Bacteroides fragilis enterotoxin upregulates intercellular adhesion molecule-1 in endothelial cells via an aldose reductase-, MAPK-, and NF-κB-dependent pathway, leading to monocyte adhesion to endothelial cells

Enterotoxigenic Bacteroides fragilis (ETBF) produces a ～ 20-kDa heat-labile enterotoxin (BFT) that plays an essential role in mucosal inflammation. Although a variety of inflammatory cells is found at ETBF-infected sites, little is known about leukocyte adhesion in response to BFT stimulation. We investigated whether BFT affected the expression of ICAM-1 and monocytic adhesion to endothelial cells (ECs). Stimulation of HUVECs and rat aortic ECs with BFT resulted in the induction of ICAM-1 expression. Upregulation of ICAM-1 was dependent on the activation of IκB kinase (IKK) and NF-κB signaling. In contrast, suppression of AP-1 did not affect ICAM-1 expression in BFT-stimulated cells. Suppression of NF-κB activity in HUVECs significantly reduced monocytic adhesion, indicating that ICAM-1 expression is indispensable for BFT-induced adhesion of monocytes to the endothelium. Inhibition of JNK resulted in a significant attenuation of BFT-induced ICAM-1 expression in ECs. Moreover, inhibition of aldose reductase significantly reduced JNK-dependent IKK/NF-κB activation, ICAM-1 expression, and adhesion of monocytes to HUVECs. These results suggest that a signaling pathway involving aldose reductase, JNK, IKK, and NF-κB is required for ICAM-1 induction in ECs exposed to BFT, and may be involved in the leukocyte-adhesion cascade following infection with ETBF.     

Statins attenuate high mobility group box-1 protein induced vascular endothelial activation : a key role for TLR4/NF-κB signaling pathway

High mobility group box-1 (HMGB1) has recently been implicated as a proinflammatory cytokine that plays critical roles in endothelial dysfunction and atherosclerosis. Atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, exerts anti-inflammatory effects in the cardiovascular system beyond its cholesterol-lowering property. The aim of our study was to investigate whether atorvastatin inhibits HMGB1-induced vascular endothelial activation, and elucidate the underlying molecular mechanism. In this study, we found that atorvastatin, at concentrations ranging from 0.1 to 10 μM, effectively and in a dose-dependent manner inhibited HMGB1-induced endothelial cells (ECs) activation. Incubation of ECs with 10 μM atorvastatin reduced adhesion molecules (ICAM-1 and E-selectin) expression concomitant with a significant inhibition in HMGB1-stimulated leukocyte-endothelial adhesion. Further experiments showed that atorvastatin markedly suppressed HMGB1-induced Toll like receptor 4 (TLR4) expression, Nuclear factor kappaB (NF-κB) nuclear translocation and DNA binding activity in ECs. Similar effects were also observed in ECs pretreated with the TLR4- specific inhibitor CLI-095, suggesting an important role of TLR4/NF-κB pathway. These findings indicate that atorvastatin attenuates HMGB1-induced vascular endothelial activation. The underlying mechanism involves, at least in part, inhibition of TLR4/NF-κB-dependent signaling pathway, which provied the new evidence for therapeutic application of statins to target inflammatory processes in cardiovascular disease.     

OxLDL induces endothelial dysfunction and death via TRAF3IP2: Inhibition by HDL3 and AMPK activators

Oxidized low-density lipoprotein (oxLDL) induces endothelial cell death through the activation of NF-κB and AP-1 pathways. TRAF3IP2 is a redox-sensitive cytoplasmic adapter protein and an upstream regulator of IKK/NF-κB and JNK/AP-1. Here we show that oxLDL-induced death in human primary coronary artery endothelial cells (ECs) was markedly attenuated by the knockdown of TRAF3IP2 or the lectin-like oxLDL receptor 1 (LOX-1). Further, oxLDL induced Nox2/superoxide-dependent TRAF3IP2 expression, IKK/p65 and JNK/c-Jun activation, and LOX-1 upregulation, suggesting a reinforcing mechanism. Similarly, the lysolipids present in oxLDL (16:0-LPC and 18:0-LPC) and minimally modified LDL also upregulated TRAF3IP2 expression. Notably, whereas native HDL3 reversed oxLDL-induced TRAF3IP2 expression and cell death, 15-lipoxygenase-modified HDL3 potentiated its proapoptotic effects. The activators of the AMPK/Akt pathway, adiponectin, AICAR, and metformin, attenuated superoxide generation, TRAF3IP2 expression, and oxLDL/TRAF3IP2-mediated EC death. Further, both HDL3 and adiponectin reversed oxLDL/TRAF3IP2-dependent monocyte adhesion to endothelial cells in vitro. Importantly, TRAF3IP2 gene deletion and the AMPK activators reversed oxLDL-induced impaired vasorelaxation ex vivo. These results indicate that oxLDL-induced endothelial cell death and dysfunction are mediated via TRAF3IP2 and that native HDL3 and the AMPK activators inhibit this response. Targeting TRAF3IP2 could potentially inhibit progression of atherosclerotic vascular diseases.     

