纤维蛋白凝胶立体培养诱导血管样结构的形成

目的探讨微血管内皮细胞(MVEC)在体外培养中形成血管样结构的细胞外基质条件，为体外组织工程血管化提供实验基础。方法在纤维蛋白原浓度为2g／L的纤维蛋白凝胶中加入不同浓度的血管内皮细胞生长因子165(VEGF165)，进行MVEC的立体培养，观察有无血管样结构形成。结果在VEGF165为10μg／L组和20μg／L组中，MVEC在立体培养中形成血管样结构。VEGF165为20μg／L组比VEGF165为10μg／L组形成血管样结构的时间更早、结构更长。VEGF165为5μg／L组和对照组均无血管样结构形成，但可见细胞团聚。结论VEGF165浓度为20μg/L或10μg／L时，MVEC在纤维蛋白凝胶立体培养中可形成血管样结构。VEGF165诱导形成血管样结构的长度与剂量有依赖关系。     

筛选有效抑制血管内皮细胞小窝蛋白-1表达的小干扰RNA及其毒性检测

目的 筛选有效抑制血管内皮细胞小窝蛋白-1(Caveolin-1)表达的小干扰RNA(small interferenceRNA,siRNA)序列,并检测转染复合物的细胞毒性. 方法 设计合成针对Caveolin-1的siRNA 3条(siRNA422,siRNA548,siRNA710)及1条带绿色荧光标记的通用阴性对照FAM-siRNA.在siRNAFect介导下转染血管内皮细胞.用实时荧光定量PCR(Real-time PCR)测定转染后血管内皮细胞中Caveolin-1 mRNA的表达,Western blot方法测定干扰后Caveolin-1蛋白表达,比较抑制率,筛选出有效抑制Caveolin-1表达的siRNA.采用磺基罗丹明B法(sulforhodamineB,SRB)分析转染复合物的细胞毒性. 结果 ①在siRNAFect相同剂量下,siRNA 20、30 nmol/L组或40 nmol/L组转染效率均达到85％以上(P＜0.01);②siRNAFect 1.3μl复合10 nmol/L或20 nmol/L siRNA组细胞存活率均达到80％以上(P＜0.05);③siRNA548对EA.hy926细胞Caveolin-1基因mRNA抑制效果最明显(P＜0.01);④与阴性对照组及siRNA422、siRNA710相比较,siRNA548干扰血管内皮细胞48 h后,Caveolin-1蛋白的表达量最低(P＜0.01). 结论 ①siRNA548对血管内皮细胞Caveolin-1表达的抑制效果最大.②siRNA浓度20 nmol/L复合siRNAFect 1.3μl转染效率高,细胞毒性低,适合转染.     

异丙酚预先给药对内毒素诱导大鼠肾小球血管内皮细胞通透性升高的影响

目的 探讨异丙酚预先给药对脂多糖(LPS)诱导大鼠肾小球血管内皮细胞通透性升高的影响.方法 分离、培养SD大鼠肾小球血管内皮细胞,以1×106/ml的密度接种于24孔培养板(200 μl/孔)和transwell小室(100 μl/室),采用随机数字表法,将其随机分为6组(n=10),正常对照组(C组)不作任何处理;脂肪乳对照组(I 组)加入10%脂肪乳4 μg/ml;异丙酚组(P组)加入异丙酚4μg/ml;LPS组(L组):加入LPS 10μg/ml;LPS+脂肪乳组(L+I组)加入10%脂肪乳4 μg/ml及LPS 10μg/ml;LPS+异丙酚组(L+P组)加入异丙酚4μg/ml 及LPS 10 μg/ml.于加入LPS前30 min加入脂肪乳或异丙酚,药物的浓度均为终浓度.加入LPS后6 h,收集细胞,采用逆转录-聚合酶链反应测定血管内皮细胞生长因子(VEGF)mRNA表达水平;收集上清液,采用酶联免疫吸附法测定VEGF浓度;测定血管内皮细胞通透性.结果 与C组比较,L组、L+I组和L+P组VEGF mRNA表达上调,上清液中VEGF浓度和血管内皮细胞通透性增加(P＜0.05),而I组和P组上述指标差异无统计学意义(P＞O.05).与L组比较,L+P组VEGF mRNA表达下凋,上清液中VEGF浓度和血管内皮细胞通透性降低(P＜0.05),L+I组上述指标差异无统计学意义(P＞0.05).上清液中VEGF浓度与血管内皮细胞通透性呈正相关(r=0.833,P＜0.05).结论 异丙酚预先给药可抑制LPS诱导大鼠肾小球血管内皮细胞通透性升高,其机制与下调VEGF表达有关.

