Culture of endothelial cells by transfection with plasmid harboring vascular endothelial growth factor

Vascular endothelial cells (ECs) are usually difficult to culture in a large scale because of their complicated requirements for cell growth. As the vascular endothelial growth factor (VEGF) is a key growth factor in the EC culture, we transfected human umbilical vein endothelial cells (HUVEC) using a plasmid containing VEGF gene and let them grow in a culture medium eliminated an important supplement, endothelail cell growth supplement (ECGS). The expression of VEGF by HUVEC tansfected with VEGF gene was not enough to stimulate the growth of HUVEC, only 40% of maximum cell density obtainable in the presence of ECGS., However, when the culture medium was supplied with 2.5 ng/mL of basic fibroblast growth factor (bFGF), a synergistic effect of VEGF and bFGF was observed. In this case, the final cell density was recovered up to about 78% of maxium value.     

p38丝裂原素激活的蛋白激酶在调节低氧诱导人内皮细胞分泌血管内皮生长因子过程中的作用

血管内皮细胞中血管内皮生长因子(vascular endothelial growthfactor,VEGF)的合成增加在促进血管新生的过程中起着非常重要的作用.然而低氧诱导VEGF分泌的细胞内信号转导机制还不是很清楚.人脐静脉内皮细胞系(ECV304)在低氧或常氧的状态下培养12～24 h后分别用实时定量PCR和Western blot的方法来检测VEGF mRNA的表达及ERK1/2和p38激酶的磷酸化水平.分泌到培养液中的VEGF蛋白用酶联免疫吸附(ELISA)的方法来检测.业已报道,ERK的抑制剂PD98059能够抑制低氧诱导的VEGF基因的表达,根据这个报道,我们发现在低氧情况下,ECV304细胞的ERK1/2磷酸化水平增高以及VEGF的合成增加等这些变化也能被PD98059所抑制.本次实验的新发现是p38激酶的激活在低氧诱导VEGF合成增加中的作用.p38激酶的抑制剂SB202190能抑制低氧诱导的VEGF合成增加.这些数据首次直接证实了p38激酶在低氧诱导人内皮细胞分泌VEGF增加过程中的作用.     

Circulating and exhaled vascular endothelial growth factor in asthmatic pregnancy

Context: Vascular endothelial growth factor (VEGF) plays a role in asthma and pathological pregnancies.

Objective: This is the first study assessing plasma and exhaled breath condensate VEGF levels in asthmatic pregnancy.

Material and methods: Thirty-one asthmatic pregnant, 29 asthmatic nonpregnant, 28 healthy pregnant and 22 healthy nonpregnant women were enrolled. Plasma was collected in all subjects, EBC in 57 volunteers for VEGF measurements.

Results: Plasma VEGF decreased in both pregnant groups (p < 0.01), without any differences between the asthmatic and the respective nonasthmatic groups (p > 0.05). VEGF was undetectable in EBC.

Conclusion: Concomitant asthma does not affect plasma VEGF during pregnancy.     

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [³H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [³H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.     

血管内皮生长因子特异性鼠源单链抗体的人源化

以抗体阻断血管生成信号来治疗实体肿瘤显示了很好的前景,但鼠源抗体首先必须经人源化改造以降低其免疫原性才能应用于人体。本研究以同源模建预测了一体人血管内皮生长因子（VEGF）特异性鼠源单链抗体E11的三维结构,以结构数据为基础并采取单个最相似框架区替代法对其进行人源化设计;合成并组装了人源化单链抗体基因并在大肠杆菌中表达,包含体形式的产物以凝胶柱色谱法复性,经ELISA检测表明,人源化后的单链抗体保持了与天然VEGF结合的活性,表明采取的人源化路线具有可行性。     

Proinflammatory cytokines upregulate mRNA expression and secretion of vascular endothelial growth factor in cultured human airway smooth muscle cells

Airflow obstruction in chronic airway disease is associated with airway and pulmonary vascular remodeling, of which the molecular mechanisms are poorly understood. Paracrine actions of angiogenic factors released by resident or infiltrating inflammatory cells following activation by proinflammatory cytokines in diseased airways could play a major role in the airway vascular remodeling process. Here, the proinflammatory cytokines interleukin (IL)-1β, and tumor necrosis factor (TNF)-α were investigated on cell cultures of human airway smooth muscle (ASM) for their effects on mRNA induction and protein release of the angiogenic peptide, vascular endothelial growth factor (VEGF). IL-1β (0.5 ng/mL) and TNF-α (10ng/mL) each increased VEGF mRNA (3.9 and 1.7 kb) expression in human ASM cells, reaching maximal levels between 16 and 24 and 4 and 8h, respectively. Both cytokines also induced a time-dependent release of VEGF, which was not associated with increased ASM growth. Preincubation of cells with 1μM dexamethasone abolished enhanced release of VEGF by TNF-α. The data suggest that human ASM cells express and secrete VEGF in response to proinflammatory cytokines and may participate in paracrine inflammatory mechanisms of vascular remodeling in chronic airway disease.