抑瘤素M在高血糖致人主动脉平滑肌细胞增殖中的作用

目的 研究高血糖对人巨噬细胞中抑瘤素M表达的影响以及抑瘤素M对人主动脉平滑肌细胞增殖的影响.方法 采用佛波酯孵育人单核/巨噬细胞系THP-1细胞,诱导其分化为巨噬细胞.应用人细胞因子抗体芯片筛选出受高血糖(15 mmol/L)调控的人巨噬细胞源性细胞因子,并经Western blot和酶联免疫吸附试验(ELISA)技术验证.运用逆转录-聚合酶链反应(RT-PCR)明确人主动脉内皮细胞、平滑肌细胞及巨噬细胞中是否存在抑瘤素M受体GP130-OSMRβ二聚体及GP130-LIFR二聚体表达.运用细胞增殖实验(XTT法)研究抑瘤素M对人主动脉平滑肌细胞增殖的影响.采用单因素方差分析进行统计学分析.结果 佛波酯孵育48 h可诱导THP-1细胞分化为巨噬细胞.细胞因子芯片结果显示高血糖可上调抑瘤素M在巨噬细胞中的表达,并为Western blot及ELISA所证实.RT-PCR显示人主动脉内皮细胞、平滑肌细胞及巨噬细胞均可表达GP130-OSMRβ二聚体及GP130-LIFR二聚体.XTT法证实抑瘤素M可促进血管平滑肌细胞增殖,与0μg/L抑瘤素M(吸光度为0.468±0.032)相比,5μg/L(吸光度为0.503±0.026)、10μg/L(吸光度为0.520±0.027)、20μg/L抑瘤素M(吸光度为0.513±0.026)人主动脉平滑肌细胞增殖均明显增加(F=10.40,P<0.05).结论 高血糖可促进巨噬细胞中抑瘤素M的表达和分泌.抑瘤素M通过与人主动脉平滑肌细胞中的受体结合而促进血管平滑肌细胞的增殖,进而部分参与糖尿病大血管病变的发生发展.     

Functional activities of receptors for tumor necrosis factor-alpha on human vascular endothelial cells

Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in the control of endothelial cell function and hence in regulating traffic of circulating cells into tissues in vivo. Stimulation of endothelial cells in vitro by TNF-alpha increases the surface expression of leukocyte adhesion molecules, enhances cytokine production, and induces tissue factor procoagulant activity. In the present study, we have examined the relative roles of the two cell surface receptors for TNF- alpha (p55 and p75) on endothelial cells, using antibodies with both agonistic and antagonistic activities. We report that anti-p55 receptor agonistic antibody Htr-9 induces the expression of tissue factor antigen and the release of interleukin-8 (IL-8) and granulocyte- macrophage colony-stimulating factor (GM-CSF). In contrast, there is very little or no activation of endothelial cell responses by an anti- p75 agonist. TNF-alpha-induced expression of tissue factor and adhesion molecules, and release of IL-8 and GM-CSF, are decreased by antibodies with antagonistic activities for either receptor, although the effect of anti-p55 antibodies is markedly greater than that of anti-p75 antibodies. The responses of endothelial cells to lymphotoxin/TNF-beta are significantly decreased by anti-p55 antagonists alone. Our data suggest that endothelial cell responses to TNF-alpha, such as expression of tissue factor and adhesion molecules for mononuclear cells, which may be important in the pathogenesis of atherosclerosis, are mediated predominantly, but not exclusively, by the p55 TNF receptor.     

Dyscoordinate expression of tumor necrosis factor-alpha by human blood monocytes and alveolar macrophages

