Endothelial Cell Barrier Disruption Research Articles

CUMP elicits inter-endothelial gap formation during Pseudomonas aeruginosa infection.

P. aeruginosa utilizes a type 3 secretion system to intoxicate host cells with the nucleotidyl cyclase ExoY. After activation by its host cell cofactor, filamentous actin, ExoY produces purine and pyrimidine cyclic nucleotides, including cAMP, cGMP, and cUMP. ExoY-generated cyclic nucleotides promote inter-endothelial gap formation, impair motility, and arrest cell growth. The disruptive activities of cAMP and cGMP during P. aeruginosa infection are established; however, little is known about the function of cUMP. Here, we tested the hypothesis that cUMP contributes to endothelial cell barrier disruption during P. aeruginosa infection. Utilizing a membrane permeable cUMP analog, cUMP-AM, we revealed that during infection with catalytically inactive ExoY, cUMP promotes inter-endothelial gap formation in cultured PMVECs and contributes to increased filtration coefficient in the isolated perfused lung. These findings indicate that cUMP contributes to endothelial permeability during P. aeruginosa lung infection.

The deubiquitinase USP40 preserves endothelial integrity by targeting the heat shock protein HSP90β

Endothelial cell (EC) barrier disruption and inflammation are the pathological hallmarks of vascular disorders and acute infectious diseases and related conditions, including the coronavirus disease 2019 (COVID-19) and sepsis. Ubiquitination plays a critical role in regulating the stability, intracellular trafficking, and enzymatic activity of proteins and is reversed by deubiquitinating enzymes (DUBs). The role of DUBs in endothelial biology is largely unknown. In this study, we report that USP40, a poorly characterized DUB, prevents EC barrier disruption through reductions in the activation of RhoA and phosphorylation of myosin light chain (MLC) and cofilin. Furthermore, USP40 reduces EC inflammation through the attenuation of NF-ĸB activation, ICAM1 expression, and leukocyte-EC adhesion. We further show that USP40 activity and expression are reduced in response to endotoxin challenge. Global depletion of USP40 and EC-targeted USP40 depletion in mice exacerbated experimental lung injury, whereas lentiviral gene transfer of USP40 protected against endotoxin-induced lung injury. Using an unbiased approach, we discovered that the protective effect of USP40 occurs through the targeting of heat shock protein 90β (HSP90β) for its deubiquitination and inactivation. Together, these data reveal a critical protective role of USP40 in vascular injury, identifying a unique mechanistic pathway that profoundly impacts endothelial function via DUBs.

Spleen Tyrosine Kinase phosphorylates VE-cadherin to cause endothelial barrier disruption in acute lung injury

Increased endothelial cell (EC) permeability is a cardinal feature of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Tyrosine phosphorylation of VE-cadherin is a key determinant of EC barrier disruption. However, the identity and role of tyrosine kinases in this context are incompletely understood. Here we report that Spleen Tyrosine Kinase (Syk) is a key mediator of EC barrier disruption and lung vascular leak in sepsis. Inhibition of Syk by pharmacological or genetic approaches, each reduced thrombin-induced EC permeability. Mechanistically, Syk associates with and phosphorylates VE-cadherin to cause EC permeability. To study the causal role of endothelial Syk in sepsis-induced ALI, we used a remarkably efficient and cost-effective approach based on gene transfer to generate EC-ablated Syk mice. These mice were protected against sepsis-induced loss of VE-cadherin and inflammatory lung injury. Notably, administration of Syk inhibitor R788 (fostamatinib), currently in phase II clinical trial for treatment of COVID-19, mitigated lung injury and mortality in mice with sepsis. These data identify Syk as a novel kinase for VE-cadherin and a druggable target against ALI in sepsis.