Abstract：

Objective To investigate the influence of propofol pretreatment on the increased glomerular endothelial cell permeability induced by lipopolysaccharide (LPS) in rats.Methods Glomerular endothelial cells isolated from SD rats were cultured in 24-well plates(200 μl/well) and transwell filters (100 μl/filter) at 1×106/ml and assigned into 6 groups (n=10 each):control group (group C) , introlipid group (group I), propofol group (group P) , LPS group (group L), LPS+introlipid group (group L+I) and LPS+propofol group (group L +P). In group I, 10% introlipid 4 μg/ml was added. In group P, 4 μg/ml propofol was added. In group L, 10 μg/ml LPS was added. In group L+I, 10% introlipid 4 fig/ml combined with 10 μg/ml LPS was added. In group L+ P, 4 μg/ml propofol combined with LPS 10 μg/ml was added. Introlipid or propofol was added 30 min before the administration of LPS and the corresponding concentrations mentioned above were all final concentrations.After 6 h incubation with LPS, the cells were collected for measurement of vascular endothelial growth factor (VEGF) mRNA expression using RT-PCR. The supernatant was collected for determination of the VEGF concentration by ELJSA. The endothelial cell permeability was determined. Results Compared with group C, the expression of VEGF mRNA was up-regulated and the VEGF concentration and endothelial cell permeability were significantly increased in L, L+I and L + P groups (P＜0.05 ) ,but no significant change was found in the parameters mentioned above in I and P groups (P＞0.05). Compared with group L, the expression of VEGF mRNA was downregulated and the VEGF concentration and endothelial cell permeability were significantly decreased in L+P group (P＜0.05), but no significant change was found in the parameters mentioned above in group L+I(P＞0.05). A positive correlation existed between the concentration of VEGF and the permeability of endothelial cells(r= 0.833,P＜0.05).Conclusion Propofol pretreatment can decrease the increased glomerular endothelial cell permeability induced by LPS probably through down-regulation of VEGF expression.     

Galectin-3对骨髓间充质干细胞来源的内皮细胞增殖的影响

目的探讨galectin-3对骨髓间充质干细胞(MSCs)来源的内皮细胞增殖的影响。方法密度梯度离心法分离纯化SD大鼠骨髓MSCs,取第3代,加入血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF),体外诱导14 d,进行免疫组织荧光染色和电镜鉴定,用低、中、高不同浓度galec-tin-3(0.1μg/mL,1.0μg/mL和5.0μg/mL)对骨髓MSCs来源的内皮细胞作用24 h;采用MTT比色法观察不同浓度galectin-3对内皮细胞增殖的影响;利用流式细胞仪检测DNA细胞周期的变化。结果 (1)中高浓度galectin-3具有显著的促进血管内皮细胞增殖的作用(P0.05);(2)流式细胞仪检测DNA细胞周期显示各组S期细胞明显增加(P0.05),中高浓度galectin-3组G2M期细胞比例明显增加(P0.05)。结论 galectin-3可以促进骨髓MSCs来源的内皮细胞增殖。     

VEGF对创伤组织中KDR,bFGF和PDGF mRNA表达的影响

目的研究VEGF对创伤组织KDR,bFGF和PDGFmRNA表达的影响,探讨其促进损伤组织修复的作用机理。方法以血管内皮细胞生长因子(VEGF165)为目的基因,构建真核表达载体pcDNA3.1/myc-hisA-VEGF165,利用纳米微囊包裹后,作用于兔耳创面。利用反转录PCR方法观察术后14天损伤组织KDR,bFGF和PDGFmRNA的表达变化。结果施与纳米微囊包裹的VEGF165实验组创面肉芽生长及上皮爬行速度明显快于对照组。反转录PCR的结果显示实验组损伤组织KDR,bFGF和PDGFmRNA的表达水平明显高于对照组(P<0.05)及正常组(P<0.01)。结论VEGF能上调损伤组织内KDR,bFGF和PDGFmRNA的表达,VEGF可能作用于其受体KDR,通过与其他细胞因子的协同作用来实现其对伤口愈合的促进作用。     

