Endothelial Hyperpermeability Research Articles

Modulation of angiopoietin-2 and Tie2: Organ specific effects of microvascular leakage and edema in mice

IntroductionCritical illness is associated with organ failure, in which endothelial hyperpermeability and tissue edema play a major role. The endothelial angiopoietin/Tie2 system, a regulator of endothelial permeability, is dysbalanced during critical illness. Elevated circulating angiopoietin-2 and decreased Tie2 receptor levels are reported, but it remains unclear whether they cause edema independent of other critical illness-associated alterations. Therefore, we have studied the effect of angiopoietin-2 administration and/or reduced Tie2 expression on microvascular leakage and edema under normal conditions. MethodsTransgenic male mice with partial deletion of Tie2 (heterozygous exon 9 deletion, Tie2+/−) and wild-type controls (Tie2+/+) received 24 or 72 pg/g angiopoietin-2 or PBS as control (n = 12 per group) intravenously. Microvascular leakage and edema were determined by Evans blue dye (EBD) extravasation and wet-to-dry weight ratio, respectively, in lungs and kidneys. Expression of molecules related to endothelial angiopoietin/Tie2 signaling were determined by ELISA and RT-qPCR. ResultsIn Tie2+/+ mice, angiopoietin-2 administration increased EBD extravasation (154 %, p < 0.05) and wet-to-dry weight ratio (133 %, p < 0.01) in lungs, but not in the kidney compared to PBS.Tie2+/− mice had higher pulmonary (143 %, p < 0.001), but not renal EBD extravasation, compared to wild-type control mice, whereas a more pronounced wet-to-dry weight ratio was observed in lungs (155 %, p < 0.0001), in contrast to a minor higher wet-to-dry weight ratio in kidneys (106 %, p < 0.05).Angiopoietin-2 administration to Tie2+/− mice did not further increase pulmonary EBD extravasation, pulmonary wet-to-dry weight ratio, or renal wet-to-dry weight ratio. Interestingly, angiopoietin-2 administration resulted in an increased renal EBD extravasation in Tie2+/− mice compared to Tie2+/− mice receiving PBS. Both angiopoietin-2 administration and partial deletion of Tie2 did not affect circulating angiopoietin-1, soluble Tie2, VEGF and NGAL as well as gene expression of angiopoietin-1, −2, Tie1, VE-PTP, ELF-1, Ets-1, KLF2, GATA3, MMP14, Runx1, VE-cadherin, VEGFα and NGAL, except for gene and protein expression of Tie2, which was decreased in Tie2+/− mice compared to Tie2+/+ mice. ConclusionsIn mice, the microvasculature of the lungs is more vulnerable to angiopoietin-2 and partial deletion of Tie2 compared to those in the kidneys with respect to microvascular leakage and edema.

3′-Sialyllactose protects against LPS-induced endothelial dysfunction by inhibiting superoxide-mediated ERK1/2/STAT1 activation and HMGB1/RAGE axis

AimEndothelial hyperpermeability is an early stage of endothelial dysfunction associated with the progression and development of atherosclerosis. 3′-Sialyllactose (3′-SL) is the most abundant compound in human milk oligosaccharides, and it has the potential to regulate endothelial dysfunction. This study investigated the beneficial effects of 3′-SL on lipopolysaccharide (LPS)-induced endothelial dysfunction in vitro and in vivo. Main methodsWe established LPS-induced endothelial dysfunction models in both cultured bovine aortic endothelial cells (BAECs) and mouse models to determine the effects of 3′-SL. Western blotting, qRT-PCR analysis, immunofluorescence staining, and en face staining were employed to clarify underlying mechanisms. Superoxide production was measured by 2′,7′-dichlorofluorescin diacetate, and dihydroethidium staining. Key findingsLPS significantly decreased cell viability, whereas 3′-SL treatment mitigated these effects via inhibiting ERK1/2 activation. Mechanistically, 3′-SL ameliorated LPS-induced ROS accumulation leading to ERK1/2 activation-mediated STAT1 phosphorylation and subsequent inhibition of downstream transcriptional target genes, including VCAM-1, TNF-α, IL-1β, and MCP-1. Interestingly, LPS-induced ERK1/2/STAT1 activation leads to the HMGB1 release from the nucleus into the extracellular space, where it binds to RAGE, while 3′-SL suppressed EC hyperpermeability by suppressing the HMGB1/RAGE axis. This interaction also led to VE-cadherin endothelial junction disassembly and endothelial cell monolayer disruption through ERK1/2/STAT1 modulation. In mouse endothelium, en face staining revealed that 3′-SL abolished LPS-stimulated ROS production and VCAM-1 overexpression. SignificanceOur findings suggest that 3′-SL inhibits LPS-induced endothelial hyperpermeability by suppressing superoxide-mediated ERK1/2/STAT1 activation and HMGB1/RAGE axis. Therefore, 3′-SL may be a potential therapeutic agent for preventing the progression of atherosclerosis.

