The vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR-1) modulates Flk-1 (VEGFR-2) signaling during blood vessel formation

Mice lacking the vascular endothelial growth factor (VEGF) receptor flt-1 (VEGFR-1) die from vascular overgrowth, caused primarily by aberrant endothelial cell division (Kearney JB, Ambler CA, Monaco KA, Johnson N, Rapoport RG, Bautch VL: Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. Blood 2002, 99:2397-2407). Because a second high-affinity VEGF receptor, flk-1, produces a positive endothelial proliferation signal, it was logical to ask whether flt-1 affects developmental blood vessel formation by modulating signaling through flk-1. Differentiated embryonic stem cell cultures lacking flt-1 (flt-1-/-) had increased flk-1 tyrosine phosphorylation, indicating that flk-1 signaling is up-regulated in the mutant background. The selective flk-1 inhibitor SU5416 partially rescued the flt-1-/- mutant phenotype, and this rescue was accompanied by a decrease in the relative amount of flk-1 tyrosine phosphorylation. Thus reduced flk-1 signal transduction can partially compensate for the lack of flt-1. The flt-1-/- mutant phenotype was also partially rescued by Flt-1/Fc, a truncated flt-1 that binds and sequesters the VEGF ligand. Taken together, these data show that down-regulation of flk-1 signaling by two different strategies partially rescues the developmental vascular overgrowth seen in the absence of flt-1, and they support a model whereby flt-1 modulates the flk-1 signal at an early point in the pathway.     

牛蒡子苷调控血管内皮生长因子抑制糖尿病视网膜病变的机制研究

目的探讨牛蒡子苷(Arc)是否通过调控血管内皮生长因子(VEGF)表达进而抑制糖尿病视网膜病变。方法体外培养人视网膜微血管内皮细胞(HRCECs),用不同浓度的Arc处理HRCECs,同时将si-VEGF或pcDNA3.1-VEGF分别转染入HRCECs,MTT法检测HRCECs增殖能力;Western blot检测p21、细胞周期蛋白D1(cyclinD1)、肿瘤坏死因子-α(TNF-α)、人单核细胞趋化蛋白-1(MCP-1)、载脂蛋白M(ApoM)、VEGF蛋白表达。结果与LG组相比,HG组HRCECs的OD值明显升高(P<0.05),cyclinD1、TNF-α、MCP-1、ApoM、VEGF水平均显著升高(P<0.05),而p21表达下调(P<0.05),Arc作用后可显著逆转HG对HRCECs增殖、cyclinD1、TNF-α、MCP-1、ApoM、VEGF及p21表达的作用,且Arc不同剂量组间均存在显著性差异(P<0.05);抑制VEGF表达可明显减弱高糖作用下的HRCECs增殖能力并降低相关炎性因子表达;过表达VEGF能逆转Arc对高糖诱导的HRCECs增殖及炎症反应的抑制作用。结论牛蒡子苷可能通过降低高糖诱导的HRCECs细胞中VEGF的高表达进而抑制HRCECs增殖,并可通过减轻炎症反应保护细胞免受损伤。     

Protective effect of vascular endothelial growth factor/vascular permeability factor 165 and 121 on glomerular endothelial cell injury in the rat

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) promotes the repair of injured vessels by stimulating angiogenesis. VEGF/VPF reportedly has cytoprotective activity but no study has shown the protective effect of VEGF/VPF on glomerular endothelial cells. We examined whether recombinant VEGF/VPF121 and VEGF/VPF165 isoforms could prevent injury of glomerular endothelial cells. Mild glomerular injury was induced in rats by an intravenous-injection of a limited dose of anti-Thy-1.1 antibody to obtain lesions similar to those found in the human disease. Recombinant VEGF/VPF165, VEGF/VPF121 or BSA was administered 4 h before the injection of the antibody, and once daily for 3 days. In the BSA-injected rats, mesangial cell lysis and endothelial cell injury in dilated capillary tufts were evident without endothelial cell apoptosis on days 1-4. Thereafter, cell proliferation and repair began and remodeling of the glomeruli was completed by day 28. Macrophages but not polymorphonuclear leukocytes accumulated significantly in the glomeruli on days 1-4. Treatment with VEGF/VPF isoform protected endothelial cells but not mesangial cells from destruction on day 1, and accelerated the repair of both types of cells, which was completed by day 18, 10 days earlier than that of the control animals. The results indicate that VEGF/VPF121 or VEGF/VPF165 can protect glomerular endothelial cells against injury, independent of apoptosis-inhibition activity, thereby promoting reconstruction of glomeruli. The protective effect of VEGF/VPF on endothelial cells suggests that it could provide therapeutic benefit for certain kidney diseases.     

