Increase In Endothelial Permeability Research Articles

Emerging understandings of the role of exosomes in atherosclerosis.

Atherosclerosis remains a major contributor to cardiovascular disease, the leading cause of global morbidity and mortality. Despite the elucidation of several molecular, biochemical, and cellular aspects that contribute to the etio-pathogenesis of atherosclerosis, much remains to be understood about the onset and progression of this disease. Emerging evidence supports a role for exosomes in the cellular basis of atherosclerosis. Indeed, exosomes of activated monocytes seem to accentuate a positive feedback loop that promotes recruitment of pro-inflammatory leukocytes. Moreover, in addition to their role in promoting proliferation and invasion of vascular smooth muscle cells, exosomes can also induce neovascularization within lesions and increase endothelial permeability, two important features of fibrous plaques. Depending on their sources and cargo, exosomes can also induce clot formation and contribute to other hallmarks of atherosclerosis. Taken together, it is becoming increasingly evident that a better understanding of exosome biology is integral to elucidating the pathogenesis of atherosclerosis, and may thus provide insight into a potentially new therapeutic target for this disease.

Perivascular B cells link intestinal angiogenesis to immunity and to the gut-brain axis during neuroinflammation

Disruption of gut barrier function and intestinal immune cell homeostasis are increasingly considered critical players in pathogenesis of extra-intestinal inflammatory diseases, including multiple sclerosis (MS) and its prototypical animal model, the experimental autoimmune encephalomyelitis (EAE). Breakdown of epithelial barriers increases intestinal permeability and systemic dissemination of microbiota-derived molecules. However, whether the gut-vascular barrier (GVB) is altered during EAE has not been reported. Here, we demonstrate that endothelial cell proliferation and vessel permeability increase before EAE clinical onset, leading to vascular remodeling and expansion of intestinal villi capillary bed during disease symptomatic phase in an antigen-independent manner. Concomitant to onset of angiogenesis observed prior to neurological symptoms, we identify an increase of intestinal perivascular immune cells characterized by the surface marker lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE-1). LYVE-1+ is expressed more frequently on B cells that show high levels of CD73 and have proangiogenic properties. B cell depletion was sufficient to mitigate enteric blood endothelial cell proliferation following immunization for EAE. In conclusion, we propose that altered intestinal vasculature driven by a specialized LYVE-1+ B cell subset promotes angiogenesis and that loss of GVB function is implicated in EAE development and autoimmunity.

Endothelium-Derived Extracellular Vesicles Expressing Intercellular Adhesion Molecules Reflect Endothelial Permeability and Sepsis Severity.

Currently, clinical indicators for evaluating endothelial permeability in sepsis are unavailable. Endothelium-derived extracellular vesicles (EDEVs) are emerging as biomarkers of endothelial injury. Platelet endothelial cell adhesion molecule (PECAM) and vascular endothelial (VE)-cadherin are constitutively expressed endothelial intercellular adhesion molecules that regulate intercellular adhesion and permeability. Herein, we investigated the possible association between EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) and endothelial permeability and sepsis severity. Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor alpha (TNF-α) directly or after pretreatment with permeability-modifying reagents such as angiopoietin-1, prostacyclin, or vascular endothelial growth factor (VEGF) to alter TNF-α-induced endothelial hyperpermeability. Endothelial permeability was measured using the dextran assay or transendothelial electrical resistance. Additionally, a prospective cross-sectional observational study was conducted to analyze circulating EDEV levels in patients with sepsis. EDEVs were examined in HUVEC culture supernatants or patient plasma (nonsepsis, n = 30; sepsis, n = 30; septic shock, n = 42) using flow cytometry. The Wilcoxon rank-sum test was used for comparisons between 2 groups. Comparisons among 3 or more groups were performed using the Steel-Dwass test. Spearman's test was used for correlation analysis. Statistical significance was set at P < .05. TNF-α stimulation of HUVECs significantly increased EDEV release and endothelial permeability. Pretreatment with angiopoietin-1 or prostacyclin suppressed the TNF-α-induced increase in endothelial permeability and inhibited the release of PECAM+ and VE-cadherin+ EDEVs. In contrast, pretreatment with VEGF increased TNF-α-induced endothelial permeability and the release of PECAM+ and VE-cadherin+ EDEVs. However, pretreatment with permeability-modifying reagents did not affect the release of EDEVs expressing inflammatory stimulus-inducible endothelial adhesion molecules such as E-selectin, intracellular adhesion molecule-1, or vascular cell adhesion molecule-1. The number of PECAM+ EDEVs on admission in the septic-shock group (232 [124, 590]/μL) was significantly higher (P = .043) than that in the sepsis group (138 [77,267]/μL), with an average treatment effect of 98/μL (95% confidence interval [CI], 2-270/μL), and the number of VE-cadherin+ EDEVs in the septic-shock group (173 [76,339]/μL) was also significantly higher (P = .004) than that in the sepsis group (81 [42,159]/μL), with an average treatment effect (ATE) of 79/μL (95% CI, 19-171/μL); these EDEV levels remained elevated until day 5. EDEVs expressing intercellular adhesion molecules (PECAM+ or VE-cadherin+ EDEVs) may reflect increased endothelial permeability and could be valuable diagnostic and prognostic markers for sepsis.

