儿童急性白血病骨髓细胞中血管内皮生长因子及其受体的表达

  目的 探讨血管内皮生长因子（VEGF）及其受体（VEGFR）在儿童急性白血病（AL）患者骨髓中的表达,分析其在化疗前后的变化。方法 采用免疫组化S-P法检测53例AL患儿治疗前后以及对照组骨髓中VEGF／VEGFR（包括Flt-1和KDR两种）变化,分析其与临床特征的关系。结果 VEGF,Flt-1,KDR在AL患儿骨髓中表达水平高于对照组。VEGF,Flt-1,KDR的表达于化疗达完全缓解（CR）后下调;化疗后未获得CR患儿VEGF,Flt-1,KDR的表达在化疗前后差异无统计学意义。骨髓中VEGF,Flt-1,KDR的表达水平在不同年龄、性别、有无髓外浸润间差异无统计学意义。结论 VEGF,Flt-1,KDR在儿童AL中呈高表达,化疗后表达明显降低,说明VEGF可能作为评价儿童AL化疗反应的指标。     

VEGF及其受体Flt-1、KDR在鼻咽癌组织中的表达及意义

背景与目的:VEGF及其受体Flt-1、KDR在肿瘤血管生成及肿瘤进展中发挥关键作用。本研究探讨鼻咽癌组织中VEGF、Flt-1和KDR表达与鼻咽癌患者临床特征及预后的关系。方法:用免疫组织化学的方法检测127例鼻咽癌标本中VEGF、Flt-1和KDR的表达情况,用SPSS10.0软件分析这三种蛋白表达的相关性、与临床特征的关系以及对患者总生存的影响。结果:VEGF、Flt-1和KDR分别表达于66.9%(85/127)、90.6%(115/127)和88.2%(112/127)的鼻咽癌组织。鼻咽癌组织中VEGF表达与Flt-1和KDR表达呈正相关(r=0.292,P<0.01;r=0.287,P<0.01),Flt-1表达与KDR表达呈正相关(r=0.723,P<0.01)。VEGF蛋白的表达与原发肿瘤的侵犯范围、淋巴结转移、临床分期有关(P=0.04,P<0.01,P=0.02),KDR蛋白表达也与原发肿瘤的侵犯范围有关(P=0.03);三种蛋白表达均与肿瘤局部复发和/或远处转移(P<0.01,P<0.01,P<0.01)、总生存不佳有关(P<0.01,P<0.01,P<0.03)。Cox多因素相关分析显示,年龄>50岁和VEGF阳性为不良预后因素(P<0.01,P<0.01)。结论:VEGF及其受体Flt-1、KDR在鼻咽癌中广泛表达,与鼻咽癌患者的临床特征和预后关系密切。     

VEGF及其受体Flt和KDR水平变化与胶质瘤关系及临床意义

 目的 探讨胶质瘤患者血清血管内皮生长因子（VEGF）及其受体Flt-1和KDR水平变化，为评价胶质瘤治疗效果，判断预后寻找一种科学的生物学标志物。方法 收集治疗前后胶质瘤患者、脑转移癌患者和健康对照者血清，进行VEGF，Flt-1和KDR水平的检测，SPSS11.5统计软件包对数据进行t检验和相关分析。结果 （1）治疗前脑胶质瘤和脑转移瘤患者血清VEGF水平明显高于健康对照组； 脑胶质瘤患者组和脑转移瘤患者组血清Flt-1水平明显高于健康对照组；脑胶质瘤患者组血清KDR水平明显高于健康对照组； 治疗后缓解的胶质瘤患者组VEGF和Flt-1水平明显低于治疗前。差异均有统计学意义。（2） VEGF与Flt-1和KDR有显著的相关性。结论 胶质瘤患者血清中VEGF及其受体的检测，可作为胶质瘤辅助诊断、治疗效果监测和预后判断的重要生物学指标。     