血管内皮因子、人表皮生长因子、碱性成纤维细胞生长因子纳米缓释剂对血管内皮细胞影响的比较

目的制备血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、人表皮生长因子(EGF)可降解缓释微球,考察其生物活性的保存情况,以及它们对血管内皮细胞的作用.方法采用改良的乳化冷凝法交联制备VEGF、bFGF、EGF的明胶缓释微球,将它们加入血管内皮细胞的培养液中,用细胞计数法、四甲基偶氮唑盐微量反应比色法(MTT法)测定细胞增殖情况.结果VEGF、bFGF、EGF的缓释微球平均粒径(11.32±3.64)μm;培养1天后各组细胞计数、吸光度(A)值差异均无显著性;5天后,VEGF20ng缓释微球组细胞计数、吸光度(A)值明显高于其它组;7天后,VEGF20ng缓释微球组值仍高于其它组,但差异无显著性.结论VEGF、bFGF、EGF的缓释微球制备工艺简便,成球性好;能较长时间地持续释放活性VEGF,bFGF、EGF,可促进血管内皮细胞的增殖,其中VEGF效果最显著.     

Oligofectamine介导转录因子SP1诱骗性寡核苷酸转染猪内皮细胞系条件的优化研究

目的确定SP1诱骗性寡核苷酸(Decoy Oligodeoxynucleotides,ODNs)在Oligofectamine介导下转染猪血管内皮细胞系SV-40-PED的最佳转染条件和效果。方法以SV-40-PED为转染对象,在6孔培养板中,接种SV-40-PED细胞2×105/孔,将4μlOligofectamine和不同浓度的SP1ODNs(2.5、5.0、7.5、10.0和12.5μl)分别溶于100μl无血清、无抗生素的DMEM培养基,室温放置20min后转染细胞,SP1ODNs最终浓度分别为50、100、150、200和250nmol/L、转染26h检测不同浓度转染情况,确定最佳SP1ODNs与Oligofectamine的比率、转染时间;流式细胞仪检测细胞内的相对荧光强度及摄取率评价转染效率;荧光显微镜观察细胞内荧光分布;测定细胞上清乳酸脱氢酶(lactate dehydrogenase,LDH)含量,观察细胞损伤情况。结果SV-40-PED在Oligofectamine的介导下可以摄取SP1ODNs。SP1ODNs终浓度为50、100、150nmol/L组细胞形态无明显变化,终浓度为200、250nmol/L组细胞收缩、变圆后逐渐恢复;SP1ODNs终浓度为250nmol/L时,转染26h时细胞形态也无明显变化,48、72h时有细胞漂浮现象。荧光下观察,转染成功各组荧光物质分布于细胞核内,其中SP1ODNs终浓度为200、250nmol/L组可见清晰核仁,浓度越高,荧光强度越强,细胞内荧光物质主要聚集于细胞核。SP1ODNs终浓度为250nmol/L时,转染72h组LDH浓度为137.12±3.92U/L,显著高于26h的49.61±17.13U/L和48h的120.26±8.42U/L,均有统计学意义(P<0.01);当转染时间为26h时,各浓度组上清液LDH值差异无统计学意义(P>0.05)。结论SP1ODNs终浓度为250nmol/L、转染26h时可以为SV-40-PED转染提供良好效果。     

