Effect of shear stress on asymmetric dimethylarginine release from vascular endothelial cells

We demonstrated recently that plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, are increased by high salt intake concomitantly with a decrease in plasma levels of NO in human hypertension. We investigated the effect of shear stress on ADMA release in 2 types of cells: transformed human umbilical vein endothelial cells (HUVECs; cell line ECV-304) and HUVECs. Exposure of ECV-304 cells and HUVECs to shear stress with the use of a cone-plate viscometer enhanced gene expression of protein arginine methyltransferase (PRMT-1), ADMA synthase. In HUVECs, the ratio of PRMT-1 to glyceraldehyde 3-phosphate dehydrogenase mRNA was increased by 2-fold by a shear stress of > or =15 dyne/cm2. A dominant-negative mutant of IkappaB kinase alpha and troglitazone at 8 micromol/L, an activator of peroxisome proliferator-activated receptor gamma, abolished the shear stress-induced increase in PRMT-1 gene expression in parallel with the blockade of nuclear factor (NF)-kappaB translocation into the nucleus. The activity of dimethylarginine dimethylaminohydrolase, the degradation enzyme of ADMA, was unchanged after shear stress < or =15 dyne/cm2 and was enhanced by 1.48+/-0.06-fold (P<0.05) by shear stress at 25 dyne/cm2. The release of ADMA was increased by 1.64+/-0.10-fold (P<0.05) by shear stress at 15 dyne/cm2 but was not affected by shear stress at 25 dyne/cm2. These results indicate that shear stress enhances gene expression of PRMT-1 and ADMA release via activation of the NF-kappaB pathway. Shear stress at higher magnitudes facilitates the degradation of ADMA, thus returning ADMA release levels to baseline.     

ARB affects nicotine-induced gene expression profile in human coronary artery endothelial cells

AIM: To investigate the effects of nicotine and nicotine plus angiotensin II receptor blocker (ARB) on the gene expression profile of human coronary artery endothelial cells (HCAECs). METHODS: The changes in gene expression profiles in HCAECs treated with nicotine and nicotine plus ARB olmesartan were analyzed by DNA microarray. In nicotine-treated HCAECs, 432 genes selected by P < 0.01 were greater than 1.5-fold compared with the untreated cells. Data were analyzed using IPA (Ingenuity^® Systems, www.ingenuity.com). RESULTS: The gene expression levels of tumor necrosis factor-α, collagen type 1, matrix metalloproteinase-10, and disintegrin and metalloprotease domain 8, which are related to “cardiovascular function and disease”, were significantly increased. In canonical pathway analyses using IPA, “atherosclerosis signaling” was strongly affected by nicotine treatment and this effect was reduced by co-incubation with ARB olmesartan. These data indicate that the deleterious cardiovascular consequences of cigarette smoking may, at least in part, be due to the nicotine-induced gene expression profile related to “atherosclerosis signaling”. CONCLUSION: The inhibitory effect of ARB against the nicotine-induced gene expression profile may possibly induce anti-atherosclerotic effects that are independent of those from lowering the blood pressure.     

IDL can stimulate atherogenic gene expression in cultured human vascular endothelial cells

Previously, we have reported that the lipoprotein fraction containing intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) isolated from diabetics stimulates an atherogenic cytokine in cultured endothelial cells. To study which lipoprotein fraction isolated from diabetics can modulate the gene expression in endothelial cells, we isolated IDL and LDL fractions from 14 type 2 diabetics and seven age- and BMI- adjusted non-diabetics. We measured the effects of the lipoproteins on mRNA expression of atherogenic molecules in cultured endothelial cells. We found that the IDL fraction stimulated monocyte chemoattractant protein-1 (MCP-1) mRNA expression in endothelial cells as time- and dose-dependent fashions, while the LDL fraction was not effective. IDL isolated from diabetics also increased not only platelet-derived growth factor B-chain, but also intercellular adhesion molecule-1 mRNA contents. Furthermore, the HbA(1c) levels in diabetics were significantly correlated with their abilities of IDL to increase MCP-1 mRNA content in the cells and the increment coincided with the increase in MCP-1 protein release into culture media. These results indicate that qualitative as well as quantitative changes in IDL fraction in diabetes are atherogenic through stimulating gene expression of atherogenic molecules in endothelial cells.     