肺癌血管内皮生长因子(VEGF)及其受体KDR、Flt1的表达与患者预后的关系

背景与目的 血管内皮生长因子（VEGF）及其受体与肺癌血管生成关系密切，但与肺癌患者预后的关系尚不明确。本研究的目的是对VEGF及其受体KDR、Flt1的表达与肺癌患者预后的关系进行探讨。方法 应用免疫组织化学PV-9000法检测75例具有完整随访资料的肺癌标本中VEGF及其受体KDR、Flt1的表达。结果 VEGF、KDR、Flt1在肺癌组织中的表达较为广泛，主要位于肿瘤细胞、血管内皮细胞、纤维母细胞胞质中，且其表达呈异质性，肺癌组织周边部及坏死区周围的肺癌细胞中表达较强，三者在肿瘤细胞中的阳性率均高于间质纤维母细胞（P〈0．01，P〈0．02，P〈0．02）。肺癌细胞及纤维母细胞VEGF、KDR和Flt1的阳性表达率在术后不同生存时间的三组间差异均具统计学意义（P〈0．01，P〈0．01，P〈0．01；P〈0．01，P〈0．01，P〈0．05）；肺癌细胞VEGF、KDR、Flt1阳性组患者的生存时间均显著低于各相应指标阴性者（P〈0．0001，P〈0．0005，P〈0．0005）。肺癌细胞中VEGF与Flt1受体的表达呈正相关（P〈0．01），纤维母细胞中VEGF与肺癌细胞中Flt1受体的表达呈正相关（P〈0．01），纤维母细胞中VEGF与KDR、Flt1表达分别呈正相关（P〈0．01，P〈0．01）。结论 VEGF可能是促进肺癌细胞生长的重要因子，主要以自分泌途径并附以旁分泌途径通过Flt1发挥作用；VEGF及受体KDR、Flt1对肺癌患者的预后有重要的影响作用。     

Caveolin-1对KDR信号通路的作用

目的：研究Caveolin-1对血管内皮生长因子（vascular endothelial growth factor，VEGF）受体KDR、p42／44MAPK的作用。方法：以腺病毒为载体介导在内皮细胞中表达外源Caveolin-1，应用Western-blotting检测KDR、p42／44MAPK的磷酸化水平。结果：与对照组相比，感染腺病毒表达外源Caveolin-1蛋白的内皮细胞中KDR1054酪氨酸残基和p42／44MAPK磷酸化水平分别降低了42．3％和43．5％。结论：Caveolin-1蛋白抑制KDR信号通路，对内皮细胞增殖可能具有抑制作用。     

Caveolin1对KDR信号通路的作用

目的:研究以小鼠结肠癌细胞CT-26RNA作为抗原体外转染经mIL-12基因修饰的树突状细胞(dendriticcells,DC),观察其诱导特异性抗肿瘤的效应。方法:小鼠骨髓细胞体外以rmGM-CSF、rmIL-4诱导培养获取树突状细胞,流式细胞术检测纯度;293细胞扩增携带mIL-12基因的重组腺病毒,体外转染树突状细胞;Trizol法提取CT-26细胞总RNA,应用Trans-Messenger体外转染mIL-12基因修饰的树突状细胞,免疫接种小鼠。ELISA法检测细胞上清及小鼠血液中mIL-12水平,LDH释放法检测小鼠体内细胞毒性T淋巴细胞(CTL)杀伤活性。结果:小鼠骨髓细胞经诱导培养后,获得大量高纯度的树突状细胞,流式细胞术检测CD11c 的树突状细胞>90%;提取的CT-26细胞总RNA体外经TransMessenger介导,转染mIL-12基因修饰的树突状细胞后,回输小鼠,可以诱导体内生成较高水平的特异性CTL活性,亲本肿瘤接种后小鼠100%长期存活,而以该RNA转染Ad-LacZ修饰DC后的对照组及RNA转染DC的对照组,诱导机体生成的特异性CTL活性显著低于实验组(P<0.01),亲本肿瘤接种后小鼠60%长期存活,DC、PBS对照组则均未诱导机体生成特异性CTL活性,小鼠无长期存活。结论:树突状细胞经小鼠结肠癌CT26细胞RNA转染和mIL-12基因修饰后免疫接种小鼠,可在体内有效提呈肿瘤抗原,诱导机体产生高水平的CTL,更有效地诱发特异性抗肿瘤效应。     