Fibrinogen Fragment X Mediates Endothelial Barrier Disruption via Suppression of VE-Cadherin

IntroductionMajor traumatic injury is associated with early hemorrhage-related and late-stage deaths due to multiple organ failure (MOF). While improvements to hemostatic resuscitation have significantly reduced hemorrhage-related deaths, the incidence of MOF among trauma patients remains high. Dysregulation of vascular endothelial cell (EC) barrier function is a central mechanism in the development of MOF; however, the mechanistic triggers remain unknown. Accelerated fibrinolysis occurs in a majority of trauma patients, resulting in high circulating levels of fibrin(ogen) degradation products, such as fragment X. To date, the relationship between fragment X and EC dysregulation and barrier disruption is unknown. The goal of this study was to determine the effects of fragment X on EC barrier integrity and expression of paracellular junctional proteins that regulate barrier function. MethodsHuman lung microvascular endothelial cells (HLMVECs) were treated with increasing concentrations of fragment X (1, 10, and 100 μg/mL), and barrier function was monitored using the xCELLigence live-cell monitoring system. Quantitative PCR (qPCR) was performed to measure changes in EC expression of 84 genes. Immunofluorescent (IF) cytostaining was performed to validate qPCR findings. ResultsFragment X treatment significantly increased endothelial permeability over time (P < 0.05). There was also a significant reduction in VE-cadherin mRNA expression in fragment X-treated HLMVECs compared to control (P = 0.01), which was confirmed by IF staining. ConclusionsFragment X may induce EC hyperpermeability by reducing VE-cadherin expression. This suggests that a targeted approach to disrupting EC-fragment X interactions could mitigate EC barrier disruption, organ edema, and MOF associated with major trauma.

Amphipathic Helical Peptide L37pA Protects against Lung Vascular Endothelial Dysfunction Caused by Truncated Oxidized Phospholipids via Antagonism with CD36 Receptor.

The generation of bioactive truncated oxidized phospholipids (Tr-OxPLs) from oxidation of cell-membrane or circulating lipoproteins is a common feature of various pathological states. Scavenger receptor CD36 is involved in lipid transport and acts as a receptor for Tr-OxPLs. Interestingly, Tr-OxPLs and CD36 are involved in endothelial dysfunction-derived acute lung injury, but the precise mechanistic connections remain unexplored. In the present study, we investigated the role of CD36 in mediating pulmonary endothelial cell (EC) dysfunction caused by Tr-OxPLs. Our results demonstrated that the Tr-OxPLs KOdia-PC, Paz-PC, PGPC, PON-PC, POV-PC, and lysophosphocholine caused an acute EC barrier disruption as revealed by measurements of transendothelial electrical resistance and VE-cadherin immunostaining. More importantly, a synthetic amphipathic helical peptide, L37pA, targeting human CD36 strongly attenuated Tr-OxPL-induced EC permeability. L37pA also suppressed Tr-OxPL-induced endothelial inflammatory activation monitored by mRNA expression of inflammatory cytokines/chemokines and adhesion molecules. In addition, L37pA blocked Tr-OxPL-induced NF-κB activation and tyrosine phosphorylation of Src kinase and VE-cadherin. The Src inhibitor SU6656 attenuated KOdia-PC-induced EC permeability and inflammation, but inhibition of the Toll-like receptors (TLRs) TLR1, TLR2, TLR4, and TLR6 had no such protective effects. CD36-knockout mice were more resistant to Tr-OxPL-induced lung injury. Treatment with L37pA was equally effective in ameliorating Tr-OxPL-induced vascular leak and lung inflammation as determined by an Evans blue extravasation assay and total cell and protein content in BAL fluid. Altogether, these results demonstrate an essential role of CD36 in mediating Tr-OxPL-induced EC dysfunction and suggest a strong therapeutic potential of CD36 inhibitory peptides in mitigating lung injury and inflammation.

Calpain Promotes LPS-induced Lung Endothelial Barrier Dysfunction via Cleavage of Talin.

Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in increased endothelial permeability and pulmonary edema. The infection of gram-negative bacteria that produce toxins like LPS is one of the major causes of ALI. LPS activates Toll-like receptor 4, leading to cytoskeleton reorganization, resulting in lung endothelial barrier disruption and pulmonary edema in ALI. However, the signaling pathways that lead to the cytoskeleton reorganization and lung microvascular EC barrier disruption remain largely unexplored. Here we show that LPS induces calpain activation and talin cleavage into head and rod domains and that inhibition of calpain attenuates talin cleavage, RhoA activation, and pulmonary EC barrier disruption in LPS-treated human lung microvascular ECs invitro and lung EC barrier disruption and pulmonary edema induced by LPS in ALI invivo. Moreover, overexpression of calpain causes talin cleavage and RhoA activation, myosin light chain (MLC) phosphorylation, and increases in actin stress fiber formation. Furthermore, knockdown of talin attenuates LPS-induced RhoA activation and MLC phosphorylation and increased stress fiber formation and mitigates LPS-induced lung microvascular endothelial barrier disruption. Additionally, overexpression of talin head and rod domains increases RhoA activation, MLC phosphorylation, and stress fiber formation and enhances lung endothelial barrier disruption. Finally, overexpression of cleavage-resistant talin mutant reduces LPS-induced increases in MLC phosphorylation in human lung microvascular ECs and attenuates LPS-induced lung microvascular endothelial barrier disruption. These results provide the first evidence that calpain mediates LPS-induced lung microvascular endothelial barrier disruption in ALI via cleavage of talin.

Basement membrane product, endostatin, as a link between inflammation, coagulation and vascular permeability in COVID-19 and non-COVID-19 acute respiratory distress syndrome.

Immune cell recruitment, endothelial cell barrier disruption, and platelet activation are hallmarks of lung injuries caused by COVID-19 or other insults which can result in acute respiratory distress syndrome (ARDS). Basement membrane (BM) disruption is commonly observed in ARDS, however, the role of newly generated bioactive BM fragments is mostly unknown. Here, we investigate the role of endostatin, a fragment of the BM protein collagen XVIIIα1, on ARDS associated cellular functions such as neutrophil recruitment, endothelial cell barrier integrity, and platelet aggregation in vitro. In our study we analyzed endostatin in plasma and post-mortem lung specimens of patients with COVID-19 and non-COVID-19 ARDS. Functionally, we investigated the effect of endostatin on neutrophil activation and migration, platelet aggregation, and endothelial barrier function in vitro. Additionally, we performed correlation analysis for endostatin and other critical plasma parameters. We observed increased plasma levels of endostatin in our COVID-19 and non-COVID-19 ARDS cohort. Immunohistochemical staining of ARDS lung sections depicted BM disruption, alongside immunoreactivity for endostatin in proximity to immune cells, endothelial cells, and fibrinous clots. Functionally, endostatin enhanced the activity of neutrophils, and platelets, and the thrombin-induced microvascular barrier disruption. Finally, we showed a positive correlation of endostatin with soluble disease markers VE-Cadherin, c-reactive protein (CRP), fibrinogen, and interleukin (IL)-6 in our COVID-19 cohort. The cumulative effects of endostatin on propagating neutrophil chemotaxis, platelet aggregation, and endothelial cell barrier disruption may suggest endostatin as a link between those cellular events in ARDS pathology.

Monitoring Changes in Human Umbilical Vein Endothelial Cells upon Viral Infection Using Impedance-Based Real-Time Cell Analysis.

Endothelial cells line the inner surface of all blood and lymphatic vessels, creating a semi-permeable barrier regulating fluid and solute exchange between blood or lymph and their surrounding tissues. The ability of a virus to cross the endothelial barrier is an important mechanism that facilitates virus dissemination in the human body. Many viruses are reported to alter endothelial permeability and/or cause endothelial cell barrier disruption during infection, which is able to cause vascular leakage. The current study describes a real-time cell analysis (RTCA) protocol, using a commercial real-time cell analyzer to monitor endothelial integrity and permeability changes during Zika virus (ZIKV) infection of the human umbilical vein endothelial cells (HUVECs). The impedance signals recorded before and after ZIKV infection were translated to cell index (CI) values and analyzed. The RTCA protocol allows the detection of transient effects in the form of cell morphological changes during a viral infection. This assay could also be useful for studying changes in the vascular integrity of HUVECs in other experimental setups.