Shear stress modulation of IL-1β-induced E-selectin expression in human endothelial cells

Background

Endothelial cells (ECs) are continuously exposed to hemodynamic forces imparted by blood flow. While it is known that endothelial behavior can be influenced by cytokine activation or fluid shear, the combined effects of these two independent agonists have yet to be fully elucidated.

Methodology

We investigated EC response to long-term inflammatory cues under physiologically relevant shear conditions via E-selectin expression where monolayers of human umbilical vein ECs were simultaneously exposed to laminar fluid shear and interleukin-1ß (shear-cytokine activation) in a parallel plate flow chamber.

Results and Conclusion

Naïve ECs exposed to shear-cytokine activation display significantly higher E-selectin expression for up to 24 hr relative to ECs activated in static (static-cytokine). Peak E-selectin expression occurred after 8–12 hr of continuous shear-cytokine activation contrary to the commonly observed 4–6 hr peak expression in ECs exposed to static-cytokine activation. Cells with some history of high shear conditioning exhibited either high or muted E-selectin expression depending on the durations of the shear pre-conditioning and the ensuing shear-cytokine activation. Overall, the presented data suggest that a high laminar shear enhances acute EC response to interleukin-1ß in naïve or shear-conditioned ECs as may be found in the pathological setting of ischemia/reperfusion injury while conferring rapid E-selectin downregulation to protect against chronic inflammation.     

A20基因转导抑制异种移植中猪内皮细胞的活化

延缓性排斥反应 (DXR)是进行异种器官移植亟待解决的问题之一 .在DXR过程中 ,核心事件是异种移植物血管内皮细胞的活化 .核转录因子 (NF κB)在这一过程中起着重要的作用 .A2 0是一个具有锌指结构的蛋白 ,它可以抑制以NF κB为核心的信号转导系统 .向猪血管内皮细胞(PAEC)中导入A2 0基因 ,经RT PCR检测 ,A2 0基因能在细胞中稳定表达 .细胞生长曲线分析表明 ,A2 0的基因转导并不影响细胞的正常生长 ;电泳迁移率变动分析 (EMSA)表明 ,A2 0基因能抑制由肿瘤坏死因子 α(TNF α)诱导的NF κB的激活 ;流式细胞术分析表明 ,A2 0基因对受NF κB调控的一个重要炎症因子———E选择素的表达抑制率达 77 2 % .A2 0基因转导可能成为克服异种移植过程中DXR的手段之一 .     

Docosahexaenoic acid attenuates VCAM-1 expression and NF-κB activation in TNF-α-treated human aortic endothelial cells

This study was conducted to test the hypothesis that n-3 polyunsaturated fatty acids are able to down-regulate expression of adhesion molecules and nuclear factor-κB (NF-κB) activation in vascular endothelial cells, in addition to reducing atherosclerotic lesions in vivo. We report here that docosahexaenoic acid (DHA) reduces atherosclerotic lesions in the aortic arteries of apolipoprotein E knockout (apoE^-/-) mice. Consistent with the observation in animal study, DHA inhibited THP-1 cell adhesion to tumor necrosis factor α (TNF-α)-activated human aortic endothelial cells (HAECs). Expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) on the cell surface of HAECs was determined by cell-surface enzyme-linked immunosorbent assay. DHA and eicosapentaenoic acid decreased VCAM-1 expression in a dose-dependent manner in TNF-α treated HAECs, while cis-linoleic acid and arachidonic acid did not have any significant effect on either VCAM-1 or ICAM-1 expression. Moreover, DHA significantly reduced VCAM-1 protein expression in the cell lysates of TNF-α-treated HAECs, as determined by Western blot analysis. In line with NF-κB signaling pathway, DHA suppressed the TNF-α-activated IκBα phosphorylation and degradation as well as IκB kinase-β phosphorylation. Subsequently, translocation of the NF-κB (p50/p65) and AP-1 (c-Fos/c-Jun) subunits was down-regulated by DHA in the nucleus of HAECs. These results suggest that DHA negatively regulates TNF-α-induced VCAM-1 expression through attenuation of NF-κB signaling pathway and AP-1 activation. This study provides evidence that DHA may contribute to the prevention of atherosclerosis and inflammatory diseases in vivo.     

