Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.

Clinical and experimental studies suggest that angiogenesis is a prerequisite for solid tumour growth. Several growth factors with mitogenic or chemotactic activity for endothelial cells in vitro have been described, but it is not known whether these mediate tumour vascularization in vivo. Glioblastoma, the most common and most malignant brain tumour in humans, is distinguished from astrocytoma by the presence of necroses and vascular proliferations. Here we show that expression of an endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF), is induced in astrocytoma cells but is dramatically upregulated in two apparently different subsets of glioblastoma cells. The high-affinity tyrosine kinase receptor for VEGF, flt, although not expressed in normal brain endothelium, is upregulated in tumour endothelial cells in vivo. These observations strongly support the concept that tumour angiogenesis is regulated by paracrine mechanisms and identify VEGF as a potential tumour angiogenesis factor in vivo.     

血管内皮生长因子对女性生殖功能的调控及其临床意义

血管内皮生长因子是一种特异性血管生长因子，在血管的发生中发挥重要作用。在卵巢、子宫内膜、胎盘等部位有丰富的VEGF及其受体的表达，对女性生殖功能发挥重要的调控作用。近年来VEGF在妇产科领域的临床研究结果表明，VEGF的异常表达与卵巢癌、妊娠高血压、子宫内膜异位症有着密切的关系。     

Homoharringtonine induces apoptosis of endothelium and down－regulates VEGF expression of K562 cells

Homoharringtonine (HHT) has currently been used successfully in the treatment of acute and chronic myeloid leukemias and has been shown to induce apoptosis of different types of leukemic cells in vitro. Emerging evidence suggests that angiogenesis may play an important role in hematological malignancies, such as leukemia. However, whether HHT can relieve leukemia by anti-angiogenesis is still unknown. We investigated the anti-angiogenesis potential of HHT with the human umbilical vein endothelial cell line (ECV304) and leukemic cell line (K562) in vitro. Cellular proliferation was determined by MTT assay and apoptosis was analyzed by flow cytometry, The mRNA expression of vascular endothelial growth factor (VEGF) was assessed by RT-PCR and VEGF protein production was detected by Western blot. Inhibition of cell proliferation and induction of apoptosis by HHT were discovered in ECV304 cells, and appeared in a dose- and time-dependent manner, Also, treatment with HHT caused down-regulation of VEGF mRNA expression in K562 cells in similar dose- and time-dependent manner and inhibition of VEGF protein production in K562 cells in response to the enhancing concentration of HHT. The results demonstrated that HHT could also induce apoptosis in endothelium and down-regulate VEGF expression in K562 cells. In conclusion, we believe HHT has anti-angiogenesis potential and speculate that HHT might exert its anti-leukemia effects via reduction of angiogenesis.     

血管内皮生长因子研究进展

血管内皮生长因子是一种有效的血管形成和血管通透性诱导因子,特异性地作用于血管内皮细胞,具有维持血管正常状态和完整性、增加血管通透性、促进血管生成的作用。在正常成人和动物组织中表达水平较低,一些代谢旺盛、血供丰富的组织中VEGF表达水平略高,一些病理情况下可以过度表达。     

A role for VEGF as a negative regulator of pericyte function and vessel maturation

Angiogenesis does not only depend on endothelial cell invasion and proliferation: it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) through their cognate receptors on endothelial cells and vascular smooth muscle cells (VSMCs), respectively. PDGF induces neovascularization by priming VSMCs/pericytes to release pro-angiogenic mediators. Although VEGF directly stimulates endothelial cell proliferation and migration, its role in pericyte biology is less clear. Here we define a role for VEGF as an inhibitor of neovascularization on the basis of its capacity to disrupt VSMC function. Specifically, under conditions of PDGF-mediated angiogenesis, VEGF ablates pericyte coverage of nascent vascular sprouts, leading to vessel destabilization. At the molecular level, VEGF-mediated activation of VEGF-R2 suppresses PDGF-Rbeta signalling in VSMCs through the assembly of a previously undescribed receptor complex consisting of PDGF-Rbeta and VEGF-R2. Inhibition of VEGF-R2 not only prevents assembly of this receptor complex but also restores angiogenesis in tissues exposed to both VEGF and PDGF. Finally, genetic deletion of tumour cell VEGF disrupts PDGF-Rbeta/VEGF-R2 complex formation and increases tumour vessel maturation. These findings underscore the importance of VSMCs/pericytes in neovascularization and reveal a dichotomous role for VEGF and VEGF-R2 signalling as both a promoter of endothelial cell function and a negative regulator of VSMCs and vessel maturation.     

Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a key process in several pathological conditions, including tumour growth and age-related macular degeneration. Vascular endothelial growth factors (VEGFs) stimulate angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells. VEGFR-3 (also known as FLT-4) is present in all endothelia during development, and in the adult it becomes restricted to the lymphatic endothelium. However, VEGFR-3 is upregulated in the microvasculature of tumours and wounds. Here we demonstrate that VEGFR-3 is highly expressed in angiogenic sprouts, and genetic targeting of VEGFR-3 or blocking of VEGFR-3 signalling with monoclonal antibodies results in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models. Stimulation of VEGFR-3 augmented VEGF-induced angiogenesis and sustained angiogenesis even in the presence of VEGFR-2 (also known as KDR or FLK-1) inhibitors, whereas antibodies against VEGFR-3 and VEGFR-2 in combination resulted in additive inhibition of angiogenesis and tumour growth. Furthermore, genetic or pharmacological disruption of the Notch signalling pathway led to widespread endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3 signals. Our results implicate VEGFR-3 as a regulator of vascular network formation. Targeting VEGFR-3 may provide additional efficacy for anti-angiogenic therapies, especially towards vessels that are resistant to VEGF or VEGFR-2 inhibitors.     

A peptide fusion protein inhibits angiogenesis and tumor growth by blocking VEGF binding to KDR

Vascular endothelial growth factor (VEGF) binding to its tyrosine kinase receptors (KDR/FLK1, Flt-1) induces angiogenesis. In search of the peptides blocking VEGF binding to its receptor KDR/FLK1 to inhibit tumorangiogenesis and growth, we screened a phage display peptide library with KDR as target protein, and some candidate peptides were isolated. In this study, we cloned the DNA fragment coding the peptide K237 from the library, into a vector pQE42 to express fusion protein DHFR-K237 in E. coli M15. The affection of fusion protein DHFR-K237 on endothelial cell proliferation and angiogenesis was investigated. In vitro, DHFR-K237 could completely block VEGF binding to KDR and significantly inhibit the VEGF-mediated proliferation of the human vascular endothelial cells. In vivo, DHFR-K237 inhibited angiogenesis in chick embryo chorioallantoric membrane and tumor growth in nude mice. These results suggest that K237 is an effective antagonist of VEGF binding to KDR, and could be a potential agent for cancer biotherapy.     

Functions of VEGF in female reproductive system

As a homodimeric glycoprotein,vascular endothelial growth factor(VEGF)is a highly specific mitogen of vascular endothelial cells.It can induce proliferation and migration,and inhibit apoptosis of endothelial cell.VEGF is involved in many processes in the female reproductive system,such as ovulation,periodical changes of endometrium,embryo implantation and development,VEGF plays important roles in some reproductive diseases,including preeclampsia and fetal hypoevolutism in uterus.Based on our studies on angiogenesis and its relevant factors in the female reproductive system these years,the functions of VEGF in female reproductive system are reviewed,and the research prospect and application of VEGF are also discussed.     