血管内皮生长因子基因对人工血管内皮细胞生长的影响

目的构建携带血管内皮生长因子(vascular endothelial growth factor,VEGF)基因腺病毒的人工血管模型。方法测定ad5-EGFP转染EAhy926转染率,观察人工血管携带腺病毒在静水中的释放情况及人工血管上内皮细胞的黏附情况,构建ad5-VEGF165-IRES-EGFP转染EAhy926,反转录聚合酶链反应(RT-PCR)半定量检测VEGF基因水平表达,酶联免疫吸附试验(ELISA)法检测VEGF蛋白水平表达,噻唑蓝(MTT)法检测内皮细胞活性。结果ad5转染EAhy926的最适感染复数(MOI)为200。人工血管上携带的病毒静水中能在人工血管上保留1 h左右,逐渐释放。扫描电镜显示内皮细胞在人工血管上吸附。聚合酶链反应半定量电泳图中,ad5-hVEGF165转染组均可见564 bp条带,而ad5-Null组和空白组均未见此条带。ad5-hVEGF165组培养液中hVEGF165蛋白浓度在各时间点均高于ad5-Null组和空白组,差异有统计学意义(P<0.01)。ad5-hVEGF165组噻唑蓝试验后第3、5、7天测得的OD值高于ad5-Null组和空白组,差异有统计学意义(P<0.01)。结论本实验成功构建携带VEGF基因腺病毒的人工血管。     

Thyrocyte release of asymmetric dimethylarginine does not account for human thyrocyte inhibition of endothelial cell cyclic GMP

BACKGROUND: The thyroid gland produces and responds to the signalling molecule nitric oxide (NO). The activity of NO synthase (NOS) may be regulated by endogenous NOS inhibitors such as asymmetric dimethylarginine (ADMA). OBJECTIVE: To investigate whether human thyrocytes are capable of regulating NOS activity via the production of ADMA. DESIGN: Human thyrocytes were incubated with human umbilical vein endothelial cells (HUVEC) in order to determine the effect on HUVEC NOS activity. HUVEC cGMP production over a 3-h period was measured as an indicator of NOS activity in the absence and presence of thyrocytes. To determine thyrocyte production of ADMA, samples of conditioned media were analysed by HPLC. RESULTS: The presence of primary human thyrocytes or immortalized human thyrocyte SGHTL-189 cells caused a significant inhibition of both basal (approximately 57% inhibition) and thrombin-stimulated (approximately 42% inhibition) HUVEC cGMP production. Both primary human thyrocytes and SGHTL-189 cells released ADMA (approximately 0. 28 microg per 10(6) thyrocytes over a 3-day period). However, excess L-arginine, the natural substrate for NOS, was unable to overcome thyrocyte inhibition of HUVEC cGMP production. CONCLUSION: These data indicate that human thyrocytes potently reduce endothelial cell cGMP concentrations, and that thyrocytes produce the endogenous NOS inhibitor, ADMA. However, the inhibition of endothelial cGMP is not mediated via thyrocyte production of a competitive NOS inhibitor.     

Interaction between human monocytes and vascular smooth muscle cells induces vascular endothelial growth factor expression