Protective cross talk between activated protein C and TNF signaling in vascular endothelial cells: implication of EPCR, noncanonical NF-κB, and ERK1/2 MAP kinases

Activated protein C (APC) is a natural anticoagulant protease that displays cytoprotective and antiinflammatory activities and has been demonstrated to reduce mortality of patients with severe sepsis. However, APC signaling is not fully understood. This study further investigated the antiinflammatory effects of APC in vascular endothelial cells (EC) and examined the cross talk between APC and TNF signaling. Analysis of the regulatory mechanisms mediated by APC on vascular human EC shows that APC impairs TNF signaling by triggering a preemptive activation of intracellular pathways. We found that APC signaling causes a moderate but significant induction of cell adhesion molecules (CAMs) including VCAM-1 at mRNA and protein levels. Activation of the noncanonical NF-κB and ERK1/2 are both pivotal to APC signaling leading to VCAM-1 expression. APC upregulates TNF receptor-associated factor 2 (TRAF2) and phosphorylates NF-κB p65 at Ser276 and Ser536 independently of IκB degradation. The ultimate protective antiinflammatory effect of APC in response to TNF is associated with a sustained activation of ERK1/2 and Akt while phosphorylation of NF-κB p65 is precluded. Inhibitors of ERK (PD98059 and U0126) abolish the antiinflammatory signal mediated by APC. Blocking antibodies and silencing assays also suggest that, in EC, protease-activated receptor 1 and endothelial protein C receptor (EPCR) both conduct ERK activation and VCAM-1 induction in response to APC. To conclude, APC protects EC by attenuating CAM expression during inflammation. APC engages a regulatory cross talk involving EPCR, ERK, and NF-κB that impairs TNF signaling.     

Dehydroepiandrosterone inhibits the activation and dysfunction of endothelial cells induced by high glucose concentration

Dehydroepiandrosterone (DHEA), an adrenal steroid, has a protective role against diabetes; however, its mechanisms of action are unknown. Here, we focus on the effect of DHEA on the activation of endothelial cells induced by a high concentration of glucose. Adhesion on U937 cells, expression of adhesion molecules, production of ROS and NO, expression of eNOS, and translocation of NF-κB were evaluated in human umbilical vein endothelial cells (HUVEC) treated with high concentrations of glucose, DHEA, or both. High concentrations of glucose (>20mM) induced an increase in adhesion, an increment in mainly E-selectin and PECAM-1 expression, as well as in ROS and NO production, eNOS expression, translocation of NF-κB, and degradation of its inhibitor IκB-α. DHEA abolished adhesion and the increase of E-selectin, ICAM-1, VCAM-1, and PECAM-1 induced by glucose. In addition, DHEA completely blocked oxidative stress and decreased translocation of NF-κB and the degradation of IκB-α induced by glucose. These results suggest that DHEA protects against the activation of endothelial cells induced by high concentrations of glucose, indicating that DHEA could be useful in the treatment of hyperglycemia and diabetes.     

CO-releasing molecules and increased heme oxygenase-1 induce protein S-glutathionylation to modulate NF-κB activity in endothelial cells

Protein glutathionylation is a protective mechanism that functions in response to mild oxidative stress. Carbon monoxide (CO) can increase the reactive oxygen species concentration from a low level via the inhibition of cytochrome c oxidase. We therefore hypothesized that CO would induce NF-κB-p65 glutathionylation and then show anti-inflammatory effects. In this study, we found that CO-releasing molecules suppress TNFα-induced monocyte adhesion to endothelial cells (ECs) and reduce ICAM-1 expression. Moreover, CO donors were further found to exert their inhibitory effects by blocking NF-κB-p65 nuclear translocation, but do so independent of IκBα degradation, in TNFα-treated ECs. In addition, p65 protein glutathionylation represents the response signal to CO donors and is reversed by the reducing agent dithiothreitol. Thiol modification of the cysteine residue in the p65 RHD region was required for the CO-modulated NF-κB activation. The suppression of p65 glutathionylation by a GSH synthesis inhibitor, BSO, and by catalase could also attenuate TNFα-induced p65 nuclear translocation and ICAM-1 expression. CO donors induce Nrf2 activation and Nrf2 siRNA suppresses CO-induced p65 glutathionylation and inhibition. Furthermore, we found that the CO donors induce heme oxygenase-1 (HO-1) expression, which increases p65 glutathionylation. In contrast, HO-1 siRNA attenuates CO donor- and hemin-induced p65 glutathionylation. Our results thus indicate that the glutathionylation of p65 is likely to be responsible for CO-mediated NF-κB inactivation and that the HO-1-dependent pathway may prolong the inhibitory effects of CO donors upon TNFα treatment of ECs.     