Involvement of NEK2 and NEK9 in LPS - induced endothelial barrier dysfunction

Endothelial hyperpermeability is the hallmark of severe lung injury, including acute respiratory distress syndrome. Despite the fact that Never In Mitosis A (NIMA)-related kinase 2 (NEK2) and NEK9 mediate fundamental cellular processes, our knowledge on their role in barrier function is limited. Herein we show that NEK2 and NEK9 inhibition suppresses LPS-induced paracellular hyperpermeability and myosin light chain 2 activation in endothelial cells. Moreover, the expression levels of both kinases were elevated in inflamed mouse lungs. Based on those findings, we raise the possibility that NEK2 and NEK9 may serve as novel therapeutic targets in lung inflammatory disease.

Everolimus-induced hyperpermeability of endothelial cells causes lung injury.

The mammalian target of rapamycin (mTOR) inhibitors, everolimus (but not dactolisib), is frequently associated with lung injury in clinical therapies. However, the underlying mechanisms remain unclear. Endothelial cell barrier dysfunction plays a major role in the pathogenesis of the lung injury. This study hypothesizes that everolimus increases pulmonary endothelial permeability, which leads to lung injury. We tested the effects of everolimus on human pulmonary microvascular endothelial cell (HPMEC) permeability and a mouse model of intraperitoneal injection of everolimus was established to investigate the effect of everolimus on pulmonary vascular permeability. Our data showed that everolimus increased human pulmonary microvascular endothelial cell (HPMEC) permeability which was associated with MLC phosphorylation and F-actin stress fiber formation. Furthermore, everolimus induced an increasing concentration of intracellular calcium Ca2+ leakage in HPMECs and this was normalized with ryanodine pretreatment. In addition, ryanodine decreased everolimus-induced phosphorylation of PKCα and MLC, and barrier disruption in HPMECs. Consistent with in vitro data, everolimus treatment caused a visible lung-vascular barrier dysfunction, including an increase in protein in BALF and lung capillary-endothelial permeability, which was significantly attenuated by pretreatment with an inhibitor of PKCα, MLCK, and ryanodine. This study shows that everolimus induced pulmonary endothelial hyper-permeability, at least partly, in an MLC phosphorylation-mediated EC contraction which is influenced in a Ca2+-dependent manner and can lead to lung injury through mTOR-independent mechanisms.

Differential roles of eNOS in late effects of VEGF-A on hyperpermeability in different types of endothelial cells

Vascular endothelial growth factor (VEGF)-A induces endothelial hyperpermeability, but the molecular pathways remain incompletely understood. Endothelial nitric oxide synthase (eNOS) regulates acute effects of VEGF-A on permeability of endothelial cells (ECs), but it remains unknown whether and how eNOS regulates late effects of VEGF-A-induced hyperpermeability. Here we show that VEGF-A induces hyperpermeability via eNOS-dependent and eNOS-independent mechanisms at 2 days after VEGF-A stimulation. Silencing of expression of the eNOS gene (NOS3) reduced VEGF-A-induced permeability for dextran (70 kDa) and 766 Da-tracer in human dermal microvascular ECs (HDMVECs), but not in human retinal microvascular ECs (HRECs) and human umbilical vein ECs (HUVECs). However, silencing of NOS3 expression in HRECs increased permeability to dextran, BSA and 766 Da-tracer in the absence of VEGF-A stimulation, suggesting a barrier-protective function of eNOS. We also investigated how silencing of NOS3 expression regulates the expression of permeability-related transcripts, and found that NOS3 silencing downregulates the expression of PLVAP, a molecule associated with trans-endothelial transport via caveolae, in HDMVECs and HUVECs, but not in HRECs. Our findings underscore the complexity of VEGF-A-induced permeability pathways in ECs and the role of eNOS therein, and demonstrate that different pathways are activated depending on the EC phenotype.