血管内皮生长因子C及其受体3在非小细胞肺癌组织中的表达及意义

目的　探讨血管内皮生长因子C(VEGF C)和受体 (VEGFR) 3在人非小细胞肺癌(NSCLC)组织中的表达及其与微血管、微淋巴管形成、淋巴转移、预后之间的关系。方法　对 76例人NSCLC及相应癌旁组织行VEGF C、VEGFR 3及CD34免疫组织化学染色链霉素抗生物素蛋白 过氧化物酶 (SP)法检测 ,进行淋巴管密度计数、微血管密度 (MVD)计数 ,并结合临床和病理资料进行分析。结果　NSCLC中 ,VEGF C的表达与肺癌分化程度负相关 (P =0 0 0 9)。VEGF C和VEGFR 3的表达水平与淋巴结转移呈正相关 (分别P =0 0 0 8,P =0 0 13) ,与淋巴浸润呈正相关 (分别P =0 0 2 7,P =0 0 2 0 )。VEGF C的表达与VEGFR 3在肺癌细胞中的表达呈正相关 (P =0 0 0 9)。VEGF C与淋巴管密度 (P =0 0 0 6 )、MVD(P =0 0 4 6 )呈正相关。淋巴管密度与淋巴结转移 (P =0 0 10 )、淋巴浸润 (P =0 0 19)、TNM分期 (P =0 0 15 )呈正相关 ,MVD与血行转移 (P <0 0 0 1)、TNM分期 (P <0 0 0 1)呈正相关。VEGF C阳性表达与生存时间、5年生存率呈负相关 (P <0 0 0 1)。结论　NSCLC中 ,VEGF C通过自分泌方式作用于受体VEGFR 3,促进肺癌组织生长 ,抑制分化。VEGF C促使肺癌内淋巴管形成 ,促进肺癌淋巴结转移。VEGF C和VEGFR 3表达增高、淋巴管密度增加     

Combined inhibition of vascular endothelial growth factor (VEGF), fibroblast growth factor and platelet-derived growth factor, but not inhibition of VEGF alone, effectively suppresses angiogenesis and vessel maturation in endometriotic lesions

BACKGROUND: Angiogenesis represents the crucial step in the pathogenesis of endometriosis, because endometriotic lesions require neovascularization to establish, proliferate and invade inside the peritoneal cavity. To elucidate the role of angiogenic factors, we investigated in vivo whether blockade of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) affects angiogenesis of ectopic endometrium. METHODS: Mechanically isolated endometrial fragments were transplanted into the dorsal skinfold chamber of hormonally synchronized hamsters. Subsequently, we analysed the effect of the VEGF inhibitor SU5416 and the combined VEGF, FGF and PDGF inhibitor SU6668 on angiogenesis of the ectopic endometrium over a time-period of 14 days using intravital fluorescence microscopy. RESULTS: Selective blockade of VEGF resulted in a slight reduction of microvessel density when compared to control animals. In contrast, combined inhibition of all three growth factors significantly suppressed angiogenesis of endometrial grafts, as indicated by a reduced size of the microvascular network and a decreased microvessel density. This was caused by an inhibition of blood vessel maturation. CONCLUSIONS: Vascularization of endometriotic lesions is not solely driven by VEGF, but depends on the cross-talk between VEGF, FGF and PDGF. Thus, the combined inhibition of these growth factors may represent a novel therapeutic strategy in the treatment of endometriosis.     

The World Health Organization reference reagent for vascular endothelial growth factor,VEGF165

Preparations of human sequence recombinant vascular endothelial growth factor-165 (VEGF₁₆₅) synthesized in Escherichia coli were formulated and lyophilized at NIBSC. Following evaluation at NIBSC, the first preparation, 01/424, has been distributed since 2002 as a NIBSC research reagent, but shows variation between ampoules in the volume and crystalline appearance of the lyophilized plug. A second preparation, 02/286, was subsequently lyophilized in a different formulation. Preparation 02/286 has now been evaluated in a collaborative study for its suitability to serve as a reference standard, and compared with preparation 01/424, by five laboratories using in vitro bioassays or immunoassays. On the basis of the results reported here, the World Health Organization (WHO) established the preparation coded 02/286 as the WHO reference reagent (RR) for human VEGF₁₆₅, with an assigned unitage of 13,000 units per ampoule. Details on ordering the WHO RR can be found at www.nibsc.ac.uk.     