Rosiglitazone attenuates NF-κB-mediated Nox4 upregulation in hyperglycemia-activated endothelial cells

Vascular complications, a major cause of morbidity and mortality in diabetic patients, are related to hyperglycemia-induced oxidative stress. Previously, we reported that rosiglitazone (RSG) attenuated vascular expression and activity of NADPH oxidases in diabetic mice. The mechanisms underlying these effects remain to be elucidated. We hypothesized that RSG acts directly on endothelial cells to modulate vascular responses in diabetes. To test this hypothesis, human aortic endothelial cells (HAECs) were exposed to normal glucose (NG; 5.6 mmol/l) or high glucose (HG; 30 mmol/l) concentrations. Select HAEC monolayers were treated with RSG, caffeic acid phenethyl ester (CAPE), diphenyleneiodonium (DPI), small interfering (si)RNA (to NF-κB/p65 or Nox4), or Tempol. HG increased the expression and activity of the NADPH oxidase catalytic subunit Nox4 but not Nox1 or Nox2. RSG attenuated HG-induced NF-κB/p65 phosphorylation, nuclear translocation, and binding to the Nox4 promoter. Inhibiting NF-κB with CAPE or siNF-κB/p65 also reduced HG-induced Nox4 expression and activity. HG-induced H(2)O(2) production was attenuated by siRNA-mediated knockdown of Nox4, and HG-induced HAEC monocyte adhesion was attenuated by treatment with RSG, DPI, CAPE, or Tempol. These results indicate that HG exposure stimulates HAEC NF-κB activation, Nox4 expression, and H(2)O(2) production and that RSG attenuates HG-induced oxidative stress and subsequent monocyte-endothelial interactions by attenuating NF-κB/p65 activation and Nox4 expression. This study provides novel insights into mechanisms by which the thiazolidinedione peroxisome proliferator-activated receptor-γ ligand RSG favorably modulates endothelial responses in the diabetic vasculature.     

AGGF1 is a novel anti-inflammatory factor associated with TNF-α-induced endothelial activation

Endothelial activation contributes to the development of vascular inflammation and subsequent vascular diseases, particularly atherosclerosis. AGGF1, a new member of angiogenic factors with a FHA and a G-patch domain, has been shown critical for the regulation of vascular differentiation and angiogenesis. In this study, we found that various inflammatory cytokines strongly induced the expression of AGGF1 in endothelial cells (ECs) and identified AGGF1 as a novel anti-inflammatory factor both in vivo and in vitro. Overexpression of AGGF1 significantly repressed the expression of pro-inflammatory molecules such as E-Selectin, ICAM-1, and IL-8 and the adhesion of monocytes onto ECs activated by TNF-α. Conversely, the knockdown of AGGF1 resulted in the increased expressions of these pro-inflammatory molecules and the enhanced monocyte-EC interaction. We further demonstrated that AGGF1 potently attenuated TNF-α triggered NF-κB pathway, as indicated by the decreased promoter activity, nuclear distribution and phosphorylation of NF-κB p65 subunit as well as the increased protein level of IκBα. This inhibitory effect of AGGF1 was further proved through blocking the phosphorylation of ERK induced by TNF-α. Finally, we showed that the FHA domain of AGGF1 was required for its anti-inflammatory effect. Thus, our findings for the first time demonstrate that AGGF1 suppresses endothelial activation responses to TNF-α by antagonizing the ERK/NF-κB pathway, which makes AGGF1 a promising therapeutic candidate for the prevention and treatment of inflammatory diseases.     

ERK5 Protein Promotes, whereas MEK1 Protein Differentially Regulates, the Toll-like Receptor 2 Protein-dependent Activation of Human Endothelial Cells and Monocytes

Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation up-regulates the expression of inflammatory mediators and of TLR2 itself and modulates important endothelial functions, including coagulation and permeability. We defined TLR2 signaling pathways in EC and tested the hypothesis that TLR2 signaling differs in EC and monocytes. We found that ERK5, heretofore unrecognized as mediating TLR2 activation in any cell type, is a central mediator of TLR2-dependent inflammatory signaling in human umbilical vein endothelial cells, primary human lung microvascular EC, and human monocytes. Additionally, we observed that, although MEK1 negatively regulates TLR2 signaling in EC, MEK1 promotes TLR2 signaling in monocytes. We also noted that activation of TLR2 led to the up-regulation of intracellularly expressed TLR2 and inflammatory mediators via NF-κB, JNK, and p38-MAPK. Finally, we found that p38-MAPK, JNK, ERK5, and NF-κB promote the attachment of human neutrophils to lung microvascular EC that were pretreated with TLR2 agonists. This study newly identifies ERK5 as a key regulator of TLR2 signaling in EC and monocytes and indicates that there are fundamental differences in TLR signaling pathways between EC and monocytes.