成人急性白血病患者细胞血管内皮生长因子及其受体FLT-1、KDR的表达

目的　探讨成人急性白血病 (AL )患者细胞血管内皮生长因子 (VEGF)及其受体 FL T- 1(fm s- like tyro-sine kinase)、KDR(kinase- dom ain insert containing receptor)的表达。方法　采用 RT- PCR方法检测骨髓单个核细胞 (BM MNCs) VEGF及其受体 FL T- 1、KDR m RNA的表达水平。结果　 (1)初发未治患者、骨髓缓解患者 (BMR)和正常对照者 BM MNCs VEGF m RNA阳性表达例数的差异无显著性 (P>0 .0 5 ) ,而 BM MNCs VEGF/β- actin相对表达量 ,在初发未治组显著高于 BMR组和正常对照组 (P值均 <0 .0 1) ,后两组之间的差异无显著性 (P>0 .0 5 ) ;AML组高于 AL L患者 (P<0 .0 5 )。 (2 )初发未治患者 BM MNCs FL T- 1、KDR m RNA阳性表达例数较 BMR及正常对照者高 (P值均 <0 .0 5 ) ,后两组之间的差异无显著性 (P>0 .0 5 ) ;AML组 BM MNCs KDR m RNA阳性表达例数高于 AL L组 (P<0 .0 5 ) ;应用 FL T- 1/β- actin、KDR/β- actin相对表达量分析与上述结果一致。 (3) BM MNCsVEGF的相对表达量与其受体 FL T- 1和 KDR的相对表达量呈相关性 (P值均 <0 .0 5 )。结论　 AL细胞 VEGF、FL T- 1和 KDR m RNA呈过表达 ;VEGF和 KDR m RNA表达强度 ,在 AML明显高于 AL L。应用半定量方法更能反映患者细胞 VEGF m RNA表达     

KSHV activation of VEGF secretion and invasion for endothelial cells is mediated through viral upregulation of emmprin-induced signal transduction

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS)-one of the most common tumors arising in the setting of immune suppression. Hallmarks of KS lesions include KSHV-infected cells of endothelial lineage and neoangiogenesis. Promigratory factors secreted in the tumor microenvironment by KSHV-infected cells promote endothelial cell (EC) migration and angiogenesis but existing therapies targeting these pathways are not widely utilized. This underscores the need for additional characterization of KSHV-host interactions relevant to EC pathogenesis to identify new therapeutic targets. We recently demonstrated that de novo infection by KSHV promotes EC invasion through upregulation of extracellular matrix metalloproteinase inducer (emmprin)-a multifunctional glycoprotein previously shown to induce tumor cell invasion and regional angiogenesis through upregulation of signal transduction and promotion of tumor-stroma interactions. This study was undertaken to determine whether EC invasion for KSHV-infected cells is induced through activation of specific signal transduction pathways and proangiogenic factors by emmprin. We found that KSHV activation of emmprin induces PI3K/Akt- and mitogen-activated protein kinase (MAPK)-dependent secretion of vascular endothelial growth factor (VEGF). Functionally, EC invasion following de novo infection is induced by emmprin-dependent PI3K/Akt and MAPK activation of VEGF. These findings support the potential utility of targeting emmprin for reducing VEGF secretion and EC migration in the KS microenvironment.     

