E-selectin Promoter Research Articles

Oxidative Stress Induces E-Selectin Expression through Repression of Endothelial Transcription Factor ERG.

Oxidative stress induces a prothrombotic state through enhancement of adhesion properties of the endothelium. E-selectin, an endothelial cell adhesion molecule, becomes a therapeutic target for venous thrombosis, whereas the regulatory mechanisms of its expression have not been fully understood. In the present study, we report that H2O2 treatment increases expression of E-selectin but decreases expression of the endothelial transcription factor ETS-related gene (ERG) in HUVECs in a dose- and time-dependent manner. In BALB/c mice treated with hypochlorous acid, E-selectin expression is increased and ERG expression is decreased in endothelial cells of the brain and lung. RNA interference of ERG upregulates E-selectin expression, whereas transfection of ERG-expressing plasmid downregulates E-selectin expression in HUVECs. Knockdown or overexpression of ERG comprises H2O2-induced E-selectin expression in HUVECs. Deletion of the Erg gene in mice results in embryonic lethality at embryonic days 10.5-12.5, and E-selectin expression is increased in the Erg-/- embryos. No chromatin loop was found on the E-selectin gene or its promoter region by capture high-throughput chromosome conformation capture. Chromatin immunoprecipitation and luciferase reporter assay determined that the -127 ERG binding motif mediates ERG-repressed E-selectin promoter activity. In addition, ERG decreases H2O2-induced monocyte adhesion. Together, ERG represses the E-selectin gene transcription and inhibits oxidative stress-induced endothelial cell adhesion.

LCC-09, a Novel Salicylanilide Derivative, Exerts Anti-Inflammatory Effect in Vascular Endothelial Cells.

ObjectiveEndothelial cell (EC) activation facilitates leukocyte adhesion to vascular walls, which is implicated in a variety of cardiovascular diseases and is a target for prevention and treatment. Despite the development of anti-inflammatory medications, cost-effective therapies with significant anti-inflammatory effects and lower organ toxicity remain elusive. The goal of this study is to identify novel synthetic compounds that inhibit EC inflammatory response with minimal organ toxicity.Methods and ResultsIn this study, we discovered LCC-09, a salicylanilide derivative consisting of the functional fragment of magnolol, 2,4-difluorophenyl, and paeonol moiety of salicylate, as a novel anti-inflammatory compound in cultured ECs and zebrafish model. LCC-09 was shown to inhibit pro-inflammatory cytokine tumor necrosis factor-α (TNFα)-induced expression of adhesion molecules and inflammatory cytokines, leading to reduced leukocyte adhesion to ECs. Mechanistically, LCC-09 inhibits the phosphorylation of signal transducer and activator of transcription 1 (STAT1), TNFα-induced degradation of NF-κ-B Inhibitor-α (IκBα) and phosphorylation of NFκB p65, resulting in reduced NFκB transactivation activity and binding to E-selectin promoter. Additionally, LCC-09 attenuated TNFα-induced generation of reactive oxygen species in ECs. Molecular docking models suggest the binding of LCC-09 to NFκB essential modulator (NEMO) and Janus tyrosine kinase (JAK) may lead to dual inhibition of NFκB and STAT1. Furthermore, the anti-inflammatory effect of LCC-09 was validated in the lipopolysaccharides (LPS)-induced inflammation model in zebrafish. Our results demonstrated that LCC-09 significantly reduced the LPS-induced leukocyte recruitment and mortality of zebrafish embryos. Finally, LCC-09 was administered to cultured ECs and zebrafish embryos and showed minimal toxicities.ConclusionOur results support that LCC-09 inhibits EC inflammatory response but does not elicit significant toxicity.

RUNX1 Enhances Leukemia Cell Engraftment in the Vascular Niche through up-Regulating E-Selectin

