Surface Expression Of E-selectin Research Articles

Probing expression of E-selectin using CRISPR-Cas9-mediated tagging with HiBiT in human endothelial cells

E-selectin is expressed on endothelial cells in response to inflammatory cytokines and mediates leukocyte rolling and extravasation. However, studies have been hampered by lack of experimental approaches to monitor expression in real time in living cells. Here, NanoLuc Binary Technology (NanoBiT) in conjunction with CRISPR-Cas9 genome editing was used to tag endogenous E-selectin in human umbilical vein endothelial cells (HUVECs) with the 11 amino acid nanoluciferase fragment HiBiT. Addition of the membrane-impermeable complementary fragment LgBiT allowed detection of cell surface expression. This allowed the effect of inflammatory mediators on E-selectin expression to be monitored in real time in living endothelial cells. NanoBiT combined with CRISPR-Cas9 gene editing allows sensitive monitoring of real-time changes in cell surface expression of E-selectin and offers a powerful tool for future drug discovery efforts aimed at this important inflammatory protein.

Prevalence of Antiphospholipid Antibodies and Association With Incident Cardiovascular Events

The prevalence of antiphospholipid antibodies (aPL) and their association with future atherosclerotic cardiovascular disease (ASCVD) risk has yet to be thoroughly investigated. To determine the association between measurements of aPL at a single time point and ASCVD risk in a diverse population. This cohort study measured 8 aPL (anticardiolipin [aCL] IgG/IgM/IgA, anti-beta-2 glycoprotein I [aβ2GPI] IgG/IgM/IgA, and antiphosphatidylserine/prothrombin [aPS/PT] IgG/IgM) by solid-phase assays in plasma from participants of the Dallas Heart Study (DHS) phase 2, a multiethnic, population-based cohort study. Blood samples were collected between 2007 and 2009. The median follow-up was 8 years. Statistical analysis was performed from April 2022 to January 2023. Associations of aPL with future ASCVD events (defined as first nonfatal myocardial infarction, first nonfatal stroke, coronary revascularization, or death from cardiovascular cause) were assessed by Cox proportional hazards models, adjusting for known risk factors, medications, and multiple comparisons. Among the 2427 participants (mean [SD] age, 50.6 [10.3] years; 1399 [57.6%] female; 1244 [51.3%] Black, 339 [14.0%] Hispanic, and 796 [32.8%] White), the prevalence of any positive aPL tested at a single time point was 14.5% (353 of 2427), with approximately one-third of those detected at a moderate or high titer; aCL IgM had the highest prevalence (156 individuals [6.4%]), followed by aPS/PT IgM (88 [3.4%]), aβ2GPI IgM (63 [2.6%]), and aβ2GPI IgA (62 [2.5%]). The IgA of aCL (adjusted hazard ratio [HR], 4.92; 95% CI, 1.52-15.98) and aβ2GPI (HR, 2.91; 95% CI, 1.32-6.41) were independently associated with future ASCVD events. The risk further increased when applying a positivity threshold of at least 40 units (aCL IgA: HR, 9.01 [95% CI, 2.73-29.72]; aβ2GPI IgA: HR, 4.09 [95% CI, 1.45-11.54]). Levels of aβ2GPI IgA negatively correlated with cholesterol efflux capacity (r = -0.055; P = .009) and positively correlated with circulating oxidized LDL (r = 0.055; P = .007). aβ2GPI IgA-positive plasma was associated with an activated endothelial cell phenotype as evidenced by increased surface expression of surface E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. In this population-based cohort study, aPL detectable by solid-phase assays were present in a substantial proportion of adults; positive aCL IgA and aβ2GPI IgA at a single time point were independently associated with future ASCVD events. Longitudinal studies with serial aPL measurements are needed to further explore these findings.

Probing Ligand-Induced Changes in Cell Surface Expression of E-Selectin Using CRISPR/Cas9-Mediated Protein Tagging with HiBiT in Primary Human Endothelial Cells

Probing Ligand-Induced Changes in Cell Surface Expression of E-Selectin Using CRISPR/Cas9-Mediated Protein Tagging with HiBiT in Primary Human Endothelial Cells

Endothelial Activation in Orientia tsutsugamushi Infection Is Mediated by Cytokine Secretion From Infected Monocytes.