Lifibrate attenuates blood-brain barrier damage following ischemic stroke via the MLCK/p-MLC/ZO-1 axis.

Dysfunction of tight junction proteins-associated damage to the blood-brain barrier (BBB) plays an important role in the pathogenesis of ischemic stroke. Lifibrate, an inhibitor of cholinephosphotransferase (CPT), has been used as an agent for serum lipid lowering. However, the protective effects of Lifibrate in ischemic stroke and the underlying mechanism have not been clearly elucidated. Here, we employed an in vivo mice model of MCAO and an OGD/R model in vitro. In the mice models, neurological deficit scores and infarct volume were assessed. Evans Blue solution was used to detect the BBB permeability. The TEER was examined to determine brain endothelial monolayer permeability. Here, we found that Lifibrate improved neurological dysfunction in stroke. Additionally, increased BBB permeability during stroke was significantly ameliorated by Lifibrate. Correspondingly, the reduced expression of the tight junction protein ZO-1 was restored by Lifibrate at both the mRNA and protein levels. Using an in vitro model, we found that Lifibrate ameliorated OGD/R-induced injury in human bEnd.3 brain microvascular endothelial cells by increasing cell viability but reducing the release of LDH. Importantly, Lifibrate suppressed the increase in endothelial monolayer permeability and the reduction in TEER induced by OGD/R via the rescue of ZO-1 expression. Mechanistically, Lifibrate blocked activation of the MLCK/ p-MLC signaling pathway in OGD/R-stimulated bEnd.3 cells. In contrast, overexpression of MLCK abolished the protective effects of Lifibrate in endothelial monolayer permeability, TEER, as well as the expression of ZO-1. Our results provide a basis for further investigation into the neuroprotective mechanism of Lifibrate during stroke.