Expression of vascular endothelial growth factor (VEGF) and VEGF receptors in tumor angiogenesis and malignancies

Angiogenesis is a process by which new blood vessels are formed from preexisting vessels. New blood vessel formation by angiogenesis involves the degradation of extra-cellular matrix combined with sprouting and migration of endothelial cells from preexisting capillaries. Solid tumors consist of several components, including normal and stromal cells, extracellular matrix, and vasculature. To grow and metastasize, tumors must stimulate the development of new vasculature through angiogenesis. Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine regeneration. VEGF is both a vascular growth factor and a vascular permeability factor. Its expression can upregulate several proangiogenic and prometa-static molecules. As a central mediator of angiogenesis, VEGF has emerged as an important target for antiangiogenic therapy. In this review, the authors describe the essential characteristics of VEGF and the VEGF family of ligands and their receptors. They also provide an overview of the central role of VEGF in physiologic and pathologic angiogenesis, directly or indirectly. This review sheds light on the importance of VEGF-targeted antiangiogenic therapy based on the monoclonal antibodies against VEGF, small interfering RNA, and therapy directed against VEGF-VEGFR kinase. It also gives a brief overview of the natural products or dietary compounds that could be used as antiangiogenic agents. Therapeutic inhibition of vessel formation could be best suited to preventive strategies aimed at the suppression of angiogenesis in primary tumors in subjects at risk or of micrometastases after surgical removal of primary tumor.     

Kaposi's sarcoma associated herpesvirus G protein-coupled receptor immortalizes human endothelial cells by activation of the VEGF receptor-2/ KDR

The G protein-coupled receptor oncogene (vGPCR) of the Kaposi's sarcoma (KS) associated herpesvirus (KSHV), an oncovirus implicated in angioproliferative neoplasms, induces angiogenesis by VEGF secretion. Accordingly, we found that expression of vGPCR in human umbilical vein endothelial cells (HUVEC) leads to immortalization with constitutive VEGF receptor-2/ KDR expression and activation. vGPCR immortalization was associated with anti-senescence mediated by alternative lengthening of telomeres and an anti-apoptotic response mediated by vGPCR constitutive signaling and KDR autocrine signaling leading to activation of the PI3K/AKT pathway. In the presence of the KS growth factor VEGF, this mechanism can sustain suppression of signaling by the immortalizing gene. We conclude that vGPCR can cause an oncogenic immortalizing event and recapitulate aspects of the KS angiogenic phenotype in human endothelial cells, pointing to this gene as a pathogenic determinant of KSHV.     

Expression of vascular endothelial growth factor (VEGF) and its two receptors in diffusely infiltrating astrocytomas and relationship to proliferative activity of tumor cells

We studied the relationships among vascular endothelial growth factor (VEGF), its receptors (Flt1 and Flk1), and MIB-1. Their expression in 47 diffusely infiltrating astrocytomas obtained at surgery or autopsy was investigated by the ABC method and analyzed quantitatively. The positive rate of VEGF in tumor cells was higher than that in endothelial cells, and Flk1 was lower in tumor cells (P<0.01, 0.01), whereas Flt1 in both tumor cells and endothelial cells was found at similar levels (P>0.05). In tumor cells, VEGF became high with increased histological grades (P<0.01), whereas both Flt1 and Flk1 were higher in grade 4 than in grades 2 and 3 (P<0.01, 0.05). VEGF, Flt1, and Flk1 in endothelial cells were also highly expressed in grade 4 (P<0.01). The distribution of MIB-1-positive nuclei in grade 4 was similar to VEGF, and the percent of positivity from grade 2 to grade 4 also increased (P<0.01). There was a linear positive correlation between VEGF and both Flt1 and Flk1 in both tumor cells and endothelial cells (P<0.01). So was the percent of positivity with VEGF, Flt1, and Flk1 in tumor cells and endothelial cells (P<0.01). The experiment suggests that VEGF may act as a growth factor for both endothelial cells and tumor cells. VEGF, Flt1, and Flk1 can be considered as indicators of the malignancy potential of diffusely infiltrating astrocytomas. The expression of VEGF and the two receptors may be affected by the proliferative activity of tumor cells.     

