E-selectin Expression In Endothelial Cells Research Articles

ESDN inhibits melanoma progression by blocking E-selectin expression in endothelial cells via STAT3

An interactive crosstalk between tumor and stroma cells is essential for metastatic melanoma progression. We evidenced that ESDN/DCBLD2/CLCP1 plays a crucial role in endothelial cells during the spread of melanoma. Precisely, increased extravasation and metastasis formation were revealed in ESDN-null mice injected with melanoma cells, even if the primary tumor growth, vessel permeability, and angiogenesis were not enhanced. Interestingly, improved adhesion of melanoma cells to ESDN-depleted endothelial cells was observed, due to the presence of higher levels of E-selectin transcripts/proteins in ESDN-defective cells. In accordance with these results, anticorrelation was observed between ESDN and E-selectin in human endothelial cells. Most importantly, our data revealed that cimetidine, an E-selectin inhibitor, was able to block cell adhesion, extravasation, and metastasis formation in ESDN-null mice, underlying a major role of ESDN in E-selectin transcription upregulation, which according to our data, may presumably be linked to STAT3. Based on our results, we propose a protective role for ESDN during the spread of melanoma and reveal its therapeutic potential.

Cell-specific and athero-protective roles for RIPK3 in a murine model of atherosclerosis.

ABSTRACTReceptor-interacting protein kinase 3 (RIPK3) was recently implicated in promoting atherosclerosis progression through a proposed role in macrophage necroptosis. However, RIPK3 has been connected to numerous other cellular pathways, which raises questions about its actual role in atherosclerosis. Furthermore, RIPK3 is expressed in a multitude of cell types, suggesting that it may be physiologically relevant to more than just macrophages in atherosclerosis. In this study, Ripk3 was deleted in macrophages, endothelial cells, vascular smooth muscle cells or globally on the Apoe−/− background using Cre-lox technology. To induce atherosclerosis progression, male and female mice were fed a Western diet for three months before tissue collection and analysis. Surprisingly, necroptosis markers were nearly undetectable in atherosclerotic aortas. Furthermore, en face lesion area was increased in macrophage- and endothelial-specific deletions of Ripk3 in the descending and abdominal regions of the aorta. Analysis of bone-marrow-derived macrophages and cultured endothelial cells revealed that Ripk3 deletion promotes expression of monocyte chemoattractant protein 1 (MCP-1) and E-selectin in these cell types, respectively. Western blot analysis showed upregulation of MCP-1 in aortas with Ripk3-deficient macrophages. Altogether, these data suggest that RIPK3 in macrophages and endothelial cells protects against atherosclerosis through a mechanism that likely does not involve necroptosis. This protection may be due to RIPK3-mediated suppression of pro-inflammatory MCP-1 expression in macrophages and E-selectin expression in endothelial cells. These findings suggest a novel and unexpected cell-type specific and athero-protective function for RIPK3.This article has an associated First Person interview with the first author of the paper.

Irisin as a mediator between obesity and vascular inflammation in Chinese children and adolescents

Background and aimThis study aims to characterize the role of Irisin in obesity and early onset metabolic and vascular sequelae in Chinese children. Furthermore, we aim to examine whether Irisin mediate endothelial cells dysfunction and vascular inflammation which eventually leads to obesity. Methods and resultsWe quantified plasma Irisin levels using enzyme-linked immunosorbent assay (ELISA) among 85 lean and 120 obese children, and assessed the association of Irisin levels with anthropometric, metabolic, cardiovascular and inflammatory parameters of obese children comparing with lean children. We further characterized the markers for endothelial cells and inflammation between obese and lean children to assess potential correlations. In addition, a potential direct effect of Irisin on the expression of endothelial adhesion molecules was assessed in human coronary artery endothelial cells. Irisin levels from obese children was significantly lower than lean children, and this reduced Irisin levels is correlated with certain physical parameters of studied children. Moreover, we identified significant inverse associations between inflammation markers of endothelial activation with Irisin levels in obese children. Multiple regression analyses confirm Irisin serves as a strong predictor of SDS-SBP, Ang-2, ICAM-1, E-selectin and hsCRP levels, independent of SDS-BMI. Lifestyle intervention results in a significant improvement of these cardiovascular and inflammatory parameters, and these were accompanied by a significant improvement of Irisin levels and weight loss. Finally, in the mediation effect model, our data showed that Irisin changes act as partial mediators of the relationship between SDS-BMI changes and changes in inflammatory and endothelial parameters for ICAM-1, E-selectin and hsCRP after controlling for covariates. Likewise, on the cellular level, Irisin inhibition hsCPR, ICAM-1 and E-selectin expression in endothelial cells, whereas Ang-2, VCAM-1 and eNOS expression were unaffected. ConclusionsOur study showed that Irisin levels change may indicate the early stages of cardiovascular disease in obese children.

