Pretreatment Of Endothelial Cells Research Articles

Antithrombin-III mitigates thrombin-mediated endothelial cell contraction and sickle red blood cell adhesion in microscale flow.

SummaryIndividuals with sickle cell disease (SCD) have persistently elevated thrombin generation that results in a state of systemic hypercoagulability. Antithrombin‐III (ATIII), an endogenous serine protease inhibitor, inhibits several enzymes in the coagulation cascade, including thrombin. Here, we utilize a biomimetic microfluidic device to model the morphology and adhesive properties of endothelial cells (ECs) activated by thrombin and examine the efficacy of ATIII in mitigating the adhesion of SCD patient‐derived red blood cells (RBCs) and EC retraction. Microfluidic devices were fabricated, seeded with ECs, and incubated under physiological shear stress. Cells were then activated with thrombin with or without an ATIII pretreatment. Blood samples from subjects with normal haemoglobin (HbAA) and subjects with homozygous SCD (HbSS) were used to examine RBC adhesion to ECs. Endothelial cell surface adhesion molecule expression and confluency in response to thrombin and ATIII treatments were also evaluated. We found that ATIII pretreatment of ECs reduced HbSS RBC adhesion to thrombin‐activated endothelium. Furthermore, ATIII mitigated cellular contraction and reduced surface expression of von Willebrand factor and vascular cell adhesion molecule‐1 (VCAM‐1) mediated by thrombin. Our findings suggest that, by attenuating thrombin‐mediated EC damage and RBC adhesion to endothelium, ATIII may alleviate the thromboinflammatory manifestations of SCD.

Inhibition of mevalonate metabolism by statins augments the immunoregulatory phenotype of vascular endothelial cells and inhibits the costimulation of CD4+ T cells

The statin family of therapeutics is widely used clinically as cholesterol lowering agents, and their effects to target intracellular mevalonate production is a key mechanism of action. In this study, we performed full transcriptomic RNA sequencing and qPCR to evaluate the effects of mevalonate on the immunoregulatory phenotype of endothelial cells (EC). We find that mevalonate-dependent gene regulation includes a reduction in the expression of multiple pro-inflammatory genes including TNFSF4 (OX40-L) and TNFSF18 (GITR-L) and a co-incident induction of immunoregulatory genes including LGALS3 (Galectin-3) and LGALS9 (Galectin-9). In functional assays, pretreatment of EC with simvastatin to inhibit mevalonate metabolism resulted in a dose-dependent reduction in the costimulation of CD45RO+ CD4+ T cell proliferation as well as IL-2, IFNγ and IL-6 production versus vehicle-treated EC. In contrast, pre-treatment of EC with L-mevalonate in combination with simvastatin reversed phenotypic and functional responses. Collectively, these results indicate that relative mevalonate metabolism by EC is critical to sustain EC-dependent mechanisms of immunity. Our findings have broad relevance for the repurposing of statins as therapeutics to augment immunoregulation and/or to inhibit local tissue pro-inflammatory cytokine production following transplantation.

Abstract P260: SARS-Cov-2 Spike Protein S1-Mediated Endothelial Injury And Pro-Inflammatory State Is Amplified By Dihydrotestosterone And Prevented By Mineralocorticoid Antagonism

Objectives: Men are disproportionately affected by the coronavirus disease-2019 (COVID-19) and experience higher mortality as compared to women. Endothelial dysfunction has been proposed as a major inciting factor of pro-inflammatory and thrombotic changes in COVID-19 infection. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial damage are not defined. Methods: Here, we determined the effects of SARS-CoV-2 spike protein-mediated endothelial damage under conditions of exposure to androgens and TNF-α. We tested the therapeutic effects of approved drugs: angiotensin receptor blockade by valsartan and mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in the plasma from men and women diagnosed with COVID-19. Results: Exposure of endothelial cells (ECs) in vitro to androgen dihydrotestosterone (DHT) exacerbated spike protein S1-mediated endothelial injury transcripts for cell adhesion molecules E-selectin, VCAM-1, and ICAM-1 and anti-fibrinolytic PAI-1 (P<0.05), as well as increased THP-1 monocyte adhesion to ECs (P=0.032). Spironolactone dramatically reduced DHT+S1-induced endothelial activation. Pre-treatment of ECs with valsartan did not increase endothelial activation by S1. TNF-α exacerbated S1-induced EC activation and spironolactone abrogated this effect. Analysis of plasma from patients hospitalized with COVID-19 showed concordant higher VCAM-1 in men as compared to women. Conclusions: Androgen exposure promoted spike protein-mediated endothelial injury by increasing markers of inflammation and thrombosis. A beneficial therapeutic effect of the FDA-approved drug spironolactone was observed, supporting a rationale for further evaluation as an adjunct treatment in COVID-19.

