Toll like Receptor 4 Is Involved in In VIvo Antiphospholipid-Mediated Thrombosis and Endothelial Cell Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 133-133
Author(s):  
Silvia S. Pierangeli ◽  
Mariano E. Vega-Ostertag ◽  
Elena Raschi ◽  
Xiaowei Liu ◽  
Maria O. Borghi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Antiphospholipid Syndrome ◽  
Cell Activation ◽  
Spontaneous Mutation ◽  
Toll Like Receptor ◽  
Endothelial Cell Activation ◽  
Toll Like Receptor 4 ◽  
Control Serum

Abstract Background: Antiphospholipid antibodies (aPL) are associated with thrombosis and pregnancy loss in patients with Antiphospholipid Syndrome (APS). aPL and bacterial lipopolysaccharide (LPS) bind to and activate endothelial cells (EC) through NFκB and p38 MAPK pathways. Recent studies suggest that aPL might interact with toll-like receptor-4 (TLR-4), the receptor for LPS. Aim: to investigate the role of TLR-4 in antiphospholipid syndrome (APS). Methods: we examined: i) the aPL effects on thrombosis and EC activation in LPS non-responsive (LPS−/−) mice that display a spontaneous mutation of TLR-4 vs LPS-responsive (LPS+/+) mice displaying wild type TLR-4, ii) the prevalence of TLR-4 Asp299gly and Thr399Ile polymorphisms - both associated with decreased response to LPS - in 110 APS patients (with arterial and/or venous thrombosis) vs 220 controls (of same ethnic background). IgGs were purified from a patient with APS (IgG-APS) and from control serum (IgG-NHS). LPS −/− and LPS +/+ mice, in groups of nine, were treated with IgG-APS or with IgG-NHS twice intraperitoneally. Size of induced thrombi and # of leukocyte (WBC) adhering to endothelial cells in the microcirculation of endothelium of the cremaster muscle of mice (as a means to measure endothelial cell activation) were determined in vivo, seventy-two hours after the first injection. TLR-4 Asp299gly & Thr399Ile polymorphisms were evaluated by Allele-Specific PCR. Results: LPS +/+ mice treated with IgG-APS produced significantly larger thrombi when compared to mice treated with IgG-NHS (2166 ± 1419 μm2 vs 1176 ± 841 μm2) and significantly larger number of WBC adherence to ECs (4.5 ± 1.9 vs 2.2 ±1.1). Thrombus size and number of adhering WBC to ECs were significantly lower in LPS −/− mice treated with IgG-APS compared to LPS +/+ mice treated with IgG-APS [thrombus size: 779 ± 628 μm2 vs 2166 ± 1419 μm2 (p<0.05) and number of adherent WBC to EC: 1.0 ± 0.5 vs 4.5 1.9 (p<0.05)], respectively. The titer of anticardiolipin antibodies in the sera of mice injected with aPL was 48.2 ± 17.1 GPL (for LPS −/− mice) and 50.8 ± 11.2 GPL (for LPS +/+ mice), respectively (NS). A significant reduction in TLR-4 Asp 299gly & Thr399Ile polymorphisms was observed in APS patients (5%) compared to controls (11.4%) (p<0.05). Conclusions: These findings strongly suggest that TLR-4 is involved in aPL interaction with endothelial cells and mediates their pathogenic effects.

Abstract MP27: Human Hypertension And Endothelial Cell Activation Promote The Formation And Activation Of Axl + Siglec-6 + Dendritic Cells Via Endothelial Release Of Growth Arrest Specific 6

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Justin P Van Beusecum ◽  
Natalia R Barbaro ◽  
Charles D Smart ◽  
David M Patrick ◽  
Cyndya A Shibao ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Dendritic Cells ◽  
Endothelial Cell ◽  
Cell Activation ◽  
Growth Arrest ◽  
Positive Association ◽  
Endothelial Cell Activation ◽  
Human Hypertension