Indoxyl sulfate enhances IL-1β-induced E-selectin expression in endothelial cells in acute kidney injury by the ROS/MAPKs/NFκB/AP-1 pathway.

Uremic toxins are considered a risk factor for cardiovascular disorders in kidney diseases, but it is not known whether, under inflammatory conditions, they affect adhesion molecule expression on endothelial cells, which may play a critical role in acute kidney injury (AKI). In the present study, in cardiovascular surgery-related AKI patients, who are known to have high plasma levels of the uremic toxin indoxyl sulfate (IS), plasma levels of IL-1β were found to be positively correlated with plasma levels of the adhesion molecule E-selectin. In addition, high E-selectin and IL-1β expression were seen in the kidney of ischemia/reperfusion mice in vivo. We also examined the effects of IS on E-selectin expression by IL-1β-treated human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. IS pretreatment of HUVECs significantly increased IL-1β-induced E-selectin expression, monocyte adhesion, and the phosphorylation of mitogen-activated protein kinases (ERK, p38, and JNK) and transcription factors (NF-κB and AP-1), and phosphorylation was decreased by pretreatment with inhibitors of ERK1/2 (PD98059), p38 MAPK (SB202190), and JNK (SP600125). Furthermore, IS increased IL-1β-induced reactive oxygen species (ROS) production and this effect was inhibited by pretreatment with N-acetylcysteine (a ROS scavenger) or apocynin (a NADPH oxidase inhibitor). Gel shift assays and ChIP-PCR demonstrated that IS enhanced E-selectin expression in IL-1-treated HUVECs by increasing NF-κB and AP-1 DNA-binding activities. Moreover, IS-enhanced E-selectin expression in IL-1β-treated HUVECs was inhibited by Bay11-7082, a NF-κB inhibitor. Thus, IS may play an important role in the development of cardiovascular disorders in kidney diseases during inflammation by increasing endothelial expression of E-selectin.

The Dual E-Selectin/CXCR4 Inhibitor, GMI-1359, Enhances Efficacy of Anti-Leukemia Chemotherapy in FLT3-ITD Mutated Acute Myeloid Leukemia