Fingolimod (FTY720), an FDA-approved sphingosine 1-phosphate (S1P) receptor agonist, restores endothelial hyperpermeability in cellular and animal models of dengue virus serotype 2 infection.

Extensive vascular leakage and shock is a major cause of dengue-associated mortality. At present, there are no specific treatments available. Sphingolipid pathway is a key player in the endothelial barrier integrity; and is mediated through the five sphingosine-1-phosphate receptors (S1PR1-S1PR5). Signaling through S1PR2 promotes barrier disruption; and in Dengue virus (DENV)-infection, there is overexpression of this receptor. Fingolimod (FTY720) is a specific agonist that targets the remaining barrier-protective S1P receptors, without targeting S1PR2. In the present study, we explored whether FTY720 treatment can alleviate DENV-induced endothelial hyperpermeability. In functional assays, in both in vitro systems and in AG129 animal models, FTY720 treatment was found effective. Upon treatment, there was complete restoration of the monolayer integrity in DENV serotype 2-infected human microvascular endothelial cells (HMEC-1). At the molecular level, the treatment reversed activation of the S1P pathway. It significantly reduced the phosphorylation of the key molecules such as PTEN, RhoA, and VE-Cadherin; and also, the expression levels of S1PR2. In DENV2-infected AG129 mice treated with FTY720, there was significant improvement in weight gain, in overall clinical symptoms, and in survival. Whereas 100% of the DENV2-infected, untreated animals died by day-10 post-infection, 70% of the FTY720-treated animals were alive; and at the end of the 15-day post-infection observation period, 30% of them were still surviving. There was a significant reduction in the Evan's-blue dye permeability in the organs of FTY720-treated, DENV-2 infected animals; and also improvement in the hemogram, with complete restoration of thrombocytopenia and hepatic function. Our results show that the FDA-approved molecule Fingolimod (FTY720) is a promising therapeutic intervention in severe dengue.

TRPC6 Deletion Enhances eNOS Expression and Reduces LPS-Induced Acute Lung Injury.

Acute lung injury (ALI) is characterized by endothelial barrier disruption and associated inflammatory responses, and transient receptor potential cation channel 6 (TRPC6)-mediated Ca2+ influx is critical for endothelial hyperpermeability. In this study, we investigated the role of TRPC6 in LPS-induced ALI, analyzed gene expression in WT and TRPC6-/- lungs using RNA sequencing, and explored the effects of TRPC6 in the LPS-induced hyperpermeability in human umbilical vein endothelial cells (HUVECs) to elucidate the underlying mechanisms. Intratracheal instillation of LPS caused edema in the mouse lungs. Deletion of TRPC6 reduced LPS-induced lung edema and decreased cell infiltration. RNA sequencing analysis suggested that downregulated cell adhesion molecules in TRPC6-/- lungs may be responsible for their resistance to LPS-induced injury. In addition, downregulation of TRPC6 significantly alleviated the LPS-induced decrease in eNOS expression in lung tissue as well as in HUVECs. Moreover, inhibition of TRPC6 with the channel antagonist larixyl led to a decrease in LPS-induced hyperpermeability and ROS production in HUVECs, which could be reversed by blocking eNOS. Our findings suggest that inhibition of TRPC6 ameliorates LPS-induced ALI, which may be achieved by acting on the cell adhesion molecule signaling pathway and participating in the regulation of eNOS levels in endothelial cells.

Oral Administration of Rhamnan Sulfate from Monostroma nitidum Suppresses Atherosclerosis in ApoE-Deficient Mice Fed a High-Fat Diet.