血管内皮生长因子与急性白血病骨髓的血管新生

目的:探讨血管内皮生长因子(VEGF)在急性白血病及其骨髓的新生血管之间的关系,为白血病的治疗寻找新的治疗方法。方法:查阅总结近15年来国内外相关文献,对VEGF的性质作用特点以及与急性白血病的关系进行综述。结果:白血病细胞表达较高的VEGF,VEGF促使血管生成和内皮细胞增生,白血病细胞与骨髓新生血管之间存在密切的关系。结论:抗VEGF和抗新生血管治疗有可能成为治疗急性白血病的新的思路和方法。     

Pathophysiological consequences of VEGF-induced vascular permeability

Although vascular endothelial growth factor (VEGF) induces angiogenesis, it also disrupts vascular barrier function in diseased tissues. Accordingly, VEGF expression in cancer and ischaemic disease has unexpected pathophysiological consequences. By uncoupling endothelial cell-cell junctions VEGF causes vascular permeability and oedema, resulting in extensive injury to ischaemic tissues after stroke or myocardial infarction. In cancer, VEGF-mediated disruption of the vascular barrier may potentiate tumour cell extravasation, leading to widespread metastatic disease. Therefore, by blocking the vascular permeability promoting effects of VEGF it may be feasible to reduce tissue injury after ischaemic disease and minimize the invasive properties of circulating tumour cells.     

Effects of vascular endothelial growth factor on angiogenesis of the endothelial cells isolated from cavernous malformations

Human cerebral cavernous malformation （CM） is a common vascular malformation of the central nervous system. We have investigated the biological characteristics of CM endothelial cells and the cellular and molecular mechanisms of CM angiogenesis to offer new insights into exploring effective measures for treatment of this disease. The endothelial cells were isolated from CM tissue masses dissected during operation and expanded in vitro. Expression of VEGFR-1 and VEGFR-2 was examined with immunocytochemical staining. Proliferation, migration and tube formation of CM endothelial cells were determined using MTT, wounding and transmigration assays, and three-dimensional collagen type I gel respectively. The endothelial cells were successfully isolated from the tissue specimens of 25 CMs dissected without dipolar electrocoagulation. The cells show the general characteristics of the vascular endothelial cells. Expression of VEGFR-1 and VEGFR-2 on the cells is higher than that on the normal cerebral microvascular endothelial cells. After treatment with VEGF, numbers of the proliferated and migrated cells, the maximal distance of cell migration and the length and area of capillary-like structures formed in the three-dimensional collagen gel increase significantly. These results demonstrate that expression of VEGFR-1 and VEGFR-2 on CM endothelial cells is up-regulated. By binding to receptors, VEGF may activate the downstream signaling pathways and promote proliferation, migration and tube formation of CM endothelial cells. VEGF/VEGFR signaling pathways play important regulating roles in CM angiogenesis.     

Bojungbangdocktang inhibits vascular endothelial growth factor induced angiogenesis via blocking the VEGF/VEGFR2 signaling pathway in human umbilical vein endothelial cells

Oriental herbal medicines have been widely used for the prevention or treatment of various diseases including cancer in Asia. However, to prove their chemo preventive efficacies in modern times, scientific evidence for those herbal medicines is required. Thus, in the present study, an effective herbal cocktail Bojungbangdocktang (BJBDT) was investigated to elucidate antiangiogenic mechanism in vitro and in vivo. BJBDT significantly inhibited vascular endothelial growth factor (VEGF) induced proliferation in HUVECs at nontoxic concentrations, despite weak cytotoxicity against human umbilical vein endothelial cells (HUVECs). BJBDT also significantly suppressed VEGF-induced migration and tube formation of HUVECs. Furthermore, BJBDT treatment resulted in pale color and low hemoglobin level in Matrigel plugs, as well as dark red color and high hemoglobin level in untreated control. Interestingly, BJBDT specifically inhibited the binding of VEGF to vascular endothelial growth factor receptor 2 (VEGFR2), but not VEGFR1. In addition, friedelin, formononetin, ginsenoside Rb1, naringin, atractyloside, diosgenin, and allantonin were identified from BJBDT by high-performance liquid chromatography (HPLC) analysis as a quality of control. Taken together, these results suggest that BJBDT is a potent angiogenesis inhibitor blocking the VEGF/VEGFR2 signaling pathway in HUVECs. Supported by the Korea Science and Engineering Foundation Grant from the Korean Government (Ministry of Science and Technology) (Grant No. R13-2007-019-00000-0)     

