Down syndrome candidate region 1,a downstream target of VEGF, participates in endothelial cell migration and angiogenesis

Vascular endothelial growth factor (VEGF) is a principal stimulator of angiogenesis. However, the downstream targets of VEGF in endothelial cells (ECs) are not entirely clarified. Survey of downstream targets of VEGF in human ECs identified a number of genes, including Down syndrome candidate region 1 (DSCR1). Here, we confirmed the inducible expression of DSCR1 in ECs by Northern and Western blottings. Moreover, VEGF-stimulated induction of DSCR1 was blocked by anti-VEGF receptor-2 monoclonal antibody (mAb), or the specific calcineurin inhibitors cyclosporin A and FK506. The expression of DSCR1 in ECs of neovessels was further shown by immunohistochemical analysis. We therefore examined whether DSCR1 played any roles in angiogenesis. The specific downregulation of DSCR1 expression by antisense oligonucleotide (AS-ODN) inhibited VEGF-stimulated migration of ECs as well as angiogenesis in vivo. AS-ODN inhibited the spreading of ECs on vitronectin, as well as on the immobilized anti-alphavbeta3 mAb, but not on anti-alphavbeta5 mAb. Moreover, AS-ODN inhibited tyrosine phosphorylation of focal adhesion kinase when ECs were plated on a vitronectin-coated dish. Immunoprecipitation followed by Western blotting showed the coimmunoprecipitation of DSCR1 and integrin alphavbeta3. These results suggest that DSCR1 is involved in angiogenesis by regulating adhesion and migration of ECs via the interaction with integrin alphavbeta3.     

由cAMP激活的鸟苷酸交换蛋白（Epac）调控内皮细胞表面膜联蛋白A2的表达

目的 本研究旨在探讨内皮细胞内Epac1是否参与调控细胞表面ANXA2的表达。方法 本研究细胞实验以人静脉内皮细胞（HUVECs）为细胞模型,采用原子动力学检测方法,结合免疫荧光,分别从原子及蛋白水平验证Epac1是否参与细胞表面ANXA2表达的调控作用。分别提取15只Epac1基因敲除小鼠和15只C57BL6野生型小鼠血浆,检测每组样品ANXA2的表达水平。结果 （1）免疫荧光测得处理组（ESI09）相对荧光强度为17.59 ± 0.69,处理组（ESI09）相对荧光强度为6.32 ± 0.32,两组间数据结果有差异且有统计学意义（P<0.01）;原子动力显微镜法测得处理组（ESI09）测得力值为35113.07 ± 9866.65 pN,对照组（DMSO）测得力值为106277.70 ± 14233.77 pN,两组间数据结果有差异且有统计学意义（P<0.01）;由以上数据可得药物性抑制内皮细胞Epac的功能使其表面ANXA2表达量下调;（2）蛋白印迹法及酶联免疫吸附法测得处理组（ESI09）和对照组（DMSO）内皮细胞分泌ANXA2有差异,药物性抑制内皮细胞Epac1功能使内皮细胞胞外分泌ANXA2能力下降低;（3）酶联免疫吸附法测得Epac1基因敲除小鼠组血浆ANXA2平均含量为138.81 ± 38.93 ng/ml,显著低于野生型小鼠组血浆ANXA2平均含量281.46 ± 49.66 ng/ml（p<0.05）。结论 Epac1参与调控内皮细胞表面ANXA2的表达。     

Pregnancy-associated plasma protein a gene expression as a target of inflammatory cytokines