We have shown that dendritic cells (DCs) from hypertensive mice convey hypertension when adoptively transferred to recipients. Recently a novel subset of DCs in humans that express Axl and Sigelc-6 + (AS DCs) have been identified which drive T cell proliferation and produce IL-1β, IL-6 and IL-23, consistent with DCs we have observed in hypertension. We hypothesized that AS cells are increased in hypertension and contribute to immune activation in this disease. We quantified circulating AS DCs by flow cytometry in normotensive (n=23) and hypertensive (n=11) subjects and found a more than 2-fold increase in circulating AS DCs in hypertensive compared to normotensive subjects (297 ± 73 vs. 108 ± 26/ml; p =0.0304). To investigate the mechanism by which AS DCs are formed in hypertension, we co-cultured human aortic endothelial cells (HAECs) undergoing either normotensive (5%) or hypertensive (10%) cyclical stretch for 48 hours with CD14 + monocytes from normotensive donors. Co-culture of monocytes with HAECs exposed to 10% stretch significantly increased AS DCs and AS DC IL-1β production when compared to 5% stretch alone as assessed by flow cytometry (21 ± 5 vs. 131 ± 32 IL-1β + AS DCs). Moreover, inhibition of Axl signaling with R248, completely abolished the production of IL-1β in AS DCs (34 ± 8 IL-1β + AS DCs). In additional experiments we found that 10% stretch caused a 50% increase in release of growth arrest 6 (GAS6), the ligand for Axl, from HAECs compared to 5% stretch. Treatment of human monocytes with GAS6 mimicked the effect of 10% stretch in promoting AS cell formation and IL-1β production. Based on the increased secretion of GAS6 from HAECs, we used a J-wire to harvest human endothelial cells from 23 additional volunteers to assess endothelial cell activation and GAS6 secretion in vivo. We found a positive association between pulse pressure and plasma GAS6 (R 2 =0.25, p =0.0079) and a striking positive association between GAS6 and ICAM-1 (R 2 =0.39, p =0.0012). These data show that secretion of GAS6 by an activated endothelial seems to promote the formation and activation of AS DCs. Thus, the interplay between endothelial-derived GAS6 and AS DCs seem to be an important mechanism in human hypertension and might be a novel therapeutic target for this disease.

scholarly journals Enhancing Antitumor Efficacy of Heavily Vascularized Tumors by RAMBO Virus through Decreased Tumor Endothelial Cell Activation

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 1040
Author(s):  
Mitra Nair ◽  
Maninder Khosla ◽  
Yoshihiro Otani ◽  
Margaret Yeh ◽  
Flora Park ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Solid Tumors ◽  
Cell Activation ◽  
Leukocyte Adhesion ◽  
Oncolytic Viruses ◽  
Endothelial Cell Activation ◽  
Microscopy Imaging ◽  
Activation Markers

Vascularization is a common pathology for many solid tumors, and therefore anti-angiogenic strategies are being investigated as a therapeutic target for treatment. Numerous studies are also being conducted regarding the effects of oncolytic viruses, including ImlygicTM, an FDA approved oncolytic herpes simplex virus-1 (oHSV) for the treatment of highly vascularized tumors such as Kaposi sarcoma (NCT04065152), and brain tumors. To our knowledge, the effects of combining oncolytic HSV with angiogenesis inhibition on endothelial cell activation has not been previously described. Here, we tested the effects of Rapid Antiangiogenesis Mediated By Oncolytic Virus (RAMBO), an oHSV which expresses a potent anti-angiogenic gene Vasculostatin on endothelial cell activation in heavily vascularized solid tumors. oHSV treatment induces endothelial cell activation, which inhibits virus propagation and oncolysis in adjacent tumor cells in vitro. Consistently, this was also observed in intravital imaging of intracranial tumor-bearing mice in vivo where infected tumor endothelial cells could efficiently clear the virus without cell lysis. Quantitative real-time PCR (Q-PCR), leukocyte adhesion assay, and fluorescent microscopy imaging data, however, revealed that RAMBO virus significantly decreased expression of endothelial cell activation markers and leukocyte adhesion, which in turn increased virus replication and cytotoxicity in endothelial cells. In vivo RAMBO treatment of subcutaneously implanted sarcoma tumors significantly reduced tumor growth in mice bearing sarcoma compared to rHSVQ. In addition, histological analysis of RAMBO-treated tumor tissues revealed large areas of necrosis and a statistically significant reduction in microvessel density (MVD). This study provides strong preclinical evidence of the therapeutic benefit for the use of RAMBO virus as a treatment option for highly vascularized tumors.