Expression of vascular endothelial growth factor in rat uterus during peri-implantation

The first distinct mark of rodent implantation is the increased vascular permeability and significant angiogenesis at the sites of blastocyst implantation, but its mechanism is not clearly defined. Vascular endothelial growth factor (VEGF) is the key mediator for angiogenesis during embryogenesis and adult span and also serves as a vascular permeability factor. The aim of this study is to explore VEGF regulation mechanism and the possible role that VEGF plays in implantation by studying the VEGF expression and angiogenesis in the rat uterus during estrous cycle, ovarioectomized and peri-implantation stages usingin situ message RNA hybridization and confocal laser scanning techniques. The results indicated that VEGF was regulated by ovarian steroid hormones. VEGF expression before implantation was localized at luminal epithelium, shifted to stroma as implantation initiated and extensively located at the decidualizing stroma region after implantation. Bandeiraea simplicifolia-1 (BS-1) agglutinin and antibody against von Willebrand factor (vWF) were used to mark the endothelial cells and blood vessels. The results showed that the active angiogenesis occurred during the implantation process and this effect was probably mediated by VEGF. The results suggest that under the regulation of ovarian steroid hormones, VEGF plays an essential role in angiogenesis and increasing vascular permeability in endometrium, which are necessary for successful implantation.     

Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis.

Inefficient vascular supply and the resultant reduction in tissue oxygen tension often lead to neovascularization in order to satisfy the needs of the tissue. Examples include the compensatory development of collateral blood vessels in ischaemic tissues that are otherwise quiescent for angiogenesis and angiogenesis associated with the healing of hypoxic wounds. But the presumptive hypoxia-induced angiogenic factors that mediate this feedback response have not been identified. Here we show that vascular endothelial growth factor (VEGF; also known as vascular permeability factor) probably functions as a hypoxia-inducible angiogenic factor. VEGF messenger RNA levels are dramatically increased within a few hours of exposing different cell cultures to hypoxia and return to background when normal oxygen supply is resumed. In situ analysis of tumour specimens undergoing neovascularization show that the production of VEGF is specifically induced in a subset of glioblastoma cells distinguished by their immediate proximity to necrotic foci (presumably hypoxic regions) and the clustering of capillaries alongside VEGF-producing cells.     

Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells

Glioblastoma is a highly angiogenetic malignancy, the neoformed vessels of which are thought to arise by sprouting of pre-existing brain capillaries. The recent demonstration that a population of glioblastoma stem-like cells (GSCs) maintains glioblastomas indicates that the progeny of these cells may not be confined to the neural lineage. Normal neural stem cells are able to differentiate into functional endothelial cells. The connection between neural stem cells and the endothelial compartment seems to be critical in glioblastoma, where cancer stem cells closely interact with the vascular niche and promote angiogenesis through the release of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (refs 5-9). Here we show that a variable number (range 20-90%, mean 60.7%) of endothelial cells in glioblastoma carry the same genomic alteration as tumour cells, indicating that a significant portion of the vascular endothelium has a neoplastic origin. The vascular endothelium contained a subset of tumorigenic cells that produced highly vascularized anaplastic tumours with areas of vasculogenic mimicry in immunocompromised mice. In vitro culture of GSCs in endothelial conditions generated progeny with phenotypic and functional features of endothelial cells. Likewise, orthotopic or subcutaneous injection of GSCs in immunocompromised mice produced tumour xenografts, the vessels of which were primarily composed of human endothelial cells. Selective targeting of endothelial cells generated by GSCs in mouse xenografts resulted in tumour reduction and degeneration, indicating the functional relevance of the GSC-derived endothelial vessels. These findings describe a new mechanism for tumour vasculogenesis and may explain the presence of cancer-derived endothelial-like cells in several malignancies.     