Scrub typhus, caused by Orientia tsutsugamushi, is a common systemic infection in Asia. Delay in diagnosis and treatment can lead to vasculitis in the visceral organs and other complications. The mechanisms that drive endothelial activation and the inflammatory response in O. tsutsugamushi infection remain unknown. In addition, the interaction between monocytes and endothelial cells is still unclear. Here we demonstrate that O. tsutsugamushi-infected human dermal microvascular endothelial cells produced moderate levels of chemokines and low levels of IL-6 and IFN-β, but not TNF or IL-1β. Recombinant TNF and cytokine-rich supernatants from infected monocytes markedly enhanced chemokine production in infected endothelial cells. We also show that TNF and monocyte supernatants, but not O. tsutsugamushi infection of endothelial cells per se, upregulated the endothelial cell surface expression of ICAM-1, E-selectin, and tissue factor. This finding was consistent with the inability of O. tsutsugamushi to induce cytokine secretion from endothelial cells. The upregulation of surface molecules after stimulation with monocyte supernatants was significantly reduced by neutralizing anti-TNF antibodies. These results suggest that endothelial cell activation and response are mainly mediated by inflammatory cytokines secreted from monocytes.

Thromboinflammation Model-on-A-Chip by Whole Blood Microfluidics on Fixed Human Endothelium.

Microfluidic devices have an established role in the study of platelets and coagulation factors in thrombosis, with potential diagnostic applications. However, few microfluidic devices have assessed the contribution of neutrophils to thrombus formation, despite increasing knowledge of neutrophils’ importance in cardiovascular thrombosis. We describe a thromboinflammation model which uses straight channels, lined with fixed human umbilical vein endothelial cells, after treatment with tumour necrosis factor-alpha. Re-calcified whole blood is perfused over the endothelium at venous and arterial shear rate. Neutrophil adhesion, platelet and fibrin thrombus formation, is measured over time by the addition of fluorescent antibodies to a whole blood sample. Fixed endothelium retains surface expression of adhesion molecules ICAM-1 and E-Selectin. Neutrophils adhere preferentially to platelet thrombi on the endothelium. Inhibitors of neutrophil adhesion and anti-inflammatory agents, such as isoquercetin, decrease neutrophil adhesion. Our model offers the advantage of the use of (1) fixed endothelium, (2) whole blood, instead of isolated neutrophils, and (3) a small amount of blood (1 mL). The characteristics of this thromboinflammation model provide the potential for further development for drug screening and point-of-care applications.

Advancing human induced pluripotent stem cell-derived blood-brain barrier models for studying immune cell interactions.

Human induced pluripotent stem cell (hiPSC)‐derived blood‐brain barrier (BBB) models established to date lack expression of key adhesion molecules involved in immune cell migration across the BBB in vivo. Here, we introduce the extended endothelial cell culture method (EECM), which differentiates hiPSC‐derived endothelial progenitor cells to brain microvascular endothelial cell (BMEC)‐like cells with good barrier properties and mature tight junctions. Importantly, EECM‐BMEC‐like cells exhibited constitutive cell surface expression of ICAM‐1, ICAM‐2, and E‐selectin. Pro‐inflammatory cytokine stimulation increased the cell surface expression of ICAM‐1 and induced cell surface expression of P‐selectin and VCAM‐1. Co‐culture of EECM‐BMEC‐like cells with hiPSC‐derived smooth muscle‐like cells or their conditioned medium further increased the induction of VCAM‐1. Functional expression of endothelial ICAM‐1 and VCAM‐1 was confirmed by T‐cell interaction with EECM‐BMEC‐like cells. Taken together, we introduce the first hiPSC‐derived BBB model that displays an adhesion molecule phenotype that is suitable for the study of immune cell interactions.

