (p = 0.29), VFD (p = 0.29) or 28-day mortality (p = 0.79). IV imatinib was well-tolerated and appeared safe. In a subgroup of patients characterized by high IL-6, TNFR1 and SP-D levels (n = 20), imatinib significantly decreased EVLWi per treatment day (- 1.17 ml/kg, 95% CI - 1.87 to - 0.44). CONCLUSIONS: IV imatinib did not reduce pulmonary edema or improve clinical outcomes in invasively ventilated COVID-19 patients. While this trial does not support the use of imatinib in the general COVID-19 ARDS population, imatinib reduced pulmonary edema in a subgroup of patients, underscoring the potential value of predictive enrichment in ARDS trials. Trial registration NCT04794088 , registered 11 March 2021. European Clinical Trials Database (EudraCT number: 2020-005447-23).

Dexmedetomidine preconditioning ameliorates lung injury induced by pulmonary ischemia/reperfusion by upregulating promoter histone H3K4me3 modification of KGF-2.

Keratinocyte growth factor (KGF)-2 has been highlighted to play a significant role in maintaining the endothelial barrier integrity in lung injury induced by ischemia-reperfusion (I/R). However, the underlying mechanism remains largely unknown. The aims of this study were to determine whether dexmedetomidine preconditioning (DexP) modulates pulmonary I/R-induced lung injury through the alteration in KGF-2 expression. In our I/R-modeled mice, DexP significantly inhibited pathological injury, inflammatory response, and inflammatory cell infiltration, while promoted endothelial barrier integrity and KGF-2 promoter activity in lung tissues. Bioinformatics prediction and ChIP-seq revealed that I/R significantly diminished the level of H3K4me3 modification in the KGF-2 promoter, which was significantly reversed by DexP. Moreover, DexP inhibited the expression of histone demethylase JMJD3, which in turn promoted the expression of KGF-2. In addition, overexpression of JMJD3 weakened the protective effect of DexP on lung injury in mice with I/R. Collectively, the present results demonstrated that DexP ameliorates endothelial barrier dysfunction via the JMJD3/KGF-2 axis.

The vascular endothelial specific IL-4 receptor alpha-ABL1 kinase signaling axis regulates the severity of IgE-mediated anaphylactic reactions.

BACKGROUND: Severe IgE-mediated, food-induced anaphylactic reactions are characterized by pulmonary venous vasodilatation and fluid extravasation, which are thought to lead to the life-threatening anaphylactic phenotype. The underlying immunologic and cellular processes involved in driving fluid extravasation and the severe anaphylactic phenotype are not fully elucidated. OBJECTIVE: We sought to define the interaction and requirement of IL-4 and vascular endothelial (VE) IL-4 receptor α chain (IL-4Rα) signaling in histamine-abelson murine leukemia viral oncogene homology 1 (ABL1)-mediated VE dysfunction and fluid extravasation in the severity of IgE-mediated anaphylactic reactions in mice. METHODS: Mice deficient in VE IL-4Rα and models of passive and active oral antigen- and IgE-induced anaphylaxis were used to define the requirements of the VE IL-4Rα and ABL1 pathway in severe anaphylactic reactions. The human VE cell line (EA.hy926 cells) and pharmacologic (imatinib) and genetic (short hairpin RNA knockdown of IL4RA and ABL1) approaches were used to define the requirement of this pathway in VE barrier dysfunction. RESULTS: IL-4 exacerbation of histamine-induced hypovolemic shock in mice was dependent on VE expression of IL-4Rα. IL-4- and histamine-induced ABL1 activation in human VE cells and VE barrier dysfunction was ABL1-dependent. Development of severe IgE-mediated hypovolemia and shock required VE-restricted ABL1 expression. Treatment of mice with a history of food-induced anaphylaxis with the ABL kinase inhibitor imatinib protected the mice from severe IgE-mediated anaphylaxis. CONCLUSION: IL-4 amplifies IgE- and histamine-induced VE dysfunction, fluid extravasation, and the severity of anaphylaxis through a VE IL-4Rα/ABL1-dependent mechanism. These studies implicate an important contribution by the VE compartment in the severity of anaphylaxis and identify a new pathway for therapeutic intervention of IgE-mediated reactions.

