Effect of vascular endothelial growth factor and epidermal growth factor on iatrogenic apoptosis in human endothelial cells

To study the effect of growth factors on iatrogenic apoptosis, we examined the influence of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) on staurosporine-induced apoptosis in primary cultures of human umbilical vein endothelial cells (HUVEC). Apoptosis was evaluated by a cell viability test, the TUNEL-POD assay and the activation of the pro-apoptotic caspase-3. Staurosporine (10-100nM) caused the activation of caspase-3. This effect was manifest after 2hr of incubation and reached its maximum after 5hr. Severe loss of viability followed within 18hr. VEGF or EGF (10-100ng/mL) added together with staurosporine decreased the activation of caspase-3. The loss of viability was 24hr delayed. The action of growth factors was observed at 1% serum concentration but also at concentration optimal for HUVEC survival (10%, v/v). Furthermore, the inhibition of PI-3 kinase (PI-3K) by wortmannin or LY294002 as well as the inhibition of MEK by PD098059 or U0126 prevented the protective effect of VEGF and EGF. Western blotting analysis showed that after 3hr of incubation with staurosporine the level of the anti-apoptotic protein Mcl-1 decreased and this effect was reverted by VEGF. It is concluded that VEGF and EGF antagonize the pro-apoptotic action of staurosporine by the combined signalling of PI-3K and ERKs pathways.     

人大肠癌组织血管内皮生长因子C及血管内皮细胞生长因子受体3和P53蛋白的表达及临床意义

目的研究血管内皮生长因子（VEGF）C及血管内皮生长因子受体（VEGFR）3和P53蛋白在大肠癌组织中的表达及其与临床病理和预后的关系。方法对89例大肠癌患者手术切除标本的癌组织、癌旁组织及正常大肠组织采用免疫组织化学方法检测VEGF—C、VEGFR-3和P53蛋白表达情况,分析其变化规律。结果VEGF—C、VEGFRG和P53在大肠癌组织中的阳性表达高于癌旁组织及正常大肠组织,差异有统计学意义[癌组织：67．4％（60／89）、55．1％（49／89）和66．3％（59／89）,癌旁组织：22．6％（12／53）、20．8％（11／53）、11．3％（6／53）,正常组织：13．2％（7／53）、11．3％（6／53）、0,P〈0．05]。DukesB、C和D期的大肠癌P53阳性率[分别为：61．1％（11／18）、76．2％（16／21）、87．1％（27／31）]均高于DukesA期[26．3％（5／19）],差异均有统计学意义（均P〈0．05）;DukesD期高于DukesB期（P〈0．05）。大肠高分化腺癌P53阳性率低于中分化和低分化腺癌,差异均有统计学意义[45．7％（16／35）比78．3％（18／23）、81．1％（25／31）,P〈0．05],中分化腺癌与低分化腺癌P53阳性率的差异无统计学意义（P〉0．05）。89例大肠癌中60例有淋巴结转移,VEGF—C和VEGFR-3阳性率分别为76．7％（46例）和63．3％（38例）;无淋巴结转移者29例,VEGF-C和VEGFRG阳性率分别为48．3％（14例）和37．9％（11例）;有淋巴结转移患者中VEGF—C和VEGFR-3阳性率明显高于无淋巴结转移者（P〈0．05）。VEGF—C在黏膜下层、肌层和浆膜层的阳性率分别为50．0％（12／24）、67．6％（25／37）和82．1％（23／28）,黏膜下层阳性率与浆膜层的差异有统计学意义（P〈0．05）;VEGFR-3的阳性率分别为29．2％（7／24）、54．1％（20／37）和78．6％（22／28）,浆膜层阳性表达率高于黏膜下层和肌层,差异有统计学意义（P〈0．05）。在大肠低、中、高分化腺癌组织中VEGF—C阳性率分别为83．9％（26／31）、69．6％（16／23）、51．4％（18／35）,VEGFR-3阳性率分别为77．4％（24／31）、56．5％（13／23）、34．3％（12／35）,VEGF-C和VEGFR-3在低分化程度阳性表达率高于高分化程度者,差异有统计学意义（P〈0．05）。结论大癌组织中VEGF—C及VEGFR~、P53的表达可能是肿瘤发生、浸润、转移的重要因素,联合检测对判断肿瘤的恶性程度、转移、预后、评价复发具有重要的临床意义。     

Recombinant human erythropoietin stimulates vascular endothelial growth factor release by glomerular endothelial cells.

