Expression of Vascular Endothelial Growth Factor-C and Vascular Endothelial Growth Factor Receptor-3 in Ovarian Epithelial Tumors

Objective： To explore the role of vascular endothelial growth factor-C （VEGF-C） in the process of angiogenesis, lymphangiogenesis and lymphatic metastasis in epithelial ovarian tumors. Methods： In situ hybridization and immunohistochemical staining for VEGF-C were performed in 30 epithelial ovarian carcinomas, 9 borderline tumors and 26 benign tumors. Endothelial cells were immunostained with anti-VEGFR-3 pAb and anti-CD31 mAb, and VEGFR-3 positive vessels and microvessel density （MVD） were assessed by image analysis. Results： VEGF-C mRNA and protein expression were detected in cytoplasm of carcinoma cells. VEGF-C mRNA and protein expression in ovarian epithelial carcinomas were significantly higher than those in borderline tumors and benign tumors （P〈0.05 or P〈0.01）. In ovarian epithelial carcinomas, VEGF-C protein expression, VEGFR-3 positive vessels and MVD were significantly higher in the cases of clinical stage Ⅲ-Ⅳ and with lymph node metastasis than those of clinical stage Ⅰ-Ⅱ and without lymph node metastasis respectively （P〈0.05 or P〈0.01）. VEGFR-3 positive vessels and MVD were significantly higher in VEGF-C protein positive tumors than negative tumors （P〈0.05）. VEGFR-3 positive vessels was significantly correlated with MVD（P〈0.01）. Conclusion： VEGF-C might play a role in lymphatic metastasis via lymphangiogenesis and angiogenesis in epithelial ovarian tumors, and VBEGF-C could be used as a biologic marker of metastasis in ovarian epithelial tumors.     

Intracrine vascular endothelial growth factor signaling in survival and chemoresistance of human colorectal cancer cells

Although the effects of vascular endothelial growth factor (VEGF) on angiogenesis and vascular function are well known, the effects of VEGF on tumor cell function remain to be elucidated. We studied phenotypic changes in human colorectal cancer (CRC) cells with homozygous deletion of VEGF alleles to determine the potential direct role of VEGF on tumor cell function. Loss of VEGF expression led to significantly decreased cell growth and increased spontaneous apoptosis in CRC cells (P<0.01). Loss of VEGF also increased the in vitro sensitivity of cells to the cytotoxic effects of the chemotherapeutic drug 5-fluorouracil, as shown by increased apoptosis (P<0.05). These effects were mediated via upregulation of the proapoptotic mediators caspase-3, cleaved PARP and Bax and downregulation of the pro-survival mediator survivin. Our findings suggest a novel and distinct function of VEGF in mediating autocrine/intracrine CRC cell survival.     

血管内皮细胞生长因子及其受体在结直肠癌中的表达

目的:探讨血管内皮细胞生长因子(WIGF)及其受体(FLT、FLK-1)在结直肠癌中的表达.方法:应用LSAB免疫组化技术,检测50例人结直肠癌组织VEGF、FLT、FLK-1和8因子相关抗原(FⅧRAg)的表达情况.结果:VEGF、FLT和FLK-1主要表达于侵袭性癌的浸润边缘和坏死组织周围.FLT表达于癌细胞膜和/或细胞浆,而FLK-1则只表达于细胞浆.新生血管多位于肿瘤组织的侵袭性边缘和间质浸润灶旁.结论:WIGF、FLT和FLK-1表达呈现出明显的异质性,并与肿瘤新生血管形成密切相关.综合分析VEGF及其受体的表达有助于对判断结直肠癌侵袭能力和新生血管形成的判断.     

Expression of vascular endothelial growth factor,mitogen-activated protein kinase and p53 in human colorectal cancer

BACKGROUND: VEGF is a growth factor involved in the regulation of angiogenesis, a process that plays a central role in tumor growth. It has been suggested that mutations of p53 and activation of the Ras/MAPK pathway may contribute to the up-regulation of VEGF expression and induction of angiogenesis. PATIENTS AND METHODS: We explored the expression of p53 and VEGF and p44MAPK phosphorylation in 43 human colorectal carcinomas, as well as in peritumoral mucosas, and in normal mucosas in order to establish a correlation between VEGF expression and either mutations of p53 or phosphorylation of p44MAPK. Overexpression of p53 in tumor tissues was interpreted as evidence of mutations. RESULTS: p53 was overexpressed in 22 out of 43 tumors; MAPK was phosphorylated in 25 out of 43 cases whereas only 4 out of 22 peritumoral mucosas showed a moderate phosphorylation of p44MAPK VEGF was up-regulated in 22 out of 43 tumors, moderately expressed in 4 out of 22 peritumoral mucosas and not detectable in normal mucosa. Immunohistochemical analysis showed the presence of a phosphorylated form of p44 MAPK only in neoplastic cells. Statistical analysis demonstrated a significant correlation between p53 and VEGF expression (p<0.03) as well as between VEGF expression and p44 MAPK phosphorylation (p<0.002). CONCLUSION: These data suggest that mutations of p53 and activation of the Ras/MAPK pathway may play a role in the induction of VEGF expression in human colorectal cancer.     