Metformin attenuates adhesion between cancer and endothelial cells in chronic hyperglycemia by recovery of the endothelial glycocalyx barrier

BackgroundEpidemiologic studies suggest that diabetes is associated with an increased risk of cancer. Concurrently, clinical trials have shown that metformin, which is a first-line antidiabetic drug, displays anticancer activity. The underlying mechanisms for these effects are, however, still not well recognized. MethodsMethods based on atomic force microscopy (AFM) were used to directly evaluate the influence of metformin on the nanomechanical and adhesive properties of endothelial and cancer cells in chronic hyperglycemia. AFM single-cell force spectroscopy (SCFS) was used to measure the total adhesion force and the work of detachment between EA.hy926 endothelial cells and A549 lung carcinoma cells. Nanoindentation with a spherical AFM probe provided information about the nanomechanical properties of cells, particularly the length and grafting density of the glycocalyx layer. Fluorescence imaging was used for glycocalyx visualization and monitoring of E-selectin and ICAM-1 expression. ResultsSCFS demonstrated that metformin attenuates adhesive interactions between EA.hy926 endothelial cells and A549 lung carcinoma cells in chronic hyperglycemia. Nanoindentation experiments, confirmed by confocal microscopy imaging, revealed metformin-induced recovery of endothelial glycocalyx length and density. The recovery of endothelial glycocalyx was correlated with a decrease in the surface expression of E-selectin and ICAM-1. ConclusionOur results identify metformin-induced endothelial glycocalyx restoration as a key factor responsible for the attenuation of adhesion between EA.hy926 endothelial cells and A549 lung carcinoma cells. General significanceMetformin-induced glycocalyx restoration and the resulting attenuation of adhesive interactions between the endothelium and cancer cells may account for the antimetastatic properties of this drug.

Enhanced angiogenic function in response to fibroblasts from psoriatic arthritis synovium compared to rheumatoid arthritis

IntroductionAngiogenesis is an early event in the pathogenesis of both psoriatic arthritis (PsA) and rheumatoid arthritis (RA); however, there are striking differences in blood vessel morphology and activation between the two arthropathies. The aim of this study was to assess if the PsA and RA joint microenvironments differentially regulate endothelial cell function.MethodsPsA and RA primary synovial fibroblasts (SFC) were isolated from synovial biopsies, grown to confluence, and supernatants harvested and termed ‘conditioned media’ (CM). Human umbilical vein endothelial cells (HUVEC) were cultured with PsA SFC or RA SFC-CM (20%). HUVEC tube formation, migration, and PBMC adhesion were assessed by matrigel tube formation, wound repair, and PBMC adhesion assays. HUVEC cell surface expression of ICAM, VCAM, and E-Selectin was assessed by flow cytometry. Transcriptome analysis of genes promoting angiogenesis was performed by real-time PCR. Finally, a MSD multiplex angiogenic assay was performed on PsA SFC and RA SFC supernatants.ResultsMacroscopic synovitis and vascularity were similar in PsA and RA patients; however, significant differences in vascular morphological pattern were recorded with tortuous, elongated vessels observed in PsA compared to straight regular branching vessels observed in RA. Transcriptome analysis showed strong upregulation of the pro-angiogenic signature in HUVEC primed with PsA SFC-CM compared to RA SFC-CM and basal control. In parallel, paired PsA SFC-CM significantly induced HUVEC tube formation compared to that of RA SFC-CM. Furthermore, PsA SFC-CM induced HUVEC migration was paralleled by a significant induction in VEGFA, PFKFB3, ICAM-1, and MMP3 mRNA expression. A significant increase in PBMC adhesion and cell surface expression of VCAM-1, ICAM-1, and E-Selectin expression was also demonstrated in PsA SFC-CM-primed HUVEC compared to RA SFC-CM. Finally, VEGF, TSLP, Flt-1, and Tie-2 expression was elevated in PsA SFC-CM compared to RA SFC-CM, with no significant difference in other pro-angiogenic mediators including MIP-3, bFGF, PIGF, and MCP-1.ConclusionPsA SFC and RA SFC secreted factors differentially regulate endothelial cell function, with soluble mediators in the PsA joint microenvironment inducing a more pro-angiogenic phenotype compared to the RA.