Although the function of runt-related transcription factor 1 (RUNX1) has been well-characterized in leukemogenesis and regarded as an ideal target in anti-leukemia strategies, neither the effect of RUNX1 inhibition therapy on the bone marrow niche cells nor its impact on the engraftment of acute myeloid leukemia (AML) cells has largely been unknown. Here we show evidence that RUNX1 inhibition attenuates the expression of E-selectin in the vascular endothelial cells of bone marrow niche, thereby disrupting the homing of AML cells to the bone marrow niche, ultimately extending the overall survival period in AML mice models. In order to investigate the effect of RUNX1 inhibition on endothelial vascular niche cells, firstly we identified and focused on E-selectin from significant affected factors, such as E-selectin, P-selectin, VCAM 1 and TIE 2 through the screening the expressions of representative surface molecules in human umbilical vein endothelial cells (HUVEC) treated with our selective RUNX inhibitor : Chb-M'. E-selectin has recently been recognized as a vital component of vascular niche and fundamentally involved in the housing of hematopoietic stem cells (HSCs). We also genetically attenuated the expression of RUNX1 in HUVEC with doxycycline-inducible short hairpin RNA (shRNA). As we have expected, the expression of E-selectin was tightly and most profoundly down-regulated both at the transcript and surface expression levels upon RUNX1 -silencing. Of note, neither Chb-M'-treatment nor shRNA-mediated RUNX1 knockdown resulted in significant change in the expressions of niche-associated surface molecules in AML-derived MV4-11 cells. In addition, neither Chb-M'-treatment nor RUNX1 knockdown resulted in significant change in the expression of osteoblastic niche components in osteosarcoma-derived HOS cells. These results suggest that RUNX1 potentially and primordially be involved in the regulation of E-selectin in the endothelial vascular niche cells. Besides, the activity of E-selectin proximal promoter was increased upon additional RUNX1 expression, while the modified E-selectin promoter which lacks RUNX consensus binding site (5'-TGTGGT-3') dramatically decreased its luciferase reporter activity, confirming that RUNX1 directly transactivates E-selectin expression. We next examined the effect of RUNX1 attenuation on E-selectin expression in murine endothelial vascular niche cells through several assays. Treatment of C56BL/6 mice for 2 weeks with either E-selectin inhibitor: andrographolide, A205804 or RUNX-inhibitor: Chb-M' showed effective decreasing of E-selectin expression on endothelial vascular niche cells. We next retrovirally-transduced MLL-ENL to c-kit+ mice bone marrow cells and obtained mice leukemic cells. Using these mice leukemic cells, we investigated the effect of Runx1 inhibition therapy on the homing ability of leukemia cells to their bone marrow niche. We studied and confirmed the effect of RUNX1 inhibition on the homing ability of leukemic cells by both in vitro static assays and in vivo mouse models. Mise treated with andrographolide, A 205804 or Chb-M' for 2 weeks, mouse leukemic cells of MLL-ENL+ mice were transplanted. All these treatments significantly decreased the number of homed leukemic cells in the bone marrow compared to the vehicle-treated mice, underscoring the importance of E-selectin in the engraftment of AML cells in the vascular niche. Consistent with these observations, conditioning bone marrow vascular niche with Chb-M' before transplantation of leukemic cells significantly prolonged the overall survival period of AML mice with Runx inhibition therapy by Chb-M' or vehicle control treatment. Our work collectively indicates that RUNX -silencing drugs such as Chb-M' not only directly control the growth of AML cells themselves, but also indirectly enhance its anti-leukemic potential through attenuating E-selectin expressions in the bone marrow vascular niche. These drugs potentially bring the leukemic stem-like cells out from deep in the bone marrow niche to the peripheral blood stream and expose these primordial leukemia cells to chemotherapeutic drugs at much higher concentrations. DisclosuresNo relevant conflicts of interest to declare.

1α, 25-dihydroxyvitamin D3 inhibits tumor necrosis factor-α induced activation of human umbilical vein endothelial cells via NF-κB signaling pathway

Objective To investigate the effect of 1α, 25-dihydroxyvitamin D3[1α, 25-(OH)2D3] on tumor necrosis factor-α (TNF-α) induced activation of human umbilical vein endothelial cells (HUVECs). The mechanism involved in this process was also studied. Methods HUVECs were cultured and treated with TNF-α (40 ng/ml), 1α, 25-(OH)2D3(10-8 mol/L), and SN50 as indicated. Vascular cell adhesion molecule (VCAM) and E-selectin were used as markers of endothelial activation, which were detected by Western blotting and realtime PCR (RT-PCR). NF-κB signaling pathway was investigated to study the mechanism. Western blotting, RT-PCR, immunofluorescence assay, and chromatin immunoprecipitation (ChIP) method were used to evaluate the effects of 1α, 25-(OH)2D3 on its early activation, nuclear transport, and binding to VCAM and E-selectin promoters. Results (1)Western blotting and RT-PCR showed that TNF-α could significantly up-regulate the expression of VCAM and E-selectin in HUVECs, which can be inhibited by specific NF-κB blocker SN50. 1α, 25-(OH)2D3 down-regulated the expression of VCAM and E-selectin induced by TNF-α. (2) Western blotting showed that TNF-α induces I-κBα phosphorylation, thereby activating NF-κB p65 subunit. Immunofluorescence showed that 1α, 25-(OH)2D3 significantly inhibited the nuclear translocation of NF-κB p65 subunit. ChIP analysis revealed that 1α, 25-(OH)2D3 inhibited the binding of NF-κB p65 to VCAM and E-selectin promoters and thus affected gene expression. Conclusions TNF-α enhanced the expression of E-selectin and VCAM in HUVECs via NF-κB signaling pathway. 1α, 25-(OH)2D3 may inhibit NF-κB early activation, nuclear transport and the binding of NF-κB p65 to VCAM and E-selectin promoters, thereby inhibiting TNF-α-induced endothelial cell activation. Key words: 1α, 25-dihydroxyvitamin D3; Endothelial activation; NF-κB signaling pathway; Adhesion molecule