Pregnancy-associated plasma protein A (PAPP-A) cleaves IGF-binding protein-4 (IGFBP-4) and appears to enhance local IGF bioavailability in response to injury. In this study we determined the effects of growth factors and cytokines involved in the healing process on PAPP-A expression in human dermal fibroblasts. There was no effect of platelet-derived growth factor, epidermal growth factor, or basic fibroblast growth factor on PAPP-A mRNA expression in these cells. However, treatment with the proinflammatory cytokines, TNFalpha and IL-1 beta, resulted in time- and dose-dependent increases in PAPP-A mRNA and protein expression (3- to 4-fold maximal effects), which were prevented by actinomycin D. On the other hand, interferon-gamma (IFN gamma) treatment markedly inhibited PAPP-A expression. IGFBP-4 proteolytic activity was increased 4-fold in medium from TNFalpha- and IL-1 beta-treated (1 nm) cells and decreased 40% in medium from IFN gamma-treated (1 nm) cells. IGF-I-stimulated [(3)H]thymidine incorporation was significantly enhanced by pretreatment with 1 nm TNFalpha, and this enhancement was blocked in the presence of protease-resistant IGFBP-4. In conclusion, PAPP-A expression is regulated by inflammatory cytokines in adult human fibroblasts, with functional consequences on IGFBP-4 protease activity and IGF-I bioavailability. These data provide a mechanism for the regulation of PAPP-A in response to injury and further implicate PAPP-A in the wound-healing processes.     

The Drosophila homolog of Down's syndrome critical region 1 gene regulates learning: implications for mental retardation

Mental retardation is the most common phenotypic abnormality seen in Down's syndrome (DS) patients, yet the underlying mechanism remains mysterious. DS critical region 1 (DSCR1), located on chromosome 21, is overexpressed in the brain of DS fetus and encodes an inhibitor of calcineurin, but its physiological significance is unknown. To study its functional importance and role in mental retardation in DS, we generated Drosophila mutants of nebula, an ortholog of human DSCR1. Here, we report that both nebula loss-of-function and overexpression mutants exhibit severe learning defects that are attributed by biochemical perturbations rather than maldevelopment of the brain. These results, combined with our data showing that the same biochemical signaling pathway is altered in human DS fetal brain tissue overexpressing DSCR1, suggest that alteration of DSCR1 expression could contribute to mental retardation in DS.     

炎症细胞因子对人脐静脉内皮细胞分泌VEGF、ICAM-1的影响

目的：探讨炎症因子自细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNFα）对人脐静脉内皮细胞（HU—VEC）分泌血管内皮生长因子（VEGF）、细胞间粘附分子1（ICAM-1）的影响。方法：人脐静脉内皮细胞在37℃、5％CO2的条件下常规培养。实验采用4～5代细胞;按1×10^5／ml密度接种于5cm^2的培养皿中。24h后换液。生长接近80％融合时进行干预。按IL-1β组、TNF—α组、IL-1β＋TNF—α组、空白对照组进行刺激（IL—1β和TNFα干预浓度均为10ng／m1）,每组12个样本;在共同培养24h后收集细胞上清液。应用酶联免疫吸附测定（ELISA）法检测细胞上清液中VEGF、ICAM—1的浓度。结果：IL—1β组、TNF—α组、IL—1β＋TNF-α组的VEGF值（ng／ml）分别为（27．91±0．63）、（13．54±1．17）和（13．05±0．11）,均明显高于对照组（4．21±0．91）,P=0．000;IL—1β组、TNF-α组、IL-1β＋TNF—α组的ICAM-1值（ng／ml）分别为（2．88±0．98）、（2．96±0．03）和（2．30±0．20）．均明显高于空白对照组（0．25±0．01）。P=0．000。结论：炎症因子IL—1β、TNF—α均可促进人脐静脉内皮细胞分泌斑块不稳定相关标志物VEGF、ICAM-1,可能是炎症在急性冠脉综合征的发生、发展中起重要作用的机制之一。     