The objective of this study was to investigate whether synthesis of vascular endothelial growth factor (VEGF), a major mitogen for vascular endothelial cells, was induced by a cell-to-cell interaction between monocytes and vascular smooth muscle cells (VSMCs). Human VSMCs and THP-1 cells (human monocytoid cell) were cocultured. VEGF levels in the coculture medium were determined by enzyme-linked immunosorbent assay. Northern blot analysis of VEGF mRNA was performed using a specific cDNA probe. Immunohistochemistry was performed to determine which types of cell produce VEGF. Adding THP-1 cells to VSMCs for 24 h increased VEGF levels of the culture media, 8- and 10-fold relative to those of THP-1 cells and VSMCs alone, respectively. Northern blot analysis showed that VEGF mRNA expression was induced in the cocultured cells and peaked after 12 h. Immunohistochemistry disclosed that both types of cell in the coculture produced VEGF. Separate coculture experiments revealed that both direct contact and a soluble factor(s) contributed to VEGF production. Neutralizing anti-interleukin (IL)-6 antibody inhibited VEGF production by the coculture of THP-1 cells and VSMCs. A cell-to-cell interaction between monocytes and VSMCs induced VEGF synthesis in both types of cell. An IL-6 mediated mechanism is at least partially involved in VEGF production by the cocultures. Local VEGF production induced by a monocyte-VSMC interaction may play an important role in atherosclerosis and vascular remodeling.     

血管内皮生长因子基因修饰对内皮细胞前列腺环素合成影响的研究

目的 :观察血管内皮生长因子 (VEGF)基因转染对内皮细胞前列腺环素 (PGI2 )合成的影响 ,了解VEGF基因修饰对内皮细胞抗凝作用的影响。方法 :用脂质体转染的方法 ,将含有人VEGF基因的真核表达质粒pCD -hVEGF16 5体外转染人脐静脉内皮细胞。原位杂交、免疫组化法检测VEGF基因的表达 ,Elisa法检测培养上清液中VEGF及 6 -keto -PGF1α的含量。应用细胞计数法观察VEGF基因对内皮细胞生长的影响。结果 :外源性VEGF基因能被脂质体有效的导入内皮细胞并呈稳定表达。在高表达VEGF的同时 ,对PGI2 的合成和分泌较对照组明显增高 (p <0 .0 5 )。导入VEGF基因明显促进内皮细胞的分裂增殖。结论 :VEGF的高表达可显著提高内皮细胞对PGI2 的释放 ,可能增强内皮细胞的抗凝功能。     

血管内皮生长因子165基因对人胃癌细胞在体外生长及其受体表达的影响

目的:将已构建的重组腺病毒Ad-VEGF165进行扩增和纯化并观察其对人胃腺癌细胞(BGC-823)在体外生长及其受体表达的影响.方法:以人胚肾293细胞对重组腺病毒Ad-VEGF165和对照病毒Ad-GFP进行包装、扩增后感染BGC-823N胞,应用MTT比色法分析VEGF165对该种细胞体外生长的影响,并以免疫细胞化学方法检测该种细胞VEGFR-2的表达情况.结果:成功扩增及纯化了重组腺病毒Ad-VEGF165及对照病毒Ad-GFP,获得约3.2×1013pfu/L滴度的重组腺病毒和2.0×1013 pfu/L滴度的对照病毒.当感染复数(MOI)为20时转导率即接近100%而无明显细胞中毒现象.MTT结果显示Ad-VEGF165组吸光度明显高于Ad-GFP组(24 h:0.960±0.0 1 vs 0.737±O.01,P＜0.01:48 h:1.321±0.03 vs0.981±0.02,P＜0.01:72 h:1.663±0.03 vs 1.207±0.01,P＜0.01)及对照组(24 h:0.960±0.01 vs 0.724±0.03.P＜0.01:48 h:1.321±0.03 vs 0.968±0.01.P＜0.01:72 h:1.663±0.03 vs 1.185±0.02,P＜0.01).另外,在该细胞株上检测到了VEGFR-2的表达,且在Ad-VEGF165组其表达明显高于Ad-GFP组(62.5%vs37.6%,P＜0.01)和对照组(62.5%vs 34.1%,P＜0.01).结论:VEGF对胃癌细胞有直接的促生长作用,并可上调其主要受体VEGFR-2的表达水平,从而进一步证实了VEGF对胃癌细胞的自分泌作用.     