Monitoring Endothelial Cell Activation In Vivo during Thrombus Formation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 294-294
Author(s):  
Ben T. Atkinson ◽  
Prathima Nandivada ◽  
Bruce Furie ◽  
Barbara C. Furie
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Intracellular Calcium ◽  
Cell Activation ◽  
Thrombus Formation ◽  
Single Cells ◽  
Calcium Mobilization ◽  
Endothelial Cell Activation ◽  
Calcium Elevation

Abstract The endothelium serves as a metabolically active interface between the blood and underlying tissues and offers response to changes in its microenvironment. The endothelium is rapidly activated in response to stimuli associated with fluctuations in shear stress, physical trauma, oxidative stress, and thrombotic and inflammatory mediators. To study endothelial cell activation we have monitored calcium mobilization in vitro in cultured endothelial cells and also in situ in the living mouse, following vessel wall injury. The addition of exogenous ADP (10 μM) or thrombin (1 U/mL) to Human Umbilical Vein Endothelial Cells (HUVECs) loaded with the calcium sensitive dye, Fluo-4 AM, is followed by rapid elevation of intracellular calcium, with a sustained peak observed within 10 sec. To further investigate the ability of the endothelium to activate in response to mediators potentially localized in the microenvironment of the endothelium during thrombus formation, we investigated the ability of IL-6 (0.1 ng/mL), IL-8 (80 ng/mL), MCP-1 (15 ng/mL), NAP-2 (10 ng/mL) and SDF-α (60 ng/mL) to mobilize calcium in HUVECs. These potential agonists were selected based on their primary roles in inflammation and thrombosis, and proposed roles in atherosclerosis and tumor angiogenesis. SDF-α and NAP-2 rapidly mobilized calcium in HUVECs, with similar maximum responses, but NAP-2 initiated a more prolonged (4-fold longer) rise in intracellular calcium. IL-6, IL-8, and MCP-1 also mobilized calcium, but the rise in intracellular calcium was almost 4-fold lower than that observed with SDF-α and NAP-2. Comparable calcium mobilization occurs in HUVECs subjected to a single pulse of a nitrogen dye-tuned laser. In addition, targeting of single cells within a confluent culture of endothelial cells initiated calcium elevation in the targeted cell and was followed by a wave of calcium elevation in surrounding cells. To determine whether this endothelial cell activation, and more specifically the calcium elevation, occurs in vivo, we conducted experiments using the laser-induced model of thrombus formation to look for calcium elevation in the arteriolar endothelium in live mice. Calcium elevation was monitored by Fluo-4AM introduced intravenously into the mouse circulation via the jugular vein. Fluo-4AM is non-specific in its uptake among cell types, and endothelial cell uptake of the dye in the cremaster muscle microcirculation was observed. Upon laser-induced injury, calcium elevation in the endothelium could be monitored by intravital microscopy. The observed calcium elevation was rapid (within 30 s) and preceded detection of platelets in the developing thrombus. These results add to the evidence of a dynamic endothelium and demonstrate that the endothelium activates rapidly prior to thrombus formation in the laser induced thrombosis model.

