Endothelial Cell Hyperpermeability Research Articles

Protective Effects of Growth Hormone-Releasing Hormone Antagonists in Interferon-γ-induced Endothelial Barrier Dysfunction

Growth Hormone - Releasing Hormone (GHRH) regulates the secretion of Growth Hormone from the anterior pituitary gland. An emerging body of evidence suggests that the activities of that neuropeptide are not limited to the GH /Insulin-like Growth Factor 1 axis, but expand towards the mediation of inflammatory processes. GHRH antagonists (GHRHAnt) were developed to oppose the activities of GHRH in malignancies, and have been associated with strong anti-inflammatory and anti-oxidative effects in a diverse variety of tissues, including the lungs. In the present study we report that GHRHAnt oppose interferon gamma (IFN-γ)-induced paracellular hyperpermeability and reactive oxygen species generation in bovine pulmonary endothelial cells. Moreover, those peptides suppress the activation of IFN-γ-triggered signal transducer and activator of transcription 3, cofilin and extracellular signal - regulated kinase 1/2. Our observations substantiate previous findings on the protective effects of GHRHAnt in endothelial inflammation, and suggest their potential application in disorders related to barrier dysfunction. Dr. Barabutis' research is supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P2O GM103424-21. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

The paracrine isthmin1 transcriptionally regulated by C/EBPβ exacerbates pulmonary vascular leakage in murine sepsis.

It is known that pulmonary vascular leakage, a key pathological feature of sepsis-induced lung injury, is largely regulated by perivascular cells. However, the underlying mechanisms have not been fully uncovered. In the present study, we aimed to evaluate the role of isthmin1, a secretory protein originating from alveolar epithelium, in the pulmonary vascular leakage during sepsis and to investigate the regulatory mechanisms of isthmin1 gene transcription. We observed an elevated isthmin1 gene expression in the pulmonary tissue of septic mice induced by cecal ligation and puncture (CLP), as well as in primary murine alveolar type II epithelial cells (ATII) exposed to lipopolysaccharide (LPS). Furthermore, we confirmed that isthmin1 derived from ATII contributes to pulmonary vascular leakage during sepsis. Specifically, adenovirus-mediated isthmin1 disruption in ATII led to a significant attenuation of the increased pulmonary microvascular endothelial cell (PMVEC) hyperpermeability in a PMVEC/ATII coculture system when exposed to LPS. In addition, adeno-associated virus 9 (AAV9)-mediated knockdown of isthmin1 in the alveolar epithelium of septic mice significantly attenuated pulmonary vascular leakage. Finally, mechanistic studies unveiled that nuclear transcription factor CCAAT/enhancer binding protein (C/EBP)β participates in isthmin1 gene activation by binding directly to the cis-regulatory element of isthmin1 locus and may contribute to isthmin1 upregulation during sepsis. Collectively, the present study highlighted the impact of the paracrine protein isthmin1, derived from ATII, on the exacerbation of pulmonary vascular permeability in sepsis and revealed a new regulatory mechanism for isthmin1 gene transcription.NEW & NOTEWORTHY This article addresses the role of the alveolar epithelial-secreted protein isthmin1 on the exacerbation of pulmonary vascular permeability in sepsis and identified nuclear factor CCAAT/enhancer binding protein (C/EBP)β as a new regulator of isthmin1 gene transcription. Targeting the C/EBPβ-isthmin1 regulatory axis on the alveolar side would be of great value in the treatment of pulmonary vascular leakage and lung injury induced by sepsis.

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.

