Protease-activated receptors upregulate cyclooxygenase-2 expression in human endothelial cells

We have previously shown that the serine protease thrombin and other G protein-coupled agonists acutely enhance synthesis and release of prostacyclin from human umbilical vein endothelial cells (HUVEC) through activation of cPLA2 alpha. Here, we show that thrombin and other physiological endothelial cell agonists upregulate COX-2 induction in HUVEC. Thrombin treatment caused a rapid and sustained increase in prostacyclin (PGI2) synthesis from HUVEC. Thrombin and a selective protease-activated receptor-1 (PAR-1) peptide (TRAP) evoked dose- and time-dependent increases in COX-2 protein expression which were equivalent to that induced by the proinflammatory cytokine IL-1 alpha. Quantitative and real-time PCR analysis showed enhanced COX-2 mRNA expression in thrombin- or TRAP-stimulated HUVEC whereas COX-1 expression was unaffected. A PAR-2 agonist peptide also induced COX-2 protein and mRNA expression with kinetics distinct from those of thrombin, and promoted PGI2 release. These results demonstrate that regulation of COX-2 induction is an important functional response of HUVEC to PAR activation and suggest that PARs promote sustained upregulation of prostanoid production in human endothelium.     

纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子表达的影响

目的观察纤维蛋白对大鼠脑血管内皮细胞血管内皮生长因子(VEGF)转录及蛋白水平表达的影响。方法大鼠脑血管内皮细胞分离后培养,加入不同浓度的纤维蛋白,通过Real-time PCR检测VEGF转录水平,应用酶联免疫方法(ELISA)定量检测培养基和细胞裂解液中的VEGF水平。结果纤维蛋白可以特异性诱导大鼠脑血管内皮细胞表达VEGF;加入不同浓度的纤维蛋白(0.03mg/ml、0.1mg/ml、0.3mg/ml和1.0mg/ml),24h后,1.0mg/ml纤维蛋白组的培养基VEGF水平显著增高(P<0.001);1.0mg/ml纤维蛋白与大鼠脑血管内皮细胞分别培养0、2、4、8、24、48h,VEGF浓度在共培养2h已经升高,8h时显著升高,在24h时仍然保持在显著升高表达水平(P<0.005),48h有所下降;Real-time PCR结果提示,大鼠脑血管内皮细胞中VEGF mRNA的上调呈现出剂量和时间依赖性增加。结论纤维蛋白可以上调大鼠血管内皮细胞中的VEGF。     

Vascular endothelial growth factor and vascular endothelial growth factor receptor-2 expression in mdx mouse brain

Recent data have demonstrated that vascular endothelial growth factor (VEGF) is expressed by subsets of neurons, coincident with angiogenesis within its developing cerebral cortex. In this study, with the aim of elucidating the mechanisms of vascular involvement during brain impairment in Duchenne muscular distrophy (DMD), we have correlated the vascular density with VEGF and VEGF receptor-2 (VEGFR-2) expression in the brain cortex of normal and mdx mouse, an animal model with a genetic defect in a region homologous with the human DMD gene. Results showed that in mdx mouse, tissue area occupied by microvessels positive to factor VIII related antigen and VEGFR-2 increased in parallel to the tissue area occupied by neurons positive to VEGF. Our data suggest that increased vascularity in the brain of mdx mouse may be due, at least in part, to proliferation of endothelial cells in response to VEGF secreted by neuronal cells.     

MG63和MG63/ADR骨肉瘤裸鼠模型血管内皮生长因子和核因子κB1的表达

背景：恶性肿瘤耐药研究中发现核因子κB1及血管内皮生长因子基因可能与肿瘤耐药密切相关。 目的：观察MG63和MG63/ADR骨肉瘤裸鼠模型中血管内皮生长因子和核因子κB1表达的差异。 方法：对数期生长的MG63细胞采用阿霉素浓度递增培养法冲击诱导建立MG63/ADR细胞株。BALB/C裸鼠随机分为2组,分别将含5.0×106个MG63和MG63/ADR细胞的0.2 mL细胞悬液采用皮下接种法注入裸鼠的一侧腋部皮下。第6周处死裸鼠取材。 结果与结论：注射MG63和MG63/ADR细胞的2组裸鼠成瘤率均为100%;免疫组织化学及RT-PCR方法检测发现MG63/ADR组瘤灶中血管内皮生长因子和核因子κB1基因和蛋白的表达均明显高于MG63组(P < 0.05)。说明血管内皮生长因子和核因子κB1表达的上调可能是导致骨肉瘤耐药的重要原因。     