Challenges and Insights in Regulation of p53 and NF‐kappaB Transcription Factors: Making the Case for Cancer Prevention from the Environmental‐Physiological Paradigm

The American Cancer Society currently reports that 42% of cancer cases are caused by avoidable risk factors, such as tobacco smoking, low fiber diets and HPV, while the remaining 58% of cases have been correlated with chronic exposure to known and unknown environmental carcinogens and endogenous inflammatory reactive oxygen species. Given the high correlation of acquired mutations in the TP53 (tumor suppression) gene in various stages of cancer progression and the associated misregulation of NF-kappaB (innate immunity and inflammation), there have been numerous efforts to deduce the mechanism of transcriptional regulation of the two families that are also associated with cell cycle regulation, cell proliferation, apoptosis, metabolism, embryonic development and somatic cell differentiation. However, there is no definitive model of p53 or NF-kappaB regulation in the nucleus that can explain how the numerous genes involved in complex cell processes are activated/repressed. The main challenges stem from the sheer structural complexity of each family member, having multiple isoforms, splice variants and post-translational modifications that subsequently interact with different epigenetic and genetic cofactors. We have reported the first crystallographic and biochemical evidence showing the structure-function relationships of key DNA-binding features and regulatory domains in p73 and RelA that play a role in gene activation and repression via DNA response element (RE) sequence recognition. While initial studies show p53 and NF-kappaB family members binding to regulatory regions for hundreds of different genes at canonical DNA RE sequences, there may be a permissive modality in sequence recognition implying the larger importance of epigenetic regulation in tumor suppression or epigenetic mis-regulation in oncogenesis. As transcription is largely regulated in response to environmental stimuli via signaling cascades, the same endogenous factors involved in regulating inflammation, wound healing and cell differentiation, e.g. gut-derived serotonin, may be key to counteracting exogenous factors in cancer onset and progression. Support or Funding Information Molecular Biophysics Training Grant, NIH Grant T32 GM008326; Ruth L. Kirschstein National Research Service Award #T32 CA009523; Diffraction data were collected at BL7-1 of the Stanford/Stanford Synchrotron Radiation Lightsource supported by the Department of Energy and National Institutes of Health [P41RR001209] Comparison of the DNA-binding residues of five structures of the p73 DBD in complex with DNA consensus sequences associated with cell-cycle arrest or apoptosis. Abdul S. Ethayathulla et al. J. Biol. Chem. 2013;288:4744–4754 DNA-binding constants of the p73 S139F DBD mutant and the ΔNp73δ isoform for GGGCA and GAACA sequences associated with cellcycle arrest and apoptosis response elements, respectively. Ciribilli et al. Nucleic Acids Res. 2013 Oct; 41(18): 8637–8653. [Figure and data generated by HT Nguyen and A Ramos] Two RelA homodimers bind tandem κB sites of E-selectin promoter asymmetrically. Maria Carmen Mulero et al. J. Biol. Chem. 2017;292:18821–18830 [Apo] activation domain of RelA inhibits DNA binding. Maria Carmen Mulero et al. J. Biol. Chem. 2017;292:18821–18830 Tandem κB sites act synergistically to activate transcription by RelA. Maria Carmen Mulero et al. J. Biol. Chem. 2017;292:18821–18830 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor κB are not correlated