Parasitic infestations as a cause of episodic angioedema

Angioedema (AO) is a localized transient acute, recurrent edema of the skin or mucous membranes. Vasoactive substances play a key role in the development of AO: histamine, tryptase, prostaglandin, bradykinin, which lead to a reversible increase in endothelial permeability. Usually, the manifestations persist from 2 hours to several days and in most cases disappear without a trace, without additional therapy. ICD-10 CODE: T 78.3 - angioedema, D 84.1 - defect in the complement system. The purpose of the study. The purpose of the study is to study the features of episodic angioedema as a manifestation of parasitic invasion Materials and methods. The clinical case of a 10-year-old boy who was hospitalized in the pediatric department as planned with complaints of recurrent swelling of the eyelids, perianal area, itchy skin, hemorrhagic rashes, abdominal pain was analyzed. These symptoms are noted periodically for 2.5 years. The child’s anamnestic data, allergoanamnesis, gender characteristics, and the structure of the main diagnoses were analyzed. The results of laboratory and instrumental research methods were evaluated (including the level of C1 inhibitor for differential diagnosis with hereditary angioedema). Results. The results of the study. For 2.5 years, the child was periodically hospitalized in various hospitals with symptoms of periorbital edema and abdominal pain. These symptoms persisted from 2 weeks to 2 months. And they were registered with a frequency of 2 months. Blood tests showed an increase in IgE by 10 times, IgG to helminths by 6 times. At normal values of the C1 inhibitor. During the EGDS, a large number of roundworms were discovered. The normal level of the C1 inhibitor, as well as the absence of similar signs of the disease in the child’s closest relatives (autosomal dominant type of inheritance) made it possible to exclude hereditary angioedema with a high degree of probability. Conclusion. The data obtained during the analysis allowed us to conclude that parasitic invasion may be the cause of angioedema in children. Blood eosinophilia, as one of the important indicators of parasitic invasion, may be absent during the long course of infection. The presence of elevated total immunoglobulin E and positive minimum levels of antibodies to helminths in the blood should alert doctors in terms of further search for parasitic invasion.

Pathophysiology of Acute Respiratory Distress Syndrome

Acute respiratory distress syndrome (ARDS) is a common condition in intensive care medicine. Various intra- and extrapulmonal causes may trigger an epithelial and endothelial permeability increase, which leads to impaired gas exchange due to fluid overload of the alveoli and transmigration of leukocytes. This results in hypoxemia and hypercapnia, as well as deleterious consequences for the macro- and microcirculation with the risk of multi-organ failure and high mortality. This review summarizes ARDS pathophysiology and clinical consequences.

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

RNA-binding protein-regulated fibronectin is essential for EGFR-activated metastasis of head and neck squamous cell carcinoma.

There is a higher expression level of epidermal growth factor receptor (EGFR) in up to 90% of advanced head and neck squamous cell carcinoma (HNSCC) tissue than in normal surrounding tissues. However, the role of RNA-binding proteins (RBPs) in EGFR-associated metastasis of HNSCC remains unclear. In this study, we reveal that RBPs, specifically nucleolin (NCL) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), correlated with the mesenchymal phenotype of HNSCC. The depletion of RBPs significantly attenuated EGF-induced HNSCC metastasis. Intriguingly, the EGF-induced EMT markers, such as fibronectin, were regulated by RBPs through the ERK and NF-κB pathway, followed by the enhancement of mRNA stability of fibronectin through the 5' untranslated region (5'-UTR) of the gene. The upregulation of fibronectin triggered the integrin signaling activation to enhance tumor cells' attachment to endothelial cells and increase endothelial permeability. In addition, the concurrence of EGFR and RBPs or EGFR and fibronectin was associated with overall survival and disease-free survival of HNSCC. The in vivo study showed that depletion of NCL, hnRNPA2B1, and fibronectin significantly inhibited EGF-promoted extravasation of tumor cells into lung tissues. The depletion of fibronectin or treatment with integrin inhibitors dramatically attenuated EGF-induced HNSCC metastatic nodules in the lung. Our data suggest that the RBPs/fibronectin axis is essential for EGF-induced tumor-endothelial cell interactions to enhance HNSCC cell metastasis.

IRHOM2 regulates inflammation and endothelial barrier permeability via CX3CL1.

