Geniposide inhibits high glucose-induced cell adhesion through the NF-KB signaling pathway in human umbilical vein endothelial cells

Aim:

To investigate whether geniposide, an iridoid glucoside extracted from gardenia jasminoides ellis fruits, inhibits cell adhesion to human umbilical vein endothelial cells (HUVECs) induced by high glucose and its underlying mechanisms.

Methods:

HUVECs were isolated from human umbilical cords and cultured. The adhesion of monocytes to HUVECs was determined using fluorescence-labeled monocytes. The mRNA and protein levels of vascular cell adhesion molecule-1 (VCAM-1) and endothelial selectin (E-selectin) were measured using real-time RT-PCR and ELISA. Reactive oxygen species (ROS) production was measured using a fluorescent probe. The amounts of nuclear factor-kappa B (NF-κB) and inhibitory factor of NF-κB (IκB) were determined using Western blot analysis. The translocation of NF-κB from the cytoplasm to the nucleus was determined using immunofluorescence.

Results:

Geniposide (10–20 μmol/L) inhibited high glucose (33 mmol/L)-induced adhesion of monocytes to HUVECs in a dose-dependent manner. This compound (5–40 μmol/L) also inhibited high glucose-induced expression of VCAM-1 and E-selectin at the gene and protein levels. Furthermore, geniposide (5–20 μmol/L) decreased ROS production and prevented IκB degradation in the cytoplasm and NF-κB translocation from the cytoplasm to the nucleus in HUVECs.

Conclusion:

Geniposide inhibits the adhesion of monocytes to HUVECs and the expression of CAMs induced by high glucose, suggesting that the compound may represent a new treatment for diabetic vascular injury. The mechanism underlying this inhibitory effect may be related to the inhibition of ROS overproduction and NF-κB signaling pathway activation by geniposide.     

Effect of cicletanine on the nitric oxide pathway in human umbilical vein endothelial cells

The purpose of this study was to evaluate the effects of cicletanine, a slightly diuretic antihypertensive drug, on human vascular endothelial cells with regard to nitric oxide, intracellular calcium concentration ([Ca2+]i), cyclic nucleotide, inositol 1,4,5-trisphosphate (IP3), and prostacyclin generation. Primary cultured human umbilical vein endothelial cells were used in this study. [Ca2+]i was measured by fura-2/AM. Cyclic adenosine monophosphate (AMP), cyclic guanosine monophosphate (GMP), IP3, and prostacyclin were measured by radioimmunoassay. Nitric oxide was measured by the Griess method. Cicletanine had no effect on [Ca2+]i. Cicletanine (10(-6)-10(-4) M) increased cyclic GMP but decreased prostacyclin generation. Cicletanine had no stimulating effect on cyclic AMP or IP3 generation. IP3 increased 45Ca release from storage sites. Cicletanine decreased prostacyclin generation via increase in cyclic GMP. Cicletanine had no stimulating effect on nitrogen oxides for 2 h after incubation but increased it after 3-24 h. Pretreatment with L-N(G)-monomethyl-arginine (L-NMMA) prevented this increase. The inhibitory effect of L-NMMA was prevented by pretreatment with L-arginine. These results indicate that nitric oxide and cyclic GMP may contribute to the antihypertensive action of cicletanine.     

Effects of statins on nitric oxide/cGMP signaling in human umbilical vein endothelial cells

Human umbilical vein endothelial cells (HUVECs) were established as in vitro models for the modulation of endothelial function and cell viability by statins. Emphasis was placed on the biphasic effects of the drugs on nitric oxide (NO) bioavailability and cytotoxicity, as well as drug interference with the interaction of endothelial NO synthase (eNOS) with caveolin-1 (Cav-1). Incubation of HUVECs with fluvastatin, lovastatin or cerivastatin for 24 h caused an approximately 3-fold upregulation of eNOS expression that was associated with increased eNOS activity and accumulation of cGMP. Cerivastatin exhibited the highest potency with an EC50 of 13.8 ± 2nM after 24 h, while having no effect after only 30 min. The effects of statins on eNOS expression were similar in control and Cav-1 knockdown cells, but the increase in eNOS activity was less pronounced in Cav-1-deficient cells. Statin-triggered cyto-toxicity occurred at ~10-fold higher drug concentrations (maximal toxicity at 1–10 µM), was sensitive to mevalonate, and was significantly enhanced in the presence of N^G-nitro-L-arginine. The overexpression of eNOS induced by clinically relevant concentrations of statins may contribute to the beneficial vascular effects of the drugs in patients. Stimulation of NO synthesis and cytotoxicity appear to share a common initial mechanism but involve distinct downstream signaling cascades that exhibit differential sensitivity to HMG-CoA reductase inhibition.     