We designed the present study to address the question of whether recombinant human erythropoietin stimulates DNA synthesis and vascular endothelial growth factor (VEGF) secretion in vitro using cultured bovine glomerular endothelial cells (GENs). Recombinant human erythropoietin dose-dependently stimulated the proliferation of GENs in culture, and this proliferative effect was inhibited by 1 microg/ml anti-VEGF antiserum. The increase in VEGF concentrations in the supernatants containing 10 U/ml rHuEpo was abolished by incubation with 10 microg/ml of anti-human rHuEPO antiserum, 0.2 microg/ml actinomycin D or 10 microg/ml cycloheximide. Taken together, rHuEpo stimulates GEN proliferation in vitro and VEGF release from these cells is associated with stimulation of RNA-dependent DNA and protein synthesis.     

Vasopressin increases vascular endothelial growth factor secretion from human vascular smooth muscle cells

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen of vascular endothelial cells which promotes neovascularization in vitro. To determine whether vasopressin induces VEGF secretion in human vascular smooth muscle cells, we performed enzyme-linked immunosorbent assays. Vasopressin potently induced a time-dependent and concentration-dependent (maximal, 10(-7) M) increase in VEGF secretion by human vascular smooth muscle cells that was maximal after 24 h. Furthermore, vasopressin also concentration-dependently caused mitogenic effect, as reflected by total protein content of cells per culture well. These vasopressin-induced VEGF secretion increase and mitogenic effect of these cells were potently inhibited by vasopressin V1A receptor antagonists, confirming this is a vasopressin V1A receptor-mediated event. These results indicate that vasopressin increases VEGF secretion in human vascular smooth muscle cells, the magnitude of VEGF secretion being temporally related to the mitogenic effect of vascular smooth muscle cells and the potency of the growth-promoting stimulus. Vasopressin-induced VEGF secretion by proliferating vascular smooth muscle cells could act as a paracrine hormone to powerfully influence the permeability and growth of the overlying vascular endothelium, vasopressin play a more fundamental role in the regulation of vascular function than has previously been recognized.     

Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor II.

AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domain III of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain III (KDR-III) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-III were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial cells. RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-III protein. Ycom1D3 bound specifically to both the soluble KDR-III and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.     

反义寡脱氧核苷酸抑制人胶质瘤细胞的血管内皮生长因子(英文)

目的:了解反义VEGF寡脱氧核苷酸(ODN)对人胶质细胞瘤系(A172细胞)VEGF表达的抑制作用。方法:应用半定量PCR、免疫组化方法分别了解细胞内VEGF mRNA和蛋白的变化,并利用ELISA检测培养上清液中VEGF蛋白的含量。结果:A172细胞经反义VEGF ODN作用后VEGF mRNA的表达量明显减少,且随其浓度的增加而明显减少,但其表达量不受正义和错义VEGF ODN的影响。当A172细胞经50μmol·L~(-1)反义VEGF ODN作用后,细胞内及上清液内VEGF蛋白水平较对照显著减少。结论:反义VEGF ODN特异地抑制A172细胞VEGF mRNA和蛋白的表达。     

Inhibitors of vascular endothelial growth factor in cancer

Angiogenesis is a complex process that is regulated by pro- and antiangiogenic factors. These factors can emanate from diverse sources including cancer cells, stromal cells, blood and extracellular matrix. Their relative contribution is likely to change with tumor type and tumor site. Vascular endothelial growth factor (VEGF) is now well confirmed as the primary and the most potent inducer of angiogenesis. To activate cellular signaling pathways, VEGF binds to receptor kinases VEGF-R1, R2 and R3. It then promotes several events required for the formation of new blood vessels, such as endothelial cell survival, proliferation, migration and vascular permeability. Activation of endothelial cells, leads to the secretion of enzymes which degrade the extracellular matrix (ECM) and hence promote metastasis. Similarly it promotes survival by inducing Bcl-2 expression on VEGF receptor positive leukemia. Besides being a potent mitogen for macrovascular cells derived from arteries, veins and lymphatics, it is also highly involved in a number of angiogenic related disorders including inflammatory diseases, rheumatoid arthritis, psoriasis, retinopathies and age related macular degeneration. Neovascularization and increased vessel permeability are being recognized as major causes of VEGF related pathogenesis. Therefore, inhibition of VEGF pathway is a strategy being widely pursued to provide new therapeutics for the treatment of VEGF related disorders. Over twenty compounds with anti-angiogenic properties ranging from VEGF neutralizing antibody, soluble receptors, receptor antagonists or tyrosine kinase inhibitors (TKIs) are either approved or are currently under clinical (phase I - III) study. This review aims to provide an updated account of how VEGF inhibitors are shaping up to become an important class of drugs used in the treatment of cancer.     