Acute lung injury (ALI) is associated with increased lung inflammation and lung permeability. The present study aimed to determine the role of inactive rhomboid-like protein 2 (iRHOM2) in ALI in lipopolysaccharide (LPS)-induced pulmonary microvascular endothelial cell model. Human pulmonary microvascular endothelial cells (HPMVECs) were transfected with small interfering RNA targeting iRHOM2 and C-X3-C motif chemokine ligand 1 (CX3CL1) overexpression plasmids and treated with LPS. Cell viability was detected using a Cell Counting Kit-8 assay, while levels of TNFα, IL-1β, IL-6 and p65 were measured by reverse transcription-quantitative PCR and western blotting. Apoptosis levels were measured using a TUNEL assay. Endothelial barrier permeability was detected, followed by analysis of zonula occludens-1, vascular endothelial-cadherin and occludin by immunofluorescence staining or western blotting. The interaction of iRHOM2 and CX3CL1 was analyzed using an immune-coprecipitation assay. Through bioinformatics analysis, it was found that CX3CL1 was upregulated in the LPS group compared with the control. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the TNF signaling pathway affected by iRHOM2 and cytokine-cytokine receptor interaction, including CX3CL1, served a key role in ALI. HPMVECs treated with LPS exhibited a decrease in cell viability and an increase in inflammation, apoptosis and endothelial barrier permeability, while these effects were reversed by iRHOM2 silencing. However, CX3CL1 overexpression inhibited the effects of iRHOM2 silencing on LPS-treated HPMVECs. The present study demonstrated a novel role of iRHOM2 as a regulator that affects inflammation, apoptosis and endothelial barrier permeability; this was associated with CX3CL1.

The Role of Txnip in Mediating Low-Magnesium-Driven Endothelial Dysfunction

Magnesium deficiency is associated with a greater risk of developing cardiovascular diseases since this cation is fundamental in regulating vascular function. This clinical evidence is sustained by in vitro studies showing that culturing endothelial cells in low concentrations of magnesium promotes the acquisition of a pro-oxidant and pro-inflammatory phenotype. Here, we show that the increase in reactive oxygen species in endothelial cells in low-magnesium-containing medium is due to the upregulation of the pro-oxidant protein thioredoxin interacting protein (TXNIP), with a consequent accumulation of lipid droplets and increase in endothelial permeability through the downregulation and relocalization of junctional proteins. Silencing TXNIP restores the endothelial barrier and lipid content. Because (i) mitochondria serve multiple roles in shaping cell function, health and survival and (ii) mitochondria are the main intracellular stores of magnesium, it is of note that no significant alterations were detected in their morphology and dynamics in our experimental model. We conclude that TXNIP upregulation contributes to low-magnesium-induced endothelial dysfunction in vitro.

SPHK1 maintains endothelial barrier function and reanneals adherens junctions, thus hastens the recovery process.

Abstract Disruption of endothelial barrier is a crucial factor in the pathogenesis of tissue inflammation, the hallmark of inflammatory diseases such as diabetes and atherosclerosis. Increased endothelial permeability occurs because of loss of cell–cell contacts and disruption of cell–extracellular matrix (ECM) adhesions. Vascular injury is associated with activation of the coagulation cascade and release of thrombin, which increases endothelial permeability by activating endothelial cell surface thrombin receptor. This increase in endothelial permeability is typically followed by a recovery period of ≈2 hours, during which barrier integrity is restored. It has been surmised that thrombin signaling stimulates intrinsic repair mechanisms that restore barrier function. To validate our finding we determined endothelial barrier function using state of the art Electric Cell-substrate Impedance Sensing (ECIS) mechanism, a measure of trans-endothelial electrical resistance (TEER) across the endothelial monolayers using TEER electrodes. Our data show that 50mM thrombin increase endothelial permeability followed by complete recovery and reannealing of adherens junction in control endothelial cells. However, SPHK1 depleted cells showed significantly decrease in barrier disruption, and barrier failed recover completely compared to control monolayer. Upon treatment with S1P in SPHK1 depleted endothelia showed completely recovery in barrier function. In conclusion, our study for the first time shows that SPHK1-S1P-S1PR1 signaling pathway is emerging as a potential therapeutic target in improving endothelial barrier function and prevent coronary artery disease. CTRE Seed Grant to Professor Nadeem Fazal, MD, PhD

Role of cholinergic anti-inflammatory pathway in the regulation of vascular endothelial permeability and inflammation in type 2 diabetes