Growth hormone-releasing hormone antagonists counteract interferon-γ – induced barrier dysfunction in bovine and human endothelial cells

GHRH regulates the secretion of GH from the anterior pituitary gland. An emerging body of evidence suggests that the activities of that neuropeptide are not limited to the GH/IGF-I axis, but they expand towards the mediation of inflammatory processes. GHRHAnt were developed to oppose the activities of GHRH in malignancies, and have been associated with strong anti-inflammatory and anti-oxidative effects in a diverse variety of tissues, including the lungs. In the present study we report that GHRHAnt oppose interferon-γ – induced paracellular hyperpermeability and reactive oxygen species generation in bovine and human pulmonary endothelial cells; and suppress interferon-γ – triggered STAT3, cofilin and ERK1/2 activation. Our observations substantiate previous findings on the protective effects of GHRHAnt in endothelial inflammation and barrier break-down.

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.

Regulation of Blood-Brain Barrier Endothelial Cell Hyperpermeability by NLRP3 Inflammasome Inhibition

The blood-brain barrier (BBB) is a semi-permeable and protective barrier of the brain, primarily composed of endothelial cells interconnected by tight junction proteins, that regulates the movement of ions and molecules between the blood and the neural matter. In pathological conditions such as traumatic brain injury (TBI), disruption of the BBB contributes to the leakage of solutes and fluids into the brain parenchyma resulting in the onset of cerebral edema and elevation of intracranial pressure. Recent studies from our lab have demonstrated that the release of the proinflammatory cytokine interleukin 1-β (IL-1β) followed by the activation of matrixmetalloproteinase-9 (MMP-9) is critical to BBB dysfunction following TBI. NLRP3 inflammasome is a multiprotein complex that is critical to the formation of IL-1β. The role of NLRP3 inflammasome in modulating BBB integrity has been recently investigated in our lab. Our results suggest that NLRP3 inflammasome activation is critical to BBB dysfunction and hyperpermeability following TBI. Once activated, the NLRP3 inflammasome is a critical component in the regulation of BBB hyperpermeability, recruiting enzymes such as caspase-1 and subsequent activation of MMP-9, which disrupt tight junction proteins necessary for BBB integrity. Thus, the NLRP3 inflammasome pathway remains a potential target for TBI therapeutics. The objective of this study was to determine the upstream regulators of NLRP3 signaling and BBB hyperpermeability, particularly to determine if extracellular ATP via P2X7R, a purinergic receptor promotes NLRP3 inflammasome activation. Extracellular ATP is a major contributor of secondary injuries following TBI. NLRP3 inflammasome pathway and its impact on BBB endothelial monolayer permeability, tight junction protein expression, and caspase-1 and MMP-9 activation following extracellular ATP-mediated barrier dysfunction was investigated in in human brain microvascular endothelial cells. The effect of benzyl ATP (BzATP)-treatment on tight junction integrity was investigated via zonula occludens-1 (ZO-1) immunofluorescence localization, ZO-1 gene expression by RT-PCR, and caspase-1 and MMP-9 activities by fluorometric assays. Pharmacological inhibition of NLRP3 (using MCC950) resulted in a decrease in BzATP-induced monolayer hyperpermeability, caspase-1 and MMP-9 activities. These effects were independent of changes in cell viability. Our results suggest that extracellular ATP promotes NLRP3 inflammasome activation and subsequent caspase-1 and MMP-9-mediated tight junction disorganization are major pathways that lead to BBB dysfunction and hyperpermeability following conditions such as traumatic brain injury. This project would not have been possible without funding from the National Institute of Health and guidance from Dr. Travis Hein and other collaborators at Texas A&amp;M. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Growth hormone-releasing hormone antagonists counteract hydrogen peroxide – induced paracellular hyperpermeability in endothelial cells

Growth Hormone-Releasing Hormone (GHRH) is a hypothalamic peptide which regulates the release of Growth Hormone from the anterior pituitary gland, and has been involved in inflammatory processes. On the other hand, GHRH antagonists (GHRHAnt) were developed to counteract those effects. Herein we demonstrate for the first time that GHRHAnt can suppress hydrogen peroxide (H2O2) - induced paracellular hyperpermeability in bovine pulmonary artery endothelial cells. Increased production of reactive oxygen species (ROS) and barrier dysfunction have been associated with the development of potentially lethal disorders, including sepsis and acute respiratory distress syndrome (ARDS). Our study supports the protective actions of GHRHAnt in the impaired endothelium, and suggests that those compounds represent an exciting therapeutic possibility towards lung inflammatory disease.