白藜芦醇诱导VEGF表达促进缺血再灌注小鼠新生血管形成

研究白藜芦醇能否诱导小鼠缺血,再灌注后缺血区域新生血管形成及其与血管内皮细胞生长因子(VEGF)表达的相关性,用90只雄性BALB/c小鼠随机分为单纯缺血组、白藜芦醇组及假手术组,采用大脑中动脉线栓法制作局灶性脑缺血模型,免疫组化法测定微血管密度,Western blot检测VEGF蛋白表达.结果白藜芦醇处理组与单纯缺血对照组微血管计数存在差异,并且有统计学意义(P<0. 05),白藜芦醇处理组在各时间点VEGF蛋白表达水平均较高(P<0.05).说明白藜芦醇可以诱导脑缺血后VEGF早期表达,促进缺血区域新生血管形成,发挥脑保护作用.     

A vascular niche and a VEGF-Nrp1 loop regulate the initiation and stemness of skin tumours

Angiogenesis is critical during tumour initiation and malignant progression. Different strategies aimed at blocking vascular endothelial growth factor (VEGF) and its receptors have been developed to inhibit angiogenesis in cancer patients. It has become increasingly clear that in addition to its effect on angiogenesis, other mechanisms including a direct effect of VEGF on tumour cells may account for the efficiency of VEGF-blockade therapies. Cancer stem cells (CSCs) have been described in various cancers including squamous tumours of the skin. Here we use a mouse model of skin tumours to investigate the impact of the vascular niche and VEGF signalling on controlling the stemness (the ability to self renew and differentiate) of squamous skin tumours during the early stages of tumour progression. We show that CSCs of skin papillomas are localized in a perivascular niche, in the immediate vicinity of endothelial cells. Furthermore, blocking VEGFR2 caused tumour regression not only by decreasing the microvascular density, but also by reducing CSC pool size and impairing CSC renewal properties. Conditional deletion of Vegfa in tumour epithelial cells caused tumours to regress, whereas VEGF overexpression by tumour epithelial cells accelerated tumour growth. In addition to its well-known effect on angiogenesis, VEGF affected skin tumour growth by promoting cancer stemness and symmetric CSC division, leading to CSC expansion. Moreover, deletion of neuropilin-1 (Nrp1), a VEGF co-receptor expressed in cutaneous CSCs, blocked VEGF's ability to promote cancer stemness and renewal. Our results identify a dual role for tumour-cell-derived VEGF in promoting cancer stemness: by stimulating angiogenesis in a paracrine manner, VEGF creates a perivascular niche for CSCs, and by directly affecting CSCs through Nrp1 in an autocrine loop, VEGF stimulates cancer stemness and renewal. Finally, deletion of Nrp1 in normal epidermis prevents skin tumour initiation. These results may have important implications for the prevention and treatment of skin cancers.     

重组人血管内皮生长因子165在大肠杆菌中的可溶性表达、纯化与体外活性评价

人血管内皮生长因子165(VEGF165)可有效促进血管新生和增加血管通透性,在伤口愈合方面有重要医疗价值。建立获取高纯度、高活性的优质重组VEGF165蛋白的方法具有重要意义。研究利用带有6组氨酸标签的二硫键形成蛋白A(Dsb A)的E.coli表达系统实现了Dsb A-VEGF165融合蛋白的可溶性表达;诱导过程中添加5%(v/v)的乙醇可显著提高工程菌中可溶性融合蛋白表达水平。融合蛋白通过Ni亲和层析粗纯,并经牛肠激酶酶切去除标签蛋白。随后利用肝素亲和层析精纯获得重组人VEGF165蛋白。非还原及还原SDS-PAGE电泳检测到分子量为约40 k Da的同源二聚体蛋白,促HUVEC细胞增殖实验显示重组蛋白具有较优的活性,EC50为13 ng/m L。研究实现了Dsb A-VEGF165的在E.coli中可溶性表达,建立了经济、高效的纯化方法,获得了高质量、高活性的重组人VEGF165蛋白。