Oral administration of rhamnan sulfate (RS), derived from the seaweed Monostroma nitidum, markedly suppresses inflammatory damage in the vascular endothelium and organs of lipopolysaccharide-treated mice. This study aimed to analyze whether orally administered RS inhibits the development of atherosclerosis, a chronic inflammation of the arteries. ApoE-deficient female mice were fed a normal or high-fat diet (HFD) with or without RS for 12 weeks. Immunohistochemical and mRNA analyses of atherosclerosis-related genes were performed. The effect of RS on the migration of RAW264.7 cells was also examined in vitro. RS administration suppressed the increase in blood total cholesterol and triglyceride levels. In the aorta of HFD-fed mice, RS reduced vascular smooth muscle cell proliferation, macrophage accumulation, and elevation of VCAM-1 and inhibited the reduction of Robo4. Increased mRNA levels of Vcam1, Mmp9, and Srebp1 in atherosclerotic areas of HFD-fed mice were also suppressed with RS. Moreover, RS directly inhibited the migration of RAW264.7 cells in vitro. Thus, in HFD-fed ApoE-deficient mice, oral administration of RS ameliorated abnormal lipid metabolism and reduced vascular endothelial inflammation and hyperpermeability, macrophage infiltration and accumulation, and smooth muscle cell proliferation in the arteries leading to atherosclerosis. These results suggest that RS is an effective functional food for the prevention of atherosclerosis.

A ligand-independent Tie2-activating antibody reduces vascular leakage in models of Clarkson disease.

Vascular dysfunction resulting from endothelial hyperpermeability is a common and important feature of critical illness due to sepsis, trauma, and other conditions associated with acute systemic inflammation. Clarkson disease [monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome (ISCLS)] is a rare, orphan disorder marked by spontaneous and recurrent episodes of hypotensive shock and peripheral edema due to widespread vascular leakage in peripheral tissues. Mortality from acute flares approaches 30% due to lack of effective therapies. We evaluated a monoclonal antibody (4E2) specific for the endothelial receptor tyrosine kinase Tie2 in ISCLS models. 4E2 activated Tie2 in ISCLS patient-derived endothelial cells and reduced baseline and proinflammatory mediator-induced barrier dysfunction. 4E2 also reduced mortality and/or vascular leakage associated with systemic histamine challenge or influenza infection in the SJL/J mouse model of ISCLS. These findings support a critical role for Tie2 dysregulation in ISCLS and highlight a viable therapeutic approach to this catastrophic disorder.

Inhibition of farnesylation of Ras-GTPases ameliorates inflammation-induced loss of endothelial barrier integrity and leukocyte transmigration

Abstract Background and Aims The family of small GTPases (Rho- and Ras-GTPases) is known to regulate endothelial cytoskeleton dynamics and hence barrier integrity. The role of Rho GTPases (RhoA and Rac1) in endothelial barrier integrity is well characterized; however, little is known about the role of Ras-GTPases. The activity of small GTPases is regulated by several mechanisms, including post-translational prenylation (farnesylation or geranylgeranylation). The Ras-GTPases are preferentially post-translationally farnesylated by farnesyl transferases (FTs). The aim of the present study was to investigate whether inhibition of farnesylation of Ras-GTPases protects loss of endothelial barrier integrity induced by inflammatory mediators. Methods and Results Treatment of human primary endothelial cells (ECs; HUVEC and lung microvascular ECs) with pharmacological inhibitor of farnesyl transferases (FTIs), LB42708 (LB) and tipifarnib, stabilized basal endothelial barrier function (albumin-flux) and abrogated thrombin-induced hyperpermeability in a concentration- and time-dependent manner reaching maximum at a concentration of 2 mmol/L after 12 h of incubation. These concentrations of the FTIs reduced farnesylation of all the Ras isoforms but not the geranylgeranylation of Rho GTPases. Likewise, LB abrogated the TNFa-induced leucocyte transendothelial migration (Trans-well assay). Interestingly, FTIs treatment caused an enhanced translocation of VE-cadherin to cell-cell junctions (confocal microscopy) without exerting any effect on the actin cytoskeleton or endothelial contractile machinery; mainly regulated by Rho GTPases. The Ca2+-switch experiments demonstrated an inhibition of VE-cadherin internalization. These data were reproduced by lentiviral-mediated stable over-expression of the prenylation-dead mutants of Ras isoforms but not RhoA or Rac1. Conclusion The novel data of the present study demonstrate that FTIs protect endothelial barrier function against inflammatory mediator-induced endothelial hyperpermeability. We propose that FTIs are novel drug candidates for the treatment of edematous conditions (such as lung edema) where endothelial barrier integrity is compromised.