The Pro-Adhesive and Reduced Angiogenic Properties of Endothelial Colony Forming Cells (ECFC) from JAK2V617F MPN Patients Are Not Related to the Expression of the JAK2 mutation in These Cells: A Potential Role of ADAM17

INTRODUCTION: JAK2V617F mutation is independently associated with a higher risk of venous thromboembolic events (VTE) in myeloproliferative neoplasms (MPN). We have previously shown that endothelial colony-forming cells (ECFCs) from JAK2V617F MPN patients do not express this mutation but exhibit a pro-adhesive phenotype with a lower in vitro angiogenic potential. Here, we explore the mechanisms through which ECFCs from JAK2V617F MPN patients behave differently from ECFCs from healthy individuals. METHODS: We selected 38 patients with newly or previously diagnosed MPN, regardless of current or prior hydroxyurea therapy, with or without a history of VTE. Plasma levels of ADAMTS13, P-selectin, soluble VCAM, soluble ICAM, PDGF-AA, PDGF-AB/BB, IL-8, IL-1β, and soluble CD40 ligand were measured through the use of a multiplex assay. We then tried to isolate ECFCs from 20 of the JAK2V617F patients as well as 14 healthy individuals. Successfully isolated ECFCs were then submitted to co-culture with red blood cells (RBC), after which RNA was extracted from ECFCs. ADAMTS13, P-selectin, sVCAM, and sICAM were measured in the supernatant from ECFCs and RBC co-culture with multiplex assay. ECFCs' surface expression of P-selectin, VCAM, ICAM, and E-selectin was measured with the use of flow cytometry. Gene expression in RNA from co-cultured ECFCs was assessed with two 84-gene RT-PCR arrays for endothelial cell biology and signal transduction pathways (RT2 Profiler PCR array, Qiagen®). RESULTS: Recruited patients had polycythemia vera (PV) (n=17), primary myelofibrosis (PMF) (n=7) and essential thrombocythemia (ET) (n=14); 34% of them had had a previous VTE (6/17 PV; 3/14 ET and 4/7 PMF). Median age was 59 years (26-88), 23% were male. Compared with JAK2wt, JAK2V617F MPN patients (n=29) showed higher median concentrations of sVCAM (1063 vs. 786.5 ng/ml, p=0.0315), sCD40L (339 vs. 166.4 ng/ml, p=0.0065), PDGF-AA (430.6 vs. 136.5 ng/ml, p=0.03) and PDGF-AB/BB (1068 vs. 365.8 ng/ml, p=0.004). Success in ECFC isolation was 9/20 (45%) in JAK2V617F MPN patients and 8/14 (57%) in healthy individuals. None of the ECFCs from JAK2V617F MPN patients expressed this mutation. In comparison with ECFCs from healthy individuals, ECFCs from JAK2V617F MPN patients did not show a higher expression of either VCAM1, ICAM, P-selectin, and E-selectin, as measured by flow cytometry after co-culture with RBCs. Multiplex assay on the supernatant of this co-culture did not show any difference in the levels of ADAMTS13, sVCAM, sICAM, and sP-selectin. RNA PCR array from the ECFCs after co-culture with RBCs showed no difference in the expression of adhesion molecules when compared with ECFCs from healthy individuals, except for the increased expression of ADAM17 (fold regulation: 5.46, p=0.006), a metalloprotease involved in the shedding of adhesion molecules, among them VCAM1. ECFCs from MPN patients did not show higher activation of the JAK-STAT pathway but had a lower expression of WNT5 (fold regulation: -2.22, p=0.03), indicating a reduced activation of the Hedgehog pathway, suitable with the reduced in vitro angiogenesis capacity of these cells. CONCLUSIONS: JAK2V617F MPNs are associated with an increase in circulating adhesion molecules, such as sVCAM, and platelet activation markers. The pro-adhesive phenotype and lower angiogenic potential of ECFCs JAK2V617F MPN patients are not related to this mutation, which is not expressed in these cells and might be related to an increased expression of ADAM17 and reduced activation of the Hedgehog pathway. Disclosures Ozelo: BioMarin: Research Funding; Pfizer: Research Funding; Takeda: Research Funding; Novo Nordisk: Research Funding; Roche: Research Funding.

Metabolic-hypoxic modulation of cytokine induction of intestinal endothelial adhesion molecules: Relevance to ischemic injury mediated necrotizing enterocolitis?