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.

Urinary trypsin inhibitor attenuates LPS-induced endothelial barrier dysfunction by upregulation of vascular endothelial-cadherin expression.

INTRODUCTION: Urinary trypsin inhibitor (UTI) decreases inflammatory cytokine levels and mortality in experimental animal models of inflammation. Here, we observed the effect of UTI on lipopolysaccharide (LPS)-induced hyperpermeability in human umbilical vein endothelial cells (HUVECs) and explored the role of vascular endothelial-cadherin (VE-cadherin) in its effect. METHODS: The effect of UTI on endothelial barrier hyperpermeability was detected by an electrical cell-substrate impedance sensing (ECIS) system and a transwell chamber system. The expression of VE-cadherin in HUVECs was examined by real-time PCR and western blot. RESULTS: We demonstrated that the alleviation of LPS-induced barrier dysfunction could be achieved by pretreatment with 3000 U/mL of UTI. VE-cadherin monoclonal antibody (mAb) could inhibit the protective effects. UTI maintained VE-cadherin expression by increasing protein stability at both the transcriptional and post-transcriptional levels. Meanwhile, VE-cadherin expression on the cell surface increased when the cells were pretreated with UTI. Furthermore, pretreatment with UTI decreased the phosphorylation of VE-cadherin at Tyr658 but not Tyr731. CONCLUSIONS: Our data show that prophylactic UTI maintains the endothelial barrier function, increases VE-cadherin expression, and inhibits the phosphorylation of VE-cadherin at Tyr658 under inflammatory conditions. It suggests a scientific and potential clinical therapeutic importance of UTI in treatment of inflammatory disorders.

Veronica linariifolia subsp. dilatata ameliorates LPS-induced acute lung injury by attenuating endothelial cell barrier dysfunction via EGFR/Akt/ZO-1 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried aerial parts of Veronica linariifolia subsp. dilatata (Nakai & Kitag.) D.Y.Hong named Shui Man Jing (SMJ) is a traditional Chinese medicine with a long history of clinical use in the treatment of chronic bronchitis and coughing up blood, however, its role on acute lung injury (ALI) has not been revealed yet. AIM OF THE STUDY: To assess the efficiency of SMJ on ALI and to investigate whether it inhibited endothelial barrier dysfunction by regulating the EGFR/Akt/ZO-1 pathway to alleviate ALI in vivo and in vitro based on the result of network pharmacology. MATERIALS AND METHODS: An in vivo model of ALI was established using inhalation of atomized lipopolysaccharide (LPS), and the effects of SMJ on ALI were evaluated through histopathological examination and inflammatory cytokines, lung histology and edema, vascular and alveolar barrier disruption. Network pharmacology was applied to predict the mechanism of SMJ in the treatment of ALI. The crucial targets were validated by RT-PCR, Western Blotting, molecular docking, immunohistochemistry and immunofluorescence methods in vivo and in virto. RESULTS: Administration of SMJ protected mice against LPS-induced ALI, including ameliorating the histological alterations in the lung tissues, and decreasing lung edema, protein content of bronchoalveolar lavage fluid, infiltration of inflammatory cell and secretion of cytokines. SMJ exerted protective effects in ALI by inhibiting endothelial barrier dysfunction in mice and bEnd.3 cell. SMJ relieved endothelial barrier dysfunction induced by LPS through upregulating the EGFR expression. SMJ also increased the phosphorylation of Akt, and ZO-1 expression both in vivo and in vitro. CONCLUSION: SMJ attenuates vascular endothelial barrier dysfunction for LPS-induced ALI via EGFR/Akt/ZO-1 pathway, and is a promising novel therapeutic candidate for ALI.

Myosin light chain kinase signaling in endothelial barrier dysfunction.