Abstract 18731: Connexin 43 Hemichannels Contribution to Ischemia-Reperfusion-Induced Hyperpermeability

Introduction: Ischemia-reperfusion (I-R) injury is a significant complication in vascular surgery or organ transplantation. I-R injury involves intracellular Ca2+ overload, nitric oxide (NO), oxidative stress. I-R injury increases endothelial vascular hyperpermeability leading to tissue edema and damage mediated by endothelial Ca2+ increase. The role of Ca2+ permeable channels in I-R is not defined. Connexin (Cx) proteins form hemichannels, allowing the exchange of molecules between intracellular and extracellular spaces. The Cx43 isoform is highly expressed in endothelial cells (EC). Endothelial Ca2+ increase promotes NO-dependent Cx43 S-nitrosylation. However, whether Cx43 hemichannels are involved in I-R-induced hyperpermeability is unknown. Hypothesis: VEGF-induced hyperpermeability causes increased NO-mediated Cx43 S-nitrosylation and opening of endothelial Cx43 hemichannels, leading to Ca2+ entry and exacerbating reperfusion damage. Methods: We used VEGF as I-R related pro-inflammatory agonist. Using primary cultures of mesenteric endothelial cells, we tested Cx43 hemichannel activity by ethidium uptake and Ca2+ influx with Fluo-4 indicator. We evaluated hyperpermeability in vitro and in vivo Results: Results indicate that VEGF stimulation increased hemichannel activity in EC, associated with a rapid rise of Ca2+ influx. Both hemichannel activation and Ca2+ increase were abolished by treatment with specific Cx43 blocker (Gap19) or blocking NO production with NG-monomethyl-L-arginine (LNMMA). Cx43 inhibition blunted VEGF-induced hyperpermeability both in vitro in primary EC culture and in vivo mouse mesenteric vascular bed. To further understand the participation of NO-mediated S-nitrosylation in the VEGF-induced hyperpermeability, we replaced cysteine 271 by serine to create a Cx43C271S mouse that fails to cause S-nitrosylation of Cx43. We demonstrated that this knockin mouse exhibits a decrease in VEGF-stimulated hyperpermeability in vivo. Conclusions: These results indicate that Cx43 hemichannels activation is critical to VEGF (I-R inflammatory environment)-induced hyperpermeability and the S-nitrosylated Cx43 is a key regulator in I-R damage. AHA support: # 932684 and #23DIVSUP1054931

HMGB1 Release Induced by EV71 Infection Exacerbates Blood-Brain Barrier Disruption via VE-cadherin Phosphorylation

PurposeEV71 (Enterovirus 71) is a major causative agent of the outbreaks of HFMD (hand, foot, and mouth disease), which is associated with neurological damage caused by permeability disruption of BBB (blood-brain barrier). HMGB1 (high-mobility group box 1) is a widely expressed nuclear protein that triggers host inflammatory responses. Our work aimed to explore the function of HMGB1 in EV71 infection and its contributions to EV71-related BBB damage. MethodsHeLa cells, HT-29 cells and AG6 mice were used to explore the translocation of HMGB1 in EV71 infection in vitro and in vivo. The roles of released HMGB1 on EV71 replication and associated inflammatory cytokines were investigated using recombinant HMGB1 in HeLa cells. The mechanisms of released HMGB1 in EV71-induced BBB injury were explored using recombinant HMGB1 and anti-HMGB1 neutralizing antibodies in monolayer HCMECs (immortalized human brain microvascular endothelial cells) and AG6 mice brain. ResultsEV71 induced HMGB1 nucleocytoplasmic translocation and extracellular release in vitro and in vivo. Released HMGB1 acted as an inflammatory mediator in EV71 infection rather than affecting viral replication in vitro. Released HMGB1 disrupted BBB integrity by enhancing VE-cadherin phosphorylation at tyrosine 685 in HCMECs, and reducing total VE-cadherin levels in HCMECs and AG6 mice in EV71 infection. And released HMGB1 induced an increase in activated astrocytes. Neutralization of HMGB1 reversed the increased endothelial hyperpermeability and phosphorylation of VE-cadherin in HCMECs. ConclusionThe inflammatory mediator HMGB1 released by EV71 exacerbated BBB disruption by enhancing VE-cadherin phosphorylation, which in turn aggravated EV71-induced neuroinflammation.