SARS-CoV-2 Spike Protein S1-Mediated Endothelial Injury and Pro-Inflammatory State Is Amplified by Dihydrotestosterone and Prevented by Mineralocorticoid Antagonism

Background: Men are disproportionately affected by the coronavirus disease-2019 (COVID-19) and experience higher mortality as compared to women. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial damage are not defined.Methods: Here, we determined the effects of SARS-CoV-2 spike protein-mediated endothelial damage under conditions of exposure to androgens and TNF-α. We tested the therapeutic effects of approved drugs: angiotensin receptor blockade by valsartan and mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in the plasma from men and women diagnosed with COVID-19.Findings: Exposure of endothelial cells (ECs) in vitro to androgen dihydrotestosterone (DHT) exacerbated spike protein S1-mediated endothelial injury transcripts for cell adhesion molecules E-selectin, VCAM-1, and ICAM-1 and anti-fibrinolytic PAI-1 (PInterpretation: Androgen exposure promotes spike protein-mediated endothelial injury by increasing markers of inflammation and thrombosis. A beneficial therapeutic effect of the FDA-approved drug spironolactone was observed, supporting a rationale for further evaluation as an adjunct treatment in COVID-19. Funding: This work was supported by the Michigan Biological Research Initiative on Sex Differences in Cardiovascular Disease, COVID-19 Ignitor Award, and University of Michigan Taubman Medical Research Institute.Declaration of Interests: No conflicts of interest, financial or otherwise, are declared by the author(s).Ethics Approval Statement: Study involving measurement of circulating endothelial injury markers in the plasma of COVID-19 patients was approved by the Institutional Review Board (HUM00180521) at the University of Michigan.

Defining an Upstream VEGF (Vascular Endothelial Growth Factor) Priming Signature for Downstream Factor-Induced Endothelial Cell-Pericyte Tube Network Coassembly.

In this work, we have sought to define growth factor requirements and the signaling basis for different stages of human vascular morphogenesis and maturation. Approach and Results: Using a serum-free model of endothelial cell (EC) tube morphogenesis in 3-dimensional collagen matrices that depends on a 5 growth factor combination, SCF (stem cell factor), IL (interleukin)-3, SDF (stromal-derived factor)-1α, FGF (fibroblast growth factor)-2, and insulin (factors), we demonstrate that VEGF (vascular endothelial growth factor) pretreatment of ECs for 8 hours (ie, VEGF priming) leads to marked increases in the EC response to the factors which includes; EC tip cells, EC tubulogenesis, pericyte recruitment and proliferation, and basement membrane deposition. VEGF priming requires VEGFR2, and the effect of VEGFR2 is selective to the priming response and does not affect factor-dependent tubulogenesis in the absence of priming. Key molecule and signaling requirements for VEGF priming include RhoA, Rock1 (Rho-kinase), PKCα (protein kinase C α), and PKD2 (protein kinase D2). siRNA suppression or pharmacological blockade of these molecules and signaling pathways interfere with the ability of VEGF to act as an upstream primer of downstream factor-dependent EC tube formation as well as pericyte recruitment. VEGF priming was also associated with the formation of actin stress fibers, activation of focal adhesion components, upregulation of the EC factor receptors, c-Kit, IL-3Rα, and CXCR4 (C-X-C chemokine receptor type 4), and upregulation of EC-derived PDGF (platelet-derived growth factor)-BB, PDGF-DD, and HB-EGF (heparin-binding epidermal growth factor) which collectively affect pericyte recruitment and proliferation. Overall, this study defines a signaling signature for a separable upstream VEGF priming step, which can activate ECs to respond to downstream factors that are necessary to form branching tube networks with associated mural cells.

A new role for host annexin A2 in establishing bacterial adhesion to vascular endothelial cells: lines of evidence from atomic force microscopy and an in vivo study