TFAM-Mediated mitochondrial transfer of MSCs improved the permeability barrier in sepsis-associated acute lung injury.

Vascular endothelial cell barrier disruption is a hallmark of sepsis-induced acute lung injury (ALI). Mesenchymal stem cells (MSCs)-based therapy has been regarded as a promising treatment for repairing injured lungs, and mitochondrial transfer was shown to be important for the therapeutic effects of MSCs. Here we investigated the ability of MSCs to modulate endothelial barrier integrity through mitochondrial transfer in sepsis-induced ALI. We found that mitochondrial transfer from MSCs to LPS-induced PMVECs through forming tunneling nanotubes (TNTs). Due to the inhibition of TNTs (using LAT-A), MSCs-mediated reparation on PMVECs functions, including cell apoptosis, MMP, ATP generation, TEER level and monolayer permeability of FITC-dextran were greatly inhibited. In addition, silencing of mitochondrial transcription factor A (TFAM) in MSCs could also partly inhibit the TNTs formation and aggravate the LPS-induced mitochondrial dysfunction and permeability barrier in PMVECs. Furthermore, the LPS-induced pulmonary edema and higher pulmonary vascular permeability were alleviated by MSCs while that of lung tissue bounced back after MSCs were pre-incubated by LAT-A and or down-regulation of TFAM. Therefore, we firstly revealed that regulation of TFAM expression in MSCs played a critical role to improve the permeability barrier of PMVECs by TNTs mediating mitochondrial transfer in sepsis-associated ALI. This study provided a new therapeutic strategy for the treatment of sepsis-induced ALI.

Overexpression of FoxM1 optimizes the therapeutic effect of bone marrow mesenchymal stem cells on acute respiratory distress syndrome

BackgroundInjury of alveolar epithelial cells and capillary endothelial cells is crucial in the pathogenesis of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Mesenchymal stem cells (MSCs) are a promising cell source for ALI/ARDS treatment. Overexpression of Fork head box protein M1 (FoxM1) facilitates MSC differentiation into alveolar type II (AT II) cells in vitro. Moreover, FoxM1 has been shown to repair the endothelial barrier. Therefore, this study explored whether overexpression of FoxM1 promotes the therapeutic effect of bone marrow-derived MSCs (BMSCs) on ARDS by differentiation of BMSCs into AT II cells or a paracrine mechanism.MethodsA septic ALI model was established in mice by intraperitoneal administration of lipopolysaccharide. The protective effect of BMSCs-FoxM1 on ALI was explored by detecting pathological variations in the lung, total protein concentration in bronchoalveolar lavage fluid (BALF), wet/dry (W/D) lung weight ratio, oxidative stress levels, cytokine levels, and retention of BMSCs in the lung. In addition, we assessed whether FoxM1 overexpression promoted the therapeutic effect of BMSCs on ALI/ARDS by differentiating into AT II cells using SPC−/− mice. Furthermore, the protective effect of BMSCs-FoxM1 on lipopolysaccharide-induced endothelial cell (EC) injury was explored by detecting EC proliferation, apoptosis, scratch wounds, tube formation, permeability, and oxidative stress, and analyzing whether the Wnt/β-catenin pathway contributes to the regulatory mechanism in vitro using a pathway inhibitor.ResultsCompared with BMSCs-Vector, treatment with BMSCs-FoxM1 significantly decreased the W/D lung weight ratio, total BALF protein level, lung injury score, oxidative stress, and cytokine levels. With the detected track of BMSCs-FoxM1, we observed a low residency rate and short duration of residency in the lung. Notably, SPC was not expressed in SPC−/− mice injected with BMSCs-FoxM1. Furthermore, BMSCs-FoxM1 enhanced EC proliferation, migration, and tube formation; inhibited EC apoptosis and inflammation; and maintained vascular integrity through activation of the Wnt/β-catenin pathway, which was partially reversed by XAV-939.ConclusionOverexpression of FoxM1 enhanced the therapeutic effect of BMSCs on ARDS, possibly through a paracrine mechanism rather than by promoting BMSC differentiation into AT II cells in vivo, and prevented LPS-induced EC barrier disruption partially through activating the Wnt/β-catenin signaling pathway in vitro.

ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis.

Diabetes mellitus is hallmarked by accelerated atherosclerosis, a major cause of mortality among patients with diabetes. Efficient therapies for diabetes-associated atherosclerosis are absent. Accelerated atherosclerosis in diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation. Here, we directly compared the effects of high glucose and oxidized LDL, revealing that high glucose induced more pronounced responses in regard to maladaptive unfolded protein response (UPR), senescence, and vascular endothelial cell barrier disruption. Ex vivo, diabetic ApoE−/− mice displayed increased levels of senescence and UPR markers within atherosclerotic lesions compared with nondiabetic ApoE−/− mice. Activated protein C pretreatment maintained barrier permeability and prevented glucose-induced expression of senescence and UPR markers in vitro. These data suggest that high glucose-induced maladaptive UPR and associated senescence promote vascular endothelial cell dysfunction, which—however—can be reversed by aPC. Taken together, current data suggest that reversal of glucose-induced vascular endothelial cell dysfunction is feasible.

Induction of endothelial barrier dysfunction by serum factors in rats subjected to traumatic brain injury and hemorrhagic shock.

Traumatic brain injury (TBI) has been associated with the development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanisms linking TBI with the development of ARDS, we characterized the effects of serum factors released following TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS. We found that serum samples from animals exposed to both controlled cortical impact (CCI) and HS, but not from sham‐operated rats induced significant barrier dysfunction in human pulmonary artery EC monolayers at 2 days post injury. Thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of the serum‐induced trans‐endothelial resistance (TER) decline caused by CCI‐HS serum, but not in later time points. However, both the early and late phases of CCI‐HS‐induced EC permeability were inhibited by heparin. The barrier disruptive effects of CCI‐HS serum were also prevented by serum preincubation with heparin‐sepharose. Pulmonary EC treated for 3 h with serum from CCI‐HS rats demonstrated a significant decline in expression of EC junctional protein, VE‐Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE‐cadherin antibody. These results suggest that exposure to CCI‐HS causes early and late‐phase barrier disruptive effects in vascular endothelium. While thrombin‐PAR1 signaling has been identified as a mechanism of acute EC permeability increase by CCI‐HS serum, the factor(s) defining long‐term EC barrier disruption in CCI‐HS model remains to be determined.

TLR7 Mediates Acute Respiratory Distress Syndrome in Sepsis by Sensing Extracellular miR-146a.

TLR7 (Toll-like receptor 7), the sensor for single-stranded RNA, contributes to systemic inflammation and mortality in murine polymicrobial sepsis. Recent studies show that extracellular miR-146a-5p serves as a TLR7 ligand and plays an important role in regulating host innate immunity. However, the role of miR-146a-5p and TLR7 signaling in pulmonary inflammation, endothelial activation, and sepsis-associated acute respiratory distress syndrome remains unclear. Here, we show that intratracheal administration of exogenous miR-146a-5p in mice evokes lung inflammation, activates endothelium, and increases endothelial permeability via TLR7-dependent mechanisms. TLR7 deficiency attenuates pulmonary barrier dysfunction and reduces lung inflammatory response in a murine sepsis model. Moreover, the impact of miR-146a-5p-TLR7 signaling on endothelial activation appears to be a secondary effect because TLR7 is undetectable in the human pulmonary artery and microvascular endothelial cells (ECs), which show no response to direct miR-146a-5p treatment invitro. Both conditioned media of miR-146a-5p-treated macrophages (Mϕ) and septic sera of wild-type mice induce a marked EC barrier disruption invitro, whereas Mϕ conditioned media or septic sera of TLR7-/- mice do not exhibit such effect. Cytokine array and pathway enrichment analysis of the Mϕ conditioned media and septic sera identify TNFα (tumor necrosis factor α) as the main downstream effector of miR-146a-5p-TLR7 signaling responsible for the EC barrier dysfunction, which is further supported by neutralizing anti-TNFα antibody intervention. Together, these data demonstrate that TLR7 activation elicits pulmonary inflammation and endothelial barrier disruption by sensing extracellular miR-146a-5p and contributes to sepsis-associated acute respiratory distress syndrome.