The World Health Organization reference reagent for vascular endothelial growth factor, VEGF165

Preparations of human sequence recombinant vascular endothelial growth factor-165 (VEGF165) synthesized in Escherichia coli were formulated and lyophilized at NIBSC. Following evaluation at NIBSC, the first preparation, 01/424, has been distributed since 2002 as a NIBSC research reagent, but shows variation between ampoules in the volume and crystalline appearance of the lyophilized plug. A second preparation, 02/286, was subsequently lyophilized in a different formulation. Preparation 02/286 has now been evaluated in a collaborative study for its suitability to serve as a reference standard, and compared with preparation 01/424, by five laboratories using in vitro bioassays or immunoassays. On the basis of the results reported here, the World Health Organization (WHO) established the preparation coded 02/286 as the WHO reference reagent (RR) for human VEGF165, with an assigned unitage of 13,000 units per ampoule.     

Hepatocyte growth factor enhances vascular endothelial growth factor-induced angiogenesis in vitro and in vivo

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis in both physiological and pathological processes. Hepatocyte growth factor (HGF) is a mesenchyme-derived mitogen that also stimulates cell migration, and branching and/or tubular morphogenesis of epithelial and endothelial cells. In the present study, we tested the hypothesis that simultaneous administration of HGF and VEGF would synergistically promote new blood vessel formation. HGF acted in concert with VEGF to promote human endothelial cell survival and tubulogenesis in 3-D type I collagen gels, a response that did not occur with either growth factor alone. The synergistic effects of VEGF and HGF on endothelial survival correlated with greatly augmented mRNA levels for the anti-apoptotic genes Bcl-2 and A1. Co-culture experiments with human neonatal dermal fibroblasts and human umbilical vein endothelial cells demonstrated that neonatal dermal fibroblasts, in combination with VEGF, stimulated human umbilical vein endothelial cells tubulogenesis through the paracrine secretion of HGF. Finally, in vivo experiments demonstrated that the combination of HGF and VEGF increased neovascularization in the rat corneal assay greater than either growth factor alone. We suggest that combination therapy using HGF and VEGF co-administration may provide a more effective strategy to achieve therapeutic angiogenesis.     

体外重组表达血管生长因子VEGF165单克隆抗体的制备及鉴定

目的:研制人VEGF165单克隆抗体(mAb),为研究VEGF165的生物学活性提供基础.方法:应用RT-PCR从脐静脉内皮细胞中克隆人VEGF165基因,克隆人原核表达载体pGEX-6P1中获得重组表达载体pGEX-6P1 -VEGF165,转化大肠杆菌BL21,经IPTG诱导表达获得重组人VEGF165蛋白,将重组蛋白纯化后免疫BALB/c小鼠,通过杂交瘤技术,制备人VEGF165高效价的mAb.通过鸡胚血管形成抑制实验、HUVEC迁移抑制实验以及HUVEC血管形成抑制实验,对获得的人VEGF165特异性mAb进行进一步鉴定.结果:成功地从脐静脉血管内皮细胞中克隆出人VEGF165基因,并在大肠杆菌表达系统中获得高效表达,以纯化重组蛋白作为免疫原免疫小鼠,筛选获得5株分泌人VEGF165特异性mAb的杂交瘤细胞株,分别命名为5A6、3F5、6H3、7D10、7A10,其中5A6、3F5、6H3、7D10分泌的mAb亚类为IgG2a,7A10分泌的mAb亚类为IgG2b,抗体轻链均为κ链.5株mAb均能抑制鸡胚血管形成、抑制HUVEC迁移和及血管形成.结论:所获得的mAb具有效价高,活性强的优点,为抗肿瘤血管研究中进一步研究VEGF165的生物学作用提供了重要的基础.     