Previous studies have shown that human alveolar macrophages produce less interleukin-1 (IL-1) in response to lipopolysaccharide (LPS) than do their precursors, blood monocytes. The purpose of this study was to compare the capacities of alveolar macrophages and blood monocytes to synthesize tumor necrosis factor (TNF) in response to LPS. Alveolar macrophages were obtained by bronchoalveolar lavage of healthy nonsmoking subjects, and blood monocytes were obtained by adherence of mononuclear cells to plastic. TNF activity was measured in supernatants and cell lysates as cytotoxicity to L929 fibroblasts (uptake of neutral red at 570 nm). TNF activity of alveolar macrophages stimulated at 10(6) cells/ml with LPS (10 micrograms/ml) for 16 h was 596 +/- 367, and of blood monocytes it was 60 +/- 84 U/ml (mean +/- SD, p less than 0.005). At no concentration of LPS and at no period of stimulation did alveolar macrophages express less TNF activity than did blood monocytes. In concurrent experiments, supernatants of LPS-stimulated alveolar macrophages contained less IL-1 activity than did blood monocytes. Lysates of both cell types contained less than 20% of total TNF activity. The TNF activity of LPS-stimulated alveolar macrophages was neutralized greater than 99% by monoclonal antibody to TNF-alpha; control monoclonal antibody OKT3 had no effect. Next, alveolar macrophages and blood monocytes were biosynthetically labeled with [3H]leucine during incubation with LPS; supernatants were immunoprecipitated with anti-TNF, and precipitates were electrophoresed on polyacrylamide gels. Autoradiographs indicated that immunoreactive TNF was produced by both blood monocytes and alveolar macrophages and that the relative molecular weights were identical (17,000).(ABSTRACT TRUNCATED AT 250 WORDS)     

Polymyxin B prevents lipopolysaccharide-induced release of tumor necrosis factor-alpha from alveolar macrophages

Polymyxin B (PmB) blocks many of the toxic effects of lipopolysaccharide by mechanisms that are not yet understood. The production of tumor necrosis factor-alpha (TNF-alpha) by isolated rat alveolar macrophages in response to lipopolysaccharide and macrophage-activating factor was blocked by PmB at concentrations of 100, 10, and 1 micrograms/ml. Gentamicin enhanced rather than inhibited TNF production at the 100-micrograms/ml concentrations and had no effect at low concentration. Similar inhibitory effects were induced by PmB in an in vivo model in which rat macrophage TNF production was stimulated by intratracheally injected lipopolysaccharide. Because many of the effects of lipopolysaccharide are mediated by TNF, this inhibition provides a mechanism to explain the protection afforded by PmB against lipopolysaccharide-induced toxicity.     

Role of NAD(P)H:quinone oxidoreductase 1 on tumor necrosis factor-alpha-induced migration of human vascular smooth muscle cells

OBJECTIVES: In a preliminary study, NAD(P)H:quinone oxidoreductase 1 (NQO1) was found to be highly expressed in cultured human aortic smooth muscle cells (HASMC) and dicumarol, a NQO1 inhibitor and a coumarin-derived natural anticoagulant, suppressed tumor necrosis factor (TNF)-alpha-induced HASMC migration. Therefore, it was hypothesized that NQO1 plays an important role in the regulation of vascular smooth muscle cells (VSMC) migration activated by TNF-alpha. METHODS AND RESULTS: Gelatin zymography, reporter gene, electrophoretic mobility shift and Western blotting assays showed that dicumarol, but not other coumarin-derived anticoagulants, inhibited TNF-alpha-induced HASMC migration and suppressed TNF-alpha-induced matrix metalloproteinase (MMP)-9 expression and secretion in a dose-dependent manner. In addition, down-regulation of NQO1 by transfection of its small interfering RNA similarly inhibited TNF-alpha-induced MMP-9 secretion, indicating that dicumarol-mediated inhibition of MMP-9 expression is due in large part to inhibition of NQO1. Down-regulation of NQO1 inhibits MMP-9 gene expression by suppressing activation of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1), well-known key elements mediating MMP-9 gene expression in its promoter, via the p38 MAPK and JNK pathways. CONCLUSION: The results of the present study demonstrate that down-regulation of NQO1 effectively suppresses TNF-alpha-induced HASMC migration through inhibition of MMP-9 expression, suggesting that NQO1 may be a potential target for the prevention of vascular disorders related to migration of VSMC.     