Understanding bacterial adhesion is challenging and critical to our understanding of the initial stages of the pathogenesis of endovascular bacterial infections. The vascular endothelial cell (EC) is the main target of Rickettsia, an obligately intracellular bacterium that causes serious systemic disease in humans and animals. But the mechanism(s) underlying bacterial adherence to ECs under shear stress from flowing blood prior to activation are unknown for any bacteria. Although host surface annexin a2 (ANXA2) has been identified to participate in efficient bacterial invasion of epithelial cells, direct evidence is lacking in the field of bacterial infections of ECs. In the present study, we employ a novel, anatomically based, in vivo quantitative bacterial-adhesion-to-vascular-EC system, combined with atomic force microscopy (AFM), to examine the role of endothelial luminal surface ANXA2 during rickettsial adherence to ECs. We also examined whether ANXA2 antibody affected binding of Staphylococcus aureus to ECs. We found that deletion of ANXA2 impeded rickettsial attachment to the ECs in vitro and blocked rickettsial adherence to the blood vessel luminal surface in vivo. The AFM studies established that EC surface ANXA2 acts as an adherence receptor for rickettsiae, and that rickettsial adhesin OmpB is the associated bacterial ligand. Furthermore, pretreatment of ECs with anti-ANXA2 antibody reduced EC surface-associated S. aureus. We conclude that the endothelial surface ANXA2 plays an important role in initiating pathogen–host interactions, ultimately leading to bacterial anchoring on the vascular luminal surface.

A New Role for Host Annexin A2 in Establishing Bacterial Adhesion to Vascular Endothelial Cell: Lines of Evidence from in vivo Study and Micro Force Spectroscopy

Background: Understanding bacterial adhesion is challenging and critical to our understanding of the initial stages of the pathogenesis of endovascular bacterial infections. The vascular endothelial cell (EC) is the main target of Rickettsia, an obligately intracellular bacteria that causes serious systemic disease in humans and animals. But the mechanism(s) underlying bacterial adherence to ECs under shear stress from flowing blood prior to activation are unknown for any bacteria. Although host surface annexin a2 (ANXA2) has been identified to participate in efficient bacterial invasion of epithelial cells, direct evidence are completely lacking in the field of bacterial infections of ECs. Methods: In the present study, we employ a novel, anatomically-based, in vivo quantitative bacterial-adhesion-to-vascular-EC system, combined with atomic force microscopy (AFM), to examine the role of endothelial luminal surface ANXA2 during rickettsial adherence to ECs. We also examined whether ANXA2 antibody affected binding of Staphylococcus aureus to ECs. Findings: We found that deletion of ANXA2 impeded rickettsial attachment to the ECs in vitro and blocked rickettsial adherence to the blood vessel luminal surface in vivo. The AFM studies established that EC surface ANXA2 acts as an adherence receptor for rickettsiae, and that rickettsial adhesin OmpB is the associated bacterial ligand. Furthermore, pretreatment of ECs with anti-ANXA2 antibody reduced EC surface-associated S. aureus. Interpretation: The endothelial surface ANXA2 plays an important role in initiating pathogen-host interactions, ultimately leading to bacterial anchoring on the vascular luminal surface. Funding: This work was supported by NIH (B.G.) and Natural Science Foundation of China (F.L.). Declaration of Interest: The authors declare that they have no conflicts of interest. Ethical Approval: All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch (UTMB).

Microtubule destabilization caused by particulate matter contributes to lung endothelial barrier dysfunction and inflammation

Exposure to particulate matter (PM) associated with air pollution remains a major public health concern, as it has been linked to significant increase in cardiopulmonary morbidity and mortality. Lung endothelial cell (EC) dysfunction is one of the hallmarks of cardiovascular events of lung exposure to PM. However, the role of PM in acute lung injury (ALI) exacerbation and delayed recovery remains incompletely understood. This study tested a hypothesis that PM augments lung injury and EC barrier dysfunction via microtubule-dependent mechanisms. Our data demonstrate that in pulmonary EC PM caused time- and dose-dependent remodeling of actin cytoskeleton and considerable destabilization of the microtubule (MT) network. These events led to the weakening of cell junctions and formation of actin stress fibers, resulting in disruption of lung EC monolayer and increased permeability. PM also caused ROS-dependent activation of MT-specific deacetylase, HDAC6. Suppression of HDAC6 activity by pharmacological inhibitors or siRNA-based depletion of HDAC6 abolished PM-induced EC permeability increase, which was accompanied by reduced activation of stress kinase signaling, inhibition of Rho cascade, decreased IL-6 production and suppressed activation of its downstream target STAT3. Pretreatment of pulmonary EC with IL-6 inhibitor led to inhibition of STAT3 activity and decreased PM-induced hyper-permeability. Because one of the major activators of Rho-GTPase, GEFH1, is localized on the MT, we examined its involvement in PM-caused EC barrier compromise. Inhibition of GEF-H1 activation significantly attenuated PM-induced permeability increase. Moreover, combined inhibition of IL-6 and GEF-H1 signaling exhibited additive protective effect. Taken together, these results demonstrate a critical involvement of MT-associated signaling in the PM-induced exacerbation of lung EC barrier compromise and inflammatory response.

