NF-κB translocation and endothelial cell activation is potentiated by macrophage-released signals co-secreted with TNF-α and IL-1β

Objective and Methods: Over-expression of the immune response can lead to pathological conditions such as septic shock or chronic inflammation. Endothelial cell activation by pro-inflammatory products of activated macrophages plays a key role in these conditions. Here we examine the response of primary human endothelial cells (HUVEC) to conditioned media (CM) obtained from LPS-activated macrophages. We further characterized the translocation of NF-B in the presence of CM by studying the degradation rate of individual IB isoforms.Results: We show that, as expected, CM induced NF-B translocation, as well as adhesion capacity in HUVEC. We further show that this response is critically dependent on TNF- and IL1 naturally present in the CM. However, both the amplitude of NF-B translocation and adhesiveness observed with CM were well beyond the saturation levels attained after the sole stimulation with recombinant TNF- and IL-1, either separately or together. Our results show that CM induced a faster degradation of the IB- and IB- isoforms than the recombinant cytokines, leading to an enhanced recruitment of NF-B activity.Conclusions: The above results suggest that the physiological context of factors co-secreted by LPS-activated macrophages enhances TNF- mediated endothelial activation.Received 5 October 2003; returned for revision 2 December 2003; accepted by A. Falus 19 May 2004     

风湿祛痛胶囊对VEGF诱导的人脐静脉内皮细胞功能的影响

目的:观察风湿祛痛胶囊对血管内皮细胞生长因子(VEGF)诱导的人脐静脉内皮细胞(HUVEC)的增殖、迁移、黏附、侵袭和管腔形成能力的影响,以及对VEGF受体2(VEGFR2)的干预作用。方法:VEGF体外诱导HUVEC,加入风湿祛痛胶囊低、中、高质量浓度(0. 02,0. 1,0. 5μg·L-1)作用后,分别采用噻唑蓝(MTT)比色法、转移小室(transwell)法、黏附实验、细胞侵袭及管腔形成实验检测HUVEC的增殖活性、迁移、黏附、侵袭及管腔形成能力,蛋白免疫印迹法(Western blot)检测细胞中VEGFR2磷酸化水平和蛋白含量,实时荧光定量聚合酶链式反应(Real-time PCR)检测VEGFR2 mRNA表达水平。结果:与正常组比较,VEGF诱导24,48 h后均能显著升高HUVEC的增殖活性(P 0. 01),诱导24 h能明显升高HUVEC的迁移、黏附、侵袭和管腔形成能力(P 0. 01),显著升高HUVEC中VEGFR2磷酸化及蛋白和mRNA表达水平(P 0. 01);与VEGF组比较,风湿祛痛胶囊低、中、高质量浓度组作用48 h均能明显抑制HUVEC增殖活性(P 0. 05,P 0. 01),作用24 h对VEGF诱导的HUVEC迁移、黏附、侵袭和管腔形成能力有明显抑制作用(P 0. 05),也能下调VEGFR2磷酸化及蛋白和mRNA表达水平(P 0. 05)。结论:风湿祛痛胶囊能降低VEGF诱导的HUVEC细胞增殖、迁移、黏附、侵袭及管腔形成能力,这一作用可能与其抑制VEGFR2的磷酸化、蛋白和mRNA表达水平有关。     

Cyclo(dehydrohistidyl-l-tryptophyl) inhibits nitric oxide production by preventing the dimerization of inducible nitric oxide synthase

Dimerization of inducible NOS has been known to be a potential therapeutic target for iNOS-mediated pathologies. Cyclic dipeptides are among the simplest peptides commonly found as by-products of food processing or metabolites of microorganisms. In this study, we found that cyclo(dehydrohistidyl-l-tryptophyl) (CDHT), a cyclic dipeptide from an unidentified fungal strain Fb956, prevents iNOS dimerization in activated microglial BV-2 cells. CDHT inhibited NO production with an IC50 of 6.5 microM in LPS-treated BV-2 cells. Western blot analysis and iNOS activity measurement of fractions from size-exclusion chromatography of cell lysates indicated that CDHT inhibits dimerization of iNOS, while it has no effect on iNOS expression or enzyme activity. The CDHT inhibition of iNOS dimerization was confirmed by partially denaturing SDS-PAGE analysis. In contrast, CDHT did not affect cGMP production in endothelial HUVEC cells, which indicates no inhibition of endothelial NOS activity. These results reveal that CDHT, one of the simplest and cyclic dipeptides, selectively inhibits NO production by inhibiting iNOS dimerization, and could be a useful therapeutic agent for inflammation-mediated diseases.     