Platelet-activating factor enhances the expression of vascular endothelial growth factor in normal human astrocytes

Sulfo-glycosaminoglycans (sGAGs) are involved in the assembly of tau in at least a subpopulation of paired helical filaments (PHFs) in Alzheimer's disease (AD). To further understand the role of sGAG molecules in the structure of PHFs, we isolated PHFs from patients with AD and treated them with heparinase. Immunoelectron microscopy and Western blotting (WB) were used later on to analyze the changes obtained. The heparinase treatment abolished Tau14 and AT8 immunodecoration (two N-terminal tau antibodies) and increased PHF-1 labeling (a C-terminal antibody). In addition, heparinase-treated filaments are more labile than control ones as demonstrated by sodium dodecyl sulfate-extraction and subsequent WB. In summary, our results demonstrate that sGAG content affects PHF conformation as well as PHF-tau solubilization.     

血管内皮生长因子表达载体的构建及在内皮祖细胞中的表达

背景：血管内皮生长因子(vascular endothelial growth factor, VEGF)具有很强的促进血管生成作用，但构建其真核表达质粒是否可转染血管内皮祖细胞并完整表达还不清楚。 目的：构建VEGF表达载体,并观察其在内皮祖细胞中的表达情况。 设计、时间及地点：观察性试验，于2007-12/2008-03在上海交通大学医学院附属新华医院科研中心实验室完成。 材料：质粒PDC315-VEGF165自备，质粒pIRES2-EGFP由美国国立卫生研究院Stanko Stojilkovic教授惠赠。 方法：运用DNA重组技术构建VEGF表达载体pIRES2-EGFP-VEGF，应用脂质体包裹的方法将载体转染猪外周血血管内皮祖细胞。 主要观察指标：采用荧光显微镜观察VEGF在内皮祖细胞内的表达，反转录-聚合酶链反应法检测VEGFmRNA表达水平， ELISA检测VEGF165蛋白表达情况。 结果：成功构建VEGF真核表达载体pIRES2-EGFP-VEGF。转染重组质粒pIRES2-EGFP-VEGF后,在内皮祖细胞内有VEGF的表达，VEGFmRNA和VEGF165 蛋白表达水平均明显增加。 结论：VEGF 表达载体转染猪外周血血管内皮祖细胞后能有效增加VEGF基因的表达，能够获得较高水平的VEGF蛋白。     

Daunorubicin inhibits gene expression of cyclooxygenase-2 in vascular smooth muscle cells.

Daunorubicin (0.1-1 microM) concentration-dependently inhibited prostacyclin production induced by interleukin-1beta (IL-1beta, 2.5 ng/ml) in cultured aortic smooth muscle cells isolated from rats. IL-1beta stimulation caused activation of nuclear factor-kappaB (NF-kappaB) and expression of cyclooxygenase-2 (COX-2) mRNA and protein, which were inhibited by daunorubicin. However, COX activity, evaluated by conversion of exogenous arachidonic acid to prostacyclin, was not affected by daunorubicin (0.1-1 microM). Protein expression of COX-1 and NF-kappaB was not affected by daunorubicin. Daunorubicin also inhibited nitric oxide (NO) production induced by IL-1beta. These results suggest that daunorubicin attenuated prostacyclin synthesis through inhibiting expression of COX-2 mRNA, which could be explained by perturbation of NF-kappaB activation.     

蛋白酶体抑制剂MG-132作用下失神经骨骼肌myf-5的表达

背景：MG-132是蛋白酶体抑制剂的一种,有报道显示其对失神经骨骼肌萎缩有延缓作用。目的：进一步验证蛋白酶体抑制剂MG-132对大鼠骨骼肌成肌调节因子myf-5基因表达的影响。方法：SD大鼠随机被分成3组,空白对照组不切断坐骨神经,仅做假手术,去神经组和去神经MG-132干预组切断坐骨神经1 cm以上,制作失神经骨骼肌动物模型,去神经MG-132干预组肌肉内注射MG-132。分别于去神经第2,7,28 d处死大鼠。用反转录聚合酶链式反应技术检测myf-5 mRNA表达情况,Western-blot检测myf-5蛋白表达的变化。结果及结论：在去神经支配后第2,7,28天,与空白对照组比较,去神经组、去神经MG-132干预组myf-5mRNA和蛋白质均表达上调(P < 0.01);去神经MG-132干预组myf-5mRNA和蛋白表达较去神经组均明显上调(P < 0.01)。结果提示蛋白酶体抑制剂MG-132可以通过抑制泛素-蛋白酶体途径来上调myf-5的表达,从而起到延缓骨骼肌萎缩的作用。     