Arctigenin Prevents Retinal Edema in a Murine Retinal Vein Occlusion Model.

Macular edema causes vision loss in patients with retinal vein occlusion (RVO) and diabetic macular edema (DME). The intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents is used for treatment; however, this therapy is invasive, and recurrence occurs in some cases. The establishment of a non-invasive treatment would help to solve these problems. Here, we focused on arctigenin, a lignan polyphenol found in burdock sprout, and has effects on inflammatory and microcirculatory when taken orally. We hypothesized that oral intake of arctigenin could be effective against retinal edema in RVO and DME. In this study, the degree of retinal edema by measuring the total retinal thickness using optical coherence tomography (OCT) and the thickness of the inner nuclear layer (INL) by hematoxylin-eosin (H&E) staining were investigated. Oral administration of arctigenin ameliorated retinal edema in an RVO murine model by inhibiting the decrease in occludin and vascular endothelial (VE)-cadherin. Moreover, in retinas with edema, arctigenin suppressed the induction of VEGF, tumor necrosis factor α (TNFα), and matrix metallopeptidase 9 (MMP9). Next, the effects of arctigenin on barrier function were assessed in human retinal microvascular endothelial cells (HRMECs) by measuring the trans-endothelial electrical resistance (TEER) and conducting fluorescein isothiocyanate (FITC)-dextran permeability assays. Arctigenin showed a protective effect against VEGF-induced barrier dysfunction. In addition, arctigenin inhibited the TNFα-mediated activation of the nuclear factor-kappaB (NF-κB)/p38 mitogen-activated protein kinase (MAPK) pathway. These results suggested that oral administration of arctigenin has beneficial effects on retinal edema by inhibiting vascular hyperpermeability in endothelial cells.

6'-Sialylactose abolished lipopolysaccharide-induced inflammation and hyper-permeability in endothelial cells.

Disruption of the endothelial barrier function and reduction in cell migration leads to endothelial dysfunction. One of the most abundant human milk oligosaccharides, 6'-sialylactose (6'-SL), is reported to exert various biological functions related to inflammatory responses. In this study, we evaluated the effects of 6'-SL on lipopolysaccharide (LPS)-induced inflammation caused by endothelial barrier damage. Our results showed that LPS at 500ng/mL strongly not only abolished cell migration but also hyperactivated MAPK and NF-κB pathways. 6'-SL suppressed LPS-induced endothelial inflammation via ERK1/2, p38, and JNK MAPK pathways. 6'-SL supported endothelial junctions by upregulating PECAM-1 expression and mRNA levels of tight junctions, such as ZO-1 and occludin, which were downregulated by LPS stimulation. It significantly inhibited the nuclear translocation of NF-κB, along with the downregulation of inflammatory cytokines, including TNF-α, IL-1β, MCP-1, VCAM-1, and ICAM-1. Furthermore, 6'-SL abolished NF-κB-mediated STAT3 in controlling endothelial migration and hyperpermeability via downregulating STAT3 activation and nuclear translocation. Finally, LPS induced over-expression of VCAM-1 and ZO-1 disassembly in both atheroprone and atheroprotective areas of mouse aorta, which were reversed by 6'-SL treatment. Altogether, our findings suggest that 6'-SL is a potent therapeutic agent for modulating inflammatory responses and endothelial hyperpermeability.

Mast Cell-Derived Proteases Induce Endothelial Permeability and Vascular Damage in Severe Fever with Thrombocytopenia Syndrome.