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.

Simultaneous Targeting of IL-1-Signaling and IL-6-Trans-Signaling Preserves Human Pulmonary Endothelial Barrier Function During a Cytokine Storm.

Systemic inflammatory diseases, such as sepsis and severe COVID-19, provoke acute respiratory distress syndrome in which the pathological hyperpermeability of the microvasculature, induced by uncontrolled inflammatory stimulation, causes pulmonary edema. Identifying the inflammatory mediators that induce human lung microvascular endothelial cell barrier dysfunction is essential to find the best anti-inflammatory treatments for critically ill acute respiratory distress syndrome patients. We have compared the responses of primary human lung microvascular endothelial cells to the main inflammatory mediators involved in cytokine storms induced by sepsis and SARS-CoV2 pulmonary infection and to sera from healthy donors and severely ill patients with sepsis. Endothelial barrier function was measured by electric cell-substrate impedance sensing, quantitative confocal microscopy, and Western blot. The human lung microvascular endothelial cell barrier was completely disrupted by IL (interleukin)-6 conjugated with soluble IL-6R (IL-6 receptor) and by IL-1β, moderately affected by TNF (tumor necrosis factor)-α and IFN (interferon)-γ and unaffected by other cytokines and chemokines, such as IL-6, IL-8, MCP (monocyte chemoattractant protein)-1 and MCP-3. The inhibition of IL-1 and IL-6R simultaneously, but not separately, significantly reduced endothelial hyperpermeability on exposing human lung microvascular endothelial cells to a cytokine storm consisting of 8 inflammatory mediators or to sera from patients with sepsis. Simultaneous inhibition of IL-1 and JAK (Janus kinase)-STAT (signal transducer and activator of transcription protein), a signaling node downstream IL-6 and IFN-γ, also prevented septic serum-induced endothelial barrier disruption. These findings strongly suggest a major role for both IL-6 trans-signaling and IL-1β signaling in the pathological increase in permeability of the human lung microvasculature and reveal combinatorial strategies that enable the gradual control of pulmonary endothelial barrier function in response to a cytokine storm.

Increased R-spondin 3 contributes to aerobic exercise-induced protection against renal vascular endothelial hyperpermeability and acute kidney injury.

Exercise training exerts protective effects against sepsis-associated multiple organ dysfunction. This study aimed to investigate whether aerobic exercise protected against sepsis-associated acute kidney injury (AKI) via modulating R-spondin 3 (RSPO3) expression. To investigate the effects of aerobic exercise on lipopolysaccharide (LPS)-induced AKI, LPS (20 mg/kg) was intraperitoneally injected after six weeks of treadmill training. To investigate the role of RSPO3 in LPS-induced AKI, wild-type (WT) or inducible endothelial cell-specific RSPO3 knockout (RSPO3EC-/- ) mice were intraperitoneally injected with 12 mg/kg LPS. RSPO3 was intraperitoneally injected 30 min before LPS treatment. Aerobic exercise-trained mice were more resistant to LPS-induced body weight loss and hypothermia and had a significant higher survival rate than sedentary mice exposed to LPS. Exercise training restored the LPS-induced decreases in serum and renal RSPO3 levels. Exercise or RSPO3 attenuated, whereas inducible endothelial cell-specific RSPO3 knockout exacerbated LPS-induced renal glycocalyx loss, endothelial hyperpermeability, inflammation, and AKI. Bioinformatics analysis results revealed significant increases in the expression of matrix metalloproteinases (MMPs) in kidney tissues of mice exposed to sepsis or endotoxaemia, which was validated in renal tissue from LPS-exposed mice and LPS-treated human microvascular endothelial cells (HMVECs). Both RSPO3 and MMPs inhibitor restored LPS-induced downregulation of tight junction protein, adherens junction protein, and glycocalyx components, thus ameliorating LPS-induced endothelial leakage. Exercise or RSPO3 reversed LPS-induced upregulation of MMPs in renal tissues. Increased renal expression of RSPO3 contributes to aerobic exercise-induced protection against LPS-induced renal endothelial hyperpermeability and AKI by suppressing MMPs-mediated disruption of glycocalyx and tight and adherens junctions.