BackgroundNecrotizing enterocolitis (NEC) triggers an intense inflammatory response in the neonatal gut associated with cytokine activation, altered nutrient status and intracellular O2-deprivation. Endothelial cell adhesion molecules (ECAMs) play critical roles in driving immune cell infiltration into inflamed gut. Currently, relationships between inflammation, metabolism and ECAM expression remain poorly understood in NEC. We studied the effects of metabolic depletion (aglycemia/ hypoxia) on TNF-α mediated ECAM expression including ICAM-1, MAdCAM-1, VCAM-1 and E-selectin, in vitro in intestinal microvascular endothelial cells (IMEC). MethodsTo study the effects of TNF-α, aglycemia and hypoxia (alone or in combination) IMECs expression of adhesion molecules was studied using cell surface ELISA and immunoblotting. ResultsTotal VCAM-1 expression was induced TNF-α and by hypoxia + TNF-α, cell surface expression was induced by hypoxia, TNF-α, TNF- α+hypoxia, and TNF- α+hypoxia and aglycemia. Total ICAM-1 increased following TNF- α, TNF- α+hypoxia, hypoxia + aglycemia, and TNF- α+hypoxia + aglycemia. Total MAdCAM-1 protein expression was significantly induced by a combination of TNF-α+hypoxia + aglycemia and cell surface expression induced by TNF- α+hypoxia. Surface expression of E-selectin was induced by TNF- α+aglycemia and TNF- α+hypoxia + aglycemia. ConclusionEnergy metabolism influences inflammation induced injury through mobilization of intestinal ECAMs, and may represent an important mechanism in NEC pathology.

High glucose-induced endothelial microparticles increase adhesion molecule expression on endothelial cells.

The experimental aim of this study was to determine, in vitro, the effects of glucose-induced EMPs on endothelial cell expression of E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 and platelet cell adhesion molecule-1 (PECAM-1). Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated in 6-well plates at a density of 5.0 × 105 cells/condition. HUVECs were incubated with media containing either 25mM d-glucose (concentration representing a hyperglycemic state) or 5mM d-glucose (normoglycemic condition) for 48h to generate EMPs. EMP identification (CD144+) and concentration were determined by flow cytometry. HUVECs (3 × 106 cells/condition) were treated with either high glucose-derived EMPs (hgEMPs) or normal glucose-derived (ngEMPs) for 24h and surface expression of E-selectin (CD62E-PE), ICAM-1 (CD54-FITC), VCAM-1 (CD106-APC) and PECAM-1 (CD31-BV) was assessed by flow cytometry and reported as mean fluorescent intensity (MFI). Hyperglycemic-derived EMPs induced significantly higher surface expression of E-selectin (2614 ± 132 vs. 2010 ± 204MFI), ICAM-1 (2110 ± 81 vs. 1688 ± 152MFI), VCAM-1 (3589 ± 431 vs. 2134 ± 386) and PECAM-1 (4237 ± 395 vs. 2525 ± 269MFI) on endothelial cells than EMPs from normoglycemic conditions. Microparticle-induced cell adhesion molecule expression provides potential novel mechanistic insight regarding the accelerated risk of atherosclerotic vascular disease associated with hyperglycemia.

Endothelial cells of extremely premature infants display impaired immune response after proinflammatory stimulation.

BackgroundEndothelial cells (ECs) exert immunological functions such as production of proinflammatory cytokines/chemokines as well as facilitation of extravasation of immune cells into infected tissue. Limited data are available on the functionality of ECs from extremely preterm neonates during infection. Accordingly, the aim of our study was to investigate the immune response of premature ECs after proinflammatory stimulation.MethodsCell adhesion receptors' expression and function, nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) signaling, and chemokine production were analyzed in umbilical cord ECs from extremely preterm and term neonates after proinflammatory stimulation.ResultsP-selectin and E-selectin surface expression as well as NFκB signaling were lower after lipopolysaccharide (LPS) stimulation in premature ECs. Preterm ECs exhibited lower, but significant, cell-adhesive functions after LPS stimulation compared with term ECs. CCL2/CXCL8 chemokine secretion was significantly upregulated after proinflammatory stimulation in both groups. CXCL10 production was significantly increased in term but not in preterm ECs upon stimulation with tumor necrosis factor compared with unstimulated ECs.ConclusionExtremely premature ECs showed partly reduced expression levels and function of cell adhesion molecules. Both NFκB signaling and chemokine/cytokine production were reduced in premature ECs. The diminished endothelial proinflammatory immune response might result in impaired infection control of preterm newborns rendering them prone to severe infection.