CGRP与VEGF对内皮细胞成血管的作用比较研究

[目的]比较降钙素基因相关肽(calcitonin gene-related peptide,CGRP)与血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)对血管内皮细胞增殖、迁移、体外成血管作用的大小.[方法]体外分离培养获取脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs).并通过血管性血友病因子(vonWillebrand factor,Vwf)与CD31抗原鉴定.实验分9组,(10-12～10-8)CGRP,(5 ～20 ng/ml) VEGF165,另设置一个空白对照组.采用细胞免疫荧光脱察HUVECs的CGRP受体(calcitonin gene-related peptide receptors,CGRPR)表达情况,alarmar Blue法动态检测各组HUVECs的增殖率,Transwell小室检测各组HUVECs的迁移能力,采用体外成管实验检测各组促血管生成作用大小.Q-PCR检测CGRP和VEGF165刺激细胞时,CGRP受体的mRNA表达情况.[结果]CGRP及VEGF165时间-浓度依赖性促进HUVECs增殖,VEGF165促内皮细胞增殖的能力强于CGRP,差异有显著性.CGRP及VEGF165都能明显促进HUVECs迁移及促进HUVECs在基质胶中形成毛细血管样结构.并且两者的最大作用基本相当.CGRP与VEGF165都能促进内皮细胞中CGRP受体表达水平增加.但是CGRP促CGRP受体的生成作用明显强于VEGF165.[结论]CGRP是强的促血管生成因子,其促血管生成的作用与低浓度(5 ～20ng/ml) VEGF165相当.     

化痰通络方含药血清对血管内皮生长因子诱导的脐静脉内皮细胞增殖的影响及其机制研究

目的:观察化痰通络方含药血清对血管内皮生长因子(VEGF)诱导的脐静脉内皮细胞(HUVEC)增殖的影响及其机制。方法:将20只新西兰兔随机分为化痰通络方高(HTTL-h)、中(HTTL-m)、低(HTTL-l)剂量组,雷公藤多苷组(TG组),生理盐水组(Sal组),每组4只。分离鉴定健康新生儿HUVEC,siRNA敲降VEGFR2、PLCG1以及MAPK1,并采用RT-PCR和及Western Blot检测VEGFR2、PLCG1和MAPK1 mRNA及蛋白水平的表达。MTT法检测经VEGF诱导的HUVEC增殖情况。结果:与Sal组比较,VEGF可显著促进HUVEC增殖,而TG或HTTL-l均可显著抑制VEGF诱导的HUVEC增殖;siRNA敲降VEGFR2后,VEGF则不能促进HUVEC增殖,同时TG也失去了对HUVEC增殖的抑制能力;siRNA敲降PLCG1后,VEGF依然可以显著促进HUVEC增殖,此时TG对VEGF刺激的HUVEC增殖失去了抑制效应,但HTTL-m和HTTL-l依然对VEGF刺激的HUVEC增殖具有显著抑制效应;siRNA敲降MAPK1后,VEGF也可显著促进HUVEC增殖,TG对VEGF刺激的HUVEC增殖具有抑制效应,同时HTTL-l也对VEGF刺激的HUVEC增殖具有显著抑制效应。结论:化痰通络方含药血清对VEGF诱导的HUVEC增殖具有抑制作用。TG抑制VEGF诱导的HUVEC增殖依赖于VEGFR2和PLCG1表达,而化痰通络方对VEGF刺激的HUVEC增殖的抑制效应则不依赖VEGF/VEGFR2/PLCG1/MAPK1信号通路。     

生长因子缓释微球的制备及其对内皮细胞的影响

目的:制备碱性成纤维细胞生长因子(bFGF)、血管内皮细胞生长因子(VEGF)可降解缓释微球,考察其生物活性的保存情况及它们对内皮细胞的作用。方法:采用改良的乳化冷凝法交联制备复合bFGF、VEGF的明胶缓释微球,将它们加入内皮细胞的培养液中,用细胞计数法、四甲基偶氮唑盐微量反应比色法(MTT法)测定细胞增殖情况。结果:复合bFGF、VEGF的缓释微球平均粒径(11.32±3.64)μm;培养1天后各组细胞计数、吸光度(A)值差异均无显著性;5天后两种生长因子缓释微球组细胞计数、吸光度(A)值明显高于对照组;7天后两种生长因子缓释微球组值仍高于其它组。结论:复合bFGF、VEGF的缓释微球制备工艺简便,具有良好的缓释活性能较长时间地持续释放活性bFGF、VEGF,可明显促进内皮细胞的增殖。     