Vasopressin induces human mesangial cell growth via induction of vascular endothelial growth factor secretion

Vasoactive hormones, growth factors, and cytokines are important in promoting mesangial cell growth, a characteristic feature of many glomerular diseases. Vascular endothelial growth factor (VEGF) is an endothelial mitogen and promoter of vascular permeability that is constitutively expressed in human glomeruli, but its role in the kidney is still unclear. In the present study, we investigated the ability of vasopressin (AVP) to stimulate VEGF secretion by and correlation with AVP-induced cell growth in human mesangial cells. AVP caused time- and concentration-dependent increases in VEGF secretion from human mesangial cells, which was in turn potently inhibited by a V_1A receptor-selective antagonist, confirming that this secretion is a V_1A receptor-mediated event. VEGF also induced mesangial cell growth which was completely inhibited on administration of an anti-VEGF neutralizing antibody. Further, AVP-induced mesangial cell growth was completely abolished by the V_1A receptor-selective antagonist and partially inhibited by an anti-VEGF neutralizing antibody. These results suggest that AVP stimulates VEGF secretion by human mesangial cells via V_1A receptors. This secreted VEGF may function as an autocrine hormone to regulate mesangial cell growth, a mechanism by which AVP might contribute to progressive glomerular diseases such as diabetic nephropathy.     

The transcription factor Nurr1 in human NT2 cells and hNT neurons

Human, neuronally committed hNT or NT2-N cells, originally derived from the Ntera2/D1 (NT2) clone after exposure to retinoic acid (RA), represent a potentially important source of cells to treat neurodegenerative diseases. Our previous in vitro experiments showed that hNT cells possess immunocytochemically detectable markers typical of dopaminergic (DA) ventral mesencephalic (VM) neurons, including tyrosine hydroxylase (TH), dopamine transporter (DAT), dopamine receptor (D2), and aldehyde dehydrogenase (AHD-2). In the current study, we sought to examine whether Nurr1, an orphan receptor of the nuclear receptor superfamily shown to be essential for the development, differentiation and survival of midbrain DA neurons, would be expressed in 3, 4, or 5 week RA-induced hNT neurons and their NT2 precursors. Our immunocytochemical analyses indicate that NT2 cells as well as hNT neurons independent of the length of RA-driven differentiation were Nurr1-immunoreactive. RT-PCR analysis confirmed the expression of Nurr1-specific mRNA in both NT2 precursors and the hNT neurons. Furthermore, immunocytochemical co-expression of Nurr1 and TH was detected in hNT neurons. The findings of this study suggest that Nurr1 may be important during the development of hNT neurons and involved in their differentiation into the dopaminergic phenotype.     

人血管内皮生长因子165基因转染兔骨髓间充质干细胞的蛋白表达

背景：骨髓间充质干细胞是最好的组织工程种子细胞来源,含有血管内皮生长因子165(vascular endothelial growth factor 165,VEGF165)不仅对血管再生和启动成骨修复有重要意义,其持续稳定的释放还能够提高新生骨的矿化程度,增强修复组织的力学性能。 目的：观察hVEGF165基因转染的兔骨髓间充质干细胞分泌血管内皮生长因子的蛋白功能。 方法：体外分离、培养兔骨髓间充质干细胞,纯化并鉴定兔骨髓间充质干细胞;免疫荧光法检测细胞表面标志;传代培养后的骨髓间充质干细胞以pcDNA3.1-VEGF165质粒和脂质体1∶3比例的混合液转染,并分为3组：转染组应用pcDNA3.1-VEGF165转染细胞,空载体转染组应用pcDNA3.1-空载体转染,未转染组不处理。通过ELISA和Western-blot检测转染后细胞中外源性血管内皮生长因子的表达。 结果与结论：转染组与其他两组比较,VEGF165蛋白含量显著增高,差异有显著性意义(P < 0.05),但空载体转染组与未转染组之间差异无显著性意义(P > 0.05),转染组不同时间点之间VEGF165蛋白含量差异均有显著性意义(P < 0.05),hVEGF165基因转染的骨髓间充质干细胞能成功分泌VEGF165蛋白。提示采用基因转染技术可将hVEGF165基因转染到骨髓间充质干细胞中并可有效表达具有生物活性的VEGF165。