Cyclooxygenase-2 is required for tumor necrosis factor-alpha- and angiotensin II-mediated proliferation of vascular smooth muscle cells

Tumor necrosis factor-alpha (TNF-alpha) and angiotensin II (Ang II) induced a transient increase in vascular smooth muscle cell (VSMC) cyclooxygenase-2 (COX-2) mRNA accumulation, without affecting COX-1 mRNA levels. The kinetics of COX-2 mRNA accumulation were similar in VSMCs challenged with either TNF-alpha or Ang II; mRNA accumulation peaked at 2 hours and decreased to control levels by approximately 6 hours. Accumulation of COX-2 mRNA was associated with a time-dependent increase of COX-2 protein expression that displayed similar kinetics in response to either TNF-alpha or Ang II. Both the increase in COX-2 mRNA accumulation and protein expression in response to either TNF-alpha or Ang II were inhibited by the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD098059. In addition, the AT(1)-selective receptor antagonist losartan attenuated the Ang II-mediated increase in COX-2 mRNA accumulation; the AT(2)-selective antagonist PD123319 had no effect. Prostacyclin I(2) synthesis was tightly coupled to expression of COX-2, whereas prostaglandin E(2) and thromboxane A(2) (TXA(2)) synthesis may be associated with differential usage of COX-1 and COX-2. The COX-2-selective inhibitors NS-398 and nimesulide and the TXA(2) receptor antagonist BMS 180,291 inhibited TNF-alpha- and Ang II-mediated increases in DNA content and cell number by approximately 95%. These findings suggest that a prostanoid derived from COX-2, possibly TXA(2), may contribute to VSMC hyperplasia in vessel injury or pathophysiological conditions associated with elevated levels of either TNF-alpha or Ang II.     

Human arterial smooth muscle cells synthesize granulocyte colony-stimulating factor in response to interleukin-1 alpha and tumor necrosis factor-alpha.

Vascular smooth muscle cells (SMC) are a major cell type comprising the walls of blood vessels. We report the synthesis of granulocyte colony-stimulating factor (G-CSF) by cultured human SMC obtained from the internal mammary artery and thoracic aorta. Interleukin-1 alpha (IL-1 alpha) greatly increased in a dose-dependent manner the amount of this cytokine produced by the SMC, with tumor necrosis factor-alpha (TNF-alpha) being less effective. Newly formed G-CSF could be detected in culture supernatants within 6 hours after IL-1 alpha or TNF-alpha treatment. Northern blot analysis of SMC stimulated with IL-1 alpha and TNF-alpha showed an increase in the amount of mRNA for G-CSF as compared with control cells. Enhanced G-CSF mRNA levels were observed when SMC were treated with cycloheximide in the absence or presence of added cytokine. In vasculitis, the walls of blood vessels become inflamed as evidenced by a leucocytic infiltrate usually dominated by polymorphonuclear neutrophil leukocytes (PMNs). G-CSF is known to stimulate PMNs, and our findings raise the possibility that G-CSF made by SMC contributes to the development of vasculitis lesions.     

Differential maturation of murine bone-marrow derived dendritic cells with lipopolysaccharide and tumor necrosis factor-alpha

Dendritic cells (DCs) play a key role in the interface between the innate and acquired immune systems. In response to both exogenous as well as endogenous signals, DCs undergo a programmed maturation to become an efficient, antigen-presenting cell. Yet little is known regarding the differential responses by endogenous versus exogenous stimuli on DC maturation. In the present report, we have compared the phenotypic, functional, and genome-wide expression responses associated with maturation by bone marrow derived DCs to either an endogenous danger signal, tumor necrosis factor-alpha (TNF-alpha), or a microbial product, bacterial lipopolysaccharide (LPS). Examination of the cell surface expression of DCs as well as cytokine production demonstrated that patterns of DC maturation varied dramatically depending upon the stimulus. Whereas LPS was highly effective in terms of inducing phenotypic and functional maturation, TNF-alpha exposure produced a phenotypically distinct DC. Gene expression patterns in DCs 6 and 24 h after LPS and TNF-alpha exposure revealed that these activation signals produce fundamentally different genomic responses. Supervised analysis revealed that the expression of 929 probe sets discriminated among the treatment groups, and the patterns of gene expression in TNF-alpha stimulated DCs were more similar to unstimulated cells at both 6 and 24 h post-stimulation than to LPS-stimulated cells at the same time points. These findings reveal that DCs are capable of a varying phenotypic response to different antigens and endogenous signals.     