人肺泡上皮细胞株A549和脐静脉内皮细胞中的内皮素-1通路(英文)

AIM: This study was designed to characterize the en-dothelin pathway in an immortalized human adenocarcino-ma-derived alveolar epithelial cell line (A549) and human umbilical vein endothelial cell line (HUVEC). METHODS: The release of BT-1 and big-ET-1 was measured in the incubation medium of both cell lines. The expression of mRNAs coding for the endothelin iso-forms (hppET-1, -2, -3), the endothelin converting enzymes (hECE-la, b, c, and d) and the hETA and hETB receptors was investigated using RT-PCR. The expression of ECE-1 mRNA in various human tissues and in A549 cells was investigated by Northern blot analysis and the subcellular localization of ECE-1 in A549 cells was investigated by immunoblotting using a polyclonal antibody. RESULTS: Under control conditions, HUVEC release both ET-1 and big-ET-1 (ratio 5 to 1) while in A549 cells the big-ET-1 levels were below the threshold of detection. The release of these two peptides was minimally affected by various inhibitors of peptidases. However, in b     

Rapid action of pesticides on cytosolic calcium concentrations in cultures of human umbilical vein endothelial cells

Persistent metabolites of pesticides such as p,p'-DDE, at environmentally relevant concentrations, have been shown to have a rapid effect on intracellular calcium [Ca2+]i concentrations in human granulosa-lutein cells. Since endocrine disrupting substances can be transferred from the maternal circulation to the fetus the present study examined whether the pesticides, kepone, o,p-DDE, p,p'-DDE and methoxychlor, could alter cytoplasmic calcium [Ca2+]cyt concentrations in human umbilical vein endothelial (HUVE) cells. Cultured HUVE cells were loaded with Fura-2 AM and changes in [Ca2+]cyt of single cells were studied using a dynamic digital Ca2+ imaging system. Kepone and methoxychlor consistently increased [Ca2+]cyt concentrations, similar to the effects of estradiol and progesterone. p,p'-DDE increased [Ca2+]cyt concentrations in 80% of experiments whereas o,p-DDE stimulated its increases in 42%. Estrone, estriol, pregnenolone and cortisol were not effective. These results demonstrate that pesticides can have a rapid effect on HUVE cells probably through a nongenomic mechanism of action.     

Testing endodontic sealers on human umbilical vein endothelial cells

The toxic potential of the endodontic sealers ingredients, especially the unreacted monomer, that can irritate the periapical tissue and interfere with the healing process, thus having a negative impact on the biocompatibility of the material. The aim of this study was to evaluate the influence of three experimental endodontic sealers on cells viability in vitro. Human umbilical vein endothelial cells (HUVEC) were used. The experiments were done with solid samples and extracts of sealers in artificial saliva and water. The experiments evaluated the cytotoxicity of the residual monomers that resulted from the tested composites. The decrease in cell viability was quantified by colorimetric measurement of formazan. The components of the sealers dissolved in artificial saliva and water were determined by high performance liquid chromatography (HPLC). The HUVEC are a novelty for testing the endodontic sealers biocompatibility, with certain advantages compared to other cell types used in the literature, e.g. HELA cells, fibroblasts. The data showed that cytotoxicity was directly linked with the unreacted monomer — 2-hydroxyethyl methacrylate (HEMA) present in these composites. Two of the three formulations had little or no cytotoxic effect, which makes them suitable for further testing in order to be used in endodontic treatment.     

多奈哌齐对人脐静脉内皮细胞损伤的防护作用研究

目的 探讨多奈哌齐对过氧化氢诱导人脐静脉内皮细胞(HUVEC)损伤的防护作用,并研究其作用机制.方法 将实验细胞平均分为对照组、模型组、多奈哌齐组,通过过氧化氢建立细胞损伤模型,采用细胞计数试剂盒( CCK-8)法检测细胞存活率,通过光镜观察细胞形态,Hoechst 33258染色观察细胞凋亡的形态学变化,蛋白质印迹测定细胞中锰超氧化物歧化酶(MnSOD)蛋白表达.结果 多奈哌齐组与模型组比较,细胞存活率明显增加,且差异有统计学意义[(60.8±12.2)％比(44.7±9.3)％,P＜0.05];多奈哌齐组皱缩细胞数量介于模型组和对照组处理组之间;而多奈哌齐组细胞凋亡数量和程度均较模型组降低,差异有统计学意义[(30.8±10.4)％比(45.1±14.1)％,P＜0.05];此外,蛋白质印迹试验结果表明,多奈哌齐组细胞MnSOD蛋白表达强于对照组.结论 多奈哌齐可以明显减少过氧化氢诱导的HUVEC损伤,提高细胞存活率.多奈哌齐对于过氧化氢诱导的内皮细胞损伤的防护作用可能通过上调MnSOD的表达来实现.     