Bone morphogenetic protein 9 (BMP9) and BMP10 enhance tumor necrosis factor-α-induced monocyte recruitment to the vascular endothelium mainly via activin receptor-like kinase 2

Bone morphogenetic proteins 9 and 10 (BMP9/BMP10) are circulating cytokines with important roles in endothelial homeostasis. The aim of this study was to investigate the roles of BMP9 and BMP10 in mediating monocyte–endothelial interactions using an in vitro flow adhesion assay. Herein, we report that whereas BMP9/BMP10 alone had no effect on monocyte recruitment, at higher concentrations both cytokines synergized with tumor necrosis factor-α (TNFα) to increase recruitment to the vascular endothelium. The BMP9/BMP10-mediated increase in monocyte recruitment in the presence of TNFα was associated with up-regulated expression levels of E-selectin, vascular cell adhesion molecule (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Using siRNAs to type I and II BMP receptors and the signaling intermediaries (Smads), we demonstrated a key role for ALK2 in the BMP9/BMP10-induced surface expression of E-selectin, and both ALK1 and ALK2 in the up-regulation of VCAM-1 and ICAM-1. The type II receptors, BMPR-II and ACTR-IIA were both required for this response, as was Smad1/5. The up-regulation of cell surface adhesion molecules by BMP9/10 in the presence of TNFα was inhibited by LDN193189, which inhibits ALK2 but not ALK1. Furthermore, LDN193189 inhibited monocyte recruitment induced by TNFα and BMP9/10. BMP9/10 increased basal IκBα protein expression, but did not alter p65/RelA levels. Our findings suggest that higher concentrations of BMP9/BMP10 synergize with TNFα to induce the up-regulation of endothelial selectins and adhesion molecules, ultimately resulting in increased monocyte recruitment to the vascular endothelium. This process is mediated mainly via the ALK2 type I receptor, BMPR-II/ACTR-IIA type II receptors, and downstream Smad1/5 signaling.

Co-Administration of Hydroxyurea and a Specific AKT2 Inhibitor Has Beneficial Effects on Acute Vaso-Occlusive Events and Survival in Sickle Cell Disease Mice

We recently demonstrated that the basal levels of AKT phosphorylation were significantly increased in neutrophils and platelets isolated from sickle cell disease (SCD) patients compared with those cells from healthy donors and that specific inhibition of AKT2 impaired neutrophil-endothelial cell and neutrophil-platelet interactions in venules of Berkeley (SCD) mice (Li et al. J Clin Invest 2014). Although hydroxyurea (HU), an inducer of fetal hemoglobin, is the main therapy for treatment of SCD, it is unclear whether the short-term treatment with HU has immediate benefits on acute vaso-occlusive events in SCD. Using real-time fluorescence intravital microscopy, we demonstrated that co-administration of HU (100 microg/g body weight, iv injection) and Akti XII (3 microg/g body weight, iv injection), an AKT2 inhibitor, efficiently reduced neutrophil adhesion and platelet-neutrophil aggregation in cremaster muscle venules of TNF-α-challenged Berkeley mice. Importantly, compared with HU or Akti XII treatment alone, treatment with both agents significantly improved blood flow rates and survival in the mice. Further, similar results were obtained in Berkeley mice challenged with hypoxia (8% oxygen for 3 hours) and subsequent reoxygenation (room air for 3 hours). As determined by histochemistry of cremaster muscle sections following intravital microscopy of Berkeley mice, the expression of endothelial E-selectin and intercellular adhesion molecule 1 (ICAM-1) was significantly decreased by treatment with either HU or Akti XII. Leukocyte transmigration in the lung section of Berkeley mice was also inhibited by either agent, and the inhibitory effect was potentiated by co-administration of HU and Akti XII. Using the plasma and isolated cells of Berkeley mice, we found that the level of plasma nitric oxide species (NOx) was significantly elevated by treatment with HU but not Akti XII and that AKT2 phosphorylation levels in activated neutrophils and platelets were reduced by treatment with Akti XII but not HU. Taken together, these results suggest that short-term treatment of Berkeley mice with either HU or Akti XII inhibits inflammatory conditions: treatment with HU significantly increases plasma NOx levels, treatment with Akti XII decreases AKT2 phosphorylation in neutrophils and platelets without affecting plasma NOx levels, and administration of either agent reduces the surface expression of ICAM-1 and E-selectin on activated endothelial cells. Thus, our results provide evidence that co-administration of HU and a specific AKT2 inhibitor has immediate benefits on acute vaso-occlusive events and survival in SCD mice exceeding those seen for single therapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract 378: Contribution of Unfolded Protein Response in Arsenic-Induced Endothelial Activation and Atherosclerois