On the detection of neutrophil-derived vascular endothelial growth factor (VEGF)

In recent years, several investigators have addressed the question of whether mature polymorphonuclear neutrophils (PMN) are able to secrete cytokines. Their studies have brought forward new and exciting discoveries, by establishing that the release of inflammatory cytokines constitutes a novel and important aspect of the neutrophil biology, thereby emphasizing that PMN should no longer be regarded as cells that only release preformed mediators. Although it is still premature to assess the true biological significance of cytokine production by neutrophils, this new aspect of neutrophil biology opens novel perspectives as to the potential role of these cells in the inflammatory and immune responses. In this context, a correct methodological analysis and a detailed molecular investigation of the mechanisms regulating cytokine production by neutrophils in vitro is a critical and fundamental step to better understand how the release of cytokines by PMN may influence pathophysiological processes in vivo. We now describe and discuss the approach that we typically used throughout most of the last decade to characterize cytokine production by human neutrophils, as illustrated herein for a protein that is expressed and released by PMN, namely, vascular endothelial growth factor (VEGF).     

Expression and prognostic relevance of vascular endothelial growth factor (VEGF) and its receptor (FLT-1) in nephroblastoma

AIMS: To investigate the prognostic relevance of vascular endothelial growth factor (VEGF) and its receptor Flt-1 in nephroblastoma and whether tumour microvessel density (MVD) immunoreactivity, determined by the CD31 antigen, is related to the expression of VEGF and Flt-1. METHODS: The expression of VEGF and Flt-1 and MVD were investigated by means of immunohistochemical analysis in 62 Wilms's tumours. Patients were treated preoperatively with chemotherapy and had a mean follow up of 5.7 years. RESULTS: In general, VEGF and Flt-1 were expressed in normal kidney parenchyma and to a variable extent in the three main components of Wilms's tumour, namely: the blastemal, epithelial, and stromal cells. In tumour tissue, 52% and 47% of blastemal cells were positive for VEGF and Flt-1, respectively. A non-significant correlation was found between the expression of VEGF and Flt-1 in blastemal and epithelial cells and the clinicopathological stage. MVD was significantly higher in VEGF and Flt-1 positive tumours than in VEGF and Flt-1 negative tumours. Univariate analysis showed that the expression of VEGF and Flt-1 in blastemal cells was indicative of clinical progression and tumour specific survival. In addition, MVD expression was indicative of clinical progression. Epithelial staining was of no prognostic value. In a multivariate analysis, VEGF protein expression by blastemal cells was an independent prognostic marker for clinical progression. CONCLUSIONS: These results indicate that VEGF and Flt-1 protein expression are closely related to MVD and seem to be an important predictor for poor prognosis in treated patients with Wilms's tumour. Therefore, the expression of these molecules in primary Wilms's tumour may be useful in identifying those patients at high risk of tumour recurrence and in guiding antiangiogenic treatment.     

甲状腺癌组织中VEGF和VEGF-C的表达及意义

目的　探讨血管内皮生长因子(VEGF)、VEGF- C在甲状腺癌中的表达及其意义。方法　应用免疫组化S P法检测44例甲状腺癌中VEGF、VEGF C的表达情况,并以17例癌旁正常甲状腺组织作对照。结果　VEGF、VEGF- C在甲状腺癌中呈高水平表达(88. 6%、81. 8% )。VEGF表达随癌组织分化程度的减低而增高, 9例死亡病例均为阳性表达;VEGF- C表达随癌组织分化程度的减低而减低,乳头状癌阳性表达(88 .9% )高于其它类型,VEGF- C阳性率在有淋巴结转移组(92. 0% )明显高于无淋巴结转移组(68. 4% ) (P<0. 05)。9例死亡病例中7例为阳性表达,且7例同时VEGF呈阳性表达。结论　VEGF、VEGF- C表达与甲状腺癌病理分型及预后可能有一定关系,VEGF- C与甲状腺癌淋巴结转移密切相关。     