Gas6 Regulates Thrombin-Induced of VCAM-1 Expression by a FoxO1 Dependent Mechanism

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5192-5192
Author(s):  
Richard Robins ◽  
Catherine A. Lemarie ◽  
Mark D. Blostein
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Venous Thrombosis ◽  
Cell Biology ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Vascular Dysfunction ◽  
Soluble Form ◽  
Endothelial Cell Activation

Abstract Abstract 5192 Forkhead proteins play a broad role in endothelial cell biology. These factors mediate cell adhesion to extracellular matrix, regulate the expression of pro-inflammatory and pro-thrombotic genes, and participate in cell repair, proliferation and apoptosis. FoxOs are known downstream targets of the PI3K/Akt signaling pathway. Phosphorylation of FoxO transcription factors results in their translocation from the nucleus to the cytoplasm, thereby inhibiting their transcriptional activity. It has recently been shown that the deletion of the three FoxO isoforms in endothelial cells protects mice from vascular dysfunction. Gas6, a member of the vitamin K-dependent family of proteins, has been shown to protect endothelial cells from apoptosis and promote endothelial cell activation in vivo. It has been shown that the expression of ICAM-1 and VCAM-1 were blunted in the absence of gas6. Interestingly, a role for VCAM-1 in the pathogenesis of venous thrombosis has been proposed. Elevated levels of the soluble form of VCAM-1 have been detected in the serum of patients with venous thrombosis. We previously demonstrated that the anti-apoptotic effect of gas6 was mediated partially through FoxO1, but overall, the signalling mechanisms occurring downstream of gas6 remain largely unknown. We hypothesize that gas6 promotes thrombin-induced VCAM-1 expression through the regulation of FoxO1 in endothelial cells. Western blot analysis demonstrated that thrombin induced time dependent phosphorylation of FoxO1 with a maximum at 30 minutes in WT (p<0. 05) but not in gas6 deficient (−/−) cells. In addition, thrombin reduced the nuclear content of FoxO in WT (p<0. 05) but not in gas6−/− endothelial cells. Using qPCR, we found that mRNA expression of VCAM-1 was increased after 30 minutes of stimulation with thrombin in WT cells (p<0. 05). More importantly, thrombin-mediated induction of VCAM-1 was blunted in gas6−/− endothelial cells. We found that FoxO1 siRNA increased basal VCAM-1 expression in WT endothelial cells. Taken together, our data demonstrate that gas6 is a crucial mediator of FoxO1 that regulates thrombin-induced VCAM-1 expression. This pathway may explain the pro-thrombotic and pro-inflammatory role of gas6. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Antithrombin protects against Plasmodium falciparum histidine-rich protein II-mediated inflammation and coagulation

2021 ◽  
Author(s):  
Indranil Biswas ◽  
Sumith R Panicker ◽  
Hemant Giri ◽  
Xiaofeng S Cai ◽  
Alireza R Rezaie
Keyword(s):  
Endothelial Cells ◽  
Plasmodium Falciparum ◽  
Endothelial Cell ◽  
Animal Models ◽  
Cell Activation ◽  
Histone H3 ◽  
Neutrophil Infiltration ◽  
Human Neutrophils ◽  
Endothelial Cell Activation

Plasmodium falciparum (Pf)-derived histidine-rich protein II (HRPII) has been shown to inhibit heparin-dependent anticoagulant activity of antithrombin (AT) and induce inflammation in vitro and in vivo. In a recent study, we showed that HRPII interacts with the AT-binding vascular glycosaminoglycans (GAGs) to not only disrupt the barrier-permeability function of endothelial cells but also inhibit the anti-inflammatory signaling function of AT. Here we investigated the mechanisms of the pro-inflammatory function of HRPII and the protective activity of AT in cellular and animal models. We found that AT competitively inhibits the GAG-dependent HRPII-mediated activation of NF-κB and expression of intercellular cell adhesion molecule 1 (ICAM1) in endothelial cells. Furthermore, AT inhibits HRPII-mediated histone H3 citrullination and neutrophil extracellular trap (NET) formation in HL60 cells and freshly isolated human neutrophils. In vivo, HRPII induced Mac1 expression on blood neutrophils, MPO release in plasma, neutrophil infiltration and histone H3 citrullination in the lung tissues. HRPII also induced endothelial cell activation as measured by increased ICAM1 expression and elevated vascular permeability in the lungs. AT effectively inhibited HRPII-mediated neutrophil infiltration, NET formation and endothelial cell activation in vivo. AT also inhibited HRPII-meditated deposition of platelets and fibrin(ogen) in the lungs and circulating level of von Willebrand factor in the plasma. We conclude that AT exerts protective effects against pathogenic effects of Pf-derived HRPII in both cellular and animal models.

scholarly journals Functional interactions of T cells with endothelial cells: the role of CD40L-CD40-mediated signals.