Abstract Despite tremendous advancement in glycemic control, anti-diabetic medications have failed to revert vascular impairment once triggered by the metabolic disorder. The cholinergic anti-inflammatory pathway (CAP) is a neurophysiological mechanism that regulates the immune system. Studies show that the CAP inhibits inflammation by suppressing cytokine synthesis via release of acetylcholine in organs of the reticuloendothelial system, including the heart, lungs and gastrointestinal tract. Upon release, acetylcholine interacts with α7 nicotinic acetylcholine receptors (α7nAChR), down-regulate pro-inflammatory cytokine synthesis and prevent tissue damage and inflammation. Although a CAP modulated the response to severe inflammation during sepsis, the role of this pathway in the regulation of type-2 diabetes (T2D)-induced increase in endothelial permeability is completely unknown. Our data showed that human coronary artery endothelial cells (HCAECs) from T2D displayed increased endothelial permeability and enhanced pro-inflammatory signaling in response to pro-coagulant thrombin and endotoxin lipopolysaccharide (LPS), respectively. We also found increased expression and activity of acetylcholinesterase enzyme, and no change in expression of α7nAChR in T2D endothelial cells (ECs) compared to ECs from normal subjects. Our research findings showed that the T2D increased the expression and activity of AChE enzyme in HCAECs, and thereby decreasing the availability of ACh at α7nAChR. In summary, our work offered novel insights and therapeutic targets that we think will lead to the development of more effective treatments with improved outcomes. Grant from CTRE Chicago State University, Chicago, IL

Donepezil prevents high glucose-induced endothelial barrier dysfunction and accelerates wound healing process in human coronary endothelial cells

Abstract The Cholinergic Anti-inflammatory Pathway (CAP) is a neurophysiological mechanism that regulates the immune system. Studies show that the CAP inhibits inflammation by suppressing cytokine synthesis via release of acetylcholine in the heart, lungs and gastrointestinal tract. Upon release, acetylcholine interacts with α7 nicotinic acetylcholine receptors (α7nAChR), down-regulate pro-inflammatory cytokine synthesis and prevent tissue damage and inflammation. Donepezil, a centrally acting reversible acetylcholinesterase inhibitor, has been used in the palliative treatment of Alzheimer’s disease (AD). We hypothesized that the donepezil prevents high glucose-induced endothelial barrier dysfunction and accelerates wound healing process in human coronary endothelial cells by preventing the destabilization of junctional proteins. We investigated the potential protective role of Donepezil in high glucose induced increase in human coronary artery endothelial (HCAE) permeability and wound healing process. Endothelial permeability was evaluated by using state of the art Electric Cell-substrate Impedance Sensing (ECIS) mechanism, a measure of trans-endothelial electrical resistance (TEER) across the endothelial monolayers using TEER electrodes. Wound healing assay was performed by employing electric signals to both wound and monitor the healing process in endothelial monolayer. Our data show that 25mM glucose concentration increased endothelial permeability and abrogated the wound healing process. Donepezil at the dose of 5 μM and 10 μM significantly prevented alteration of endothelial barrier function and enhances wound healing process in the presence of high glucose concentration.

Donepezil is emerging as a potential therapeutic treatment in improving endothelial barrier function to prevent and treat coronary artery disease in Diabetes Mellitus.

Abstract Current treatments have shown limited success in reversing DM induced vascular endothelial barrier dysfunction. Vascular endothelium forms the inner most lining of the blood vessels, regulates variety of biological processes such as angiogenesis, wound healing and host defense mechanisms. During inflammatory conditions, such as diabetes mellitus and myocardial infarction endothelial cell-cell junctions start to disrupt because of the internalization of the junctional proteins, such as vascular endothelial (VE) cadherin. This leads to the formation of minute inter-endothelial gaps, and the infiltration of protein-rich fluid and immune cells in the interstitial space. If remains unchecked, the persistent buildup of edema underlying the endothelial lining sets the stage for the serious life-threatening complications. Therefore, we hypothesized that the donepezil prevents high glucose-induced endothelial barrier dysfunction by preventing the destabilization of junctional proteins. We investigated the potential protective role of Donepezil in high glucose induced increase in human coronary artery endothelial (HCAE) permeability and wound healing process. Wound healing assay was performed by employing electric signals to both wound and monitor the healing process in endothelial monolayer. To investigate the signaling mechanism involved in the protection offered by Donepezil, we found that Donepezil prevented loss of VE-cadherin in endothelial junctions, and abrogated stress fiber formation in endothelial cell exposed to high-glucose solution. In conclusion, our study shows that Donepezil maintained endothelial barrier function and improved wound healing process in endothelial cells exposed to high glucose. Chicago State University CTRE grant to Professor Nadeem Fazal, MD, PhD