Piceatannol attenuates homocysteine-induced endoplasmic reticulum stress and endothelial cell damage via heme oxygenase-1 expression

A growing body of evidence implicates endoplasmic reticulum (ER)-induced cellular dysfunction and apoptosis as important factors to a variety of diseases. In endothelial cells (ECs), the sulfur-containing amino acid homocysteine (Hcy) causes EC apoptosis and reactive oxygen species (ROS) generation through induction of ER stress. Here, we have investigated whether piceatannol (Pic), a resveratrol analog, could protect ECs against Hcy-induced apoptosis, oxidative stress and ER stress, with specific emphasis on heme oxygenase-1 (HO-1). In human ECs, we determined the effects of Hcy and Pic on annexin V positivity, glucose-regulated protein 78kDa (GRP78) and C/EBP homologous protein (CHOP) expression, X-box binding protein 1 (Xbp-1) mRNA slicing, and ROS-sensitive dihydroethidium (DHE) oxidation. Hcy increased annexin V-positive cells, DHE oxidation, GRP78 and CHOP expression and Xbp-1 mRNA splicing, indicating that Hcy induces apoptosis, oxidative stress and ER stress. Pretreatment of ECs with Pic significantly inhibited Hcy-induced apoptosis, ROS generation and ER stress. Pic also increased HO-1 expression via activation of nuclear factor-E2-related factor 2 (Nrf2). Interestingly, the inhibitory effects of Pic on Hcy-induced apoptosis, ROS generation and ER stress were abolished by down-regulation of HO-1 expression, while mimicked by treatment of ECs with the HO-1 inducer hemin. Overall, these results suggest that Pic may protect ECs against Hcy-induced apoptosis, oxidative stress and ER stress via Nrf2-dependent HO-1 expression.

Anti-ß2GPI Antibodies Induce Oxidative and Nitrative Stress in Endothelial Cells through Activation of Discrete Nox Isoforms and eNOS Uncoupling

IntroductionAntiphospholipid syndrome (APS) is characterized by thrombosis and recurrent fetal loss in patients with circulating antiphospholipid antibodies (APLAs). These antibodies react with β2 glycoprotein I (β2 GPI) on cell surfaces leading to cellular activation and dysfunction. Previous studies have shown that oxidative stress is associated with the pathophysiology of APS and that NADPH oxidase (NOX)-derived ROS act as intermediates in signaling pathways leading to cellular activation. However, detailed mechanisms and isoforms of NOX involved in ROS generation in response to anti-β2GPI antibodies and their contribution to the pathophysiology of APS have not been clearly delineated. In the present study we explored the role of NOX isoforms in endothelial cell (EC) activation and ROS generation in response to anti-β2GPI antibodies.MethodsControl IgG was isolated using protein A/G, and anti-β2GPI antibodies were affinity-purified from sera of patients with APS or rabbits immunized with β2GPI. EC were treated with β2GPI and either control IgG or anti-β2GPI antibodies in the presence or absence of the NOX inhibitor diapocynin, or following treatment with siRNA specific for NOX1, NOX2, or NOX4. Oxidative stress was induced in EC by treating cells with 250 µM H2O2. Activation of EC was assessed by measuring E-selectin expression on the cell surface, or levels of mRNA encoding E-selectin and other markers of cellular activation. Fluorescent dyes (CM-H2DCFDA and CellROX Deep Red) or chemiluminescent substrates were used to measure ROS generation. Cell surface proteins were labeled with EZ-Link Sulfo-NHS-Biotin and separated from total cell lysate using Streptavidin Plus Ultralink resin. Protein expression of NOX1, NOX2, NOX4, ANXA2, 3-nitrotyrosine, and thioredoxin reductase 1 (TrxR1) were analyzed by western blot. Detection of eNOS monomers and dimers was performed using cold SDS-PAGE and immunblot. Immunoprecipitation of TrxR1 was performed using protein A/G agarose beads.ResultsROS generated by EC in response to β2GPI and anti-β2GPI antibodies but not control IgG were detected both intracellularly and in conditioned medium. ROS generation was correlated with significantly increased expression of NOX2 but not NOX1 or NOX4 in EC exposed to β2GPI and anti-β2GPI antibodies. Consistently, pre-treatment of EC with diapocynin, a NOX inhibitor, inhibited EC activation by anti-β2GPI antibodies, as did siRNA knockdown of NOX2 and NOX4; however, activation was not blocked by NOX1 siRNA. Exposure of EC to anti-β2GPI antibodies increased the eNOS monomer/dimer ratio, suggesting eNOS uncoupling, and also increased 3-nitrotyrosine expression, suggesting increased production of peroxynitrite leading to nitrative stress. Compared to control IgG, Immunoprecipitation with TrxR1 pulled down more 3-nitrotyrosine protein suggesting nitration of TrxR1 tyrosine residue which leads to loss of its function.Conclusionsβ2GPI and anti-β2GPI antibodies stimulate ROS generation in EC, primarily through NOX2. Anti-β2GPI antibodies also induce nitrative stress and eNOS uncoupling. eNOS uncoupling is promoted by nitration and inactivation of TrxR1, and results in superoxide generation. Taken together these studies suggest that anti-β2GPI antibodies induce oxidative and nitrative stress in EC, and suggest new targets for directed therapy of APS. DisclosuresNo relevant conflicts of interest to declare.