罗格列酮下调肺腺癌细胞血管内皮细胞生长因子的表达

目的探讨过氧化物酶增殖物活化受体γ(PPARγ)配体罗格列酮(RSG)对肺腺癌血管内皮细胞生长因子(VEGF)表达的调节作用,旨在进一步揭示PPARγ抑制肺腺癌血管生成的机制。方法体外培养人肺腺癌细胞株A549,给予不同浓度RSG作用24 h后,用免疫细胞化学检测VEGF蛋白的表达;逆转录聚合酶链反应(RT-PCR)检测PPARγ、VEGF mRNA的表达。结果RT-PCR及免疫细胞化学结果显示A549细胞存在PPARγmRNA和蛋白的表达。RSG呈浓度依赖方式上调PPARγ的表达;1.25μmol.L-1RSG处理组VEGF mRNA表达明显下调,10μmol.L-1RSG下调作用最强,≥20μmol.L-1RSG下调作用减弱,100μmol.L-1RSG对VEGF mRNA表达下调与对照组比较无统计学意义;PPARγ阻断剂GW 9662明显减弱了RSG下调VEGF的作用(P<0.01或P<0.05,n=6)。结论PPARγ的活化可抑制肺腺肿瘤新生血管的生成,其机制可能是通过部分PPARγ途径下调VEGF的表达而实现的。提示PPARγ可能是肺腺癌血管生成治疗的1个新分子靶点。     

Influence of dichlorodiphenylchloroethylene on vascular endothelial growth factor and insulin-like growth factor in human and rat ovarian cells

1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p′-DDE, DDE), a metabolite of DDT is a persistent hormonally active environmental toxicant present in human serum and follicular fluid. The objective of this study was to investigate the effects of DDE on the expression of the ovarian vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF-1) in primary cultures of human granulosa cells and in the rat ovary. Granulosa cells were obtained at the time of oocyte retrieval for in vitro fertilization and cultured with environmentally relevant concentrations of DDE. Immature female rats were treated with 100 μg DDE/kg body weight or vehicle at 28 and 31 days of age and then euthanized at 50 days of age for collection of ovarian tissue. Expression of VEGF, the VEGF receptor fetal liver kinase (Flk-1) and IGF-1 were determined by Western blotting analysis of protein lysates from granulosa cell cultures and by immunohistochemistry in the rat ovary. DDE at concentrations of 100–1000 ng/mL increased the expression of VEGF, Flk-1 and IGF-1 in vitro in primary cultures of human granulosa cells, with the highest expression occurring at 1000 ng/mL. Similarly, acute administration of DDE resulted in a significant increase in immunoreactive VEGF, Flk-1 and IGF-1 in the rat ovary. We conclude that DDE, at levels, which have been detected in humans, alters the expression of the ovarian growth factors VEGF and IGF-1 both in vivo and in vitro. This alteration in expression of growth factors may lead to altered ovarian function as seen in polycystic ovaries and impaired fertility.     

Targeting vascular endothelial growth factor in angina therapy

Despite tremendous success of growth factor therapy in animal models, clinical trials have demonstrated minimal success. Vascular endothelial growth factors are perhaps the most potent inducers of angiogenesis in these animal models. This review outlines the biology of vascular endothelial growth factors in the context of myocardial angiogenesis with an emphasis on its effects on the endothelium. It also provides an overview of delivery strategies and summarises the preclinical and clinical evidence relating to exogenous growth factor delivery for myocardial angiogenesis with an emphasis on the key future challenges.     

血管内皮生长因子165诱导人血管内皮细胞多药耐药表型

目的：研究血管内皮生长因子165（VEGF165）对血管内皮细胞药物敏感性的影响。方法：人真皮微血管内皮细胞（HDMEC）与抗癌药物和VEGF165混合培养,MTT法分析药敏变化,琼脂糖电泳检测细胞凋亡,RT-PCR、免疫印迹法分析HDMEC产生多药耐药表型的机制。结果：VEGF165体外诱导HDMEC对多种抗癌药物耐药,如表柔比星、顺铂、足叶乙甙、丝裂霉素C、长春新碱、CPT-11、泰素等,其机制与VEGF165诱导HDMEC上调表达多药耐药相关蛋白和肺耐药相关蛋以及下调表达Bax蛋白有关。结论：VEGF165诱导血管内皮细胞的多药耐药表型,提示化疗时抗癌药物的抗血管生成活性可能取决于肿瘤微环境。     

Insulin-like growth factor binding protein-7 (IGFBP7) blocks vascular endothelial cell growth factor (VEGF)-induced angiogenesis in human vascular endothelial cells