Serum from rats subjected to traumatic brain injury and hemorrhagic shock induces profound endothelial barrier dysfunction

IntroductionTraumatic brain injury (TBI) has been associated with development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanism linking TBI with development of ARDS, we characterized effects of serum factors released during TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS.MethodsThe adult male rat polytrauma model consisted of controlled cortical impact (CCI)–induced TBI followed by 30 minutes of hemorrhage shock (HS) induced by blood withdrawal. The HS phase was followed by a 1‐hour “prehospital” Hextend fluid resuscitation phase and then a 1‐hour “hospital phase” when shed blood was reinfused. 48 hours after the TBI, blood was drawn from right ventricle and serum samples were obtained. Serum was collected from the TBI or sham rats, and its effects on barrier properties of pulmonary endothelial cell culture were assessed by measurements of transendothelial electrical resistance (TER) using the electrical cell impedance sensor and visualization of fluorescent tracer penetration through EC monolayer using XPerT assay.ResultsWe found pronounced barrier‐disruptive effects of serum samples from animals exposed to controlled cortical impact (CCI) and HS, but not from sham operated rats. Rapid and sustained decrease in EC monolayer transendothelial resistance was observed within minutes after serum addition and remained below basal TER levels after incubation with CCI‐HS sera. Thrombin has been implicated in the early‐phase barrier disruptive activity of CCI‐HS serum, as both thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of serum‐induced TER decline, but did not affect TER decline at later time points. These findings suggest that thrombin elevation may play a role in early‐phase barrier disruptive activity of CCI‐HS serum. However, both the early and late phases of CCI‐HS‐induced EC permeability were inhibited by heparin. The late phase barrier disruptive effect of CCI‐HS was also prevented by serum preincubation with heparin‐sepharose. Pulmonary EC treated with serum from CCI‐HS rats for 3 hours have demonstrated significant decline in expression of EC junctional protein, VE‐Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE‐cadherin antibody.ConclusionThese results suggest that exposure to CCI‐HS leads to production of humoral factors causing early and late‐phase barrier disruptive effects in lung vascular endothelium. While thrombin‐PAR1 signaling has been identified as mechanism of acute EC permeability increase by CCI‐HS serum, the factor(s) defining long‐term EC barrier disruption in CCI‐HS model remain to be determined.

Calcium Signaling in Trauma: Increased Intracellular Calcium Flux in the Severely Injured

IntroductionThe pathogenesis of organ dysfunction following traumatic injury involves endothelial barrier disruption, and plasma from severely injured patients increases endothelial permeability in vitro. Many endothelial cell (EC) functions entail changes in intracellular calcium (Ca2+) concentrations, and multiple inflammatory mediators have been shown to induce EC barrier disruption in a Ca2+ dependent manner. However, study of Ca2+ signaling in trauma remains limited. Our objective was to investigate the role of intracellular Ca2+ signaling in trauma pathology. We hypothesized that ex vivo plasma from injured patients would induce intracellular EC Ca2+ flux and do so to a greater magnitude in more severely injured patients.MethodsBlood was collected from injured patients at a single, urban Level I Trauma Center. Patients were categorized by minimal injury without shock (Group 1: ISS&lt;15 + base excess &gt;‐6mEq/L; n=3) and severe injury with shock (Group 2: ISS&gt;15 + base excess &lt;‐6mEq/L; n=4). Human cerebral microvascular endothelial cells were grown on culture inserts and pre‐incubated with Fluo‐4 Ca2+ indicator. Plasma was diluted to concentrations previously used for in vitro permeability assays and flowed over EC’s under physiologic conditions for five minutes. Confocal microscopy was used to visualize and quantify intracellular Ca2+ flux. Thrombin was used as a positive control for inducing Ca2+ flux. We used Prism software to calculate areas under curves (AUC) and unpaired t‐tests to compare group means.ResultsCompared to Group 1 plasma, Group 2 induced greater calcium flux, which was sustained for a longer period of time, corresponding to greater AUC for total intracellular calcium flux (P&lt;0.05, Figure).ConclusionsEx vivo plasma from more severely injured trauma patients increases intracellular Ca2+ flux in EC’s. These data suggest Ca2+signaling may play a role in trauma‐induced endotheliopathy and present as a therapeutic target. Our study lays the groundwork for additional investigation to elucidate sources of Ca2+ in this setting as well as identify plasma mediators of both Ca2+ flux and endothelial permeability.