血管生成因子对心脏微血管内皮细胞神经调节蛋白1表达和分泌的影响

目的探讨血管内皮生长因子(VEGF)、血管形成蛋白1(Ang-1)和Ang-2对人源性心脏微血管内皮细胞(HCMEC)的神经调节蛋白1(NRG-1)表达和NRG-1β分泌的影响。方法培养HCMEC至4~5代,在正常培养条件下,将其分为正常组、VEGF处理组、Ang-1处理组和Ang-2处理组,与HCMEC共孵育24h,收集细胞和培养液,用蛋白印迹法和细胞酶联免疫吸附法分别检测NRG-1蛋白表达和NRG-1β分泌。蛋白印迹法检测酪氨酸激酶受体(ErbB2、ErbB3和ErbB4)蛋白表达。结果ErbB2、ErbB3和ErbB4均表达于HCMEC。与正常组比较,VEGF处理组和Ang-1处理组NRG-1[(1.44±0.05)、(1.18±0.04)vs(1.05±0.06)]表达和NRG-1β[(534.07±6.89)ng/L、(505.19±22.82)ng/L vs(380.71±14.96)ng/L]分泌显著增加,差异有统计学意义(P<0.05),而Ang-2处理组NRG-1表达和NRG-1β分泌显著降低(P<0.05)。结论VEGF和Ang-1显著增加HCMEC的NRG-1表达和NRG-1β分泌,Ang-2显著减少HCMEC的NRG-1表达和NRG-1β分泌。     

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。     

The orphan nuclear receptor, NOR-1, a target of beta-adrenergic signaling, regulates gene expression that controls oxidative metabolism in skeletal muscle

beta 1-3-Adrenoreceptor (AR)-deficient mice are unable to regulate energy expenditure and develop diet-induced obesity on a high-fat diet. We determined previously that beta2-AR agonist treatment activated expression of the mRNA encoding the orphan nuclear receptor, NOR-1, in muscle cells and plantaris muscle. Here we show that beta2-AR agonist treatment significantly and transiently activated the expression of NOR-1 (and the other members of the NR4A subgroup) in slow-twitch oxidative soleus muscle and fast-twitch glycolytic tibialis anterior muscle. The activation induced by beta-adrenergic signaling is consistent with the involvement of protein kinase A, MAPK, and phosphorylation of cAMP response element-binding protein. Stable cell lines transfected with a silent interfering RNA targeting NOR-1 displayed decreased palmitate oxidation and lactate accumulation. In concordance with these observations, ATP production in the NOR-1 silent interfering RNA (but not control)-transfected cells was resistant to (azide-mediated) inhibition of oxidative metabolism and expressed significantly higher levels of hypoxia inducible factor-1alpha. In addition, we observed the repression of genes that promote fatty acid oxidation (peroxisomal proliferator-activated receptor-gamma coactivator-1alpha/beta and lipin-1alpha) and trichloroacetic acid cycle-mediated carbohydrate (pyruvate) oxidation [pyruvate dehydrogenase phosphatase 1 regulatory and catalytic subunits (pyruvate dehydrogenase phosphatases-1r and -c)]. Furthermore, we observed that beta2-AR agonist administration in mouse skeletal muscle induced the expression of genes that activate fatty acid oxidation and modulate pyruvate use, including PGC-1alpha, lipin-1alpha, FOXO1, and PDK4. Finally, we demonstrate that NOR-1 is recruited to the lipin-1alpha and PDK-4 promoters, and this is consistent with NOR-1-mediated regulation of these genes. In conclusion, NOR-1 is necessary for oxidative metabolism in skeletal muscle.     

Gax基因双向调节低氧性肺动脉内皮细胞增殖及影响HIF-1α基因表达的研究

目的观察Gax基因对低氧性肺动脉内皮细胞（PAECs）增殖和低氧诱导因子-1α（HIF-1α）基因表达的影响,为进一步研究Gax基因调节低氧性肺动脉高压（HPH）的作用与机制奠定基础。方法取大鼠肺动脉,用酶消化法获取PAECs并进行原代培养;PAECs分4组：未转染常氧对照组（常氧组）、未转染低氧处理组（低氧组）、Ad—SGal转染再行低氧处理组（Ad—SGal＋低氧组）、Ad—Gax转染再行低氧处理组（Ad—Gax＋低氧组）。分别在常氧（21％O2）和低氧（2．5％O2）1h、3h、6h和12h各时相点,采用3H-胸腺嘧啶核苷（3H-TdR）掺入法检测PAECs增殖;使用RT—PCR和Weatern blot方法分别检测PAECs中HIF-1α mRNA和蛋白表达水平。结果①PAECs的3H-TdR掺入量：与常氧组同时相点比较,低氧组和Ad—pGaJ＋低氧组均显著升高（P均〈0．01）,在低氧6h达最大值;Ad．Gax＋低氧组与常氧组同时相点比较均显著升高（P均〈0．01）,但与低氧组比较却均明显降低（P〈0．01、P〈0．05）,到低氧6h降幅最大;②在低氧处理6h,与常氧组比较,低氧组和Ad—BGal＋低氧组HIF-1α mRNA和蛋白表达均明显上调;与低氧组或Ad—BGal＋低氧组比较,Ad—Gax＋低氧组HIF-1α mRNA和蛋白表达皆显著下调,差异有统计学意义（P〈0．05、P〈0．01）。低氧早期内皮细胞异常增殖加速,而此时增强Gax基因的表达可抑制细胞的异常增殖;随着低氧时间的不断延长,细胞增殖受到抑制,而此时增强内皮细胞中Gax基因表达却又促进细胞增殖,以此来维持细胞的数量。结论Gax基因对维持内皮细胞数量的稳态具有双向调节作用。增强Gax基因的表达能下调低氧诱导的HIF-1α mRNA和蛋白表达,这可能与Gax基因抑制低氧性内皮细胞异常增殖的机制相关。     