Schisandrin A ameliorates increased pulmonary capillary endothelial permeability accompanied with sepsis through inhibition of RhoA/ROCK1/MLC pathways.

Sepsis is a systemic inflammatory response, and vascular leakage associated with acute lung injury (ALI) is an important pathophysiological process during sepsis. Schisandrin A (SchA) is a bioactive lignan which has been reported to have the anti-inflammatory effects in many studies, while whether SchA can ameliorate ALI-related vascular leakage caused by sepsis is unknown. To evaluate the role and the underlying mechanism of SchA in increase of pulmonary vascular permeability induced by sepsis. The effect of SchA on pulmonary vascular permeability was examined in rat acute lung injury model. The effect of SchA on skin vascular permeability of mice was investigated through Miles assay. MTT assay was performed to detect the cell activity, and transwell assay was used to detect the effect of SchA on cell permeability. The effects of SchA on junction proteins and RhoA/ROCK1/MLC signaling pathway were manifested by immunofluorescence staining and western blot. The administration of SchA alleviated rat pulmonary endothelial dysfunction, relieved increased permeability in the mouse skin and HUVECs induced by lipopolysaccharide (LPS). Meanwhile, SchA inhibited the formation of stress fibers, reversed the decrease of expression of ZO-1 and VE-cadherin. Subsequent experiments confirmed that SchA inhibited RhoA/ROCK1/MLC canonical pathway in rat lungs and HUVECs induced by LPS. Moreover, overexpression of RhoA reversed the inhibitory effect of SchA in HUVECs, which suggested that SchA protected the pulmonary endothelial barrier by inhibiting RhoA/ROCK1/MLC pathway. In summary, our results indicate that SchA ameliorates the increase of pulmonary endothelial permeability induced by sepsis through inhibition of RhoA/ROCK1/MLC pathway, providing a potentially effective therapeutic strategy for sepsis.

Soluble Urokinase Plasminogen Activator Receptor Contributes to ANCA-positive IgG-mediated Glomerular Endothelial Activation through TLR4 Pathway.

The soluble urokinase plasminogen activator receptor (suPAR), a biomarker of inflammation, has been found to be a potential prognostic factor of renal function progression. Our previous study showed that plasma suPAR levels were significantly associated with disease activity and prognosis in patients with antineutrophil cytoplasmic autoantibody-associated vasculitis (AAV). This study aimed to explore whether urokinase plasminogen activator receptor (uPAR) participated in MPO-ANCA-induced glomerular endothelial cell (GEnC) injury, which is one of the most important aspects in the pathogenesis of AAV. GEnC activation and injury were analyzed by measuring the mRNA levels of ICAM-1 and VCAM-1. Permeability experiments were performed to detect endothelial monolayer activation in number. The expression of TLR4 was detected. In addition, TLR4 siRNA and TLR4 inhibitors were employed to determine its role. Bioinformatics methods were used for further analysis. Compared with a single stimulation, uPAR could further increase the expression of ICAM-1 and VCAM-1 mRNA levels, increase endothelial monolayer permeability and impair tight junctions in GEnCs stimulated with MPO-ANCA-positive IgG. The expression of TLR4 was upregulated by uPAR and MPO-ANCApositive IgG stimulation. TLR4 siRNA significantly reduced the expression of ICAM-1 and VCAM-1 mRNA levels induced by uPAR and MPO-ANCA-positive IgG. The TLR4 antagonist significantly downregulated the levels of ICAM-1 mRNA in cells and sICAM-1 in the supernatants of GEnCs treated with uPAR plus MPOANCA- positive IgG. PLAUR is a core gene in bioinformatics analysis. uPAR protein can enhance the GEnC activation and injury induced by MPO-ANCA-positive IgG through the TLR4 pathway, indicating that suPAR may be involved in the pathogenesis of AAV and that su- PAR might be regarded as a potential therapeutic target.