Aberrant activation of the FMS-like tyrosine kinase-3 (FLT3) is driven by internal tandem duplication (ITD) mutations in the FLT3 gene, which are commonly observed in patients with acute myeloid leukemia (AML). Hence, FLT3 represents an attractive therapeutic target in AML (Weisberg et al., 2002). Indeed, several small molecule FLT3 inhibitors including sorafenib have showed encouraging efficacy in reducing leukemia blasts in the peripheral blood in FLT3 mutated AML patients. However, these agents have little effect on leukemic stem cells in the bone marrow (BM) microenvironment (Borthakur et al., 2011; Fathi and Chabner, 2011; Zhang et al., 2008).The BM microenvironment is enriched with cytokines and adhesion molecules, such as CXCR4 and E-selectin, which are believed to provide AML cells protection against chemotherapeutic agents (Horacek et al., 2013; Peled and Tavor, 2013). In fact, treatment with sorafenib markedly upregulated CXCR4 levels in FLT3 -mutated cells. In addition, leukemia cells can activate endothelial cells (EC) that induce adhesion of a sub-set of the leukemia cells through E-selectin. The adherent AML cells are sequestered in a nonproliferative state that further protects them from chemotherapy (Pezeshkian et al., 2013). Therefore, blocking CXCR4 and E-selectin in parallel could theoretically eliminate the protection provided by the interaction of leukemic cells with their BM microenvironment and enhance effectiveness of chemotherapy in FLT3-mutant AML patients. In the present study, we evaluated the effectiveness of a dual CXCR4 and E-selectin antagonist, GMI-1359 (GlycoMimetics, Inc., Rockville, MD), in targeting FLT3-ITD-mutant AML in vitro and in vivo. High levels of CXCR4 expression were observed in several human and murine AML cell lines, which was further increased in hypoxic (i.e., 1% oxygen) conditions that mimic the BM microenvironment. These FLT3 -ITD leukemic cell lines also expressed hypoxia-responsive, functional E-selectin ligands identified by reactivity with an antibody (HECA452) that binds the same carbohydrate epitope required for binding to E-selectin. One such E-selectin ligand CD44 increased in FLT3 -ITD cells cultured in hypoxia compared to those cultured in normoxia (i.e. 21% oxygen). In addition, hypoxia also enhanced CXCR4 expression on mesenchymal stem cells (MSC) and EC such as HUVEC. In hypoxic co-cultures of the FLT3 -ITD-mutant leukemia cells MV4-11 or MOLM14 with MSCs and ECs (i.e., HUVEC or TeloHAEC), the presence of the dual E-selectin/CXCR4 inhibitor GMI-1359 effectively reduced leukemic cell adhesion by ~ 50% to the MSC/EC feeder layer compared to the PBS-treated control (p<0.05), even in the presence of TNFa, which induces E-selectin expression in EC. However, an E-selectin specific inhibitor only reduced adhesion of MV4-11 and MOLM14 by ~ 20%. GMI-1359 markedly abrogated the protection provided by the BM microenvironment (i.e., hypoxia and/or MSC and EC) of Baf3-FLT3 -ITD leukemic cells treated with the FLT3 inhibitor sorafenib. Apoptosis was induced in 36.6%, 35.6% and 48.9% of leukemic cells cultured with sorafenib alone, sorafenib and an E-selectin inhibitor or sorafenib and GMI-1359, respectively.The significance of these in vitro findings were studied in vivo. Female SCID beige mice were injected iv with MV4-11 and followed for survival. Beginning 14 days post tumor injection, cohorts of mice (n=10/group) were treated with saline, GMI-1359 (40 mg/kg), standard chemotherapy cytarabine plus daunorubicin, or a combination of GMI-1359 and chemotherapy. Combined treatment of mice with GMI-1359 (40 mg/kg) and chemotherapy demonstrated a profound survival benefit compared to controls or chemotherapy alone at day 135 after leukemia cell injection (i.e., 67% vs. 11% or 30%, p=0.0011 and 0.0406, respectively). Single agent treatment with GMI-1359 was statistically indistinguishable from saline alone or chemotherapy alone. In a separate cohort of MV4.11-engrafted mice, the single administration of GMI-1359 increased circulating WBC and leukemic MV4-11cells, which persisted for at least 8 hrs. This effect was consistent with GMI-1359 disrupting the protective effects of the tumor microenvironment and mobilizing MV4-11 cells from the BM niche.. These findings provide the pre-clinical basis for the evaluation of GMI-1359 in patients with FLT3 -mutant AML. [Display omitted] DisclosuresZhang:Karyopharm: Research Funding. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Laminar shear stress elicit distinct endothelial cell E-selectin expression pattern via TNFα and IL-1β activation.

The ability to discriminate cell adhesion molecule expression between healthy and inflamed endothelium is critical for therapeutic intervention in many diseases. This study explores the effect of laminar flow on TNFα-induced E-selectin surface expression levels in human umbilical vein endothelial cells (HUVECs) relative to IL-1β-induced expression via flow chamber assays. HUVECs grown in static culture were either directly (naïve) activated with cytokine in the presence of laminar shear or pre-exposed to 12 h of laminar shear (shear-conditioned) prior to simultaneous shear and cytokine activation. Naïve cells activated with cytokine in static served as control. Depending on the cell shear history, fluid shear is found to differently affect TNFα-induced relative to IL-1β-induced HUVEC expression of E-selectin. Specifically, E-selectin surface expression by naïve HUVECs is enhanced in the 8-12 h activation time range with simultaneous exposure to shear and TNFα (shear-TNFα) relative to TNFα static control whereas enhanced E-selectin expression is observed in the 4-24 h range for shear-IL-1β treatment relative to IL-1β static control. While exposure of HUVECs to shear preconditioning mutes shear-TNFα-induced E-selectin expression, it enhances or down-regulates shear-IL-1β-induced expression dependent on the activation period. Under dual-cytokine-shear conditions, IL-1β signaling dominates. Overall, a better understanding of E-selectin expression pattern by human ECs relative to the combined interaction of cytokines, shear profile and history can help elucidate many disease pathologies.