1995 ◽  
Vol 182 (6) ◽  
pp. 1857-1864 ◽  
Author(s):  
M J Yellin ◽  
J Brett ◽  
D Baum ◽  
A Matsushima ◽  
M Szabolcs ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Cell Activation ◽  
Endothelial Cell Activation ◽  
Cd40 Expression

CD40 is expressed on a variety of cells, including B cells, monocytes, dendritic cells, and fibroblasts. CD40 interacts with CD40L, a 30-33-kD activation-induced CD4+ T cell surface molecule. CD40L-CD40 interactions are known to play key roles in B cell activation and differentiation in vitro and in vivo. We now report that normal human endothelial cells also express CD40 in situ, and CD40L-CD40 interactions induce endothelial cell activation in vitro. Frozen sections from normal spleen, thyroid, skin, muscle, kidney, lung, or umbilical cord were studied for CD40 expression by immunohistochemistry. Endothelial cells from all tissues studied express CD40 in situ. Moreover, human umbilical vein endothelial cells (HUVEC) express CD40 in vitro, and recombinant interferon gamma induces HUVEC CD40 upregulation. CD40 expression on HUVEC is functionally significant because CD40L+ Jurkat T cells or CD40L+ 293 kidney cell transfectants, but not control cells, upregulate HUVEC CD54 (intercellular adhesion molecule-1), CD62E (E-selectin), and CD106 (vascular cell adhesion molecule-1) expression in vitro. Moreover, the kinetics of CD40L-, interleukin 1-, or tumor necrosis factor alpha-induced CD54, CD62E, and CD106 upregulation on HUVEC are similar. Finally, CD40L-CD40 interactions do not induce CD80, CD86, or major histocompatibility complex class II expression on HUVEC in vitro. These results demonstrate that CD40L-CD40 interactions induce endothelial cell activation in vitro. Moreover, they suggest a mechanism by which activated CD4+ T cells may augment inflammatory responses in vivo by upregulating the expression of endothelial cell surface adhesion molecules.

scholarly journals Expression of Recombinant β2GPI and Site-Directed Mutants Provides Insight into the Pathogenesis of Antiphospholipid Syndrome

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1226-1226
Author(s):  
Ravi Kumar Alluri ◽  
Suman Kundu ◽  
Jack Su ◽  
Sekhar Kambakam ◽  
Edward Yu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Molecular Modeling ◽  
Cell Lines ◽  
Antiphospholipid Syndrome ◽  
Cell Activation ◽  
Full Length ◽  
Antibody Specificity ◽  
Wild Type ◽  
Endothelial Cell Activation