The nuclear factor κB (NF-κB) transcription factor family regulates genes involved in cell proliferation and inflammation. The promoters of these genes often contain NF-κB-binding sites (κB sites) arranged in tandem. How NF-κB activates transcription through these multiple sites is incompletely understood. We report here an X-ray crystal structure of homodimers comprising the RelA DNA-binding domain containing the Rel homology region (RHR) in NF-κB bound to an E-selectin promoter fragment with tandem κB sites. This structure revealed that two dimers bind asymmetrically to the symmetrically arranged κB sites at which multiple cognate contacts between one dimer to the corresponding DNA are broken. Because simultaneous RelA-RHR dimer binding to tandem sites in solution was anti-cooperative, we inferred that asymmetric RelA-RHR binding with fewer contacts likely indicates a dissociative binding mode. We found that both κB sites are essential for reporter gene activation by full-length RelA homodimer, suggesting that dimers facilitate DNA binding to each other even though their stable co-occupation is not promoted. Promoter variants with altered spacing and orientation of tandem κB sites displayed unexpected reporter activities that were not explained by the solution-binding pattern of RelA-RHR. Remarkably, full-length RelA bound all DNAs with a weaker affinity and specificity. Moreover, the transactivation domain played a negative role in DNA binding. These observations suggest that other nuclear factors influence full-length RelA binding to DNA by neutralizing the transactivation domain negative effect. We propose that DNA binding by NF-κB dimers is highly complex and modulated by facilitated association-dissociation processes.

A novel endothelial-derived anti-inflammatory activity significantly inhibits spontaneous choroidal neovascularisation in a mouse model.

BackgroundEndothelial cells (EC) grown on collagen particles inhibit intimal hyperplasia in animal models when applied perivascularly, and this effect appears to be, at least in part, the result of EC-derived soluble factors that suppress local vascular inflammation. To elucidate the molecular basis of the therapeutic effects of EC grown on collagen particles, the anti-inflammatory activity of conditioned medium from these cells was characterized.MethodsHuman aortic EC (HAEC) and, for chromatin immunoprecipitation assays, human umbilical vein EC (HUVEC) were treated with tumor necrosis factor alpha (TNFα) in the presence of conditioned medium generated by HAEC grown on collagen particles (ECPCM), and the anti-inflammatory effects were evaluated by analysing the expression of the inflammation-related adhesion molecules E-selectin and vascular cell adhesion molecule-1 (VCAM-1). The therapeutic activity of ECPCM was studied using the mouse strain JR5558, which develops spontaneous choroidal neovascularisation (CNV) lesions driven by local inflammation.ResultsECPCM significantly suppressed TNFα-induced expression of E-selectin and VCAM-1. ECPCM did not affect the mRNA stability of the two genes, but suppressed TNFα-induced binding of the p65 subunit of NF-kB transcription factor to E-selectin and VCAM-1 promoters. In vivo, systemic ECPCM treatment significantly reduced the CNV area and the recruitment of activated macrophages to the lesions. Characterization of the molecule responsible for the anti-inflammatory activity in ECPCM indicates that it is unlikely to be a protein and that it is not any of the better characterized EC-derived anti-inflammatory molecules.ConclusionsMedium conditioned by HAEC grown on collagen particles exhibits significant anti-inflammatory activity via inhibition of genes that mediate inflammatory responses in EC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13221-016-0036-4) contains supplementary material, which is available to authorized users.

Crucial Role of the Aryl Hydrocarbon Receptor (AhR) in Indoxyl Sulfate-Induced Vascular Inflammation.

The aryl hydrocarbon receptor (AhR), a ligand-inducible transcription factor mediating toxic effects of dioxins and uremic toxins, has recently emerged as a pathophysiological regulator of immune-inflammatory conditions. Indoxyl sulfate, a uremic toxin, is associated with cardiovascular disease in patients with chronic kidney disease and has been shown to be a ligand for AhR. The aim of this study was to investigate the potential role of AhR in indoxyl sulfate-induced leukocyte-endothelial interactions. Endothelial cell-specific AhR knockout (eAhR KO) mice were produced by crossing AhR floxed mice with Tie2 Cre mice. Indoxyl sulfate was administered for 2 weeks, followed by injection of TNF-α. Leukocyte recruitment to the femoral artery was assessed by intravital microscopy. Vascular endothelial cells were transfected with siRNA specific to AhR (siAhR) and treated with indoxyl sulfate, followed by stimulation with TNF-α. Indoxyl sulfate dramatically enhanced TNF-α-induced leukocyte recruitment to the vascular wall in control animals but not in eAhR KO mice. In endothelial cells, siAhR significantly reduced indoxyl sulfate-enhanced leukocyte adhesion as well as E-selectin expression, whereas the activation of JNK and nuclear factor-κB was not affected. A luciferase assay revealed that the region between －153 and －146 bps in the E-selectin promoter was responsible for indoxyl sulfate activity via AhR. Mutational analysis of this region revealed that activator protein-1 (AP-1) is responsible for indoxyl sulfate-triggered E-selectin expression via AhR. AhR mediates indoxyl sulfate-enhanced leukocyte-endothelial interactions through AP-1 transcriptional activity, which may constitute a new mechanism of vascular inflammation in patients with renal disease.