Apoptosis of vascular smooth muscle cells is induced by Fas ligand derived from endothelial cells.

Although Fas-mediated cell death may play a role in atherogenesis, causal data in support of this hypothesis are lacking. The present study investigated the possibility that endothelial cells are involved in vascular smooth muscle cell (VSMC) apoptosis via the Fas-FasL pathway, and hence in atherogenesis. FACS analysis detected FasL on the surface of human umbilical vein endothelial cells (HUVECs) and immunofluorescence staining of the HUVECs demonstrated high levels of FasL in the intracellular compartment. FasL was down-regulated 4 h after tumor necrosis factor (TNFalpha) treatment, coinciding with maximal surface expression of the adhesion molecules vascular cell adhesion molecule-1 and E-selectin. However, the down-regulation of FasL expression was transient, as surface expression returned within 24 h of TNFalpha treatment. When cocultured with VSMCs, the FasL-expressing EC could kill the VSMCs in a manner that could be blocked by recombinant Fas-Fc, deployed as a soluble receptor for Fas. Moreover, when human coronary arteries were studied with immunohistochemistry using G247-4 monoclonal antibody for the detection of FasL, few FasL positive EC were observed in diffuse intimal thickening. In contrast, endothelium overlying the plaque showed prominent and uniform expression of FasL. These findings suggest that the Fas/FasL pathway can be used by EC to induce VSMC apoptosis in the atherosclerotic lesion.     

Dihydrotestosterone decreases tumor necrosis factor-alpha and lipopolysaccharide-induced inflammatory response in human endothelial cells

CONTEXT: An increasing body of evidence suggests that testosterone may exert beneficial effects on the development of atherosclerosis. It was suggested that testosterone may act after conversion into estradiol and activation of the estrogen receptors; however, a direct role of androgens on the vascular wall has been proposed. OBJECTIVE: We investigated the effects of dihydrotestosterone on the proinflammatory response observed in human endothelial cells. DESIGN: Human endothelial cells isolated from umbilical cords were incubated with lipopolysaccharide or TNFalpha in the presence or absence of dihydrotestosterone (DHT). mRNA and cellular proteins were processed for gene expression studies, and transient transfection experiments were performed to investigate molecular mechanisms involved in the effects observed. SETTING: These studies took place at the Department of Pharmacological Sciences, University of Milan, Milan, Italy. RESULTS: Lipopolysaccharide and TNFalpha induced VCAM-1 and ICAM-1 mRNA and protein expression, as detected by real-time quantitative PCR, fluorescence-activated cell sorting, and confocal microscopy, but this effect was inhibited when cells were incubated with DHT. In addition, DHT inhibited mRNA expression of IL-6, MCP-1, CD40, TLR4, PAI-1, and Cox-2 and the release of cytokines and chemokines such as GRO, granulocyte-macrophage colony-stimulating factor, and TNF. The DHT effect was counteracted by bicalutamide, an antagonist of the androgen receptor. Furthermore, when cells were cotransfected with a Cox-2 promoter or a 3X-NF-kappaB luciferase reporter vector and a plasmid expressing the human androgen receptor, DHT treatment inhibited the increase of the luciferase activity observed with TNFalpha. CONCLUSION: DHT could positively regulate endothelial function through the control of the inflammatory response mediated by nuclear factor-kappaB in endothelial cells.     

Human blood-derived macrophages induce apoptosis in human plaque-derived vascular smooth muscle cells by Fas-ligand/Fas interactions