Targeting non-canonical nuclear factor-κB signalling attenuates neovascularization in a novel 3D model of rheumatoid arthritis synovial angiogenesis.

Angiogenesis is crucial in RA disease progression. Lymphotoxin β receptor (LTβR)-induced activation of the non-canonical nuclear factor-κB (NF-κB) pathway via NF-κB-inducing kinase (NIK) has been implicated in this process. Consequently, inhibition of this pathway may hold therapeutic potential in RA. We describe a novel three-dimensional (3D) model of synovial angiogenesis incorporating endothelial cells (ECs), RA fibroblast-like synoviocytes (RAFLSs) and RA synovial fluid (RASF) to further investigate the contributions of NF-κB in this process. Spheroids consisting of RAFLSs and ECs were stimulated with RASF, the LTβR ligands LTβ and LIGHT, or growth factor bFGF and VEGF, followed by quantification of EC sprouting using confocal microscopy and digital image analysis. Next, the effects of anginex, NIK-targeting siRNA (siNIK), LTβR-Ig fusion protein (baminercept) and a novel pharmacological NIK inhibitor were investigated. RASF significantly promoted sprout formation, which was blocked by the established angiogenesis inhibitor anginex (P < 0.05). LTβ and LIGHT induced significant sprouting (P < 0.05), as did bFGF/VEGF (P < 0.01). siNIK pre-treatment of ECs led to reductions in LTβR-induced vessel formation (P < 0.05). LTβR-Ig not only blocked LTβ- or LIGHT-induced sprouting, but also RASF-induced sprouting (P < 0.05). The NIK inhibitor blocked angiogenesis induced by LTβ, LIGHT, growth factors (P < 0.05) and RASF (P < 0.01). We present a novel 3D model of synovial angiogenesis incorporating RAFLSs, ECs and RASF that mimics the in vivo situation. Using this system, we demonstrate that non-canonical NF-κB signalling promotes neovascularization and show that this model is useful for dissecting relative contributions of signalling pathways in specific cell types to angiogenic responses and for testing pharmacological inhibitors of angiogenesis.

OR13 Mitogen-activated protein kinase P38 and C-JUN NH2-terminal kinase regulate hla class II antibody-stimulated activation of human aortic endothelial cells

Aim Transplant recipients developing donor specific HLA antibodies (DSA) are at a higher risk for acute and chronic antibody (Ab) mediated rejection (AMR). HLA Ab contribute to the process of AMR by binding to the HLA molecules on endothelial cells (EC) and triggering signal transduction pathways leading to EC activation, adhesion molecule expression, proliferation and migration. DSA against HLA class I appear earlier, while class II DSA tends to prevail late post transplantation and associate with chronic graft dysfunction. Ligation of HLA-I with Ab on EC and smooth muscle cells have been shown to contribute to the process of transplant vasculopathy. However, the mechanism(s) underlying how HLA-II Ab contribute to chronic rejection remain unknown. Method Primary human aortic EC were infected with recombinant adenovirus pAd/PL-DEST encoding CIITA (Ad-CIITA) an HLA-II transactivator, or pretreated with TNF-alpha/IFN-gamma. HLA-II expression was determined by flow cytometry. Ad-CIITA infected or cytokine-treated EC were stimulated with anti-HLA-II Ab, phosphorylation was detected by Western Blot, EC proliferation was measured by BrdU incorporation and analyzed by flow cytometry. Results Infection of EC with Ad-CIITA or pretreatment of EC with TNF-alpha and IFN-gamma induced a marked increase in HLA-II antigen expression and CIITA protein presentation. Ligation of HLA-II antigens on Ad-CIITA infected EC or TNF-alpha/IFN-gamma pretreated EC stimulated a significant increase in phosphorylation of MAPK p38 and JNK. Ligation of HLA on Ad-CIITA infected EC or TNF-alpha/IFN-gamma pretreated EC with class II mAb also stimulated cell proliferation. Pretreatment of class II positive EC with pharmacological inhibitors of p38 (SB203580) or JNK (SP600125) or siRNA inhibited HLA-II Ab-stimulated EC proliferation. Conclusions In this study we found that Ad-CIITA infection or TNF-alpha/IFN-gamma treatment can induce HLA-II expression on EC. Ligation of class II with Ab on EC triggers intracellular signal transduction promoting cell proliferation via a MAPK dependent pathway. Our results provide information on novel mechanisms of HLA-II Ab-mediated activation of EC. Disrupting activation of MAPK p38 or JNK may provide new therapeutic targets to prevent AMR.