青蒿琥酯诱导人脐静脉内皮细胞凋亡

目的　探讨青蒿琥酯对人脐静脉内皮细胞(HUVEC)的诱导凋亡作用 ,以及这种诱导作用与内皮细胞增殖及细胞内游离Ca2 + 的关系。方法　采用MTT比色法测定青蒿琥酯对HUVEC增殖的影响 ,Fura 2 /AM荧光标记法测定HUVEC胞内游离Ca2 + 浓度 ([Ca2 + ]i) ,应用吖啶橙 /溴化乙啶荧光双染色、DNA琼脂糖凝胶电泳、流式细胞术检测细胞凋亡。结果　青蒿琥酯可抑制HUVEC增殖 ,明显诱导HUVEC凋亡 ,并呈浓度依赖性和时间依赖性效应关系。在发生凋亡的同时伴有 [Ca2 + ]i 显著升高。结论　青蒿琥酯可诱导HUVEC凋亡 ,其机制可能与[Ca2 + ]i 升高有关     

人脐静脉血管内皮细胞容量激活和钙激活氯离子通道(英文)

目的:研究人脐静脉血管内皮细胞的氯离子通道及其不同的调节机制。方法:全细胞膜片箝技术。结果:13.5％和27％的低渗液体灌流细胞激活一外向电流(I_(Cl·vol))。该电流有弱的外向整流特性,无明显时间和电压依赖性;电流大小为(58±5)pA/pF;分别为(20±3),(58±4)增加细胞内钙或胞外应用ATP能激活I_(Cl,Ca)。该电流幅值较小(23±5)pA/pF;外向整流特性明显,在正电压下缓慢激活。两种电流均被DIDS及维拉帕米抑制。在同一个细胞上,在激活I_(Cl,Ca)的基础上灌流低渗液体可进一步激活I_(Cl,vol)。结论:HUVEC表达有两种氯通道,改变细胞容积可激活I_(Cl,vol),而增加细胞内钙则诱导出I_(Cl,Ca)。     

Propofol protects human umbilical vein endothelial cells from cisplatin-induced injury

The anticancer drug cisplatin can up-regulate endothelial adhesion molecule expression, and trigger vascular endothelial injury. Propofol, an intravenous anesthetic, can inhibit endothelial adhesion molecule expression in some situations. Here, we explored whether and how propofol improved cisplatin-induced up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells. Compared with control group, cisplatin reduced endothelial nitric oxide synthase dimer/monomer ratio, activated protein kinase C and enhanced endothelial nitric oxide synthase-Thr⁴⁹⁵ phosphorylation, decreased nitric oxide production, augmented intercellular adhesion molecule 1 expression and monocyte-endothelial adhesion. These cisplatin-mediated effects were attenuated by propofol treatment. Nω-Nitro-L-arginine methyl ester hydrochloride, a nitric oxide synthase inhibitor, inhibited the effect of propofol on cisplatin-induced intercellular adhesion molecule 1 expression. Propofol improved cisplatin-mediated tetrahydrobiopterin reduction and nitrotyrosine overexpression. Compared with control group, cisplatin and PMA, a protein kinase C activator, both increased endothelial nitric oxide synthase-Thr⁴⁹⁵ phosphorylation, while propofol and GFX, a protein kinase C inhibitor, both decreased cisplatin-induced endothelial nitric oxide synthase-Thr⁴⁹⁵ phosphorylation. Our data indicated that propofol, via reducing cisplatin-induced endothelial nitric oxide synthase uncoupling and endothelial nitric oxide synthase-Thr⁴⁹⁵ phosphorylation, restored nitric oxide production, intercellular adhesion molecule 1 expression and monocyte-endothelial interaction.     

Apoptosis of human umbilical vein endothelial cells induced by artesunate

Artesunate (ART), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua, is an effective novel antimalarial drug. The present study investigated the apoptotic activity of artesunate in cultured human umbilical vein endothelial cell (HUVEC) by means of nuclear staining, DNA agarose gel electrophoresis, and flow cytometry. The observations also indicated that artesunate induced apoptosis of HUVEC in a concentration-dependent and time-dependent manner. A Western immunoblot analysis showed down-regulation of the bcl-2 protein and up-regulation of the bax protein in the artesunate-treated HUVEC. Ca2+ in cells was evaluated by fluorescent spectrophotometer using Fura 2-AM as probe. These results suggest that artesunate may be a potential apoptosis-inducing agent for endothelial cells.     