Sirtuin 1 stabilization by HuR represses TNF-α- and glucose-induced E-selectin release and endothelial cell adhesivenessin vitro: relevance to human metabolic syndrome

Chronic inflammation and hyperglycaemia, typical features of metabolic diseases, trigger endothelial damage and release of E-selectin, a marker of endothelial activation. In the present study, we investigated molecular pathways involved in the regulation of endothelial cell activation induced by tumour necrosis factor-α (TNF-α) and high glucose. In cultured human umbilical vein endothelial cells (HUVECs), we studied the role of HuR, an ELAV (embryonic lethal, abnormal vision, Drosophila) family RNA-binding protein, and Sirtuin 1 (SIRT1) on E-selectin release and cell adhesion at different glucose concentrations. HuR expression and binding to SIRT1 were also analysed ex vivo in peripheral blood mononuclear cells (PBMCs) of subjects with and without the metabolic syndrome (MS), by immunoprecipitation (IP) of the ribonucleoprotein (RNP) complex. We found that SIRT1 overexpression prevented TNF-α- and high-glucose-dependent nuclear factor-κB (NF-κB)-p65 acetylation, E-selectin promoter activity, E-selectin release and adhesion of THP-1 cells to HUVECs. The same was mimicked by HuR overexpression, which binds and stabilizes SIRT1 mRNA. Importantly, in PBMCs of individuals with MS compared with those without, SIRT1 expression was lower, and the ability of HuR to bind SIRT1 mRNA was significantly reduced, while plasma E-selectin was increased. We conclude that post-transcriptional stabilization of SIRT1 by HuR represses inflammation- and hyperglycaemia-induced E-selectin release and endothelial cell activation. Therefore, increasing SIRT1 expression represents a strategy to counter the accelerated vascular disease in metabolic disorders.

Cell Density Impacts Epigenetic Regulation of Cytokine-Induced E-Selectin Gene Expression in Vascular Endothelium

Growing evidence suggests that the phenotype of endothelial cells during angiogenesis differs from that of quiescent endothelial cells, although little is known regarding the difference in the susceptibility to inflammation between both the conditions. Here, we assessed the inflammatory response in sparse and confluent endothelial cell monolayers. To obtain sparse and confluent monolayers, human umbilical vein endothelial cells were seeded at a density of 7.3×103 cells/cm2 and 29.2×103 cells/cm2, respectively, followed by culturing for 36 h and stimulation with tumor necrosis factor α. The levels of tumor necrosis factor α-induced E-selectin protein and mRNA expression were higher in the confluent monolayer than in the sparse monolayer. The phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase or nuclear factor-κB activation was not involved in this phenomenon. A chromatin immunoprecipitation assay of the E-selectin promoter using an anti-acetyl-histone H3 antibody showed that the E-selectin promoter was highly and specifically acetylated in the confluent monolayer after tumor necrosis factor α activation. Furthermore, chromatin accessibility real-time PCR showed that the chromatin accessibility at the E-selectin promoter was higher in the confluent monolayer than in the sparse monolayer. Our data suggest that the inflammatory response may change during blood vessel maturation via epigenetic mechanisms that affect the accessibility of chromatin.

Krüppel-Like Factor-11, a Transcription Factor Involved in Diabetes Mellitus, Suppresses Endothelial Cell Activation via the Nuclear Factor-κB Signaling Pathway

Endothelial cell (EC) inflammatory status is critical to many vascular diseases. Emerging data demonstrate that mutations of Krüppel-like factor-11 (KLF11), a gene coding maturity-onset diabetes mellitus of the young type 7 (MODY7), contribute to the development of neonatal diabetes mellitus. However, the function of KLF11 in the cardiovascular system still remains to be uncovered. In this study, we aimed to investigate the role of KLF11 in vascular endothelial inflammation. KLF11 is highly expressed in vascular ECs and induced by proinflammatory stimuli. Adenovirus-mediated KLF11 overexpression inhibits expression of tumor necrosis factors-α-induced adhesion molecules. Moreover, small interfering RNA-mediated KLF11 knockdown augments the proinflammatory status in ECs. KLF11 inhibits promoter activity of adhesion molecules induced by tumor necrosis factor-α and nuclear factor-κB p65 overexpression. Mechanistically, KLF11 potently inhibits nuclear factor-κB signaling pathway via physical interaction with p65. Furthermore, KLF11 knockdown results in increased binding of p65 to vascular cell adhesion molecule-1 and E-selectin promoters. At the whole organism level, KLF11(-/-) mice exhibit a significant increase in leukocyte recruitment to ECs after lipopolysaccharide administration. Taken together, our data demonstrate for the first time that KLF11 is a suppressor of EC inflammatory activation, suggesting that KLF11 constitutes a novel potential molecular target for inhibition of vascular inflammatory diseases.