Epidemiological and animal studies suggest that exposure to arsenic contaminated water exacerbates atherosclerosis. However, the mechanisms by which arsenic exerts its atherogenic effects are not known. We observed that chronic (1 μM; 4 days) or acute (20 μM; 2-6 hours) exposure of sodium arsenite to human umbilical vein endothelial cells (HUVEC) increased the surface expression of adhesion molecules ICAM-1, VCAM-1 and E-Selectin by 1.2-1.5-fold; leukocyte adhesion by 1.5-3.5 fold; leukocyte trans-endothelial migration by 1.7-2.5-fold; and mRNA expression of IL-8 by 5-25-fold. Similarly, exposure of human aortic endothelial cells and mouse aortic endothelial cells to arsenic also caused endothelial activation. Arsenic also enhanced the unfolded protein response (UPR) in endothelial cells as evident by the phosphorylation of IRE-1 and activation of its downstream proteins JNK and NF-kappaB; nuclear translocation of ATF6, and increased expression of molecular chaperones GRP78 and HERP. SiRNA-mediated knockdown of IRE-1 attenuated arsenic-induced NF-kappaB activation and IL-8 expression, but did not affect JNK phosphorylation. Adenoviral transfection with ATF-6 upregulated GRP78 and decreased the expression of IL-8. Similarly, pre-incubation of endothelial cells with phenyl butyric acid (PBA; 5 mM, 16h), a chemical chaperone of protein folding, significantly (P<0.05) prevented arsenic-induced leukocyte adhesion, trans-endothelial migration and IL-8 expression. Feeding of apoE-null mice with PBA for 16 weeks inhibited the arsenic-induced UPR in atherosclerotic lesions; expression of adhesion molecules on endothelial cells lining the atherosclerotic lesions; lesional and systemic inflammation; and prevented the arsenic-induced exacerbation of lesion formation by 90% (P<0.05). Together, these data suggest that arsenic causes endothelial activation and exacerbates atherosclerosis by triggering UPR and chemical chaperones of protein folding prevent arsenic-induced exacerbation of atherogenesis.

Exacerbation of Intracranial Aneurysm and Aortic Dissection in Hypertensive Rat Treated With the Prostaglandin F–Receptor Antagonist AS604872

Intracranial aneurysm (IA) and aortic dissection are both complications of hypertension and characterized by degeneration of the media. Given the involvement of prostaglandin F2α and its receptor, FP, in extracellular matrix remodeling in a mouse model of pulmonary fibrosis, here we induced hypertension and IA in rats by salt loading and hemi-lateral ligation of renal and carotid arteries and examined effects of a selective FP antagonist, AS604872, on these vascular events. AS604872 significantly accelerated degeneration of the media in both cerebral artery and aorta as evidenced by thinning of the media and disruption of the elastic lamina and promoted IA and aortic dissection. Notably, AS604872 induced expression of pro-inflammatory genes such as E-selectin in lesions and significantly enhanced macrophage infiltration. Suppression of surface expression of E-selectin with cimetidine prevented macrophage infiltration and aortic dissection. Thus, AS604872 exacerbates vascular inflammation in hypertensive rats and facilitates IA and aortic dissection. These results demonstrate that both IA and aortic dissection are caused by chronic inflammation of the arterial wall, which is worsened by AS604872, cautioning that other FP antagonists may share such deleterious actions in vascular homeostasis and suggesting that AS604872 can be used to make models of these vascular diseases with extensive degeneration.