重组血管内皮生长因子165-PE38融合基因的构建及其对人脐静脉内皮细胞的抑制作用

目的 构建血管内皮生长因子(VEGF)基因和铜绿假单胞菌外毒素(PE38)基因真核融合表达载体,观察其表达产物对人脐静脉内皮细胞(HUVECs)的影响.方法 通过聚合酶链反应(PCR)获得VEGF165基因片段,通过Hind Ⅲ和EcoR Ⅰ双酶切pRB391质粒获得PE38基因.构建两融合基因的真核表达载体plRES2-VEGF165-PE38-EGFP,转染293细胞后用逆转录(RT)-PCR及ELISA检测VEGF165-PE38融合蛋白在293细胞的表达,并检测转染细胞的培养上清对HUVECs的选择性细胞毒作用.结果 成功构建VEGF165-PE38融合基因真核表达载体,其可在293细胞表达,ELISA检测表明空质粒转染组、无转染组和重组质粒转染组VEGF浓度分别为(269.0±23.6)、(306.0±29.3)和(1390.0±136.6)ng/L.CCK-8和TUNEL检测表明重组质粒细胞转染上清对HUVECs具有较强的细胞抑制效应,凋亡细胞率分别为8.34%、7.69%和39.88%,差异有统计学意义(P<0.05).结论 VEGF 165-PE38融合基因构建,表达及其功能的初步研究,为肿瘤血管内皮细胞的靶向治疗及临床应用奠定了基础.     

同型半胱氨酸对血管生成的影响及叶酸的作用

目的通过研究不同浓度同型半胱氨酸（homocysteine,HCY）对人脐静脉内皮细胞血管生成的影响及叶酸（folicacid,FA）对其的保护作用。方法实验分6组,0、1、10、100μmol／L同型半胱氨酸,10μmol／L同型半胱氨酸＋100μmol／L叶酸,100μmol／L叶酸,进行体外血管生成能力实验,Transwell迁移能力检测。Western-blot检查血管生成及迁移相关蛋白表达。结果同型半胱氨酸能明显抑制血管生长因子的表达,降低人脐静脉内皮细胞的成血管环能力。同型半胱氨酸能明显抑制迁移相关蛋白表达,降低人脐静脉内皮细胞的迁移运动能力。叶酸能改善同型半胱氨酸的抑制作用。结论同型半胱氨酸通过抑制血管生长因子、ROCKl／2的表达,从而抑制血管内皮细胞分裂增殖、血管环生成及迁移能力,导致内皮功能障碍,而叶酸能改善这种抑制作用。     

Vascular endothelial growth factor stimulates a novel calcium-signaling pathway in vascular smooth muscle cells

BACKGROUND: Vascular endothelial growth factor (VEGF) is a potent vascular mitogen that selectively stimulates vascular smooth muscle cell (VSMC) migration through an unknown mechanism while having no effect on VSMC proliferation. It is known that VSMC migration and proliferation are dependent on the second messenger Ca2+ and, in particular, mitogen-stimulated Ca2+ influx. We hypothesized that the selective effect of VEGF on VSMC migration versus proliferation was a result of differential VEGF-stimulated Ca2+ signaling pathways. METHODS: Primary cultured human aortic smooth muscle cells (VSMCs) were grown to subconfluency and assigned to the following experimental groups: no stimulation, stimulation with platelet-derived growth factor-BB (PDGF-BB) (20 ng/mL) as positive control, and stimulation with VEGF165 (40 ng/mL). Total increase in [Ca2+]cyt and intracellular calcium release was quantified with the use of a fura-2 fluorescence assay. Assays for the following receptors VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and PDGFR-beta were performed by immunoprecipitation, while PLCgamma1, Akt 1/2, and phospholamban B phosphorylation were assessed with Western immunoblotting. RESULTS: VSMCs stimulated with VEGF165 exhibited no intracellular Ca2+ release, compared with a 75 +/- 30 nmol/L intracellular calcium release after PDGF-BB stimulation, (P < .02) VEGF165-stimulated VSMCs in Ca2+-containing media exhibited 192 +/- 26 nmol/L increase in [Ca2+]cyt, compared with 354 +/- 54 nmol/L increase after PDGF-BB stimulation (P < .02). VEGF165 did not phosphorylate PLCgamma1 after 1, 5, or 10 minutes of treatment. VEGF165 treatment did not result in PI3-K/Akt activation at 1-, 5-, or 10-minute time points. Calmodulin-dependent kinase II (CaMKII) was activated by both VEGF165 and PDGF-BB after 1 and 5 minutes of stimulation. The presence of the receptors VEGFR-1, VEGFR-2, and PDGFR-beta was confirmed in all experimental groups. CONCLUSIONS: VEGF induces extracellular calcium influx but no intracellular calcium release in VSMCs. This lack of intracellular Ca2+ release stems from the inability of VEGF165 to activate the PLCgamma1 cascade and IP3 receptor-mediated Ca2+ release. The lack of PI3-K/Akt activation at these time points indicates a novel extracellular Ca2+ influx pathway sufficient to activate CaMKII. A paradigm relating extracellular Ca2+ influx to CaMKII activation and migration is suggested and may account for the selective effects of VEGF on VSMC migration.     