Prognostic value of expression of vascular endothelial growth factor and its flt-1 and KDR receptors in stage I non-small-cell lung cancer

BACKGROUND: Angiogenesis plays an important role in tumorigenesis and has attracted interest as a potential target in cancer treatment. PATIENTS AND METHODS: We examined the prognostic value of the expression of vascular endothelial growth factor (VEGF) and of the VEGF receptors (VEGFRs) fms-like tyrosine kinase receptor-1 (flt-1) and kinase insert domain-containing receptor (KDR) in non-small-cell lung cancer (NSCLC). Sixty patients with surgical stage I NSCLC who had not undergone induction therapy or adjuvant therapy were selected from among 170 patients with NSCLC who had undergone surgery from January to December 1995. Specimens obtained at surgical resection were subjected to immunohistological staining, and the relationship between postoperative outcome and the expression of VEGF and its receptors was investigated. All patients included in the analysis had been followed up for 5 years or longer or until death. RESULTS: Patients with tumors expressing VEGF or KDR tended to have poorer outcomes, and VEGF expression and KDR expression were positively correlated. In contrast, flt-1 expression was not correlated with VEGF expression or outcome. Outcomes were poor in patients with tumors positive for both VEGF and VEGFRs. Multivariate analysis identified expression of both flt-1 and KDR and VEGF and KDR as possible independent prognostic factors. CONCLUSIONS: Our results suggest that expression of VEGF and VEGFR are associated with a poor prognosis via autocrine and paracrine growth stimulation of cancer cells. Moreover, tumors expressing both flt-1 and KDR may have greater malignant potential and are associated with a poor prognosis.     

Expression of vascular endothelial growth factor and its receptor, KDR/Flk-1, in soft tissue sarcomas

The aim of the present study was to evaluate the association of vascular endothelial growth factor (VEGF) and its receptor, KDR/Flk-1, with tumor grade, microvessel density (MVD) and clinical outcome in patients with soft tissue sarcomas (STSs). Tissue specimens of 28 patients with STSs were analyzed immunohistochemically using specific monoclonal antibodies. Tissue samples were obtained prior to any treatment. Half of the STSs exhibited strong expression of VEGF that was associated statistically significantly with high tumor grade. Strong expression of KDR/Flk-1 was detected in only 2 sarcomas. No association was demonstrated between VEGF and KDR/Flk-1 expression. MVD was significantly associated with tumor grade and was higher in sarcomas with strong VEGF expression. Limited data on clinical outcome precluded solid analyses for an association with disease progression. This study provides further evidence on the role of VEGF and MVD in tumor aggressiveness in STSs.     

Pancreatic carcinoma cells express neuropilins and vascular endothelial growth factor, but not vascular endothelial growth factor receptors

BACKGROUND: Neuropilins (NRPs) are characterized as coreceptors of vascular endothelial growth factor (VEGF). In the current study, the authors assessed the expression of NRPs, VEGF, and vascular endothelial growth factor receptors (VEGFRs), as well as VEGF-induced cell proliferation, in pancreatic carcinoma cell lines and tissue specimens. METHODS: Human pancreatic carcinoma cell lines (Panc-1 and MIA PaCa-2), normal human pancreatic ductal epithelial cells (HPDE), and human umbilical vein endothelial cells (HUVECs) were cultured. Human pancreatic adenocarcinoma tissue specimens were also studied. Expression levels of NRPs, VEGFRs, and VEGF were determined by real-time polymerase chain reaction analysis and immunostaining. Cell proliferation was examined using a [3H]thymidine incorporation assay. RESULTS: Both NRP-1 and NRP-2 were expressed in Panc-1 cells, HPDE cells, and HUVECs but were expressed minimally in MIA PaCa-2 cells. Panc-1 expressed 30 times more NRP-1 mRNA than NRP-2 mRNA. NRP-1 levels in Panc-1 cells were 5.3 times higher than in HPDE cells but were similar to NRP-1 levels in HUVECs. NRP-2 levels in Panc-1 cells were similar to NRP-2 levels in HPDE cells but lower than NRP-2 levels in HUVECs. Expression of all three VEGFRs was observed only in HUVECs. However, VEGF mRNA was detected in all cell types except for HUVECs. NRP-1 immunoreactivity levels were much higher than NRP-2 immunoreactivity levels in Panc-1 and human pancreatic adenocarcinoma tissue specimens, whereas VEGFRs were not detected in either of these two settings. In response to VEGF165, [3H]thymidine incorporation in Panc-1 cells increased significantly (by 61%; P < 0.01). A monoclonal antibody against human NRP-1 significantly blocked VEGF-induced cell proliferation in Panc-1 cells. CONCLUSIONS: The pancreatic carcinoma cell line Panc-1 and adenocarcinoma tissue specimens expressed high levels of NRP-1 and VEGF, but not VEGFRs, and exogenous VEGF significantly increased NRP-1-mediated, but not VEGFR-mediated, Panc-1 cell proliferation. These data suggested that NRP-1 may be involved in the pathogenesis of pancreatic carcinoma.     