The Effect of Resuscitation Fluids on Neutrophil-Endothelial Cell Interactions in Septic Shock

Fluid resuscitation is essential in the treatment of septic shock. This study examined the effect of resuscitative fluids (RFs) on sepsis-induced neutrophil-endothelial cell interactions. The RFs studied were 0.9% saline (NS), Ringer's lactate (RL), 7.5% saline and dextran-70 (DHS), 5% albumin (AL), and 6% hydroxyethyl starch (HS). Platelets and neutrophils were obtained from normal volunteers, and plasma was obtained from patients with septic shock. Microslides coated with human umbilical endothelial vein cell (HUVEC) and platelet-neutrophil solutions were primed with septic plasma with/without the RF. Neutrophil rolling velocity, leukoaggregation, and neutrophil adherence were determined. Separately, platelet-neutrophil solutions and endothelial cells were exposed to septic plasma with/without RFs, and cellular activation, neutrophil superoxide production, and endothelial cell E-selectin expression were assessed. Ringer's lactate decreased neutrophil rolling velocity and increased aggregation and adherence. Normal saline had no effect on these parameters. Hydroxyethyl starch and AL increased neutrophil rolling velocity and decreased adherence and aggregation when HUVECs were preincubated with the RF. Dextran-70 and 7.5% saline decreased neutrophil-endothelial cell interactions in both HUVECs and platelet/neutrophil preincubated experiments. Ringer's lactate increased activation of neutrophils and platelets, whereas AL decreased their activation. Other than NS, all the RFs increased neutrophil superoxide production. Ringer's lactate increased endothelial cell E-selectin release, whereas AL and HS both decreased its release. These data suggest that fluids used in the resuscitation of septic shock vary in their effects on sepsis-induced neutrophil-endothelial cell interactions. Ringer's lactate amplifies the effects of sepsis, while NS appears to have minimal impact. Dextran-70 and 7.5% saline, AL, and HS in varying degrees decrease sepsis-related neutrophil-endothelial cell interactions and activation.

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.

Inhibition of CD40L induced E-selectin expression in endothelial cells through extracellular signal-regulated kinase signaling pathway

To investigate the regulatory effects of atorvastatin on CD40L induced E-selectin expression in endothelial cells and the association thereof with signal pathway. Human umbilical vein endothelial cells (HUVECs) were obtained from umbilical cord newly delivered and incubated with CD40L for 24 hours with or without pretreated by atorvastatin of the concentrations of 0.1, 1, or 10 micromol/L. The protein and mRNA levels of E-selectin were detected by flow cytometry and reverse transcription polymerase chain reaction (RT-PCR) respectively. The extracellular signal regulated kinase (ERK) 1/2 activation was analyzed by Western blotting. The E-selectin mRNA expression level of the HUVECs treated by atorvastatin was lower than that of the CD40L stimulation group by 33.33% when the atorvastatin concentration was 1 micromol/L, and was lower by 45.16% when the atorvastatin concentration was 10 micromol/L. The E-selectin protein expression level of the HUVECs treated by atorvastatin was lower than that of the CD40L stimulation group by 48.68% when the atorvastatin concentration was 1 micromol/L, and was lower by 70.25% when the atorvastatin concentration was 10 micromol/L. The phosphorylation level of ERK1/2 was enhanced by CD40L stimulation and the CD40L induced phosphorylation of ERK1/2 decreased by 81% +/- 6%, 73% +/- 5%, and 41% +/- 5% respectively after atorvastatin stimulation. Atorvastatin decreases the CD40L induced E-selectin expression in endothelial cells while atorvastatin at 0.1-10 micromol/L concentration-dependently through inhibiting the activation of ERK1/2.