吗啡对人乳腺癌MDA-MB-231细胞增殖及血管内皮生长因子表达的影响

目的探究吗啡对三阴性乳腺癌细胞(人乳腺癌MDA-MB-231细胞)增殖及血管内皮生长因子(VEGF)表达的影响,并对PI3K-AKT-c-Myc信号通路的激活在此过程中的作用进行初步探讨。方法将人乳腺癌MDA-MB-231细胞随机分为阴性对照组(N组)、1μmol/L吗啡组(1μM组)、10μmol/L吗啡组(10μM组)以及100μmol/L组(100μM组),分别予无血清培养基、含1μmol/L吗啡的无血清培养基、含10μmol/L吗啡的无血清培养基及含100μmol/L吗啡的无血清培养基处理。处理后对各组细胞行MTS法检测细胞增殖情况,并行Western Blot实验检测细胞内VEGF、c-Myc的表达及AKT蛋白磷酸化水平。结果与N组相比,不同浓度吗啡处理对人乳腺癌MDA-MB-231细胞增殖、细胞内VEGF、c-Myc的表达及AKT蛋白磷酸化均有促进作用,其中以10μM组作用最为显著(P0.05)。结论 10μmol/L吗啡可促进人乳腺癌MDA-MB-231细胞增殖及VEGF的表达,此过程可能与激活细胞内PI3K-AKT-c-Myc信号通路有关。     

降钙素基因相关肽对人脐静脉血管内皮细胞增殖和迁移的影响及机制研究

目的组织工程骨的神经化能有效促进支架材料内血管生成,修复骨缺损。研究降钙素基因相关肽(calcitonin gene-related peptide,CGRP)对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖与迁移的作用,进一步揭示组织工程骨的神经化促血管生成机制。方法体外分离获取HUVECs,并通过血管性血友病因子(von Willebrand factor,vWF)与CD31抗原鉴定,取第1代细胞用于实验。实验分为6组,分别以0(A组)、1×10—12(B组)、1×10—11(C组)、1×10—10(D组)、1×10—9(E组)、1×10—8mol/L(F组)浓度CGRP干预HUVECs。采用细胞免疫荧光观察HUVECs的CGRP1受体(CGRP1 receptor,CGRP1R)表达情况,AlarmarBlue法动态检测各组HUVECs增殖率,Transwell小室检测各组HUVECs的迁移能力,ELISA法检测HUVECs分泌VEGF的水平,Westernblot法检测其局部黏着斑激酶(focal adhesion kinase,FAK)的表达。结果分离的细胞通过形态学及vWF、CD31免疫荧光鉴定为HUVECs,并可见CGRP1R在细胞质和细胞膜表达。CGRP呈时间-浓度依赖性刺激HUVECs增殖;B～F组各时间点细胞增殖能力均高于A组(P<0.05),F组各时间点细胞增殖能力最高。B～F组迁移细胞数均显著高于A组(P<0.05),最大增幅达3倍以上。B～F组VEGF分泌量均显著高于A组(P<0.05);C、D组促进细胞分泌VEGF的能力最强。Western blot检测示,与A组相比,B～F组CGRP刺激HUVECs 3、7、10 d后,FAK表达明显增加(P<0.05)。结论 CGRP对HUVECs的增殖和迁移有直接促进作用,可能作用机制为CGRP能促进VEGF分泌和增加FAK的表达。     