ABSTRACTSevere fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever acquired by tick bites. Whether mast cells (MCs), the body’s first line of defense against pathogens, might influence immunity or pathogenesis during SFTS virus (SFTSV) infection remained unknown. Here, we found that SFTSV can cause MC infection and degranulation, resulting in the release of the vasoactive mediators, chymase, and tryptase, which can directly act on endothelial cells, break the tight junctions of endothelial cells and threaten the integrity of the microvascular barrier, leading to microvascular hyperpermeability in human microvascular endothelial cells. Local activation of MCs (degranulation) and MC-specific proteases-facilitated endothelial damage were observed in mouse models. When MC-specific proteases were injected subcutaneously into the back skin of mice, signs of capillary leakage were observed in a dose-dependent manner. MC-specific proteases, chymase, and tryptase were tested in the serum collected at the acute phase of SFTS patients, with the higher level significantly correlated with fatal outcomes. By performing receiver operator characteristic curve (ROC) analysis, chymase was determined as a biomarker with the area under the curve value of 0.830 (95% CI = 0.745 to 0.915) for predicting fatal outcomes in SFTS. Our findings highlight the importance of MCs in SFTSV-induced disease progression and outcome. An emerging role for MCs in the clinical prognosis and blocking MC activation as a potential drug target during SFTSV infection was proposed.IMPORTANCE We revealed a pathogenic role for MCs in response to SFTSV infection. The study also identifies potential biomarkers that could differentiate patients at risk of a fatal outcome for SFTS, as well as novel therapeutic targets for the clinical management of SFTS. These findings might shed light on an emerging role for MCs as a potential drug target during infection of other viral hemorrhagic fever diseases with similar host pathology as SFTS.

Andrographolide‐Mediated Protection of Barrier Integrity and Permeability is Mediated Through NLRP3 Inflammasome Inhibition in Blood‐Brain Barrier Endothelial Cells

The blood‐brain barrier (BBB) that consists of the inter‐endothelial tight junctions, acts as the major blood‐brain interface and regulator of brain microvascular hyperpermeability. In the brain, the loss of BBB integrity and hyperpermeability leads to cerebral edema formation and intracranial pressure elevation in traumatic brain injury and ischemic strokes. Recent studies demonstrated that reactive oxygen species are potential signals for the NLR family pyrin domain containing 3 (NLRP3) inflammasome activation that leads to interleukin‐1β (IL‐1β) secretion. IL‐1β plays a major role in promoting BBB hyperpermeability in various brain pathologies, including TBI. Furthermore, recent studies from our lab have demonstrated a tri‐phasic role for hydrogen peroxide (H2O2) in BBB endothelial cells where it promotes, angiogenesis, hyperpermeability and apoptosis in a tri‐phasic and concentration dependent manner. Hydrogen peroxide plays a major role physiologically as the second messenger and pathologically as an inducer of oxidative stress and associated cellular signaling. We hypothesized that in BBB endothelial cells, H2O2‐induced barrier dysfunction and hyperpermeability are NLRP3 inflammasome‐dependent. The main objective of our study was to evaluate the role of NLRP3 inflammasome signaling in H2O2‐mediated barrier dysfunction and hyperpermeability in human brain microvascular endothelial cells and to test whether andrographolide, a labdane diterpenoid possessing potent anti‐inflammatory properties will attenuate such effects. Th effect of H2O2 treatment on tight junction integrity/permeability was studied using zonula occludens‐1 (ZO‐1) immunofluorescence localization, ZO‐1 immunoblot analysis, Transwell monolayer permeability assay using FITC‐dextran (10‐kDa) as a fluorescent marker and cell viability/apoptosis assay. The effect of NLRP3 inhibitor (MCC950) and andrographolide on H2O2‐induced hyperpermeability was studied using Transwell permeability assay. Our results show that, CRISPR/Cas‐9‐mediated knockdown of ZO‐1 resulted in monolayer hyperpermeability demonstrating the significance of ZO‐1 in regulating barrier functions. CRISPR‐based activation of NLRP3 induced monolayer hyperpermeability whereas CRISPR/Cas‐9‐based NLRP3 knockdown had no significant effect on permeability. H2O2 (10µM) induced monolayer hyperpermeability and the effect was decreased by MCC950 and andrographolide treatment. Hydrogen peroxide induced cathepsin B (an activator of the NLRP3 pathway) activity significantly, and the effect was decreased by andrographolide treatment. H2O2‐mediated barrier dysfunction and hyperpermeability were not due to changes in cell viability/apoptosis. These results suggest that NLRP3 is a mediator of H2O2‐induced barrier dysfunction and hyperpermeability in human BBB endothelial cells and andrographolide is an effective inhibitor of this pathway.