Abstract Introduction: Antiphospholipid syndrome (APS) is an autoimmune disorder caused by "antiphospholipid" antibodies (aPL) directed against β2-glycoprotein I (β2GPI). In the presence of β2GPI, aPL activate endothelial cells through TLR4-mediated and other pathways. Previous studies suggest that a region encompassing amino acids 39-43 of β2GPI domain 1 comprises an important binding site for pathogenic aPL. However, due to difficulty in expressing full-length recombinant β2GPI, antibody specificity has only been tested using individual, recombinant β2GPI domains. Further definition of antibody specificity using intact recombinant β2GPI provides a new approach for diagnostic and mechanistic studies. In the present study we describe the expression of full-length, wild-type recombinant β2GPI and site-directed mutants encompassing the putative β2GPI binding region in domain 1. Binding of APS patient-derived anti-β2GPI antibodies to these proteins and their ability to support anti-β2GPI-mediated endothelial cell activation was determined. Methods: Full length APOH cDNA with a modified signal peptide (spm) was cloned into the lentiviral vector pLenti CMV Puro DEST. We generated two mutants spanning the 39 to 43 amino acid region: SEGVG (R39S; G40E; M42V; R43G) and AAGMA (R39A; G40A; R43A). The final recombinant plasmids were termed as pDEST-spmAPOH-WT; pDEST-spmAPOH-SEGVG and pDEST-spmAPOH-AAGMA. Lentivirus was produced using the Lentiviral Gateway Expression kit using GP2-293 (HEK) cells. Stable cell lines were generated by transducing HEK cells with APOH-lentivirus and selecting against puromycin. Stable cell lines were transferred to serum-free media and grown in suspension. Cell culture supernatants containing secreted β2GPI were filtered and concentrated, and recombinant β2GPI was purified using a hitrap-heparin column. Anti-β2GPI antibody binding to plasma-derived and rβ2GPI was determined using a standard β2GPI ELISA and SPR (Surface Plasmon Resonance) on a Biacore 3000. Biosensor analysis was performed by crosslinking β2GPI to carboxymethyl-dextran coated sensor chips using amine coupling. Anti-β2GPI antibodies at concentrations ranging from 1-15,000 nM, were flowed through channels until equilibrium binding was achieved, at which point dissociation was assessed over a 10 minute interval. The BIAevaluation program was used to calculate association and dissociation rates. Endothelial cell activation was assessed by measuring cell surface E-selectin expression after incubating cells with control IgG or anti-β2GPI antibodies in the presence or absence of wild-type or mutant rβ2GPI. Molecular modeling was performed using PyMol. Results: After transduction with lentivirus encoding β2GPI with a modified signal peptide, HEK-293 cells were able to express and efficiently secrete rβ2GPI and site-directed mutants. Using a standard β2GPI-ELISA and SPR we found no significant differences in binding affinity of aPL towards plasma-derived and wild-type rβ2GPI. However, in the anti-β2GPI ELISA, binding of patient-derived aPL (APS21) to both mutants was reduced by ~70% compared to wild-type rβ2GPI. Consistent with binding specificity for a region encompassing aa 39-43, the Kd for binding of APS21 anti-β2GPI antibodies to wild-type and rβ2GPI-SEGVG determined by SPR was 20 nM and 5000 nM, respectively. Moreover, the ability of patient-derived APS21 anti-β2GPI antibodies to activate endothelial cells was reduced by >60% in the presence of rβ2GPI-SEGVG compared to wild-type rβ2GPI. Molecular modeling of β2GPI demonstrated that a mutation in the aa 39-43 region is predicted to cause a change from a net positive to a net negative charge without any structural change (Figure 1). Conclusion: These studies are the first to assess binding of human anti-β2GPI antibodies to rβ2GPI and site-directed mutants expressed in mammalian cells. No significant differences between binding of aPL to plasma derived and wild-type rβ2GPI were observed. APS21 aPL has specificity towards epitope 39-43 of β2GPI domain 1 and its binding affinity to rβ2GPI-SEGVG was significantly reduced. Functional studies demonstrate the importance of β2GPI aa 39 to 43 in supporting endothelial cell activation by anti-β2GPI antibodies. These recombinant proteins should facilitate further studies concerning the role of aPL-β2GPI interactions in the diagnosis and pathogenesis of APS. Disclosures No relevant conflicts of interest to declare.

Possible Involvement of Toll-Like Receptor 4 in Endothelial Cell Activation of Larger Vessels in Response to Lipopolysaccharide

Pathobiology ◽  
2002 ◽  
Vol 70 (1) ◽  
pp. 18-25 ◽  
Author(s):  
Naoki Hijiya ◽  
Kensuke Miyake ◽  
Sachiko Akashi ◽  
Keiko Matsuura ◽  
Yasunori Higuchi ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Activation ◽  
Toll Like Receptor ◽  
Endothelial Cell Activation ◽  
Toll Like Receptor 4
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