MRSA-induced endothelial permeability and acute lung injury are attenuated by FTY720 S-phosphonate.

Disruption of the lung endothelial barrier is a hallmark of acute respiratory distress syndrome (ARDS), for which no effective pharmacologic treatments exist. Prior work has demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine-1-phosphate (S1P) and FTY720, exhibits potent endothelial cell (EC) barrier protective properties. In this study, we investigated the in vitro and in vivo efficacy of Tys against methicillin-resistant Staphylococcus aureus (MRSA), a frequent bacterial cause of ARDS. Tys-protected human lung EC from barrier disruption induced by heat-killed MRSA (HK-MRSA) or staphylococcal α-toxin and attenuated MRSA-induced cytoskeletal changes associated with barrier disruption, including actin stress fiber formation and loss of peripheral VE-cadherin and cortactin. Tys-inhibited Rho and myosin light chain (MLC) activation after MRSA and blocked MRSA-induced NF-κB activation and release of the proinflammatory cytokines, IL-6 and IL-8. In vivo, intratracheal administration of live MRSA in mice caused significant vascular leakage and leukocyte infiltration into the alveolar space. Pre- or posttreatment with Tys attenuated MRSA-induced lung permeability and levels of alveolar neutrophils. Posttreatment with Tys significantly reduced levels of bronchoalveolar lavage (BAL) VCAM-1 and plasma IL-6 and KC induced by MRSA. Dynamic intravital imaging of mouse lungs demonstrated Tys attenuation of HK-MRSA-induced interstitial edema and neutrophil infiltration into lung tissue. Tys did not directly inhibit MRSA growth or viability in vitro. In conclusion, Tys inhibits lung EC barrier disruption and proinflammatory signaling induced by MRSA in vitro and attenuates acute lung injury induced by MRSA in vivo. These results support the potential utility of Tys as a novel ARDS therapeutic strategy.

PEA prevented early BBB disruption after cerebral ischaemic/reperfusion (I/R) injury through regulation of ROCK/MLC signaling

Palmitoylethanolamide (PEA) offers a strong protection against BBB disruption and neurological deficits after cerebral ischaemic/reperfusion (I/R) injury. To date, these BBB protective effects of PEA are mainly attributed to PPARα-mediated actions. However, whether PEA protects against BBB disruption through direct regulation of cytoskeletal microfilaments remains unknown. Here, we identified PEA as a Rho-associated protein kinase (ROCK2) inhibitor (IC50 = 38.4 ± 4.8 μM). In vitro data suggested that PEA reduced the activation of ROCK/MLC signaling and stress fiber formation within microvascular endothelial cells (ECs) after oxygen-glucose deprivation (OGD), and consequently attenuated early (0–4 h) EC barrier disruption. These actions of PEA could not be blocked by the PPARα antagonist GW6471. In summary, the present study described a previously unexplored role of PEA as a ROCK2 inhibitor, and propose a PPARα-independent mechanism for pharmacological effects of PEA.