Celecoxib modulates adhesion of HT29 colon cancer cells to vascular endothelial cells by inhibiting ICAM-1 and VCAM-1 expression

BACKGROUND AND PURPOSE: Cyclooxygenase-2 (COX-2) is highly expressed during inflammation and can promote the progression of colorectal cancer. Interactions between cancer cells and vascular endothelial cells are key events in this process. Recently, the selective COX-2 inhibitor, celecoxib, was shown to inhibit expression of the adhesion molecules, ICAM-1 and VCAM-1, in the human colon cancer cell line HT29 and to inhibit adhesion of HT29 cells to FCS-coated plastic wells. Here, we evaluated the effects of celecoxib on adhesion of HT29 cells to human umbilical vein endothelial cells (HUVEC), mediated by ICAM-1 and VCAM-1, to assess further the potential protective effects of celecoxib on cancer development. EXPERIMENTAL APPROACH: Celecoxib was incubated for 4 h with HT29 cells and HUVEC and adhesion was quantified by a computerized micro-imaging system. Expression analysis of ICAM-1 and VCAM-1 cell adhesion molecules was performed by western blot. KEY RESULTS: Celecoxib (1 nM-10 microM) inhibited, with the same potency, adhesion of HT29 cells to resting HUVEC or to HUVEC stimulated by tumour necrosis factor-alpha (TNF-alpha), mimicking inflammatory conditions. Analysis of ICAM-1 and VCAM-1 expression showed that celecoxib inhibited expression of both molecules in TNF-alpha-stimulated HUVEC, but not in resting HUVEC; inhibition was concentration-dependent and maximal (about 50%) at 10 microM celecoxib. CONCLUSIONS AND IMPLICATIONS: In conclusion, our data show that celecoxib inhibits HT29 cell adhesion to HUVEC and expression of ICAM-1 and VCAM-1, in stimulated endothelial cells. These effects may contribute to the chemopreventive activity of celecoxib in the development of colorectal cancer.     

rTNF_(-α)、IFN_(γ)对人脐静脉内皮细胞表达粘附分子的影响

为了研究细胞因子ｒＴＮＦ α、ＩＦＮγ对内皮细胞表达粘附分子的影响,及ｒＩＦＮγ对ＴＮＦ α的协同效应。采用ｒＴＮＦ α、ＩＦＮγ诱导培养的人脐静脉内皮细胞（ＨＵＶＥＣ）,以Ｃｅｌ ＥＬＩＳＡ法检测粘附分子ＩＣＡＭ １、ＥＬＡＭ １和ＶＣＡＭ １的表达。结果表明,ｒＴＮＦ α以浓度和时间依赖的方式诱导ＨＵＶＥＣ表达ＩＣＡＭ １、ＥＬＡＭ １和ＶＣＡＭ １,ＩＦＮγ促进ｒＴＮＦ α诱导的ＶＣＡＭ １表达。ＩＦＮ α对ｒＴＮＦ α诱导效应无影响。提示ｒＴＮＦ α可诱导ＨＵＶＥＣ表达粘附分子,而ＩＦＮγ可促进ｒＴＮＦ α诱导的ＨＵＶＥＣ表达ＶＣＡＭ １。     

Cytoprotective role of astaxanthin against glycated protein/iron chelate‐induced toxicity in human umbilical vein endothelial cells