Heparin Decreases in Tumor Necrosis Factor α (TNFα)-induced Endothelial Stress Responses Require Transmembrane Protein 184A and Induction of Dual Specificity Phosphatase 1

Despite the large number of heparin and heparan sulfate binding proteins, the molecular mechanism(s) by which heparin alters vascular cell physiology is not well understood. Studies with vascular smooth muscle cells (VSMCs) indicate a role for induction of dual specificity phosphatase 1 (DUSP1) that decreases ERK activity and results in decreased cell proliferation, which depends on specific heparin binding. The hypothesis that unfractionated heparin functions to decrease inflammatory signal transduction in endothelial cells (ECs) through heparin-induced expression of DUSP1 was tested. In addition, the expectation that the heparin response includes a decrease in cytokine-induced cytoskeletal changes was examined. Heparin pretreatment of ECs resulted in decreased TNFα-induced JNK and p38 activity and downstream target phosphorylation, as identified through Western blotting and immunofluorescence microscopy. Through knockdown strategies, the importance of heparin-induced DUSP1 expression in these effects was confirmed. Quantitative fluorescence microscopy indicated that heparin treatment of ECs reduced TNFα-induced increases in stress fibers. Monoclonal antibodies that mimic heparin-induced changes in VSMCs were employed to support the hypothesis that heparin was functioning through interactions with a receptor. Knockdown of transmembrane protein 184A (TMEM184A) confirmed its involvement in heparin-induced signaling as seen in VSMCs. Therefore, TMEM184A functions as a heparin receptor and mediates anti-inflammatory responses of ECs involving decreased JNK and p38 activity.

Pathophysiological Concepts in Multiple Sclerosis and the Therapeutic Effects of Hydrogen Sulfide

Introduction:Multiple sclerosis (MS) is generally known as a manageable but not yet curable autoimmune disease affecting central nervous system. A potential therapeutic approach should possess several properties: Prevent immune system from damaging the brain and spinal cord, promote differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes to produce myelin, prevent the formation of fibronectin aggregates by astrocytes to inhibit scar formation, and enhance function of healthy endothelial cells (ECs).Methods:To determine if an increase in sulfur contents through H2S, a potent antioxidant known to induce protective autophagy in cells, could provide the above desired outcomes, peripheral blood mononuclear cells (PBMNCs), OCPs, astrocytes, and ECs were treated with NaHS (50 μM) in vitro.Results:Transmigration assay using EC monolayer showed that serotonin increased migration of PBMNC while pretreatment of EC with NaHS inhibited the migration induced by serotonin treatment. NaHS upregulated proteins involved in immune system response and downregulated PBMNCs- and EC-related adhesion molecules (LFA-1 and VCAM-1). Furthermore, it had a cell expansion inducing effect, altering EC morphology. The effects of NaHS on OPCs and astrocytes were studied compared to mTOR inhibitor rapamycin. In NaHS treated astrocytes the induced fibronectin production was partially inhibited while rapamycin almost fully inhibited fibronectin production. NaHS slowed but did not inhibit the differentiation of OCPs or the production of myelin compared to rapamycin.Conclusion:The in vitro results point to the potential therapeutic application of hydrogen sulfide releasing molecules or health-promoting sulfur compounds in MS.

Preconditioning with Endoplasmic Reticulum Stress Ameliorates Endothelial Cell Inflammation

Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability.