细胞外信号调节蛋白激酶介导醛固酮诱导的肾小球系膜细胞增殖

Objective To determine the role of extracellular signal-regulated kinases (ERK1/2) in aldosterone-induced rat mesangial cells (RMCs) proliferation. Methods RMCs were obtained from intact glomeruli of 4- to 6-week-old Sprague-Dawley rats and characterized according to published methods. RMCs between passages 5 and passages 10 were used. Protein levels of mineralocorticoid receptor(MR) in RMCs were analyzed by Western blotting. The cells were divided into the following groups: control group, PD98059(10 ?滋mol/L) group, eplerenone (1 ?滋mol/L) group, aldosterone (100 nmol/L) group, aldosterone (100 nmol/L) ＋PD98059 (10 ?滋mol/L) group, aldosterone(100 nmol/L)＋eplerenone (1 ?滋mol/L) group. ERK1/2 activity was measured by Western blotting. Cell proliferation of RMCs was evaluated by [3H]-thymidine uptake measurements. Results MR protein expression in RMCs was confirmed by Western blotting. Aldosterone activated ERK1/2, and the maximal ERK1/2 activation induced by aldosterone was at a concentration of 100 nmol/L. Aldosterone (100 nmol/L)-induced activation of ERK1/2 peaked at 10 minutes (P<0.05). Pretreatment with a selective MR antagonist eplerenone (1 ?滋mol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased [3H]-thymidine incorporation of RMCs (135%±8% of controls, P<0.05). Cellular proliferation induced by aldosterone could be prevented by pretreatment with eplerenone or an ERK (MEK) inhibitor PD988059. Conclusion Aldosterone induces RMCs proliferation through MR and ERK1/2 activation, which may contribute to the pathogenesis of glomerular mesangial injury.     

Basic fibroblast growth factor induces expression of VEGF receptor KDR through a protein kinase C and p44/p42 mitogen-activated protein kinase-dependent pathway.

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are angiogenic molecules whose combined mitogenic activity is potently synergistic. However, the molecular mechanism underlying this synergy is incompletely understood. We examined whether VEGF and bFGF affect expression of each other or alter expression of the VEGF receptor KDR in retinal capillary endothelial cells. In addition, we investigated the intracellular signaling mechanisms involved in this response. VEGF-induced [3H]thymidine uptake was tightly correlated with KDR mRNA and protein concentrations, suggesting that increased KDR expression might account for VEGF's synergistic activity in the presence of bFGF. bFGF (10 ng/ml) induced KDR mRNA expression within 4 h and attained a 4.0-fold increase after 24 h. KDR protein expression was increased 7.5-fold after 48 h. VEGF (= 50 ng/ml) did not alter bFGF, VEGF, or KDR mRNA expression under serum-deprived conditions. In contrast, VEGF increased KDR mRNA expression 87% under growth conditions and 2.9-fold under serum-deprived conditions in the presence of bFGF. The protein kinase C (PKC) agonist phorbol myristate acetate (PMA) induced KDR mRNA expression 5.1-fold at 100 nmol/l. bFGF increased p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation within 5 min, reaching a maximum within 15 min and remaining significantly elevated for >6 h. bFGF-induced MAPK phosphorylation and KDR mRNA expression were almost completely inhibited by 5 micromol/l GFX, a non-isoform-selective PKC inhibitor. MAPK inhibitor PD98059 reduced KDR mRNA expression 72% at concentrations that inhibited bFGF-induced MAPK phosphorylation 100%, suggesting that pathways in addition to MAPK might also be involved. Inhibitors of the beta isoform of PKC (LY333531), protein kinase A (PKA) (H89), and phosphotidylinositol (PI) 3 kinase (wortmannin) had no significant effect. These data suggest that bFGF stimulates KDR expression through a PKC and p44/p42 MAPK-dependent pathway not primarily involving the beta isoform of PKC, PKA, or PI-3 kinase. Since bFGF induces VEGF expression and since increased KDR expression potentiates VEGF action, resulting in additional KDR expression and marked mitogenic activity, these data provide a novel mechanistic explanation for the angiogenic synergy between VEGF and bFGF.