ENOS Location is a Fundamental Mechanism for Inactivation of Inflammation‐induced Hyperpermeability

Inflammation disrupts the endothelial barrier and increases microvascular permeability (hyperpermeability), leading to tissue edema. Mechanisms for the onset of hyperpermeability have been the focus of many studies. However, the major pathological consequences are related to impairment in terminating hyperpermeability, rather than its onset, and to sustained inflammation. Therefore, we are innovatively studying mechanisms involved in the inactivation of hyperpermeability and restoration of endothelial barrier and normal microvascular permeability. We demonstrated that agonist‐induced translocation of eNOS (endothelial nitric oxide synthase) from cell membrane to cytosol initiates hyperpermeability. We hypothesize that agonist signaling for hyperpermeability also initiates a delayed increase in [cAMP], causing translocation of eNOS and Epac1 (exchange protein activated by cAMP) from cytosol to the cell membrane, which inactivates hyperpermeability. We tested whether platelet‐activating factor (PAF) induced hyperpermeability first and in delayed manner increased cAMP concentration to selectively stimulate Epac1 leading to translocation of eNOS from cytosol back to the cell membrane. In addition, we studied whether vasodilator‐stimulated phosphoprotein (VASP) is involved in the inactivation of hyperpermeability since PAF‐induced hyperpermeability in VASP‐KO endothelial cells remains elevated after Epac1 stimulation. We used human microvascular endothelial cells (HMVEC) and ECV‐304 cells transfected with green fluorescent protein (GFP)‐conjugated eNOS or the constructs GFPeNOSG2A (which anchors eNOS to the cytoplasm, ECV‐GFPeNOS‐G2A) and GFPeNOS‐CAAX (which anchors eNOS to the plasma membrane, ECV‐GFPeNOS‐CAAX) to study the restoration of endothelial barrier after hyperpermeability induced by PAF or vascular endothelial growth factor (VEGF). The time‐correlation between [NO] and [cAMP] in PAF or VEGF‐stimulated HMVEC and ECV‐eNOSGFP indicates that NO increase precedes [cAMP] increase. The increase in [cAMP] in HMVEC and ECV‐eNOSGFP correlates with a close proximity between eNOS, Epac1 and VASP, and both eNOS and Epac1 translocate to the cell membrane. None of these observations occur when eNOS is anchored to the cell membrane or cytosol. Furthermore, stimulation of Epac1 in HMVEC and ECV‐eNOSGFP inactivates their hyperpermeability response to PAF or VEGF. Stimulation of Epac1 in ECV‐GFPeNOS‐CAAX, after PAF application, inhibits hyperpermeability, while Epac1 stimulation does not reduce PAF‐elicited hyperpermeability in ECV‐GFPeNOS‐G2A. Stimulation of Epac1, after application of PAF, in the in vivo male and female hamster cheek pouch prevents hyperpermeability. In conclusion, our results support the concept that eNOS, cAMP, Epac1 and VASP work in synchrony to terminate hyperpermeability by translocating eNOS back to the cell membrane, and the overarching concept that location of eNOS is an important determinant in the regulation of microvascular permeability.