Expression of the vascular endothelial growth factor receptor-3 (VEGFR-3) and its ligand VEGF-C in human colorectal adenocarcinoma

Vascular endothelial growth factors (VEGF) are secreted by many tumor types, and are believed to affect tumor growth by promoting angiogenesis through binding to their receptors present on vascular endothelium. Recently, mRNA for VEGF-C the ligand for VEGFR-3, was found to be up-regulated in colorectal adenocarcinoma (CRC). The aim of this work was to determine: 1) the distribution of VEGF-C and VEGFR-3 in CRC, and 2) the biological significance of such expression. Sections of formalin-fixed and paraffin-embedded tissues from 56 CRC were immunohistochemically stained for VEGF-C and VEGFR-3. The type and percent of positive cells was recorded. Survival analysis was performed using the Kaplan-Meier method. All CRC were positive for VEGF-C which was present in the cancer cells themselves, as well as in stromal cells. Normal colon epithelium was usually negative. Only ten (17%) of the 56 CRC completely lacked VEGFR-3 expression. VEGFR-3 immunoreactivity was detected in <25% of the cancer cells in 22 cases and in >25% of the cells in 34 cases. Expression of VEGFR-3 in >25% of the cancer cells was associated with significantly poorer overall survival (p<0.05), but not with lymph node metastasis or depth of tumor invasion. Our results suggest that VEGFs promote cancer growth not only by stimulating angiogenesis, but also by acting on receptors present on the cancer cells themselves.     

Vascular endothelial growth factor receptor-1 contributes to resistance to anti-epidermal growth factor receptor drugs in human cancer cells

PURPOSE: The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor-resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies. EXPERIMENTAL DESIGN: We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways. RESULTS: Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor-resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents. CONCLUSIONS: This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.     

Fetal stromal-dependent paracrine and intracrine vascular endothelial growth factor-a/vascular endothelial growth factor receptor-1 signaling promotes proliferation and motility of human primary myeloma cells

Induction of neoangiogenesis plays an important role in the pathogenesis of multiple myeloma. However, the mechanism by which expression of vascular endothelial growth factor (VEGF)-A and its receptors modulate the interaction of multiple myeloma cells with stromal cells is not known. Here, we describe a novel in vitro coculture system using fetal bone stromal cells as a feeder layer, which facilitates the survival and growth of human primary multiple myeloma cells. We show that stromal-dependent paracrine VEGF-A signaling promotes proliferation of human primary multiple myeloma cells. Primary multiple myeloma cells only expressed functional VEGF receptor (VEGFR)-1, but not VEGFR-2 or VEGFR-3. VEGFR-1 expression was detected in the cytoplasm and the nuclei of proliferating multiple myeloma cells. Inhibition of VEGFR-1 abrogated multiple myeloma cell proliferation and motility, suggesting that the functional interaction of VEGF-A with its cognate receptor is essential for the growth of primary multiple myeloma cells. Collectively, our results suggest that stromal-dependent paracrine and intracrine VEGF-A/VEGFR-1 signaling contributes to human primary multiple myeloma cell growth and therefore, VEGFR-1 blockade is a potential therapeutic strategy for the treatment of multiple myeloma.     

Quantitative analysis of basic fibroblast growth factor and vascular endothelial growth factor in human colorectal cancer.

Tumour growth is angiogenesis dependent. Some authors suggest a prognostic role of microvessel count in colorectal cancer. We tested the role of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in the switch to the angiogenic phenotype in 35 patients with colorectal cancer at different stages of disease. We evaluated the two angiogenic factors, by enzyme-linked immunosorbent assay (ELISA), in tumour, peritumoral mucosa, pathological mesenteric and peripheral blood. We used ten endoscopic intestinal biopsies and ten peripheral blood samples from healthy subjects as control. bFGF was significantly lower in tumour tissues and in peritumoral mucosas than in healthy mucosas, whereas VEGF was up-regulated in tumours but not in peritumoral mucosa. Both angiogenic factors were greatly increased in mesenteric blood. VEGF tumour and serum levels were significantly correlated with the stage of disease. bFGF tumour and serum concentration were not correlated with the stage of disease. The high levels of bFGF in mesenteric blood suggest that this growth factor might be abnormally released from tumour tissue and peritumoral mucosa and could function as an early effector in the switch to the angiogenic phenotype. In contrast, VEGF, whose levels show a significant correlation with the stage of disease, could act in a following step, supporting tumour progression.     