Increased Endothelial Inflammation, sTie-2 and Arginase Activity in Umbilical Cords Obtained from Gestational Diabetic Mothers

ObjectiveThe aim of this study was to determine subclinical inflammation in umbilical vein derived endothelial cells (HUVECs) obtained from Asian Indian subjects with gestational diabetes (GDM) and to determine levels of angiogenic factors and arginase activity in their cord blood. MethodsThis case-control study included 38 control and 30 GDM subjects. Subjects were confirmed as GDM based on 75g oral glucose tolerance test (OGTT) conducted in the second trimester of pregnancy. Angiogenic markers and arginase activity were measured in cord blood by ELISA and colorimetric methods respectively. Endothelial inflammation was assessed through adhesion of PKH26-labelled leukocytes onto HUVEC monolayer obtained from the study groups. Gene and surface expression of adhesion molecules were confirmed via reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry respectively. ResultsThe study revealed increased adhesion of leukocytes to HUVECs isolated from GDM subjects compared to controls. HUVECs of babies born to GDM mothers had increased surface and mRNA expression of E-selectin. sTie2 levels were significantly higher in the cord blood for GDM subjects (3869 ± 370 ng/L) compared to controls (3045 ± 296 ng/L). Furthermore, arginase activity was higher in cord blood of GDM mothers as opposed to the control group (7.75 ± 2.4 µmoles of urea/ml/hour vs 2.88 ±0.49 µmoles of urea/ml/hour; p-value= 0.019). Spearman’s correlation analysis revealed positive correlation of cord blood arginase activity with glucose intolerance (ρ=0.596, p=0.004) and post load glucose values (ρ=0.472, p=0.031) of mothers observed during the second trimester of pregnancy.ConclusionsHUVECs derived from Asian Indian GDM mothers, exhibit signs of sub-clinical endothelial inflammation along with increased levels of sTie2 and arginase activity in their cord blood serum.

S141 BMP9 is required for LPS-mediated neutrophil recruitment to PAH-patient derived blood outgrowth endothelial cells with BMPR-II mutations

IntroductionHeterozygous mutations in the gene encoding bone morphogenetic protein (BMP) receptor II (BMPR-II) are present in >70% of patients with heritable pulmonary arterial hypertension (hPAH) and 15–26% of idiopathic PAH...

Discovery of Plumericin as a novel potent NF-κB inhibitor from Himatanthus sucuuba

The transcription factor nuclear factor kappa B (NF-κB) plays a crucial role in the regulation of the inflammatory response and contributes to the development of various diseases (1). Thus, its inhibition is considered a promising approach to combat inflammation (2). In this study, we applied a bioactivity-guided approach to identify NF-κB inhibitors from the stem bark of Himatanthus sucuuba, an Amazonian plant traditionally used to treat inflammation-related disorders. We identified the spirolactone iridoid plumericin as a potent NF-κB inhibitor. Plumericin inhibits NF-κB activation in TNF-α stimulated HEK293 cells stably transfected with a NF-kB-driven luciferase reporter gene (293/NF-κB-luc cells), suppresses TNF-α-induced surface expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin in endothelial cells, and reduces neutrophil recruitment in thioglycollate-induced peritonitis in mice. The effect of plumericin on the NF-κB signalling reveals to be a direct inhibition of the upstream kinase IKK-β. Consequently, it abolishes TNF-α-induced IκB phosphorylation and subsequent degradation. These findings might contribute to the development of promising anti-inflammatory leads and provide scientific evidence for the traditional use of Himatanthus sucuuba against inflammatory diseases.

LUNG TUMOUR SECRETED VEGF AND TNF-α INDUCE EXPRESSION OF ADHESION MOLECULES ON CEREBRAL ENDOTHELIAL CELLS

IntroductionLung tumours frequently metastasise to the brain; this involves specific adhesive interactions between tumour cells and cerebral endothelial cells. Vascular endothelial growth factor (VEGF) and tumour necrosis factor- α (TNF-α)...