Activation of endothelial TRPM2 exacerbates blood-brain barrier degradation in ischemic stroke.

Damage of the blood-brain barrier (BBB) is a hallmark of brain injury during the early stages of ischemic stroke. The subsequent endothelial hyperpermeability drives the initial pathological changes and aggravates neuronal death. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable nonselective cation channel activated by oxidative stress. However, whether TRPM2 is involved in BBB degradation during ischemic stroke remains unknown. We aimed to investigate the role of TRPM2 in BBB degradation during ischemic stroke and the underlying molecular mechanisms. Specific deletion of Trpm2 in endothelial cells using Cdh5 Cre produces a potent protective effect against brain injury in mice subjected to middle cerebral artery occlusion (MCAO), which is characterized by reduced infarction size, mitigated plasma extravasation, suppressed immune cell invasion, and inhibited oxidative stress. In vitro experiments using cultured cerebral endothelial cells (CECs) demonstrated that either Trpm2 deletion or inhibition of TRPM2 activation attenuates oxidative stress, Ca2+ overload, and endothelial hyperpermeability induced by oxygen-glucose deprivation (OGD) and CD36 ligand thrombospondin-1 (TSP1). In transfected HEK293T cells, OGD and TSP1 activate TRPM2 in a CD36-dependent manner. Noticeably, in cultured CECs, deleting Trpm2 or inhibiting TRPM2 activation also suppresses the activation of CD36 and cellular dysfunction induced by OGD or TSP1. In conclusion, our data reveal a novel molecular mechanism in which TRPM2 and CD36 promote the activation of each other, which exacerbates endothelial dysfunction during ischemic stroke. Our study suggests that TRPM2 in endothelial cells is a promising target for developing more effective and safer therapies for ischemic stroke.

Endothelial Permeability and the Angiopoietin/Tie2 System Following Mild and Severe COVID-19

Endotheliopathy plays a role in the development of acute kidney and lung injury in COVID-19, probably due to inflammation, endothelial permeability, vascular leakage and edema formation. This study examined alterations in the circulation of patients with mild and severe COVID-19 on in vitro endothelial permeability and its relation to the endothelial angiopoietin/Tie2 system, which is involved in the regulation of endothelial permeability. Plasma was obtained from COVID-19 patients admitted to the ward (n = 14) or ICU (n = 20) at admission and after 1 and 2 weeks and healthy controls (n = 5). Human kidney and lung endothelial cells were exposed to patient plasma and treated with recombinant angiopoietin-1. In vitro endothelial barrier function was assessed using electric cell-substrate impedance sensing. Circulating markers of the angiopoietin/Tie2 system, endothelial dysfunction and glycocalyx degradation were measured by ELISA. Plasma from COVID-19 patients reduced endothelial resistance compared to healthy controls, but COVID-19 plasma-induced drop in endothelial resistance did not differ between ward and ICU patients. Circulating angiopoietin-2, soluble Tie2 and soluble Tie1 levels increased over time in ICU patients, whereas levels remained stable in ward patients. The increase in angiopoietin-2 was able to predict 90-day mortality (AUC = 0.914, p < 0.001). Treatment with recombinant angiopoietin-1 did not restore COVID-19 plasma-induced hyperpermeability. In conclusion, these results suggest that indirect effects of the virus represented in the circulation of COVID-19 patients induced endothelial hyperpermeability irrespective of disease severity and changes in the endothelial angiopoietin/Tie2 system. Nonetheless, angiopoietin-2 might be of interest in the context of organ injury and patient outcome in COVID-19.