Microvascular barrier dysfunction is a serious problem that occurs in many inflammatory conditions, including sepsis, trauma, ischemia-reperfusion injury, cardiovascular disease, and diabetes. Barrier dysfunction permits extravasation of serum components into the surrounding tissue, leading to edema formation and organ failure. The basis for microvascular barrier dysfunction is hyperpermeability at endothelial cell-cell junctions. Endothelial hyperpermeability is increased by actomyosin contractile activity in response to phosphorylation of myosin light chain by myosin light chain kinase (MLCK). MLCK-dependent endothelial hyperpermeability occurs in response to inflammatory mediators (e.g., activated neutrophils, thrombin, histamine, tumor necrosis factor alpha, etc.), through multiple cell signaling pathways and signaling molecules (e.g., Ca(++) , protein kinase C, Src kinase, nitric oxide synthase, etc.). Other signaling molecules protect against MLCK-dependent hyperpermeability (e.g., sphingosine-1-phosphate or cAMP). In addition, individual MLCK isoforms play specific roles in endothelial barrier dysfunction, suggesting that isoform-specific inhibitors could be useful for treating inflammatory disorders and preventing multiple organ failure. Because endothelial barrier dysfunction depends upon signaling through MLCK in many instances, MLCK-dependent signaling comprises multiple potential therapeutic targets for preventing edema formation and multiple organ failure. The following review is a discussion of MLCK-dependent mechanisms and cell signaling events that mediate endothelial hyperpermeability.

The leading role of microtubules in endothelial barrier dysfunction: disassembly of peripheral microtubules leaves behind the cytoskeletal reorganization.

Disturbance of the endothelial barrier is characterized by dramatic cytoskeleton reorganization, activation of actomyosin contraction and, finally, leads to intercellular gap formation. Here we demonstrate that the edemagenic agent, thrombin, causes a rapid increase in the human pulmonary artery endothelial cell (EC) barrier permeability accompanied by fast decreasing in the peripheral microtubules quantity and reorganization of the microtubule system in the internal cytoplasm of the EC within 5 min of the treatment. The actin stress-fibers formation occurs gradually and the maximal effect is observed relatively later, 30 min of the thrombin treatment. Thus, microtubules reaction develops faster than the reorganization of the actin filaments system responsible for the subsequent changes of the cell shape during barrier dysfunction development. Direct microtubules depolymerization by nocodazole initiates the cascade of barrier dysfunction reactions. Nocodazole-induced barrier disruption is connected directly with the degree of peripheral microtubules depolymerization. Short-term loss of endothelial barrier function occurs at the minimal destruction of peripheral microtubules, when actin filament system is still intact. Specifically, we demonstrate that the EC microtubule dynamics examined by time-lapse imaging of EB3-GFP comets movement has changed under these conditions: microtubule plus ends growth rate significantly decreased near the cell periphery. The microtubules, apparently, are the first target in the circuit of reactions leading to the pulmonary EC barrier compromise. Our results show that dynamic microtubules play an essential role in the barrier function in vitro; peripheral microtubules depolymerization is necessary and sufficient condition for initiation of endothelial barrier dysfunction.

The Role of Sphingolipids in Regulating Vascular Permeability in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease that causes scarring and fibrotic transformation of the lung parenchyma, resulting in the progressive loss of respiratory function and, often, death. Current treatments that target profibrotic factors can slow the rate of progression but are unable to ultimately stop it. In the past decade, many studies have shown that increased vascular permeability may be both a predictive and perpetuating factor in fibrogenesis. Consequently, there is a search for therapeutic targets to try and modulate vascular permeability in fibrotic lungs. One such class of targets that show great promise is sphingolipids. Sphingolipids are common in cell membranes and are increasingly recognized as critical to many cell signaling pathways, including those that affect the integrity of the vascular endothelial barrier. In this focused review we look at sphingolipids, particularly the sphingosine-1-phosphate (S1P) axis and its effects on vascular permeability, and how those effects may affect the pathogenesis of IPF. We further examine existing S1P modulators and their potential efficacy as therapeutics for IPF.

PAR1 regulates sepsis-induced vascular endothelial barrier dysfunction by mediating ERM phosphorylation via the RhoA/ROCK signaling pathway.