抗KDR胞外Ⅲ区单抗抑制血管内皮细胞增殖的研究

目的研制能够封闭血管内皮生长因子(VEGF)受体KDR的单克隆抗体,探讨其体外抑制VEGF165诱导的生物学活性。方法以原核表达的KDR胞外Ⅲ区融合蛋白免疫Balb／c小鼠,采用传统杂交瘤技术制备抗KDR胞外Ⅲ区单克隆抗体,采用ELISA和FACS方法鉴定其抗原结合特异性,采用免疫沉淀和[^3H]-TdR掺入的方法分析该单克隆抗体阻断VEGF165刺激内皮细胞表面KDR酪氨酸激酶受体磷酸化,抑制VEGF165岱诱导内皮细胞增殖的活性。结果抗KDR胞外Ⅲ区单抗Ycom1D3不仅能与可溶性KDR结合,亦可与细胞表面表达的KDR结合,并可竞争性抑制VEGF165与可溶性KDR的相互作用,阻断VEGF165刺激内皮细胞表面KDR酪氨酸激酶受体磷酸化,显著抑制由VEGF165诱导脐静脉内皮细胞的增殖。结论抗KDR胞外Ⅲ区单抗Ycom1D3可通过封闭KDR而抑制VEGF活性,在肿瘤及其他血管新生疾病治疗中具有潜在的应用前景。     

Apoptotic effects of signal transduction inhibitors on human tumor cells with different PTEN expression

An important mechanism of antitumoral targeted therapies is the induction of apoptosis in tumor cells. Tamoxifen and trastuzumab (Herceptin), respectively, are able to trigger apoptosis in human breast cancer cells. But, frequently altered apoptotic signal cascades, for instance through PTEN mutations, help tumor cells to escape antitumoral therapy. We studied to what extent the apoptotic effect of signal-transduction inhibitors is dependent on PTEN expression. PTEN expression was analysed by Western blot analysis in tumor cell lines of the breast (BT-474, MCF-7, MDA-MB-231), ovary (BG-1, SK-OV-3) and endometrium (Ishikawa, HEC-1A). Apoptotic effects of tamoxifen, trastuzumab, ZD1839 (Iressa) and different mitogen-activated protein kinase (MAP) inhibitors were measured after 24 h of treatment. Cellular apoptosis was determined by the detection of cytoplasmic histone-DNA complexes. The tested tumor cell lines exhibited a different PTEN expression, ranging from a high expression (ovarian cancer cell line BG-1 and BT-474 breast cancer cells) to a total absence of PTEN expression (endometrial Ishikawa cells). The apoptotic effect of receptor-targeting drugs (tamoxifen, trastuzumab, ZD1839) was dependent both on receptor expression and PTEN expression. When cells were treated with MAPK inhibitors, no correlation between PTEN expression and the apoptosis rate was observed. Our data underline the importance of PTEN expression regarding the induction of apoptosis through various targeted therapies.