Substance P-induced cyclooxygenase-2 expression in human umbilical vein endothelial cells

Substance P (SP) is a neuropeptide involved in neurogenic inflammation and an agonist for NK(1), NK(2), and NK(3) receptors. SP induces prostaglandin (PG) production in various cell types, and these eicosanoids are responsible for numerous inflammatory and vascular effects. Cyclooxygenase (COX) are needed to convert arachidonic acid to PGs. The study evaluated the effect of SP on COX expression in human umbilical vein endothelial cells (HUVEC). COX-2 protein expression was upregulated by SP with a peak at 100 nM and at 20 h; in the same experimental conditions COX-1 protein expression was unchanged. A correlation between COX-2 expression and PGI(2) and PGE(2) release was detected. Dexamethasone (DEX) inhibited SP-mediated COX-2 expression. Mitogen-activated protein kinases (MAPK) p38 and p42/44 were activated by SP, whereas SB202190 and PD98059, inhibitors of these kinases, blocked COX-2 expression. 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone (DFU), an experimental selective COX-2 inhibitor, blocked SP-induced PG release. By RT-PCR and Western blot analysis, we demonstrated that NK(1) and NK(2) but not NK(3) receptors are present on HUVEC. Selective NK(1) and NK(2) agonists, namely [Sar(9), Met(O(2))(11)]SP and [beta-Ala(8)] NKA(4-10), upregulated COX-2 protein expression and PG production, whereas senktide (Suc-Asp-Phe-MePhe-Gly-Leu-Met-NH(2)), a selective NK(3) agonist, was ineffective in this respect. The NK(1) selective antagonist L703,606 ((cis)-2-(diphenylmethyl)-N-((2-iodophenyl)-methyl)-1-azabicyclo(2.2.2)octan-3-amine) and the NK(2) selective antagonist SR 48,968 ((S)-N-methyl-N-(4-(4-acetylamino-4-phenylpiperidino)-2-(3,4 dichlorophenyl)butyl) benzamide) competitively antagonised SP-induced effects. The study shows HUVEC to possess functional NK(1) and NK(2) receptors, which mediate the ability of SP to induce expression of COX-2 in HUVEC, thus showing a previously-undetected effect of SP on endothelial cells.     

Desensitization of agonist-stimulated prostacyclin release in human umbilical vein endothelial cells

Prostacyclin (PGI2) release was studied in perfused columns of human umbilical vein endothelial cells cultured on microcarrier beads. Substantial homologous desensitization of PGI2 release occurred when cells were exposed to agonist for 2 min after a previous exposure; the extent depended on the concentration and duration of the first challenge. Recovery from exposure to ATP or bradykinin was complete in less than 80 min; recovery from thrombin was incomplete after greater than 80 min, and this was apparently related to its proteolytic activity. Experiments with ibuprofen, a reversible inhibitor of cyclo-oxygenase, demonstrated that homologous desensitization did not involve inactivation of cyclo-oxygenase. ATP and bradykinin did not induce heterologous desensitization. Thrombin and trypsin induced cross-desensitization, but neither agonist significantly reduced responses to ATP or bradykinin, suggesting that a common proteolytic mechanism is responsible for their ability to induce PGI2 synthesis. We conclude that desensitization of PGI2 release in response to physiological agonists is generally agonist-specific and involves modulation of molecular events at or close to the receptors involved, rather than inactivation of prostanoid biosynthesis.     

三氧化二砷对肺癌A549细胞及人脐静脉内皮细胞Notch通路的影响

目的探索三氧化二砷(As2O3)对肺癌A549细胞和血管内皮细胞Notch通路相关分子表达的影响。方法体外培养人肺癌A549细胞和人脐静脉内皮细胞(HUVECs),分别用0(空白对照)、0.5、1.0、2.0、4.0、8.0μmol·L-1的As2O3进行干预,24 h后收集细胞,定量PCR检测A549细胞Dll4 m RNA的水平、HUVECs Dll4和Notch-1 m RNA的水平。结果 A549细胞的Dll4 m RNA水平在0~2.0μmol·L-1组逐渐升高,2.0~8.0μmol·L-1组逐渐降低,且8.0μmol·L-1组的表达水平显著低于空白对照组(P<0.01);HUVECs的Dll4 m RNA水平在0~1.0μmol·L-1组逐渐升高,1.0~8.0μmol·L-1组逐渐降低,且4.0和8.0μmol·L-1组的表达水平显著低于空白对照组(P<0.01);HUVECs的Notch-1 m RNA水平随给药浓度的增加而升高,各浓度组均高于空白对照组(P<0.01)。结论在体外条件下,As2O3在较低浓度能促进A549细胞和HUVECs Dll4 m RNA表达,而在较高浓度则抑制其表达;As2O3能浓度依赖性提高HUVECs的Notch-1 m RNA表达水平。