Sepsis begins with vascular endothelial barrier breakdown and causes widespread organ failure. Protease-activated receptor 1 (PAR1) is an important target for modulating vascular endothelial permeability; however, little research has been undertaken in sepsis, and its putative molecular mechanism remains unknown. The vascular endothelial permeability was examined by detecting FITC-dextran flux. F-actin was examined by immunofluorescence (IF). PAR1, ERM phosphorylation, and RhoA/ROCK signaling pathway expression in lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs) line were examined by IF and Western blot. To develop the sepsis model, cecal ligation and puncture (CLP) were conducted. The PAR1 inhibitor SCH79797 was utilized to inhibit PAR1 expression in vivo. Vascular permeability in main organs weres measured by Evans blue dye extravasation. The pathological changes in main organs were examined by HE staining. The expression of PAR1, ERM phosphorylation, and the RhoA/ROCK signaling pathway was examined using IF, immunohistochemical and WB in CLP mice. In vitro, in response to LPS stimulation of HUVECs, PAR1 mediated the phosphorylation of ERM, promoted F-actin rearrangement, and increased endothelial hyperpermeability, all of which were prevented by inhibiting PAR1 or RhoA. Additionally, inhibiting PAR1 expression reduced RhoA and ROCK expression. In vivo, we showed that inhibiting PAR1 expression will reduce ezrin/radixin/moesin (ERM) phosphorylation to relieve vascular endothelial barrier dysfunction and thereby ameliorate multiorgan dysfunction syndrome (MODS) in CLP-induced septic mice. This study revealed that PAR1-mediated phosphorylation of ERM induced endothelial barrier dysfunction, which in turn led to MODS in sepsis, and that the RhoA/ROCK signaling pathway underlay these effects.

Steen solution protects pulmonary microvascular endothelial cells and preserves endothelial barrier after lipopolysaccharide-induced injury.

OBJECTIVES: Acute respiratory distress syndrome represents the devastating result of acute lung injury, with high mortality. Limited methods are available for rehabilitation of lungs affected by acute respiratory distress syndrome. Our laboratory has demonstrated rehabilitation of sepsis-injured lungs via normothermic ex vivo and in vivo perfusion with Steen solution (Steen). However, mechanisms responsible for the protective effects of Steen remain unclear. This study tests the hypothesis that Steen directly attenuates pulmonary endothelial barrier dysfunction and inflammation induced by lipopolysaccharide. METHODS: Primary pulmonary microvascular endothelial cells were exposed to lipopolysaccharide for 4 hours and then recovered for 8 hours in complete media (Media), Steen, or Steen followed by complete media (Steen/Media). Oxidative stress, chemokines, permeability, interendothelial junction proteins, and toll-like receptor 4-mediated pathways were assessed in pulmonary microvascular endothelial cells using standard methods. RESULTS: Lipopolysaccharide treatment of pulmonary microvascular endothelial cells and recovery in Media significantly induced reactive oxygen species, lipid peroxidation, expression of chemokines (eg, chemokine [C-X-C motif] ligand 1 and C-C motif chemokine ligand 2) and cell adhesion molecules (P-selectin, E-selectin, and vascular cell adhesion molecule 1), permeability, neutrophil transmigration, p38 mitogen-activated protein kinase and nuclear factor kappa B signaling, and decreased expression of tight and adherens junction proteins (zonula occludens-1, zonula occludens-2, and vascular endothelial-cadherin). All of these inflammatory pathways were significantly attenuated after recovery of pulmonary microvascular endothelial cells in Steen or Steen/Media. CONCLUSIONS: Steen solution preserves pulmonary endothelial barrier function after lipopolysaccharide exposure by promoting an anti-inflammatory environment via attenuation of oxidative stress, toll-like receptor 4-mediated signaling, and conservation of interendothelial junctions. These protective mechanisms offer insight into the advancement of methods for in vivo lung perfusion with Steen for the treatment of severe acute respiratory distress syndrome.

Circulating Exosomes from Alzheimer's Disease Suppress Vascular Endothelial-Cadherin Expression and Induce Barrier Dysfunction in Recipient Brain Microvascular Endothelial Cell.