MicroRNA-223 inhibits tissue factor expression in vascular endothelial cells

Objective: Atherosclerosis is a chronic inflammatory process, in which vascular endothelial cells (ECs) become dysfunctional owing to the effects of chemical substances, such as inflammatory factor and growth factors. Tissue factor (TF) expression is induced by the above chemical substances in activated ECs. TF initiates thrombosis on disrupted atherosclerotic plaques which plays an essential role during the onset of acute coronary syndromes (ACS). Increasing evidences suggest the important role of microRNAs as epigenetic regulators of atherosclerotic disease. The aim of our study is to identify if microRNA-223 (miR-223) targets TF in ECs. Methods and results: Bioinformatic analysis showed that TF is a target candidate of miR-223. Western blotting analysis revealed that tumor necrosis factor α (TNF-α) increased TF expression in aorta of C57BL/6J mice and cultured ECs (EA.hy926 cells and HUVEC) after 4 h treatment. In TNF-α treated ECs, TF mRNA was also increased measured by real-time PCR. Real-time PCR results showed that miR-223 levels were downregulated in TNF-α-treated aorta of C57BL/6J mice and cultured ECs. Transfection of ECs with miR-223 mimic or miR-223 inhibitor modified TF expression both in mRNA and protein levels. Luciferase assays confirmed that miR-223 suppressed TF expression by binding to the sequence of TF 3′-untranslated regions (3′UTR). TF procoagulant activity was inhibited by overexpressing miR-223 with or without TNF-α stimulation. Conclusions: MiR-223-mediated suppression of TF expression provides a novel molecular mechanism for the regulation of coagulation cascade, and suggests a clue against thrombogenesis during the process of atherosclerotic plaque rupture.     

Interleukin-1 induces the expression of vascular endothelial growth factor in human pericardial mesothelial cells

Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells. We have studied the production of VEGF by human pericardial mesothelial cells. Mesothelial cells were separated by scraping the pericardial surface during cardiac surgery and cultured. When stimulated with interleukin (IL)-1α, pericardial mesothelial cells expressed VEGF mRNA and protein in concentration- and time-dependent manners. Hypoxia was also found to enhance mesothelial VEGF mRNA expression. The cells expressed mRNA for Flt-1 (VEGF receptor 1) and Flk-1 (VEGF receptor 2), and exogenous VEGF was found to have migration-promoting activity on cultured cells. We conclude that pericardial mesothelial cells express VEGF, which may serve as an autocrine growth-regulatory mechanism.     

Digital gene expression profile analysis of glycated low-density lipoprotein impacted on human umbilical vein endothelial cells

正Objective To study the effect of glycated low-densi-ty lipoprotein(gly-LDL)on human vascular endothelial cell on gene level.Methods Under the condition of asepsis and with antioxidants,incubated 2 mg/ml of LDL with 20 mmol/L of glucose in sealed tubes overlaid with     

血管内皮生长因子基因转染对肝细胞移植的影响

目的探讨VEGF基因转染对移植后血管组织形成的作用及对移植细胞增殖的影响.方法SD大鼠pcDNA3VEGF121转染肝细胞移植10d后取样,行微血管密度(MVD)计数及增殖细胞核抗原(PCNA)染色.结果在体外VEGF转染肝细胞能产生足够的转基因蛋白诱导内皮细胞的增殖.而体内可形成大量移植细胞克隆及再生肝组织.各组MVD计数差异并无显著性,P>005;在VEGF基因转染组PCNA指数较pcDNA3对照组与非转染组显著升高,为13.13±275、475±1.58和4.63±1.41,P<0.01.提示在体内移植血管组织形成存在多重因素的影响.而VEGF基因表达可促进移植细胞增殖和再生肝组织形成.结论VEGF间接体内基因转染是诱导移植肝再生一有效方法.     