Human atherosclerotic plaques that rupture are characterized by relatively low vascular smooth muscle cell (VSMC) and high inflammatory cell contents. Ruptured plaques also contain higher numbers of apoptotic VSMCs than do stable lesions, suggesting that VSMC apoptosis may promote plaque rupture. We examined the ability of human monocytes/macrophages to induce apoptosis of VSMCs derived from human carotid plaque, aortic media, and coronary media. Macrophages, but not T lymphocytes, induced a dose-dependent apoptosis of VSMCs, which required monocyte maturation to macrophages and direct cell-cell contact/proximity. VSMC apoptosis was inhibited by neutralizing antibodies to Fas-ligand (Fas-L) or an Fas-Fc fusion protein, indicating the requirement for membrane-bound Fas and Fas-L. Monocyte maturation was associated with increased surface expression of Fas-L, coincident with the onset of cytotoxicity. VSMCs expressed surface Fas, which was increased in plaque VSMCs, and plaque VSMCs also underwent Fas-induced apoptosis. We conclude that human macrophages potently induce human VSMC apoptosis, which requires direct cell-cell interactions and is in part dependent on Fas/Fas-L interactions. Macrophage-induced VSMC apoptosis may therefore directly promote plaque rupture.     

亲脂素增加血管平滑肌细胞的脂质集聚

目的 观察乙酰化的低密度脂蛋白(AcLDL)对血管平滑肌细胞亲脂素(adipophilin)表达的影响及亲脂素对血管平滑肌细胞的AcLDL摄取及脂质集聚的影响.从而探讨其在糖尿病大血管病变发生中的作用.方法 以不同浓度AcLDL干预人血管平滑肌细胞(HVSMCs).应用Northern印迹及Western印迹技术检测AcLDL对亲脂素表达的影响;以RNA干扰技术、流式细胞仪、酶法和油红O染色检测亲脂素对HVSMCs的脂质集聚及AcLDL摄取的影响.结果 AcLDL呈剂量依赖性地增加HVSMCs的亲脂素的表达,沉默亲脂素基因使HVSMCs对AcLDL的摄取(降低38.7%,P<0.05)和脂质集聚能力下降(甘油三酯、总胆固醇分别下降30.6%和29.8%,均P<0.01).结论 亲脂素促进HVSMCs摄取AcLDL,增加HVSMCs脂质集聚,这可能是亲脂素促进动脉粥样硬化的机制之一.     

亲脂素增加血管平滑肌细胞的脂质集聚

目的 观察乙酰化的低密度脂蛋白(AcLDL)对血管平滑肌细胞亲脂素(adipophilin)表达的影响及亲脂素对血管平滑肌细胞的AcLDL摄取及脂质集聚的影响.从而探讨其在糖尿病大血管病变发生中的作用.方法 以不同浓度AcLDL干预人血管平滑肌细胞(HVSMCs).应用Northern印迹及Western印迹技术检测AcLDL对亲脂素表达的影响;以RNA干扰技术、流式细胞仪、酶法和油红O染色检测亲脂素对HVSMCs的脂质集聚及AcLDL摄取的影响.结果 AcLDL呈剂量依赖性地增加HVSMCs的亲脂素的表达,沉默亲脂素基因使HVSMCs对AcLDL的摄取(降低38.7%,P<0.05)和脂质集聚能力下降(甘油三酯、总胆固醇分别下降30.6%和29.8%,均P<0.01).结论 亲脂素促进HVSMCs摄取AcLDL,增加HVSMCs脂质集聚,这可能是亲脂素促进动脉粥样硬化的机制之一.     

亲脂素增加血管平滑肌细胞的脂质集聚

目的 观察乙酰化的低密度脂蛋白(AcLDL)对血管平滑肌细胞亲脂素(adipophilin)表达的影响及亲脂素对血管平滑肌细胞的AcLDL摄取及脂质集聚的影响.从而探讨其在糖尿病大血管病变发生中的作用.方法 以不同浓度AcLDL干预人血管平滑肌细胞(HVSMCs).应用Northern印迹及Western印迹技术检测AcLDL对亲脂素表达的影响;以RNA干扰技术、流式细胞仪、酶法和油红O染色检测亲脂素对HVSMCs的脂质集聚及AcLDL摄取的影响.结果 AcLDL呈剂量依赖性地增加HVSMCs的亲脂素的表达,沉默亲脂素基因使HVSMCs对AcLDL的摄取(降低38.7%,P<0.05)和脂质集聚能力下降(甘油三酯、总胆固醇分别下降30.6%和29.8%,均P<0.01).结论 亲脂素促进HVSMCs摄取AcLDL,增加HVSMCs脂质集聚,这可能是亲脂素促进动脉粥样硬化的机制之一.