OR56: PROTEIN TYROSINE KINASES SRC AND PI3K REGULATE HLA-II ANTIBODY-INDUCED SURVIVAL PROTEIN EXPRESSION IN ENDOTHELIAL CELLS

Antibody-mediated rejection (AMR) is caused by the development of donor-specific antibodies against polymorphic HLA molecules expressed by the transplanted organ. AMR is a sequential process leading to arterial and parenchymal damage, and ultimately transplant vasculopathy (TV) and graft dysfunction. Endothelial cell (EC) apoptosis followed by vascular repair and remodeling contribute to chronic rejection and development of TV. The pro-survival/anti-apoptotic molecules expressed by EC are members of the Bcl-2 protein family including Bcl-2 and Bcl-xL. The aim of this study was to determine whether HLA-II Ab triggers anti-apoptotic protein expression in EC. HLA-II expression was induced in primary human aortic EC by recombinant adenovirus pAd/PL-DEST encoding CIITA (Ad-CIITA), HLA-II transactivator, or treatment with TNFα and IFNγ. HLA-II expression was determined by flow cytometry, phosphorylation of protein kinase and expression of Bcl-2 and Bcl-xL was measured by Western Blot. Infection of EC with Ad-CIITA or pretreatment of EC with cytokines induced a marked increase in HLA-II expression, including DP, DQ, and DR. Ligation of HLA-II antigens on Ad-CIITA infected or cytokine pretreated EC stimulated a significant increase in phosphorylation of Src, PI3K and activation of mTORC1 pathway including increased phosphorylation of S6K and S6RP. Class II ligation also triggered phosphorylation of the mTORC2-targets Akt and ERK. To determine if class II-mediated mTORC2 activation regulates anti-apoptotic protein function we studied the effect of HLA-II ligation on Bcl-2 and Bcl-xL expression. Stimulation of EC with HLA-II Ab increased Bcl-2 and Bcl-xL expression at 24 h which was completely inhibited by pretreatment with the pharmacological inhibitors rapamycin and KU targeting mTORC2. Pretreatment of EC with the MEK inhibitor UO126 blocked HLA-II Ab-induced BclxL expression, but not Bcl-2 expression indicating ERK is upstream of Bcl-xL but not Bcl-2. Ligation of HLA-II with Ab on EC triggered intracellular signal cascades promoting cell survival via two distinct pathways. These studies support a role for HLA class II-mediated pro-survival signaling in the setting of chronic rejection in which antibodies promote endothelial cell survival contributing to TV.

Microtubule dynamics control HGF-induced lung endothelial barrier enhancement.

Microtubules (MT) play a vital role in many cellular functions, but their role in peripheral actin cytoskeletal dynamics which is essential for control of endothelial barrier and monolayer integrity is less understood. We have previously described the enhancement of lung endothelial cell (EC) barrier by hepatocyte growth factor (HGF) which was associated with Rac1-mediated remodeling of actin cytoskeleton. This study investigated involvement of MT-dependent mechanisms in the HGF-induced enhancement of EC barrier. HGF-induced Rac1 activation was accompanied by phosphorylation of stathmin, a regulator of MT dynamics. HGF also stimulated MT peripheral growth monitored by time lapse imaging and tracking analysis of EB-1-decorated MT growing tips, and increased the pool of acetylated tubulin. These effects were abolished by EC pretreatment with HGF receptor inhibitor, downregulation of Rac1 pathway, or by expression of a stathmin-S63A phosphorylation deficient mutant. Expression of stathmin-S63A abolished the HGF protective effects against thrombin-induced activation of RhoA cascade, permeability increase, and EC barrier dysfunction. These results demonstrate a novel MT-dependent mechanism of HGF-induced EC barrier regulation via Rac1/PAK1/stathmin-dependent control of MT dynamics.

Monocyte Recruitment to HLA Class I Antibody-Activated Endothelial Cells Is Dependent Upon mTOR.