BACKGROUND: Blood-brain barrier (BBB) breakdown is a crucial aspect of Alzheimer's disease (AD) progression. Dysfunction in BBB is primarily caused by impaired tight junction and adherens junction proteins in brain microvascular endothelial cells (BMECs). The role of adherens junctions in AD-related BBB dysfunction remains unclear. Exosomes from senescent cells have unique characteristics and contribute to modulating the phenotype of recipient cells. However, it remains unknown if and how these exosomes cause BMEC dysfunction in AD. OBJECTIVE: This study aimed to investigate the impact of AD circulating exosomes on brain endothelial dysfunction. METHODS: Exosomes were isolated from sera of AD patients and age- and sex-matched cognitively normal controls using size-exclusion chromatography. The study measured the biomechanical nature of BMECs' endothelial barrier, the lateral binding forces between live BMECs. Paracellular expressions of the key adherens junction protein vascular endothelial (VE)-cadherin were visualized in BMEC cultures and a 3D BBB model using human BMECs and pericytes. VE-cadherin signals were also examined in brain tissues from AD patients and normal controls. RESULTS: Circulating exosomes from AD patients reduced VE-cadherin expression levels and impaired barrier function in recipient BMECs. Immunostaining analysis demonstrated that AD exosomes damaged VE-cadherin integrity in a 3D microvascular tubule formation model. The study found that AD exosomes weakened BBB integrity depending on their RNA content. Additionally, diminished microvascular VE-cadherin expression was observed in AD brains compared to controls. CONCLUSION: These findings highlight the significant role of circulating exosomes from AD patients in damaging adherens junctions of recipient BMECs, dependent on exosomal RNA.

Significance of Pulmonary Endothelial Injury and the Role of Cyclooxygenase-2 and Prostanoid Signaling.

The endothelium plays a key role in the dynamic balance of hemodynamic, humoral and inflammatory processes in the human body. Its central importance and the resulting therapeutic concepts are the subject of ongoing research efforts and form the basis for the treatment of numerous diseases. The pulmonary endothelium is an essential component for the gas exchange in humans. Pulmonary endothelial dysfunction has serious consequences for the oxygenation and the gas exchange in humans with the potential of consecutive multiple organ failure. Therefore, in this review, the dysfunction of the pulmonary endothel due to viral, bacterial, and fungal infections, ventilator-related injury, and aspiration is presented in a medical context. Selected aspects of the interaction of endothelial cells with primarily alveolar macrophages are reviewed in more detail. Elucidation of underlying causes and mechanisms of damage and repair may lead to new therapeutic approaches. Specific emphasis is placed on the processes leading to the induction of cyclooxygenase-2 and downstream prostanoid-based signaling pathways associated with this enzyme.

Piceatannol alleviates glucolipotoxicity induced vascular barrier injury through inhibition of the ROS/NF-kappa B signaling pathway.

Vascular barrier dysfunction is considered as the initial and critical event in atherosclerosis progression. Recent studies have revealed that treatment with piceatannol (PIC) alleviates both acute and chronic responses to vascular injury. We investigated whether PIC treatment would have beneficial effects on glucolipotoxicity-induced endothelial barrier dysfunction. Target proteins of PIC were identified from several online databases. Then, we confirmed the effect of PIC on endothelial barrier function. PIC treatment mitigated the impairment of endothelial cell motility, adhesion and migration ability associated with high glucose/lipid stimulation. PIC stabilized cytoskeletal reorganization and expression of cell cytoskeletal associated proteins GTPase. PIC reversed changes in critical vascular junction proteins and thus preserved endothelial barrier function and permeability. Finally, we confirmed that reducing of nuclear factor kappa B (NF-κB)/p65 activation and elimination of reactive oxygen species (ROS) were involved in the protective effect of PIC against glucolipotoxicity-induced vascular barrier injury. We identify PIC as a promising therapeutic strategy for glucolipotoxicity-induced endothelial barrier injury.

Exosomally Targeting microRNA23a Ameliorates Microvascular Endothelial Barrier Dysfunction Following Rickettsial Infection.