SOCS3–microtubule interaction via CLIP-170 and CLASP2 is critical for modulation of endothelial inflammation and lung injury

Proinflammatory cytokines such as IL-6 induce endothelial cell (EC) barrier disruption and trigger an inflammatory response in part by activating the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. The protein suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of JAK-STAT, but its role in modulation of lung EC barrier dysfunction caused by bacterial pathogens has not been investigated. Using human lung ECs and EC-specific SOCS3 knockout mice, we tested the hypothesis that SOCS3 confers microtubule (MT)-mediated protection against endothelial dysfunction. SOCS3 knockdown in cultured ECs or EC-specific SOCS3 knockout in mice resulted in exacerbated lung injury characterized by increased permeability and inflammation in response to IL-6 or heat-killed Staphylococcus aureus (HKSA). Ectopic expression of SOCS3 attenuated HKSA-induced EC dysfunction, and this effect required assembled MTs. SOCS3 was enriched in the MT fractions, and treatment with HKSA disrupted SOCS3–MT association. We discovered that—in addition to its known partners gp130 and JAK2—SOCS3 interacts with MT plus-end binding proteins CLIP-170 and CLASP2 via its N-terminal domain. The resulting SOCS3–CLIP-170/CLASP2 complex was essential for maximal SOCS3 anti-inflammatory effects. Both IL-6 and HKSA promoted MT disassembly and disrupted SOCS3 interaction with CLIP-170 and CLASP2. Moreover, knockdown of CLIP-170 or CLASP2 impaired SOCS3–JAK2 interaction and abolished the anti-inflammatory effects of SOCS3. Together, these findings demonstrate for the first time an interaction between SOCS3 and CLIP-170/CLASP2 and reveal that this interaction is essential to the protective effects of SOCS3 in lung endothelium.

Endothelial eNAMPT amplifies pre-clinical acute lung injury: efficacy of an eNAMPT-neutralising monoclonal antibody.

RationaleThe severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target.MethodsWild-type C57BL/6J or endothelial cell (EC)-cNAMPT−/− knockout mice (targeted EC NAMPT deletion) were exposed to either a lipopolysaccharide (LPS)-induced (“one-hit”) or a combined LPS/ventilator (“two-hit”)-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were used in vitro and in vivo.ResultsImmunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models. In vitro human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. In vivo studies in wild-type and EC-cNAMPT−/− mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models.ConclusionsThese findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker and NAMPT genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.

Abstract 17144: miR-146a-5p Induces Pulmonary Barrier Disruption via Macrophage-Derived TNFa

Introduction: Sepsis-induced acute lung injury (ALI) has substantial morbidity and mortality. Diffused alveolar damage as a result of increased endothelial cell (EC) permeability is a hallmark of ALI. We have previously demonstrated that certain uridine-rich extracellular (ex) miRNAs such as miR-146a activate innate immune response and possess a potent proinflammatory property, but their effect on EC function is largely unknown. Hypothesis: Pulmonary EC barrier function is impaired by miR-146a-5p. Methods: Conditioned media (CM) were collected from macrophages (Mφ) cultures treated with lipofectamine (lipo) or miR-146a-5p (50 nM) for 24 h. EC barrier function was analyzed in human pulmonary arterial endothelial cells (HPAECs) by transendothelial electric resistance (TER) using an electric cell-substrate impedance sensing system. Results: Direct miR-146a-5p treatment of HPAECs did not cause any change in TER even though it induced robust IL-6 and CXCL2 production in Mφ. To test whether miR-146a-5p affects EC function indirectly, we incubated HPAECs with miR146a-CM and observed a more than 50% reduction in TER ( Fig. A ). Lipo-CM had no such effect. This barrier effect was accompanied by VE-cadherin internalization and disrupted VE-cadherin lining following 8 hours of incubation ( Fig. B) . To determine the potential mediators in the CM responsible for the EC barrier disruption, we performed a cytokine array analysis of 111 cytokines in the CM and identified 15 differentially expressed cytokines. Among them, we found that TNFα neutralizing Ab (1μg/ml), but not control IgG (1μg/ml), partially blocked the miR-146a-CM-induced EC barrier disruption with improved VE-Cadherin integrity ( Fig. B ). Conclusion: Our data demonstrated miR-146a-5p induces HPAEC barrier dysfunction via an indirect mechanism involving Mφ production of TNFα.