ALDH2 attenuates LPS-induced increase of brain microvascular endothelial cell permeability by promoting fusion and inhibiting fission of the mitochondria

To investigate the effect of aldehyde dehydrogenase 2 (ALDH2) on lipopolysaccharide (LPS)- induced damage of mouse brain microvascular endothelial barrier and explore the role of mitochondrial fusion and fission in maintaining the integrity of endothelial barrier. Mouse brain microvascular endothelial cells were treated with 1 μg/ mL LPS for 24 h with or without pretreatment with 20 μmol/mL Alda-1 (a ALDH2 agonist) for 1 h. The changes in cell viability were assessed using cell counting Kit-8 (CCK8) assay, and the cell permeability was evaluated using transendothelial cell resistance (TEER) and FITC-Dextran assay. The level of oxidative stress in the cells was assessed by detecting the levels of malondialdehyde (MDA) and superoxide dismutase (SOD), and the content of reactive oxygen species (ROS) was detected using a superoxide anion fluorescent probe (DHE). Western blotting was performed to detect the expressions of ALDH2, tight junction proteins ZO-1 and occludin, and mitochondrial fusion- and division-related proteins Mfn2, OPA1, Drp1 and Fis1. Compared with the untreated cells, the cells treated with LPS showed significantly decreased TEER, increased FITC-dextran leakage, MDA content and ROS production, decreased SOD activity expressions of ALDH2, ZO-1, occludin, Mfn2 and OPA1, and increased expressions of Drp1 and Fis1 (P < 0.05). Pretreatment with Alda-1 prior to LPS exposure strongly suppressed the increase of endothelial cell membrane permeability, reduced ROS production, increased the expressions of ALDH2, ZO-1, occludin, OPA1 and Mfn2, and lowered the expressions of Drp1 and Fis1 (P < 0.05). ALDH2 can alleviate LPS-induced damage of brain microvascular endothelial cell barrier by inhibiting the mitochondrial ROS production and promoting mitochondrial fusion and inhibiting mitochondrial fission.

FSAP Protects against Histone-Mediated Increase in Endothelial Permeability In Vitro.

Factor-VII-activating protease (FSAP) is involved in the regulation of hemostasis and inflammation. Extracellular histones play a role in inflammation and the conversion of latent pro-FSAP into active FSAP. FSAP has been shown to regulate endothelial permeability, but the mechanisms are not clear. Here, we have investigated the effects of FSAP on endothelial permeability in vitro. A mixture of histones from calf thymus stimulated permeability, and the wild-type (WT) serine protease domain (SPD) of FSAP blocked this effect. WT-SPD-FSAP did not influence permeability on its own, nor that stimulated by thrombin or vascular endothelial growth factor (VEGF)-A165. Histones induced a large-scale rearrangement of the junction proteins VE-cadherin and zona occludens-1 from a clear junctional distribution to a diffuse pattern. The presence of WT-SPD-FSAP inhibited these changes. Permeability changes by histones were blocked by both TLR-2 and TLR4 blocking antibodies. Histones upregulated the expression of TLR-2, but not TLR-4, in HUVEC cells, and WT-SPD-FSAP abolished the upregulation of TLR-2 expression. An inactive variant, Marburg I (MI)-SPD-FSAP, did not have any of these effects. The inhibition of histone-mediated permeability may be an important function of FSAP with relevance to sepsis, trauma, and stroke and the need to be investigated further in in vivo experiments.