Expression of vascular adhesion molecules on human endothelia in autoimmune thyroid disorders.

Cellular activation and expression of certain adhesion molecules within vascular endothelium is a critical event in leucocyte recruitment and emigration. A wide array of different adhesion receptors has been identified to mediate the interaction between endothelial cells (EC) and leucocyte subpopulations. In this study, the tissue expression of E-selectin, P-selectin, CD31, and endoglin endothelial cell adhesion molecules was studied on thyroid tissue from patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). We found an up-regulated expression of E-selectin in EC in GD and HT thyroids, specifically in those areas more severely inflamed, with no reactivity in control thyroids. P-selectin was basally expressed in postcapillary venules in control glands, with an increased expression in HT and GD glands. On the other hand, increased CD31 expression was found on perifollicular, small and large venule EC from GD and HT glands, that correlated with the severity of mononuclear infiltration. In addition, CD31 expression was observed in some intrathyroidal macrophages and T cells in close proximity to CD31+ EC. Furthermore, a markedly enhanced expression of endoglin, a transforming growth factor-beta binding protein, was mainly located on perifollicular EC and EC from small venules as well as in adjacent macrophages from GD and HT thyroid glands. This enhanced expression of E- and P-selectins, CD31 and endoglin by thyroid EC in GD and HT may reflect their ability to regulate leucocyte trafficking and activation.

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.

Endogenous glucocorticoids control neutrophil mobilization from bone marrow to blood and tissues in non-inflammatory conditions.

We have shown that endogenous glucocorticoids control neutrophil mobilization in the absence of inflammation. In this study the role of the glucocorticoid receptor (GR) in the physiological control of neutrophil mobilization was investigated, focusing on the specific mechanisms for mature neutrophils in bone marrow, circulating neutrophils and endothelial cells. Male Wistar rats were treated with RU 38486 or adrenalectomized. Cell numbers in bone marrow and circulation were morphologically quantified and expressions of L-selectin determined by flow cytometry. Expressions of P-selectin, E-selectin, PECAM-1, VCAM-1 and ICAM-1 were measured by immunohistochemistry on vessels of cremaster muscle and their mRNA levels quantified in primary cultured endothelial cells. NF-kappaB activity in neutrophils and endothelium was quantified by EMSA. RU 38486 treatment altered the maturation phases of neutrophilic lineage and reduced expression of L-selectin in mature neutrophils from bone marrow; increased the number of neutrophils in the circulation and elevated the expression of L-selectin in these cells. P-selectin and E-selectin expression in endothelial cells was unchanged by adrenalectomy or RU 38486 treatment. Membrane expressions, mRNA levels of ICAM-1, VCAM-1 and PECAM-1 and NF-kappaB translocation into the nucleus were higher in the endothelium of adrenalectomized and RU 38486 treated rats. Endogenous glucocorticoids, through activation of GR on neutrophils, physiologically control the rolling behaviour of these cells and, by modulating endothelial functions, affect their adhesiveness. The molecular mechanism induced by activated GR is different in each cell, as NF-kappaB translocation was only altered in endothelial cells.

Shear stress-induced activation of the AMP-activated protein kinase regulates FoxO1a and angiopoietin-2 in endothelial cells

Phosphorylation of forkhead box O (FoxO) transcription factors induces their nuclear exclusion and proteosomal degradation. Here, we investigated the effect of fluid shear stress on FoxO1a in primary cultures of human endothelial cells and the kinases that regulate its phosphorylation. Shear stress (12 dynes/cm2) elicited the phosphorylation, nuclear exclusion, and degradation of FoxO1a. Inhibition of Akt signalling using either a dominant negative (DN) mutant of Akt or downregulation of Gab1 largely failed to affect the shear stress-induced changes in FoxO1a, while a DN-AMP-activated protein kinase (AMPK) abrogated its shear stress-induced phosphorylation and degradation. Similar effects were observed using the AMPK inhibitor compound C. Moreover, in an in vitro assay, the AMPK directly phosphorylated FoxO1a. As FoxO1a regulates the expression of angiopoietin-2 (Ang-2), we determined the role of shear stress and the AMPK in this phenomenon. Not only did the DN-AMPK increase the expression of Ang-2 in cells maintained under static conditions, it also abrogated the shear stress-induced decrease in FoxO1a and Ang-2 protein levels. Functionally, Ang-2 sensitizes endothelial cells to the effects of tumour necrosis factor (TNF)-alpha, and DN-AMPK increased basal endothelial cell E-selectin expression and permeability as well as the increase induced by TNF-alpha. These data indicate that the AMPK activated by fluid shear stress is a novel regulator of FoxO1a phosphorylation and protein levels. Moreover, as the AMPK-dependent phosphorylation and degradation of FoxO1a attenuates Ang-2 expression and protects against the pro-inflammatory actions of TNF-alpha, this kinase may be a useful target to prevent the progression of vascular diseases.

Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cells and its inhibition by shear stress

AbstractE-selectin is a major adhesion molecule expressed by endothelial cells (ECs), which are exposed to shear stress and neighboring smooth muscle cells (SMCs). We investigated the mechanisms underlying the modulation of EC E-selectin expression by SMCs and shear stress. SMC coculture induced rapid and sustained increases in expression of E-selectin and phosphorylation of interleukin-1 (IL-1) receptor-associated kinase glycoprotein-130, as well as the downstream mitogen-activated protein kinases (MAPKs) and Akt. By using specific inhibitors, dominant-negative mutants, and small interfering RNA, we demonstrated that activations of c-Jun-NH2-terminal kinase (JNK) and p38 of the MAPK pathways are critical for the coculture-induced E-selectin expression. Gel shifting and chromatin immunoprecipitation assays showed that SMC coculture increased the nuclear factor-κB (NF-κB)–promoter binding activity in ECs; inhibition of NF-κB activation by p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced E-selectin promoter activity. Protein arrays and blocking assays using neutralizing antibodies demonstrated that IL-1β and IL-6 produced by EC/SMC cocultures are major contributors to the coculture induction of EC signaling and E-selectin expression. Preshearing of ECs at 12 dynes/cm2 inhibited the coculture-induced EC signaling and E-selectin expression. Our findings have elucidated the molecular mechanisms underlying the SMC induction of EC E-selectin expression and the shear stress protection against this SMC induction.

The inhibition of TNF- α-induced E-selectin expression in endothelial cells via the JNK/NF- κB pathways by highly N-acetylated chitooligosaccharides

Chitooligosaccharides (COS) have been shown to regulate various cellular and biological functions. However, the effect of COS on inflammatory responses of the cells remains unclear. We investigated the regulatory effect of highly N-acetylated COS (NACOS) on tumor necrosis factor- α (TNF- α)-induced endothelial cell (EC) E-selectin expression, which is crucial for leukocyte recruitment. ECs were kept as controls or pre-treated with NACOS for different times, and then stimulated with TNF- α for 4 h. The results show that pre-treating ECs with NACOS inhibited the TNF- α-induced E-selectin expression in a dose- and time-dependent manner. This NACOS-mediated inhibition in E-selectin expression was regulated at the transcriptional level, but not due to changes in mRNA stability. Stimulation of ECs with TNF- α-induced rapid increases in the phosphorylation of their mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated kinase (ERK), c-Jun-NH 2-terminal kinase (JNK), and p38 MAPK]; the inhibitor for JNK (i.e., SP600125), but not those for ERK (i.e., PD98059) and p38 MAPK (i.e., SB203580), attenuated this TNF-α-induced E-selectin expression. Pre-treating ECs with NACOS inhibited the TNF- α-induced JNK activation, suggesting that JNK was involved in the inhibitory effect of NACOS on TNF- α-induced E-selectin expression. Pre-treating ECs with NACOS inhibited the TNF- α-induced p65 and p50 mRNA expressions. Gel shifting and chromatin immunoprecipitation assays showed that NACOS blocked the TNF- α-induced increases in the binding activity and in vivo promoter binding of nuclear factor- κB (NF- κB) in ECs. Our findings provide a molecular mechanism by which NACOS inhibit TNF- α-induced E-selectin expression in ECs, and a basis for using NACOS in pharmaceutical therapy against inflammation.

Macrophage release of transforming growth factor beta1 during resolution of monosodium urate monohydrate crystal-induced inflammation.