HOXA9 methylation by PRMT5 is essential for endothelial cell expression of leukocyte adhesion molecules.

The induction of proinflammatory proteins in stimulated endothelial cells (EC) requires activation of multiple transcription programs. The homeobox transcription factor HOXA9 has an important regulatory role in cytokine induction of the EC-leukocyte adhesion molecules (ELAM) E-selectin and vascular cell adhesion molecule 1 (VCAM-1). However, the mechanism underlying stimulus-dependent activation of HOXA9 is completely unknown. Here, we elucidate the molecular mechanism of HOXA9 activation by tumor necrosis factor alpha (TNF-α) and show an unexpected requirement for arginine methylation by protein arginine methyltransferase 5 (PRMT5). PRMT5 was identified as a TNF-α-dependent binding partner of HOXA9 by mass spectrometry. Small interfering RNA (siRNA)-mediated depletion of PRMT5 abrogated stimulus-dependent HOXA9 methylation with concomitant loss in E-selectin or VCAM-1 induction. Chromatin immunoprecipitation analysis revealed that PRMT5 is recruited to the E-selectin promoter following transient HOXA9 binding to its cognate recognition sequence. PRMT5 induces symmetric dimethylation of Arg140 on HOXA9, an event essential for E-selectin induction. In summary, PRMT5 is a critical coactivator component in a newly defined, HOXA9-containing transcription complex. Moreover, stimulus-dependent methylation of HOXA9 is essential for ELAM expression during the EC inflammatory response.

Nicotine-mediated induction of E-selectin in aortic endothelial cells requires Src kinase and E2F1 transcriptional activity

Smoking is highly correlated with enhanced likelihood of atherosclerosis by inducing endothelial dysfunction. In endothelial cells, various cell-adhesion molecules including E-selectin, are shown to be upregulated upon exposure to nicotine, the addictive component of tobacco smoke; however, the molecular mechanisms underlying this induction are poorly understood. Here we demonstrate that nicotine-induced E-selectin transcription in human aortic endothelial cells (HAECs) could be significantly blocked by α7-nAChR subunit inhibitor, α-BT, Src-kinase inhibitor, PP2, or siRNAs against Src or β-Arrestin-1 (β-Arr1). Further, chromatin immunoprecipitations show that E-selectin is an E2F1 responsive gene and nicotine stimulation results in increased recruitment of E2F1 on E-selectin promoter. Inhibiting E2F1 activity using RRD-251, a disruptor of the Rb–Raf-1 kinase interaction, could significantly inhibit the nicotine-induced recruitment of E2F1 to the E-selectin promoter as well as E-selectin expression. Interestingly, stimulation of HAECs with nicotine results in increased adhesion of U937 monocytic cells to HAECs and could be inhibited by pre-treatment with RRD-251. Similarly, depletion of E2F1 or Src using RNAi blocked the increased adhesion of monocytes to nicotine-stimulated HAECs. These results suggest that nicotine-stimulated adhesion of monocytes to endothelial cells is dependent on the activation of α7-nAChRs, β-Arr1 and cSrc regulated increase in E2F1-mediated transcription of E-selectin gene. Therefore, agents such as RRD-251 that can target activity of E2F1 may have potential therapeutic benefit against cigarette smoke induced atherosclerosis.

High Glucose-treated Macrophages Augment E-Selectin Expression in Endothelial Cells

E-selectin expression by endothelial cells (ECs) is crucial for leukocyte recruitment during the inflammatory response. Macrophage accumulation and serum E-selectin elevation are features of type 2 diabetes mellitus. However, the interactions between macrophages and ECs in regulating vascular endothelial function are not clearly understood. We investigated the mechanisms underlying the modulation of EC E-selectin expression by high glucose (HG)-treated macrophages. Macrophage-conditioned media (MCM) were prepared from HG-treated macrophages. EC stimulation with HG-MCM induced increases the expression and secretion of E-selectin. By using specific inhibitors and small interfering RNAs, we demonstrate that the activation of the JNK and p38 MAPK pathways are critical for HG-MCM-induced E-selectin expression. Transcription factor ELISA and chromatin immunoprecipitation assays further showed that HG-MCM increases the NF-κB- and AP-1 DNA-binding activities in ECs. The inhibition of NF-κB and AP-1 activation by specific siRNAs blocks the HG-MCM-induced E-selectin promoter activity and expression. Protein arrays and blocking assays using neutralizing antibodies demonstrated that macrophage inflammatory protein 1α and 1β in HG-MCM are major mediators for the induction of EC E-selectin expression. These data support the hypothesis that E-selectin up-regulation stimulated by macrophages may play an active role in atherogenesis in the HG condition and suggest a new mechanism by which arterial disease is accelerated in diabetes.