Insulin-like growth factor binding protein-7 (IGFBP7) and vascular endothelial growth factor (VEGF) are expressed in vascular endothelial cells in several tumor types. In this study, we examined the effect of IGFBP7 on VEGF-induced tube formation in cultured human umbilical vein endothelial cells (HUVECs) and its potential action in the modulation of VEGF signaling in vascular cells. IGFBP7 treatment suppressed VEGF-induced tube formation, proliferation, and the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) 1/2 in HUVECs. IGFBP7 attenuated VEGF-enhanced cyclooxygenase (COX)-2 and VEGF mRNA expression, and prostaglandin E₂ secretion. Knocking down endogenous IGFBP7 enhanced COX-2 and VEGF mRNA expression. A significant increase in IGFBP7-induced caspases was not observed in the presence of VEGF. These findings indicate that IGFBP7 can modulate the stimulatory effect of VEGF on angiogenesis by interfering with VEGF expression as well as VEGF signaling and not by inducing apoptosis.     

Therapies directed at vascular endothelial growth factor

The inhibition of angiogenesis through vascular endothelial growth factor (VEGF) receptor targeting is a strategy that is relatively tumour selective. The high selectivity achieved with neutralising antibodies, soluble receptors and ribozymes reduces the risk of adverse reactions not related to VEGF inhibition itself. Small-molecule, orally-active protein kinase inhibitors provide an attractive alternative for chronic therapy, although specifically targeting a small subset of protein kinases from the ~ 550 expressed in mammalian cells is a challenge. Current efforts have resulted in promising clinical data for several synthetic VEGF receptor kinase inhibitors, of which PTK787/ZK222584 and ZD6474 are proceeding into large size clinical trials. It seems likely that blockers of the VEGF signalling pathway will be unsuitable for monotherapy, and that their role will be as an adjunct to additional antiangiogenic agents together with directly-acting antitumour agents or radiation therapy. Caution is needed with combinations of antiVEGF therapies and cytotoxic agents, as coadministration of cytotoxic agents with either the kinase inhibitor SU5416 or the VEGF antibody avastin appears to be associated with bleeding and thrombotic adverse events.     

Therapies directed at vascular endothelial growth factor

The inhibition of angiogenesis through vascular endothelial growth factor (VEGF) receptor targeting is a strategy that is relatively tumour selective. The high selectivity achieved with neutralising antibodies, soluble receptors and ribozymes reduces the risk of adverse reactions not related to VEGF inhibition itself. Small-molecule, orally-active protein kinase inhibitors provide an attractive alternative for chronic therapy, although specifically targeting a small subset of protein kinases from the approximately 550 expressed in mammalian cells is a challenge. Current efforts have resulted in promising clinical data for several synthetic VEGF receptor kinase inhibitors, of which PTK787/ZK222584 and ZD6474 are proceeding into large size clinical trials. It seems likely that blockers of the VEGF signalling pathway will be unsuitable for monotherapy, and that their role will be as an adjunct to additional antiangiogenic agents together with directly-acting antitumour agents or radiation therapy. Caution is needed with combinations of anti-VEGF therapies and cytotoxic agents, as coadministration of cytotoxic agents with either the kinase inhibitor SU5416 or the VEGF antibody avastin appears to be associated with bleeding and thrombotic adverse events.     

Relationship between prostaglandin E2 and vascular endothelial growth factor (VEGF) in angiogenesis in human vascular endothelial cells

To address the role of prostaglandin E2 (PGE2) in tube formation of endothelial cells and the relationships between the action of PGE2 and vascular endothelial growth factor (VEGF), cultured human umbilical vein endothelial cells (HUVECs) were used to evaluate tube formation on Matrigel and the expression of angiogenesis-related genes. PGE2 treatment stimulated the tube-like formation of HUVECs. Whereas VEGF-induced tube formation was significantly suppressed by ETYA, an inhibitor of arachidonic acid metabolism, or SU5614, an inhibitor of VEGF-receptor tyrosine kinase, the stimulatory effect of PGE2 was observed in the presence of ETYA or SU5614. Thus, PGE2 counteracted both ETYA- and SU5614-induced blockage of angiogenesis in the presence of VEGF. VEGF induced cyclooxygenase (COX) -2 mRNA expression in HUVECs and increased the PGE2 concentration in the medium. PGE2 treatment enhanced the expression of VEGF mRNA. These findings suggest that PGE2 directly stimulates angiogenesis, apart from VEGF signaling, and further induces VEGF expression in HUVECs. In addition, the effect of VEGF on angiogenesis may be mediated, in part, by PGE2 secretion.     