Astaxanthin (ASX), a red carotenoid pigment with no pro‐vitamin A activity, is a biological antioxidant that occurs naturally in a wide variety of plants, algae and seafoods. This study investigated whether ASX could inhibit glycated protein/iron chelate‐induced toxicity in human umbilical‐vein endothelial cells (HUVEC) by interfering with ROS generation in these cells. Glycated fetal bovine serum (GFBS) was prepared by incubating fetal bovine serum (FBS) with high‐concentration glucose. Stimulation of cultured HUVECs with 50 mm 1 mL of GFBS significantly enhanced lipid peroxidation and decreased antioxidant enzyme activities and levels of phase II enzymes. However, preincubation of the cultures with ASX resulted in a marked decrease in the level of lipid peroxide (LPO) and an increase in the levels of antioxidant enzymes in an ASX concentration‐dependent manner. These results demonstrate that ASX could inhibit LPO formation and enhance the antioxidant enzyme status in GFBS/iron chelate‐exposed endothelial cells by suppressing ROS generation, thereby limiting the effects of the AGE–RAGE interaction. The results indicate that ASX could have a beneficial role against glycated protein/iron chelate‐induced toxicity by preventing lipid and protein oxidation and increasing the activity of antioxidant enzymes. Copyright © 2009 John Wiley & Sons, Ltd.     

Discovery of a novel COX-2 inhibitor as an orally potent anti-pyretic and anti-inflammatory drug: design, synthesis, and structure-activity relationship

Cyclooxygenase (COX) has been considered as a significant pharmacological target because of its pivotal roles in the prostaglandin biosynthesis and following cascades that lead to various (patho)physiological effects. Non-steroidal anti-inflammatory drugs (NSAIDs) that suppress COX activities have been used clinically for the treatment of fever, inflammation, and pain; however, nonselective COX inhibitors exhibit serious side-effects such as gastrointestinal damage because of their inhibitory activities against COX-1. Thus, COX-1 is constitutive and expressed ubiquitously and serves a housekeeping role, while COX-2 is inducible or upregulated by inflammatory/injury stimuli such as interleukin-1β, tumor necrosis factor-α, and lipopolysaccharide in macrophage, monocyte, synovial, liver, and lung, and is associated with prostaglandin E₂ and prostacyclin production that evokes or sustains systemic/peripheral inflammatory symptoms. Also, hypersensitivity of aspirin is a significant concern clinically. Hence, design, synthesis, and structure–activity relationship of [2-{[(4-substituted)-pyridin-2-yl]carbonyl}-(6- or 5-substituted)-1H-indol-3-yl]acetic acid analogues were investigated to discover novel acid-type COX-2 inhibitor as an orally potent new-class anti-pyretic and anti-inflammatory drug. As significant findings, compounds 1–3 demonstrated potent COX-2 inhibitory activities with high selectivities for COX-2 over COX-1 in human cells or whole-blood in vitro, and demonstrated orally potent anti-pyretic activity against lipopolysaccharide-induced systemic-inflammatory fever model in F344 rats. Also compound 1 demonstrated orally potent anti-inflammatory activity against edema formation and a suppressive effect against PGE₂ production in carrageenan-induced peripheral-inflammation model on the paw of SD rats. These results suggest that compounds 1–3 are potential agents for the treatment of inflammatory disease and are useful for further pharmacological COX-2 inhibitor investigations.     

麝香酮对氧化应激损伤人血管内皮细胞凋亡的影响

目的:观察麝香酮对过氧化氢(H2O2)诱导人血管内皮细胞(HUVEC)凋亡的保护效应,并探讨其作用机制。方法:运用H2O2建立血管内皮细胞凋亡模型,继而用低、中、高不同剂量的麝香酮进行干预,并用MTT法检测细胞增殖,Hoechst 33258荧光染色及流式细胞技术检测细胞凋亡,激光共聚焦显微镜测定Ca2+浓度和线粒体膜电位(ΔΨm)的变化。结果:H2O2诱导HUVEC细胞凋亡,中、高剂量的麝香酮对HUVEC细胞增殖均有明显促进作用;H2O2组与正常组相比ΔΨm显著下降,Ca2+浓度明显升高(P<0.01);中、高剂量组与H2O2组相比ΔΨm则显著升高,Ca2+浓度明显降低(P<0.01)。结论:麝香酮可通过稳定线粒体ΔΨm,减轻细胞通透性,减少Ca2+内流,从而抑制H2O2所致的HUVEC细胞凋亡。