SMAD3 is a Mediator of TGF‐β ‐induced Barrier Dysfunction and Hyperpermeability in Microvascular Endothelial Cells

The endothelial transcription factor ETS (Erythroblast‐transformation specific)‐related gene (ERG) regulates canonical TGFβ‐SMAD signaling, by repressing SMAD3 activity. SMAD proteins function as important mediators of intracellular signal transduction of TGF‐β. Following their phosphorylation by TGF‐β receptor‐I, Receptor‐regulated SMADs (including SMAD2 and SMAD3 proteins) form a heteromeric complex with co‐SMAD and then translocate into the nucleus where they bind and regulate the expression of multiple target genes. Recent studies from our lab have demonstrated that ERG is a critical regulator of permeability in human lung microvascular endothelial cells (HLMECs) where ERG gene activation provided protection to the barrier. We have hypothesized that the protective effects of ERG on barrier integrity may be mediated via inhibiting TGF‐β/SMAD3 signaling pathway. To study the role of SMAD3 in microvascular endothelial barrier functions and permeability, HLMECs were grown on Transwell inserts and other cell culture platforms. Monolayer permeability was evaluated based on FITC‐albumin extravasation and measurement of its fluorescence intensity. TGF‐β (1‐50 ng/ml) induced a dose‐dependent increase in monolayer hyperpermeability. TGF‐β‐induced hyperpermeability was attenuated by a SMAD3 pharmacological inhibitor (SIS3) or CRISPR/cas9‐mediated knockdown of SMAD3. Immunoblot analysis demonstrated highly efficient SMAD3 knockdown by SMAD3 CRISPR/Cas9. TGF‐β treatment resulted in significant increase in matrixmetalloproteinase‐9 (MMP‐9) activity and the effect was decreased by SMAD3 inhibition. MMP‐9 is known to proteolytically breakdown the adherens junction proteins in microvascular endothelial cells, resulting in hyperpermeability. These results demonstrate that SMAD3 is a mediator of TGF‐β signaling‐induced loss of endothelial barrier integrity and permeability in microvascular endothelial cells. It also suggests that one of the downstream effects of TGFβ‐SMAD signaling is the activation of MMP‐9 which mediates loss of adherens junctional integrity.

The protective effects of Mirtazapine against lipopolysaccharide (LPS)-induced brain vascular hyperpermeability

ABSTRACT Sepsis is mainly characterized by severe inflammation triggered by infection, and sepsis-associated encephalopathy (SAE) is defined as brain damage caused by sepsis. Disruption of the blood-brain barrier (BBB) triggered by injured brain microvascular endothelial cells (BMECs) and damaged tight junction (TJ) structure is closely associated with the pathogenesis of SAE. The present research proposed to evaluate the potential therapeutic effects of Mirtazapine, a central presynaptic α2 receptor antagonist, on LPS-induced BBB disruption. The mice were administered with normal saline and 10 mg/kg Mirtazapine for 8 consecutive days, and from day 6, the experiment group of mice received LPS for 2 days to induce SAE. We found that the increased BBB permeability, elevated concentrations of inflammatory factors in brain tissues, and downregulated zonula occludens −1 (ZO-1) were observed in LPS-stimulated mice, all of which were reversed by 10 mg/kg Mirtazapine. In the in vitro assay, bEnd.3 brain endothelial cells were treated with 1 μM LPS in the absence or presence of Mirtazapine (25, 50 μM). We found that LPS-treated cells had significantly declined transendothelial electrical resistance (TEER), increased monolayer permeability, elevated production of inflammatory factors, and downregulated ZO-1. However, 25 and 50 μM Mirtazapine ameliorated all these LPS- induced aberrations. Mirtazapine also mitigated the decreased level of NF-E2–related factor 2 (Nrf2) in LPS-challenged endothelial cells. The protective effect of Mirtazapine on endothelial permeability against LPS was significantly abolished by the knockdown of Nrf2. Collectively, we concluded that Mirtazapine exerted protective effects on LPS-induced endothelial cells hyperpermeability by upregulating Nrf2.