肝癌组织血管内皮生长因子表达水平的免疫组化研究

目的　旨在研究血管内皮生长因子 (VEGF)与肝癌微血管形成、生长和转移诸方面的关系。方法　对临床 3 6例肝癌术后癌组织 ,以免疫组织化学法研究VEGF在肿瘤组织的胞内分布及其表达 ;并以ELISA法测定癌灶、癌旁及远癌组织中的VEGF蛋白的表达水平。结果　癌组织中VEGF阳性表达率为 63 .9% ;无包膜或包膜不完整组VEGF阳性表达率与有包膜组存在显著差异 ;肝癌伴有远处转移组VEGF阳性表达水平显著高于无转移组 (P <0 .0 1) ,癌灶组织中VEGF的表达水平明显高于癌旁、远癌组织(P <0 .0 1)。结论资料提示VEGF在肝癌组织中高度表达 ,它在HCC的血管形成、肿瘤发展和转移过程中起重要作用 ,提示癌组织中VEGF过度表达是反映肿瘤侵袭生长及转移潜能的有效指标     

血管内皮生长因子基因转移对6-羟多巴胺诱导的多巴胺能细胞损伤的保护作用

目的探讨血管内皮生长因子(VEGF)基因转移对6-羟多巴胺(6-OHDA)诱导的多巴胺能细胞损伤的保护作用。方法用腺病毒载体携带VEGF165基因(Ad-VEGF165)感染PC12细胞,并设腺病毒携带的β-半乳糖苷酶基因(Ad-LacZ)感染组和磷酸盐缓冲液(PBS)处理组作对照,基因转移成功后,用6-OHDA处理细胞,另设正常对照(不加6-OHDA)。四甲基偶氮唑盐法(MTT)测定细胞活性,免疫荧光细胞化学法检测细胞酪氨酸羟化酶(TH)表达,高效液相色谱检测细胞分泌功能。结果Ad-VEGF165感染组细胞吸光度A_(570)(0.31±0.07),高于Ad-LacZ感染组(0.15±0.07)和PBS处理组(0.13±0.05),但低于正常对照组(0.55±0.10),分别与各组比较,差异均有统计学意义(均为P<0.01);免疫荧光染色显示Ad-VEGF组细胞胞浆平均荧光强度(86.75±21.62)高于Ad-LacZ组(51.53±1 7.49)及PBS组(54.19±15.82),但低于正常对照组(110.39±24.21),分别与各组比较,差异均有统计学意义(均为(P<0.05);Ad-VEGF感染组细胞培养液中多巴胺和去甲肾上腺素含量也高于Ad-LacZ感染组和PBS处理组。结论腺病毒介导的VEGF基因转移对6-OHDA诱导的多巴胺能细胞损伤具有保护作用。     

cDNA microarray analysis of the gene expression profile of VEGF-activated human umbilical vein endothelial cells

Vascular endothelial growth factor (VEGF) is one of the most important factors that stimulate angiogenesis and vascular permeability. To clarify the role of VEGF, we analysed a human cDNA chip containing 7267 human genes to identify genes induced by VEGF in human umbilical vein endothelial cells (HUVECs). One hundred thirty-nine cDNAs, including ninety-nine previously known and forty poorly characterized or novel sequences, were increased more than two-fold by VEGF within twenty-four hours of stimulation. Among them, only five are known to regulate angiogenesis: cyclooxygenase 2 (COX2), heparin-binding epidermal growth factor-like growth factor, early growth response 1 (EGR 1), CYR61, and angiopoietin 2. Fifty-three genes induced within the first two hours were thought to be directly induced by VEGF. Of these, Down syndrome candidate region 1 (maximum induction = 6.1-fold) was the most profoundly induced, followed by Mif1 (KIAA0025; 5.5-fold), COX2 (4.7-fold), EGR 3 (3.7-fold), EGR 2 (3.2-fold), bactericidal/permeability-increasing protein (3.1-fold), and CD1B antigen, b polypeptide (3.1-fold). In addition to the genes mentioned above, there were many poorly characterized or novel genes induced by VEGF. Further analysis of these genes may aid in the elucidation of the molecular mechanisms of angiogenesis or vascular permeability stimulated by VEGF. This revised version was published online in June 2006 with corrections to the Cover Date.