Aim: A hallmark of antibody (Ab)-mediated rejection is the presence of intravascular monocytes in the allograft. The purpose of this study was to identify the role of the mTOR signaling pathway in HLA class I Ab-mediated monocyte recruitment to endothelial cells (EC). Methods: Static and flow-based (ibidi) adhesion assays were used to assess monocyte (MM6) recruitment to HLA-activated primary human aortic endothelial cells. mTOR inhibition was characterized using pharmacological inhibitors (KU 0063794 and sirolimus) at different time points and doses in endothelial cells and monocytes. Results were verified using siRNA knock-down (ko) of rictor and raptor, components of the mTOR pathway. Cell-based ELISA was performed to quantitate Weibel-Palade Body exocytosis; protein phosphorylation was measured by Western blot and quantitated by densitometry. An established murine cardiac transplant model was used to determine the effect of sirolimus treatment on acute AMR. Briefly, Rag1 ko animals were transplanted with an H-2d mismatched heart transplant and received passive transfer of anti-donor Dd+Kd IgG antibodies. Results: Pretreatment of EC for 24 hours with sirolimus or Ku reduced monocyte adherence to EC by > 80%, whereas 2- hour mTOR inhibition resulted in partial inhibition of monocyte binding. siRNA ko of mTOR using siRNA yielded comparable results significantly decreasing monocyte adherence. siRNA against raptor and rictor revealed that both mTORC1 and mTORC2 are involved in HLA-mediated monocyte recruitment. Inhibition of mTOR had no effect on P-selectin induction. Instead, flow-based adhesion assays revealed that mTOR inhibition impairs the ability of EC to support firm adhesion of tethered monocytes by affecting late stage ICAM-1 clustering and its downstream signaling effects. mTOR inhibition in monocytes using sirolimus and Ku significantly impaired monocyte binding to activated endothelium. A reduction in endothelial damage and infiltrating mononuclear cells was observed in vivo in recipients of heart allografts treated with sirolimus. Conclusion: We found that mTOR blockade prevents monocyte recruitment to HLA-activated EC through mTORC1 and mTORC2 signaling pathways. In EC, mTOR inhibition prevents ICAM-1 clustering and blunts firm adhesion of monocytes. Our results provide novel insights into the mechanisms underlying monocyte recruitment in the setting of AMR. DISCLOSURE:Salehi, S.: Grant/Research Support, Pfizer. Reed, E.: Grant/Research Support, Pfizer.

Porphyromonas gingivalis-impaired innate immune response via NLRP3 proteolysis in endothelial cells

Porphyromonas gingivalis (Pg) is involved in the link between periodontal diseases and atherosclerosis worsening. In periodontal cells, Pg modifies IL-1β expression via the NLRP3 inflammasome pathway activation. Our aim was to investigate NLRP3 inflammasome activation in endothelial cells (ECs) after Pg infection and Pg-LPS stimulation. In both situations, RT-PCR experiments demonstrated an increase of the NLRP3 mRNA level that can be potentiated by pre-treatment of ECs with 5 mM ATP. However, Western blotting analysis revealed that Pg infection induced a proteolysis of NLRP3 protein and a major decrease of the native protein. After ATP pre-treatment and/or Pg-LPS stimulation, this proteolysis was not observed, while NLRP3 protein levels were increased. Proteolysis of the NLRP3 protein was not observed with heat-killed Pg and inhibition of ECs protein synthesis with cycloheximide did not abolish the NLRP3 protein degradation induced by Pg infection in ATP pre-treated cells. Additionally, significant increases of secreted IL-1β were measured after ATP pre-treatment and/or Pg-LPS stimulation, but not after Pg infection. These data showed that Pg and Pg-LPS differentially controlled the NLRP3 inflammasome pathway in ECs, and suggested a novel potential mechanism developed by Pg to reduce IL-1β secretion and to escape host immune response.

AT1 receptor blocker potentiates shear-stress induced nitric oxide production via modulation of eNOS phosphorylation of residues Thr495 and Ser1177

We tested the hypothesis that AT1R blockade modulates the shear stress-induced (SS) synthesis of nitric oxide (NO) in endothelial cells (EC). The AT1R blocker Candesartan in the absence of the ligand angiotensin II (ang II) potentiated SS-induced NO synthesis accompanied by increased p-eNOSSer1177 and decreased p-eNOSThr495. Candesartan also inhibited SS-induced ERK activation and increased intracellular calcium transient in a time-dependent manner. To confirm the role of ERK to modulate p-eNOSThr495 and calcium to modulate p-eNOSSer1177, the MEK inhibitor U0126 and the calcium chelator BAPTA-AM were used, respectively. Pre-treatment of EC with U0126 completed abrogated basal and SS-induced ERK activation, inhibited p-eNOSThr495 and increased NO production by SS. On the other hand, pre-treatment of EC with BAPTA-AM decreased the effects of SS alone or in combination with Candesartan to induce p-eNOSSer1177 and partially inhibited the effects of Candesartan to potentiate NO release by SS. The AT1R blockers Losartan and Telmisartan were also tested but only Telmisartan potentiated NO synthesis and blocked SS-induced AT1R activation. Altogether, we provide evidence that Candesartan and Telmisartan potentiate SS-induced NO production even in the absence of the ligand ang II. This response requires both the inhibition of eNOS phosphorylation at its inhibitory residue Thr495 as well as the increase of eNOS phosphorylation at its excitatory residue Ser1177. In addition, the response is associated with inhibition of SS-induced ERK activation as well as increasing intracellular calcium transient. One may speculate that these yet undescribed events may contribute to the benefits of ARBs in cardiovascular diseases.