Anti-high mobility group box 1 monoclonal antibody suppressed hyper-permeability and cytokine production in human pulmonary endothelial cells infected with influenza A virus.

ObjectiveHigh mobility group box-1 (HMGB1) has been reported to be involved in influenza A virus-induced acute respiratory distress syndrome (ARDS). We studied the efficacy of an anti-HMGB1 mAb using an in vitro model of TNF-α stimulation or influenza A virus infection in human pulmonary microvascular endothelial cells (HMVECs).MethodsVascular permeability of HMVECs was quantified using the Boyden chamber assay under tumor necrosis factor-α (TNF-α) stimulation or influenza A virus infection in the presence of anti-HMGB1 mAb or control mAb. The intracellular localization of HMGB1 was assessed by immunostaining. Extracellular cytokine concentrations and intracellular viral mRNA expression were quantified by the enzyme-linked immunosorbent assay and quantitative reverse transcription PCR, respectively.ResultsVascular permeability was increased by TNF-α stimulation or influenza A infection; HMVECs became elongated and the intercellular gaps were extended. Anti-HMGB1 mAb suppressed both the increase in permeability and the cell morphology changes. Translocation of HMGB1 to the cytoplasm was observed in the non-infected cells. Although anti-HMGB1 mAb did not suppress viral replication, it did suppress cytokine production in HMVECs.ConclusionAnti-HMGB1 mAb might be an effective therapy for severe influenza ARDS.

Overexpressed Neuropilin-1 in Endothelial Cells Promotes Endothelial Permeability through Interaction with ANGPTL4 and VEGF in Kawasaki Disease.

Disrupted endothelial permeability plays a crucial role in the vasculitis pathogenesis of Kawasaki disease (KD), which leads to pathological vascular leak and facilitates inflammatory cell infiltration in vascular lesions; however, the mechanisms involved in the development of endothelial barrier dysfunction during KD vasculitis are still largely unclear. Here, we found that sera from patients with KD can induce endothelial cell (EC) hyperpermeability compared to sera from healthy controls. We observed that serum vascular endothelial growth factor (VEGF) levels were increased in KD patients and sera from KD patients upregulated the expression of VEGF receptor 2 (VEGFR2) and neuropilin-1 (NRP1) in human coronary artery endothelial cells (HCAECs). Intriguingly, compared with silence of VEGFR2 in HCAECs, NRP1 silence resulted in a marked decrease in EC permeability. Furthermore, soluble NRP1 (sNRP1) remarkably reduced the stimulation of EC permeability by sera from KD patients compared with bevacizumab treatment. Importantly, we showed that besides VEGF, angiopoietin-like-4 (ANGPTL4), a NRP1-binding vasoactive factor, was also increased in KD and contributed to the EC permeability in KD conditions. In addition, levels of both ANGPTL4 and VEGF were inversely correlated with albumin levels in the serum of KD patients. Collectively, the data demonstrated that overexpressed NRP1, along with upregulated VEGFR2, in HCAECs treated with KD sera promotes endothelial permeability via interaction with the increased ANGPTL4 and VEGF in KD. Neutralization of hyperpermeability factors by sNRP1 may be a novel therapeutic strategy for KD vasculitis.

DENV NS1 and MMP-9 cooperate to induce vascular leakage by altering endothelial cell adhesion and tight junction.