血管内皮生长因子受体在胶质瘤中的表达

目的 研究人脑胶质瘤组织中血管内皮生长因子受体(KDR、Flt-1)mRNA的表达,同时分析肿瘤间质血管增生的情况,并探讨两之间的关系。方法 采用半定量逆转录-聚合酶链反应(RT-PCR)方法,对49例人脑胶质瘤手术切除标本,U251等3株胶质瘤细胞、人类脐静脉内皮细胞(HUVEC)以及12例脑外伤内减压术中得到的正常脑组织检测了KDR、Flt-1 mRNA表达水平。另外,用免疫组化SP法对胶质瘤及正常脑组织的微血管密度(MVD)进行了分析。结果 胶质瘤组织中的KDR、Flt-1 mRNA表达水平明显增高,分别达59．2％、63．2％,随着病理分级的提高其表达水平有升高趋势,HUVEC及3株胶质瘤细胞亦表达水平较高。胶质瘤中的微血管密度(MVD)与胶质瘤级别有关,KDR、Flt-1 mRNA的表达同MVD呈正相关。结论 KDR、Flt-1、MVD对胶质瘤的恶性生物学行为评估有重要意义,血管内皮生长因子受体的高表达与肿瘤组织血管增生有关,在胶质瘤侵袭过程中发挥重要作用。     

造血细胞表达血管内皮生长因子及其受体基因的研究

目的 探控血管内皮生长因子（VEGF）及其受体Flt-1和KDR基因在造血细胞中的表达。方法 提取正常外周血、骨髓单个核细胞及血液肿瘤细胞系的总RNA,以β2-微球蛋白基因为内标,用单定量逆转录聚合酶链反应（RT-PCR）分析VEGF,Flt-1和KDR基因的表达。结果 在22份正常外周血和22份非恶性骨髓标本中,分别有18份和19份表达,VEGF基因,而10种人类血液肿瘤细胞系均有VEGF基因的高度表达。在12份外周血中,有4份检测到Flt-1基因表达,但均未见有KDR基因表达;在22份骨髓中,分别有9份和14份表达Flt-1基因或KDR基因。10种血液肿瘤细胞系有6种表达Flt-1基因,而KDR基因表达仅见于Raji细胞。血小板也有VEGF和Flt-1基因的低度表达。结论 VEGF作为血管生成的介导物,可能具有调节正常造血细胞和血液肿瘤细胞生长的作用。     

欧芹素乙对人脐静脉内皮细胞的保护作用及对血管内皮生长因子表达的影响

目的:研究欧芹素乙对内皮细胞的保护作用;溶血磷脂酰胆碱(LPC)对人脐静脉内皮细胞株HUVEC细胞中血管内皮生长因子(VEGF)表达的影响以及欧芹素乙的影响.方法:应用四唑盐(MTT)法检测溶血磷脂酰胆碱对HUVEC细胞的毒性作用及欧芹素乙的保护作用;应用基础酶联免疫吸附试验(ELISA)检测各组条件培养基中VEGF蛋白含量;采用RT-PCR法及Reahime PCR方法检测溶血磷脂酰胆碱对VEGF mRNA的表达及欧芹素乙的影响.结果:MTT检测结果显示,溶血磷脂酰胆碱对HUVEC细胞具有较强的生长抑制作用,而欧芹素乙对溶血磷脂酰胆碱所致的细胞增殖抑制具有较好的保护作用.ELISA结果显示,HUVEC细胞暴露于溶血磷脂酰胆碱后,VEGF蛋白含量明显升高;加入欧芹素乙后剂量依赖性地降低VEGF蛋白的表达.RT-PCR结果显示,溶血磷脂酰胆碱可以增加3种VEGF异构体的转录水平,其中VEGF165的表达显著增加,欧芹素乙可剂量依赖性地抑制溶血磷脂酰胆碱引起的VEGF mRNA的高表达.结论:欧芹素乙对溶血磷脂酰胆碱引起的细胞损伤有明显的保护作用;欧芹素乙可抑制溶血磷脂酰胆碱所诱导的HUVEC细胞中VEGF蛋白及VEGF mRNA的高表达,对内皮细胞起到保护作用.     

Variants of the angiogenic factor vascular endothelial cell growth factor

Vascular endothelial growth factor (VEGF) is an endothelial specific growth factor that can induce blood vessel formation. The patent claims the identification of three novel isoforms of VEGF-A, namely VEGF-A₁₃₈, VEGF-A₁₆₂ and VEGF-A₁₈₂. These novel isoforms are discussed for their potential use in the treatment of cardiovascular diseases.