Decreased Expression of KLF2 and KLF4 Induced by Antiphospholipid Antibodies Promotes NF-Kb-Mediated Endothelial Cell Activation and Is Modulated by CBP/p300

AbstractAbstract 4315Antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy loss in the presence of antiphospholipid antibodies (APLA). These antibodies are directed primarily against phospholipid-bound β2-glycoprotein I (β2GPI). Anti-β2GPI antibodies activate endothelial cells, enhancing the expression of adhesion molecules and tissue factor, and the secretion of proinflammatory cytokines. Krüppel-like factors (KLF) regulate endothelial cell inflammatory responses. KLF2 and KLF4 mediate anti-atherosclerotic and anti-inflammatory effects in endothelial cells, and we have hypothesized that alterations in the expression or activity of KLF2 or KLF4 may modulate the endothelial cell response to APLA. In preliminary studies, we have observed that endothelial cell activation induced by APLA/anti-β2GPI antibodies inhibits the expression of KLF2 and KLF4, and as demonstrated by our laboratory and others, is accompanied by activation of NF-kB. However, forced expression of KLF2 or KLF4 by plasmid-mediated transfection of endothelial cells inhibits neither the phosphorylation of ser536 of the p65 subunit of NF-kB, nor the nuclear translocation of p65 in response to APLA/anti-β2GPI antibodies. Despite the lack of effect on forced KLF2 or KLF4 expression in endothelial cells on p65 phosphorylation, expression of either of these factors inhibits NF-κB transcriptional activity with corresponding inhibition of cellular activation as measured by inhibition of cell-surface E-selectin expression as well as E-selectin promoter activity. Inhibition of NF-kB transcriptional activity by KLF2 and KLF4 appears to be due to recruitment of the CBP/p300 cofactor away from NF-kB by KLF2 or KLF4, since augmenting the cellular pool of CBP/p300 by transfection restores NF-κB activity and endothelial cell activation responses. Similarly, treatment of APLA-activated endothelial cells with CBP/p300 siRNA inhibits NF-kB transcriptional activity regardless of the levels of KLF2 or KLF4. These data suggest that APLA inhibit KLF expression and that these changes promote the acquisition of a prothrombotic endothelial cell phenotype. CBP/p300 may serve as a molecular switch that determines the relative antithrombotic activities of KLFs versus the prothrombotic, inflammatory responses induced by NF-kB in APLA/anti-β2GPI antibody activated endothelial cells. Disclosures:No relevant conflicts of interest to declare.

Protein arginine methyltransferase 5 (PRMT5) contributes positively to the induction of the E‐selectin gene in endothelial cells (EC) by binding to HOXA9

Multiple transcription factors, including the homeobox protein HOXA9, participate in cytokine induction of the EC‐leukocyte adhesion molecule E‐selectin in EC. HOX proteins often require other activators/co‐activators to affect rates of transcription. We have recently reported that HOXA9 associates with the type II protein arginine methyltransferase PRMT5 in TNF‐a‐activated EC. Although the cellular distribution and the protein level of PRMT5 were unaltered in EC in response to TNF, nuclear PRMT5 transiently associated with HOXA9. The kinetics of this binding was consistent with the association of these proteins with the E‐selectin promoter, as shown by chromatin immunoprecipitation (ChIP) assays. Specific depletion of PRMT5 protein by siRNA led to a significant decrease in both E‐selectin promoter activity and E‐selectin gene induction by TNF. Binding of HOXA9 to its recognition sequence was required for PRMT5 recruitment to the promoter, but not vice‐versa. Moreover, TNF‐dependent transient simultaneous association of both PRMT5 and a symmetrically dimethylated arginine (SDMA)‐containing protein was readily detected by ChIP. This latter protein appears to be distinct from HOXA9. These results demonstrate that HOXA9 recruits PRMT5 to the E‐selectin promoter where it acts as a transcriptional co‐activator. (NIH HL29582)

PPARα and PPARγ effectively protect against HIV‐induced inflammatory responses in brain endothelial cells