Leptin mediates protective effects on the vasculature

Abstract Introduction and purpose Lipodystrophy (LD) syndromes are characterized by the loss of adipose tissue resulting in metabolic complications and accelerated atherosclerosis. The systemic concentration of the adipokine leptin is reduced in LD as a result of adipose tissue deficiency. A therapeutical option to treat LD is the substitution of leptin, which improves metabolic complications and reduces mortality. However, the vascular effects of leptin remain largely unknown. Here we analyze the direct effects of leptin on the vascular system and the development of atherosclerosis. Methods and results Treatment of human endothelial cells (ECs) with leptin reduced endothelial inflammation and the process of endothelial-to-mesenchymal transition (EndMT) (CNN1, −41.4%, p&amp;lt;0.05, n=4). In addition, leptin administration prevented the EndMT-induced increase of endothelial permeability. The protective effect of leptin on EndMT was confirmed in vivo in a combined lipodystrophic and atherosclerosis-prone mouse model (LDLR−/−; aP2-nSrebp1c). Treatment of the mice with leptin (3.0 mg/kg body weight daily for 8 weeks) decreased EndMT. Leptin showed no effect on plaques size but reduced the protrusion of plaques in atherosclerotic areas of the aortic roots (−31%, p&amp;lt;0.05, n=4–6). Cytokine screening revealed an increase of the growth differentiation factor 15 (GDF15) in serum of LD patients (+26.2%, p&amp;lt;0.05, n=53–58) and in ECs after EndMT (+138%, p&amp;lt;0.05, n=6743–10920). This increase was reversed using leptin treatment in ECs undergoing EndMT, in the LD mice model, and in LD patients after 4 weeks of leptin administration. Indeed, treatment of endothelial cells with GDF15 induced EndMT (CNN1, +7.7-fold-control, p&amp;lt;0.05, n=3), and impaired EC barrier function. Neutralizing antibodies targeting GDF15 inhibited EndMT-mediated expression of mesenchymal genes (CNN1, −54%, p&amp;lt;0.05, n=4). The treatment of ECs with serum from LD patients induced EndMT and the increase of mesenchymal marker expression was inhibited with additional administration with neutralizing antibodies targeting GDF15 (CNN1, −28%, p&amp;lt;0.05, n=3). Conclusion Our findings indicate that EndMT is part of the cardiovascular disease progression in lipodystrophy syndromes. Leptin treatment has direct protective vascular effects by preventing inflammation, EndMT, and maintaining endothelial integrity. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)

Inhibiting P2Y12 receptor relieves LPS-induced inflammation and endothelial dysfunction.

BackgroundAcute lung injury (ALI) is characterized by abnormal inflammatory response without effective therapies. P2Y12 receptor (P2Y12R) plays a vital role in inflammatory response. This study intends to explore whether P2Y12R antagonists can inhibit LPS‐induced inflammatory injury of human pulmonary microvascular endothelial cells (HPMVECs) and endothelial cell dysfunction.MethodsUsing a cell model of ALI, the role of P2Y12R was investigated in LPS‐induced HPMVECs. The expression of P2Y12R was detected by RT‐qPCR and Western blot analysis assay and TNF‐α, IL‐1β, and IL‐6 levels were analyzed by RT‐qPCR. NO levels were also analyzed through NO kit. The levels of NF‐κB p65, P‐IκB‐α, and IκB‐α, as well as p‐AKT and eNOS levels were detected by Western blot analysis assay. Wound healing assay was performed to evaluate HPMVECs migration. FITC‐dextran was used to evaluate endothelial cell permeability, and the analysis of adherens junction protein VE‐cadherin and endothelial cell tight junction proteins ZO‐1, Claudin 5 and Occludin expression was performed by RT‐qPCR and Western blot analysis assay.ResultsIn vitro, LPS increased the expression levels of P2Y12R and pro‐inflammatory mediators (TNF‐α, IL‐1β, and IL‐6), followed by a decrease in HPMVECs migration. In addition, LPS led to an increase in endothelial cell permeability. P2Y12R antagonists Ticagrelor or clopidogrel treatment significantly reversed these effects of LPS.ConclusionThe inhibitor of P2Y12R was able to decrease inflammatory response, promote migration and improve endothelial cell function and permeability, suggesting a key role of P2Y12R in ALI.