Spotted fever group rickettsioses caused by Rickettsia (R) are devastating human infections, which mainly target microvascular endothelial cells (ECs) and can induce lethal EC barrier dysfunction in the brain and lungs. Our previous evidence reveals that exosomes (Exos) derived from rickettsial-infected ECs, namely R-ECExos, can induce disruption of the tight junctional (TJ) protein ZO-1 and barrier dysfunction of human normal recipient brain microvascular endothelial cells (BMECs). However, the underlying mechanism remains elusive. Given that we have observed that microRNA23a (miR23a), a negative regulator of endothelial ZO-1 mRNA, is selectively sorted into R-ECExos, the aim of the present study was to characterize the potential functional role of exosomal miR23a delivered by R-ECExos in normal recipient BMECs. We demonstrated that EC-derived Exos (ECExos) have the capacity to deliver oligonucleotide RNAs to normal recipient BMECs in an RNase-abundant environment. miR23a in ECExos impairs normal recipient BMEC barrier function, directly targeting TJ protein ZO-1 mRNAs. In separate studies using a traditional in vitro model and a novel single living-cell biomechanical assay, our group demonstrated that miR23a anti-sense oligonucleotide-enriched ECExos ameliorate R-ECExo-provoked recipient BMEC dysfunction in association with stabilization of ZO-1 in a dose-dependent manner. These results suggest that Exo-based therapy could potentially prove to be a promising strategy to improve vascular barrier function during bacterial infection and concomitant inflammation.

Acadesine alleviates acute pancreatitis-related lung injury by mediating the barrier protective function of pulmonary microvascular endothelial cells.

Severe acute pancreatitis (SAP) is a condition characterized by highly fatal acute inflammation and is usually associated with multiple organ dysfunction syndrome. Acute lung injury (ALI) is the most common complications of SAP, which is the accelerator of other organ dysfunction caused by SAP and the primary cause of early death due to SAP. Acadesine, an adenosine analog and an AMPK activator, has been discovered to modulate glucose and lipid metabolism, and inhibit the production of pro-inflammatory cytokines and iNOS. However, its role in SAP-ALI and its mechanism remains unclear and need to be explored. Herein, we discovered that acadesine mitigated the generation of reactive oxygen species (ROS) in human pulmonary microvascular endothelial cells (HPMECs), alleviated apoptosis and recovered barrier integrity, thereby contributing to anti-inflammatory effects in vitro and in vivo. Moreover, Nrf2 deficiency partially eliminated the effects of acadesine-induced antioxidant effects and thus weakened the protective effects on cells and Nrf2-knockout (Nrf2-/-) mice. This study demonstrates that acadesine attenuated SAP-ALI associated inflammation and tissue damage by modulating the Nrf2-dependent antioxidant pathway by triggering AMPK. These findings are of great significance for the treatment of SAP-related lung injury.

Sufentanil ameliorates oxygen-glucose deprivation/reoxygenation-induced endothelial barrier dysfunction in HCMECs via the PI3K/Akt signaling pathway.

Ischemic heart disease, a chronic myocardial damage disease caused by coronary artery ischemia, is the leading cause of death worldwide. The aim of the present study was to explore the efficacy of sufentanil in myocardial ischemia/reperfusion (I/R) injury. Oxygen and glucose deprivation/reoxygenation (OGD/R) was utilized to induce human cardiac microvascular endothelial cells (HCMECs) to simulate myocardial I/R injury in vitro. The Cell Counting Kit-8 assay was used to detect the effects of sufentanil on HCMECs and OGD/R-induced HCMECs. The TUNEL, lactate dehydrogenase (LDH) activity, immunofluorescence and in vitro permeability assays, were used to assess apoptosis, LDH activity, VE-cadherin protein expression levels and endothelial barrier function in OGD/R-induced HCMECs, respectively. Moreover, western blotting was performed to assess the protein expression levels of apoptosis, endothelial barrier function and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt)-related proteins. The results demonstrated that sufentanil had no significant influence on the viability of HCMECs but increased the viability of OGD/R-induced HCMECs in a dose-dependent manner. Furthermore, sufentanil inhibited cell apoptosis and permeability of OGD/R-induced HCMECs but enhanced the protein expression levels of tight junction proteins, including ZO-1, Occludin, VE-cadherin and Claudin-5. Sufentanil was also demonstrated to activate the PI3K/Akt signaling pathway. In addition, the use of LY294002, an inhibitor of the PI3K/Akt signaling pathway, partially abolished the protective effects of sufentanil on apoptosis, permeability and tight junction protein expression levels. These results indicated that sufentanil ameliorated OGD/R-induced endothelial barrier dysfunction in HCMECs, potentially via the PI3K/Akt signaling pathway. The present study therefore suggested that sufentanil may serve as a novel therapeutic option for the improvement of myocardial I/R injury.