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors which down-regulate inflammatory signaling pathways. Therefore, we hypothesized that alterations of PPAR functions can contribute to human immunodeficiency virus-1 (HIV-1)-induced dysfunction of brain endothelial cells. Indeed, treatment with HIV-1 transactivator of transcription (Tat) protein decreased PPAR transactivation in brain endothelial cells. We next stably over-expressed PPARalpha and PPARgamma in a newly developed cell line of human brain endothelial cells (hCMEC/D3 cells). Tat-induced up-regulation of inflammatory mediators, such as interleukin (IL)-1beta, tumor necrosis factor-alpha, CCL2, and E-selectin were markedly attenuated in hCMEC/D3 over-expressing PPARalpha or PPARgamma. These results were confirmed in CCL2 and E-selectin promoter activity studies. Similar protective effects were observed in hCMEC/D3 after activation of PPARgamma by exogenous PPAR agonists (dPGJ(2) and rosiglitazone). PPAR over-expression also prevented Tat-induced binding activity and transactivation of nuclear factor-kappaB. Importantly, increased PPAR activity attenuated induction of IL-1beta, tumor necrosis factor-alpha, CCL2, and E-selectin in hCMEC/D3 cells co-cultured with HIV-1-infected Jurkat cells. The protective effects of PPAR over-expression were reversed by the antagonists of PPARalpha (MK886) or PPARgamma (GW9662). The present data suggest that targeting PPAR signaling may provide a novel therapeutic approach to attenuate HIV-1-induced local inflammatory responses in brain endothelial cells.

Angiostatin K1-3 induces E-selectin via AP1 and Ets1: a mediator for anti-angiogenic action of K1-3.

Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1-3 included. We previously showed that K1-3 was the most potent angiostatin to induce E-selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1-3-induced E-selectin expression and investigate the role of E-selectin in the anti-angiogenic action of K1-3. Quantitative real time RT-PCR and Western blotting analyses confirmed a time-dependent increase of E-selectin mRNA and protein induced by K1-3. Subcellular fractionation and immunofluorescence microscopy showed the co-localization of K1-3-induced E-selectin with caveolin 1 (Cav1) in lipid rafts in which E-selectin may behave as a signaling receptor. Promoter-driven reporter assays and site-directed mutagenesis showed that K1-3 induced E-selectin expression via promoter activation and AP1 and Ets-1 binding sites in the proximal E-selectin promoter were required for E-selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1-3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E-selectin by K1-3. A modulatory role of E-selectin in the anti-angiogenic action of K1-3 was manifested by both overexpression and knockdown of E-selectin followed by cell proliferation assay. We show that K1-3 induced E-selectin expression via AP1 and Ets-1 binding to the proximal E-selectin promoter (-356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E-selectin as a novel target for the anti-angiogenic therapy.

Conditional expression of microRNA against E-selectin inhibits leukocyte–endothelial adhesive interaction under inflammatory condition

Human E-selectin, an endothelial adhesion molecule, is induced in the brain arteries by cerebral ischemia and participates in the infiltration of leukocytes that cause inflammatory reaction leading to brain damage. To prevent leukocyte infiltration in the brain, we designed gene therapeutic constructs to suppress E-selectin expression. The constructs were composed of microRNAs (miR-E1 and miR-E2) complementary to the human E-selectin cDNA, which were directed by a minimum cis-element of the human E-selectin promoter. Transfection in human aorta endothelial cells (HAECs) with these constructs revealed that the E-selectin promoter was sufficiently activated in response to tumor necrosis factor-α (TNF-α), and miR-E1 and miR-E2 could suppress E-selectin expression resulting in the significant inhibition of leukocyte adhesion. These results suggested that the combination of the E-selectin promoter and microRNAs could allow the restricted expression of transgenes in activated endothelial cells and diminish leukocyte recruitment.

Human Immunodeficiency Virus Type 1 Tat Protein Inhibits the SIRT1 Deacetylase and Induces T Cell Hyperactivation

Symptoms of T cell hyperactivation shape the course and outcome of HIV-1 infection, but the mechanism(s) underlying this chronic immune activation are not well understood. We find that the viral transactivator Tat promotes hyperactivation of T cells by blocking the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase SIRT1. Tat directly interacts with the deacetylase domain of SIRT1 and blocks the ability of SIRT1 to deacetylate lysine 310 in the p65 subunit of NF-kappaB. Because acetylated p65 is more active as a transcription factor, Tat hyperactivates the expression of NF-kappaB-responsive genes, a function lost in SIRT1-/- cells. These results support a model where the normal function of SIRT1 as a negative regulator of T cell activation is suppressed by Tat during HIV infection. These events likely contribute to the state of immune cell hyperactivation found in HIV-infected individuals.