It has previously been shown that as monocytes differentiate into macrophages, they lose the ability to secrete proinflammatory cytokines in response to monosodium urate monohydrate (MSU) crystals. The purpose of this study was to investigate whether MSU crystals induce macrophages to secrete antiinflammatory factor instead. Human monocyte or macrophage isolates were prepared from samples obtained from healthy volunteer donors either by differentiation of blood monocytes in vitro or by collecting cells from skin blisters during the early or late phase of the dermal inflammatory response to cantharidin. Monocyte or macrophage isolates were then incubated with MSU crystals for 24 hours, and culture supernatants were assayed for candidate antiinflammatory mediators (by enzyme-linked immunosorbent assay) and for the capacity to activate or suppress endothelial cell E-selectin expression and secondary neutrophil recruitment under shear flow. Analysis of supernatants from in vitro-differentiated macrophages revealed that transforming growth factor beta1 (TGFbeta1) was induced following MSU crystal stimulation (mean +/- SEM 1.50 +/- 0.24 ng/ml/10(6) cells), but there was no evidence of interleukin-10 (IL-10), IL-1 receptor antagonist, or tumor necrosis factor (TNF) receptor p55 release. Macrophage TGFbeta1 significantly suppressed endothelial cell E-selectin expression and secondary neutrophil capture on endothelial monolayers stimulated with supernatants from MSU-treated monocytes. Leukocytes isolated from resolving (40-hour) skin blisters similarly elaborated TGFbeta1 when challenged with MSU crystals (0.66 +/- 1.3 ng/ml/10(5) CD14+ cells). In contrast, cells isolated from acute (16-hour) skin blisters secreted TNFalpha (0.49 +/- 0.08 ng/ml/10(5) CD14+ cells) but no detectable TGFbeta1. These data provide further support for the concept that differentiated macrophages play a protective role in the pathophysiology of gout, and they identify macrophage TGFbeta1 as a mediator of paracrine suppression during the resolution phase of inflammation.

Bleomycin Induces E-selectin Expression in Cultured Umbilical Vein Endothelial Cells by Increasing Its mRNA Levels through Activation of NF-κB/Rel

Treatment with bleomycin (BLM) often results in the development of acute lung injury and subsequent fibrosis by mechanisms that are not well understood. It is hypothesized that active oxygen species and proteases generated by inflammatory cells that accumulate in the bronchoalveolus are responsible and that BLM-induced E-selectin expression on the endothelial surface has an essential role as a trigger in the accumulation of inflammatory cells. We aimed to understand the mechanisms of BLM-induced E-selectin expression in endothelial cells. The E-selectin antigen was induced on the surface of cultured human umbilical vein endothelial cells (HUVECs) exposed to BLM in a dose- and time-dependent manner, with an increase in mRNA levels. The binding of nuclear proteins to oligonucleotides containing the NF-κB/Rel or AP-1 binding motif of the promoter of the human E-selectin gene significantly increased in BLM-treated HUVECs compared with control cells. The increased E-selectin antigen levels induced by BLM were abrogated by pretreatment with MG132 (10 μM) or PDTC (100 μM), in parallel with inhibition of the NF-κB/Rel activation and nuclear translocation, although no inhibition of the AP-1 activation was observed. Supershift assays indicated that NF-κB/Rel bound with the NF-κB/Rel binding motif contained p65, p50, and c-Rel subunits. The AP-1 activation by BLM was inhibited by pretreatment of the cells with SB203580, although BLM-induced expression of E-selectin was not attenuated. These results suggest that BLM can directly induce E-selectin expression with an increase in transcription in endothelial cells through activation and nuclear translocation of NF-κB/Rel without mediation of inflammatory cytokines.