Dengue virus (DENV) is a mosquito-borne pathogen that causes a spectrum of diseases including life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Vascular leakage is a common clinical crisis in DHF/DSS patients and highly associated with increased endothelial permeability. The presence of vascular leakage causes hypotension, circulatory failure, and disseminated intravascular coagulation as the disease progresses of DHF/DSS patients, which can lead to the death of patients. However, the mechanisms by which DENV infection caused the vascular leakage are not fully understood. This study reveals a distinct mechanism by which DENV induces endothelial permeability and vascular leakage in human endothelial cells and mice tissues. We initially show that DENV2 promotes the matrix metalloproteinase-9 (MMP-9) expression and secretion in DHF patients' sera, peripheral blood mononuclear cells (PBMCs), and macrophages. This study further reveals that DENV non-structural protein 1 (NS1) induces MMP-9 expression through activating the nuclear factor κB (NF-κB) signaling pathway. Additionally, NS1 facilitates the MMP-9 enzymatic activity, which alters the adhesion and tight junction and vascular leakage in human endothelial cells and mouse tissues. Moreover, NS1 recruits MMP-9 to interact with β-catenin and Zona occludens protein-1/2 (ZO-1 and ZO-2) and to degrade the important adhesion and tight junction proteins, thereby inducing endothelial hyperpermeability and vascular leakage in human endothelial cells and mouse tissues. Thus, we reveal that DENV NS1 and MMP-9 cooperatively induce vascular leakage by impairing endothelial cell adhesion and tight junction, and suggest that MMP-9 may serve as a potential target for the treatment of hypovolemia in DSS/DHF patients.

Glycolysis is integral to histamine-induced endothelial hyperpermeability.

Histamine-induced vascular leakage is a core process of allergic pathologies, including anaphylaxis. Here, we show that glycolysis is integral to histamine-induced endothelial barrier disruption and hyperpermeability. Histamine rapidly enhanced glycolysis in endothelial cells via a pathway that involved histamine receptor 1 and phospholipase C beta signaling. Consistently, partial inhibition of glycolysis with 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) prevented histamine-induced hyperpermeability in human microvascular endothelial cells, by abolishing the histamine-induced actomyosin contraction, focal adherens junction formation, and endothelial barrier disruption. Pharmacologic blockade of glycolysis with 3PO in mice reduced histamine-induced vascular hyperpermeability, prevented vascular leakage in passive cutaneous anaphylaxis and protected from systemic anaphylaxis. In conclusion, we elucidated the role of glycolysis in histamine-induced disruption of endothelial barrier integrity. Our data thereby point to endothelial glycolysis as a novel therapeutic target for human pathologies related to excessive vascular leakage, such as systemic anaphylaxis.

Piezo1 acts upstream of TRPV4 to induce pathological changes in endothelial cells due to shear stress

The ion channels Piezo1 and TRPV4 have both, independently, been implicated in high venous pressure– and fluid shear stress–induced vascular hyperpermeability in endothelial cells. However, the mechanism by which Piezo1 and TRPV4 channels execute the same function is poorly understood. Here we demonstrate that Piezo1 regulates TRPV4 channel activation in endothelial cells and that Piezo1-mediated TRPV4 channel opening is a function of the strength and duration of fluid shear stress. We first confirmed that either fluid shear stress or the Piezo1 agonist, Yoda1, led to an elevation in intracellular calcium ([Ca2+]i) and that application of the Piezo1 antagonist, GsMTx4, completely blocked this change. We discovered that high and prolonged shear stress caused sustained [Ca2+]i elevation that was blocked by inhibition of TRPV4 channel opening. Moreover, Piezo1 stimulated TRPV4 opening through activation of phospholipase A2. TRPV4-dependent sustained [Ca2+]i elevation was responsible for fluid shear stress–mediated and Piezo1-mediated disruption of adherens junctions and actin remodeling. Blockade of TRPV4 channels with the selective TRPV4 blocker, HC067047, prevented the loss of endothelial cell integrity and actin disruption induced by Yoda1 or shear stress and prevented Piezo1-induced monocyte adhesion to endothelial cell monolayers. These findings demonstrate that Piezo1 activation by fluid shear stress initiates a calcium signal that causes TRPV4 opening, which in turn is responsible for the sustained phase calcium elevation that triggers pathological events in endothelial cells. Thus, deleterious effects of shear stress are initiated by Piezo1 but require TRPV4.