PAK1 contributes to cerebral ischemia/reperfusion injury by regulating the blood-brain barrier integrity

Globally, stroke is one of the leading causes of death and significant contributors to disability. Gaining a thorough comprehension of the underlying pathogenic processes is essential for stroke treatment and prevention. In this study, we investigated the role of p21-activated kinase 1 (PAK1) in stroke by using oxygen-glucose deprivation (OGD) and transient middle cerebral artery occlusion and reperfusion (tMCAO/R) models. We reported that focal ischemia and reperfusion affect the PAK1 expression and activity levels. We further demonstrated that PAK1 is responsible for the endothelial hyperpermeability that occurs in the early stages of ischemia and reperfusion. Additionally, inhibition of PAK1 was discovered to alleviate blood-brain barrier disruption and protect against brain injury induced by tMCAO/R. Mechanistically, we provide the evidence that PAK1 regulates the formation of stress fibers and expression of surface junctional proteins. Together, our findings reveal a pathogenic function of PAK1 in stroke.

Ceapin-A7 potentiates lipopolysaccharide-induced endothelial injury.

Barrier dysfunction is the hallmark of severe lung injury, including acute respiratory distress syndrome. Efficient medical countermeasures to counteract endothelial hyperpermeability do not exist, hence the mortality rates of disorders related to barrier abnormalities are unacceptable high. The unfolded protein response is a highly conserved mechanism, which aims to support the cells against endoplasmic reticulum stress, and ATF6 is a protein sensor that triggers its activation. In the current study, we investigate the effects of ATF6 suppression in LPS-induced endothelial inflammation. Our observations suggest that Ceapin-A7, which is an ATF6 suppressor, potentiates LPS-induced STAT3 and JAK2 activation. Hence ATF6 activation may serve as a new therapeutic possibility toward diseases related to barrier dysfunction.

ERRα protects against sepsis-induced acute lung injury in rats

BackgroundSepsis-induced acute lung injury (ALI) is associated with poor survival rates. The identification of potential therapeutic targets for preventing sepsis-induced ALI has clinical importance. This study aims to investigate the role of estrogen-related receptor alpha (ERRα) in sepsis-induced ALI.MethodsLipopolysaccharide (LPS) was used to simulate sepsis-induced ALI model in rat pulmonary microvascular endothelial cells (PMVECs). The effects of ERRα overexpression and knockdown on LPS-induced endothelial permeability, apoptosis and autophagy were determined by horseradish peroxidase permeability assay, TdT-mediated dUTP Nick End Labeling (TUNEL) assay, flow cytometry, immunofluorescence staining, RT-PCR and Western Blotting. The rat model with sepsis-induced ALI was established by cecal ligation and puncture in anesthetized rats to verify the results of in vitro experiments. Animals were randomly assigned to receive intraperitoneal injection of vehicle or ERRα agonist. Lung vascular permeability, pathological injury, apoptosis and autophagy were examined.ResultsOverexpression of ERRα ameliorated LPS-induced endothelial hyperpermeability, degradation of adherens junctional molecules, upregulation of bax, cleaved caspase 3 and cleaved caspase 9 levels, downregulation of anti-apoptotic protein Bcl-2 level, and promoted the formation of autophagic flux, while the knockdown of ERRα exacerbated LPS-induced apoptosis and inhibited the activation of autophagy. Administration of ERRα agonist alleviated the pathological damage of lung tissue, increased the levels of tight junction proteins and adherens junction proteins, and decreased the expression of apoptosis-related proteins. Promoting the expression of ERRα significantly enhanced the process of autophagy and reduced CLP-induced ALI. Mechanistically, ERRα is essential to regulate the balance between autophagy and apoptosis to maintain the adherens junctional integrity.ConclusionERRα protects against sepsis-induced ALI through ERRα-mediated apoptosis and autophagy. Activation of ERRα provides a new therapeutic opportunity to prevent sepsis-induced ALI.