血管内皮细胞生长因子促进成年小鼠胸腺细胞的增殖与分化

目的 研究血管内皮生长因子（VEGF121）在免疫系统的表达及其对小鼠胸腺细胞的作用。为了解VEGF在免疫系统中的功能提供实验依据。方法 构建pCDI-VEGF真核表达载体，转染COS-7细胞，收获上清检测其对小鼠胸腺细胞的促增殖效应和CD4，CD8分子表达，用抗VEGF单克隆抗体及不同信号转导抑制剂进行阻断实验。通过RT-PCR方法检测人VEGFmRNA在胸腺，脾脏等免疫器官的表达。结果 实验证明VEGFmRNA可以在多种免疫组织中表达。VEGF121真核表达蛋白可明显促进小鼠胸腺细胞增殖和分化。此作用可被VEGF的单克隆抗体所封闭；不同的信号转导抑制剂具有不同的效应。结论 VEGF参与了免疫系统的功能，对小鼠胸腺细胞的增殖分化有促进作用。可能是内源性的免疫系统的调节因子。     

Immunolocalisation of vascular endothelial growth factor (VEGF) in human neonatal growth plate cartilage

Angiogenesis is essential for the replacement of cartilage by bone during growth and repair. In order to obtain a better understanding of the mechanisms regulating vascular invasion at sites of endochondral ossification we have investigated the expression of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), by chondrocytes in human neonatal growth plates. VEGF was absent from chondrocytes in the resting zone and only weakly expressed by occasional chondrocytes in the proliferating region. In the hypertrophic zone the number of chondrocytes stained and the intensity of staining for VEGF increased with chondrocyte hypertrophy, maximum expression of VEGF being observed in chondrocytes in the lower hypertrophic and mineralised regions of the cartilage. These observations provide the first demonstration of the presence of VEGF in situ in developing human bone and are consistent with in vitro observations demonstrating the upregulation of proangiogenic growth factor production with increasing chondrocyte hypertrophy. The presence of numerous small blood vessels and vascular structures in the subchondral region where VEGF expression was maximal indicates that VEGF produced by hypertrophic chondrocytes may play a key role in the regulation of vascular invasion of the growth plate.     

人VEGF165基因真核表达质粒的构建及其对血管内皮细胞增殖的影响

目的 :构建人VEGF16 5基因的真核表达质粒pBudCE4 .1/VEGF16 5 ,观察其在血管内皮细胞 (VEC)中的表达和表达产物对VEC增殖的影响。方法 :用RT PCR法从引产胎儿心脏组织中克隆VEGF16 5基因 ,并将其克隆至真核表达质粒pBud CE4 .1中 ,对重组真核表达质粒pBudCE4 .1/VEGF16 5进行酶切鉴定和测序。以脂质体转染法将pBudCE4 .1/VEGF16 5导入VEC中 ,用Northernblot和免疫细胞化学染色法 ,分别从mR NA水平和蛋白质水平检测它们在转染的VEC中的表达 ,并检测表达产物对VEC增殖的影响。结果 :人VEGF16 5基因的RT PCR产物为 5 76bp。测序结果显示 ,扩增的VEGF16 5基因的序列与基因文库中登录的序列完全一致。经HindⅢ和BamHI酶切鉴定证实 ,VEGF16 5基因已成功地克隆至真核表达质粒pBudCE4 .1中。以其转染血管内皮细胞 (VEC)后 ,经Northernblot杂交和免疫细胞化学染色法检测均证实VEGF16 5基因表达。表达产物对VEC增殖有明显的促进作用。结论 :所构建的pBudCE4 .1/VEGF16 5真核表达质粒可在VEC中表达 ,表达产物可明显促进VEC增殖 ,为通过VEGF16 5基因转染防治移植器官内血管的狭窄奠定了基础     

Analysis of vascular endothelial growth factor (VEGF) functional variants in rheumatoid arthritis

The vascular endothelial growth factor (VEGF) is one of the most important pro-angiogenic mediators related to inflammation-associated synovial angiogenesis. The aim of this study was to asses the role of -1154 G-->A (rs1570360) and -634 G-->C (rs2010963) VEGF gene functional variants with rheumatoid arthritis (RA). The population under study was composed of a total of 753 unrelated RA patients and 801 healthy controls. The VEGF -1154 G-->A and -634 G-->C polymorphism genotyping was performed by real-time polymerase chain reaction technology, using TaqMan 5' allelic discrimination assay. No evidence of association was observed between the -1154 G-->A and the -634 G-->C VEGF polymorphisms, or inferred VEGF haplotypes with RA susceptibility or clinical manifestations. Our results suggest that the analyzed VEGF promoter polymorphisms may not play a relevant role in RA pathogenesis in our population.