Bleomycin Induces E-selectin Expression in Cultured Umbilical Vein Endothelial Cells by Increasing Its mRNA Levels through Activation of NF-κB/Rel

Treatment with bleomycin (BLM) often results in the development of acute lung injury and subsequent fibrosis by mechanisms that are not well understood. It is hypothesized that active oxygen species and proteases generated by inflammatory cells that accumulate in the bronchoalveolus are responsible and that BLM-induced E-selectin expression on the endothelial surface has an essential role as a trigger in the accumulation of inflammatory cells. We aimed to understand the mechanisms of BLM-induced E-selectin expression in endothelial cells. The E-selectin antigen was induced on the surface of cultured human umbilical vein endothelial cells (HUVECs) exposed to BLM in a dose- and time-dependent manner, with an increase in mRNA levels. The binding of nuclear proteins to oligonucleotides containing the NF-κB/Rel or AP-1 binding motif of the promoter of the human E-selectin gene significantly increased in BLM-treated HUVECs compared with control cells. The increased E-selectin antigen levels induced by BLM were abrogated by pretreatment with MG132 (10 μM) or PDTC (100 μM), in parallel with inhibition of the NF-κB/Rel activation and nuclear translocation, although no inhibition of the AP-1 activation was observed. Supershift assays indicated that NF-κB/Rel bound with the NF-κB/Rel binding motif contained p65, p50, and c-Rel subunits. The AP-1 activation by BLM was inhibited by pretreatment of the cells with SB203580, although BLM-induced expression of E-selectin was not attenuated. These results suggest that BLM can directly induce E-selectin expression with an increase in transcription in endothelial cells through activation and nuclear translocation of NF-κB/Rel without mediation of inflammatory cytokines.

Poly ADP ribose-polymerase inhibitors prevent the upregulation of ICAM-1 and E-selectin in response to Th1 cytokine stimulation.

We studied the role of poly-ADP-ribose polymerase (PARP) in the mobilization of ICAM-1, VCAM-1, and E-selectin by TNF-alpha and IL-1beta in cultured human endothelial cells. Enzyme linked immunosorbent analysis (ELISA) was used to assess if ICAM-1, VCAM-1, and E-selectin were expressed at the cell surface, and if PARP inhibition (using the selective PARP inhibitor GPI 6150) blocked the induced expression. Endothelial cell adhesion molecule expression was evaluated at 4 and at 24 h after cytokine stimulation. At 4 h ICAM-1 and E-selectin, but not VACM-1, were stimulated by both IL-1beta and TNF-alpha. Blocking PARP via GPI 6150 only affected TNF-alpha induced E-selectin expression at 4 hours. ICAM-1, VCAM-1, and E-selectin expression were all stimulated by both IL-1beta and TNF-alpha in the 24 h assays. PARP inhibition with GPI 6150 blocked the IL-1beta mediated stimulation of both ICAM-1 and E-selectin expression, and blocked TNF-alpha stimulation of ICAM-1 expression at 24 h. These experiments suggest that specific PARP inhibition may provide a novel method of controlling leukocyte dependent inflammation through the reduction of ICAM-1 and E-selectin expression in endothelial cells in response to cytokines.

Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils.

We previously have demonstrated that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified low density lipoprotein (MM-LDL), activates endothelial cells to bind monocytes. 1-Palmitoyl-2- (5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1- palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), which are present in OxPAPC, MM-LDL, and atherosclerotic lesions, were shown to have a major role in the activation of endothelial cells. We now demonstrate that these two highly similar molecules have dramatically different effects on leukocyte endothelial interactions. POVPC is a potent regulator of monocyte-specific endothelial interactions. Treatment of endothelial cells with POVPC increased monocyte binding by inducing the surface expression of the connecting segment 1 domain of fibronectin; no increase in neutrophil binding was observed. In addition, POVPC strongly inhibited lipopolysaccharide-mediated induction of neutrophil binding and expression of E-selectin protein and mRNA. This inhibition was mediated by a protein kinase A-dependent pathway, resulting in down-regulation of NF-kappaB-dependent transcription. In contrast, PGPC induced both monocyte and neutrophil binding and expression of E-selectin and vascular cell adhesion molecule 1. We present evidence to suggest that the two phospholipids act by different novel receptors present in Xenopus laevis oocytes and that POVPC, but not PGPC, stimulates a cAMP-mediated pathway. At concentrations equal to that present in MM-LDL, the effect of POVPC dominates and inhibits PGPC-induced neutrophil binding and E-selectin expression in endothelial cells. In summary, our data provide evidence that both POVPC and PGPC are important regulators of leukocyte-endothelial interactions and that POVPC may play a dominant role in a number of chronic inflammatory processes where oxidized phospholipids are known to be present.