糖基化终产物对人脐静脉内皮细胞血管内皮生长因子mRNA及蛋白表达的影响

目的研究糖基化终产物（AGEs）对人脐静脉山皮细胞血管内皮生长凼子（VEGF）mRNA及蛋白表达的影响，探讨AGEs在糖尿病动脉粥样硬化中的作用。方法将人脐静脉内皮细胞株ECV304用BSA及小同浓度的AGEs孵育24h及400mg／L的AGEs孵育0、12、24及36h，采用原位杂交及Westem blot方法检测VEGFmRNA及蛋白的表达。结果人脐静脉内皮细胞在100、200及400mg／LAGEs孵育24h后，VEGFmRNA及蛋白表达均显著高于BSA对照组（P〈0．05），在用400mg／LAGEs分别孵育12．24及36h后，各组内皮细胞VEGF mRNA及蛋白表达量也明显高于0h对照组（P〈O．05）。结论AGEs可增加人脐静脉内皮细胞VEGF mRNA及蛋白的表达，且与浓度和时间呈正相关。     

缺氧条件下血管活性因子对血管内皮细胞中血管内皮生长因子表达的影响

目的　探讨在缺氧条件下人脐静脉血管内皮细胞血管内皮生长因子 (VEGF)表达及缩血管活性物质内皮素 ,舒血管活性物质NO和NO抑制剂LNNA对VEGF基因表达的影响。方法　体外培养人脐静脉血管内皮细胞 ,经缺氧及血管活性物质处理 ,Northern杂交、酶联免疫检测和计算机图像分析等观察VEGFmRNA和蛋白表达水平。结果　缺氧 6h内皮细胞可见VEGF表达。ET可促进VEGFmRNA的表达 ,NO可明显抑制VEGFmRNA的表达 ,NO抑制剂LNNA也影响VEGFmRNA的表达。ELISA检测VEGF蛋白水平分别为 6h组 (8 2± 1 1) μg/L ,ET +6h组 (9 37± 1 0 2 ) μg/L ,NO +6h组 (2 86± 0 91) μg/L ,LNNA +6h组 (14 75± 1 87)μg/L。 结论　缺氧可诱导人脐静脉血管内皮细胞分泌VEGF并受血管活性物质的调控 ,ET促进其表达 ,NO抑制其表达。     

rpt-1, an intracellular protein from helper/inducer T cells that regulates gene expression of interleukin 2 receptor and human immunodeficiency virus type 1.

The Rpt-1 (for regulatory protein, T-lymphocyte, 1) gene, selectively expressed by resting but not by activated CD4+ inducer T cells, encodes an intracellular protein (rpt-1, Mr 41,000) that down-regulates gene expression directed by the promoter region of the gene encoding interleukin 2 receptor alpha chain and by the long terminal repeat of human immunodeficiency virus type 1. The data reported here suggest that rpt-1 levels may be inversely correlated with activation of CD4+ T cells and human immunodeficiency virus replication leading to clinical symptoms of the acquired immunodeficiency syndrome.