Induction of neuropilin-1 and vascular endothelial growth factor by epidermal growth factor in human gastric cancer cells

The epidermal growth factor receptor (EGF-R) pathway plays a pivotal role in the progression of human gastric cancer. The angiogenic factor vascular endothelial growth factor (VEGF) has been shown to be induced by EGF in various cancer cell lines. Neuropilin-1 (NRP-1) acts as a coreceptor for VEGF-165 and increases its affinity for VEGF receptor 2 (VEGFR-2) in endothelial cells. Furthermore, NRP-1 has been found to be expressed by tumour cells and has been shown to enhance tumour angiogenesis and growth in preclinical models. We examined the expression of NRP-1 mRNA and EGF-R protein in seven human gastric cancer cell lines. NRP-1 expression was expressed in five of seven cell lines, and EGF-R expression closely mirrored NRP-1 expression. Moreover, in EGF-R-positive NCI-N87 and ST-2 cells, EGF induced both NRP-1 and VEGF mRNA expression. C225, a monoclonal antibody to EGF-R, blocked EGF-induced NRP-1 and VEGF expression in NCI-N87 cells in a dose-dependent manner. The treatment of NCI-N87 cells with EGF resulted in increases in phosphorylation of Erk1/2, Akt, and P38. Blockade of the Erk, phosphatidylinositol-3 kinase/Akt, or P38 pathways in this cell line prevented EGF induction of NRP-1 and VEGF. These results suggest that regulation of NRP-1 expression in human gastric cancer is intimately associated with the EGF/EGF-R system. Activation of EGF-R might contribute to gastric cancer angiogenesis by a mechanism that involves upregulation of VEGF and NRP-1 expression via multiple signalling pathways.     

Expression of vascular endothelial growth factor receptor-3 by lymphatic endothelial cells is associated with lymph node metastasis in prostate cancer.

PURPOSE: The molecular mechanisms underlying lymph node metastasis are poorly understood, despite the well-established clinical importance of lymph node status in many human cancers. Recently, vascular endothelial growth factor (VEGF)-C and VEGF-D have been implicated in the regulation of tumor lymphangiogenesis and enhancement of lymphatic invasion via activation of VEGF receptor-3. The purpose of this study was to determine the expression pattern of the VEGF-C/VEGF-D/VEGF receptor-3 axis in prostate cancer and its relationship with lymph node metastasis. EXPERIMENTAL DESIGN: The expression pattern of VEGF-C, VEGF-D, and VEGF receptor-3 in localized prostate cancer specimens (n = 37) was determined using immunohistochemistry. RESULTS: Widespread, heterogeneous staining for VEGF-C and VEGF-D was observed in all cancer specimens. Intensity of VEGF-C staining was lower in benign prostate epithelium than in adjacent carcinoma, whereas no difference between benign epithelium and carcinoma was observed for VEGF-D staining. VEGF receptor-3 immunostaining was detected in endothelial cells of lymphatic vessels in 18 of 37 tissue samples. The presence of VEGF receptor-3-positive vessels was associated with lymph node metastasis (P = 0.0002), Gleason grade (P < 0.0001), extracapsular extension (P = 0.0382), and surgical margin status (P = 0.0069). In addition, VEGF receptor-3 staining highlighted lymphatic invasion by VEGF-C-positive/VEGF-D-positive carcinoma cells. CONCLUSIONS: Together, these results suggest that paracrine activation of lymphatic endothelial cell VEGF receptor-3 by VEGF-C and/or VEGF-D may be involved in lymphatic metastasis. Thus the VEGF-C/VEGF-D/VEGF receptor-3 signaling pathway may provide a target for antilymphangiogenic therapy in prostate cancer.     

血管内皮生长因子在非小细胞肺癌中的表达及临床相关性研究

 目的　研究血管内皮生长因子（VEGF）表达在肺良性病变与肺癌组织中的差异及其与非小细胞肺癌（NSCLC）临床病理特征、预后的关系，探讨其在NSCLC生长中的意义。方法　应用免疫组织化学染色法，检测手术切除的60份肺癌组织与30份肺良性病变组织中VEGF和微血管密度（MVD）的表达，并进行统计学分析。结果　肺癌组织中VEGF阳性率（63 %，38／60）和MVD（45.13±10.27）均高于肺良性病变组织（27 %，8／30，33±6.49）（P＜0.05，P＜0.01）；VEGF表达、MVD计数分别与NSCLC有无淋巴结转移（P＜0.05，P＜0.01）、TNM分期（P＜0.05，P＜0.01）密切相关；与VEGF阴性相比，VEGF阳性者NSCLC组织中MVD显著增高（P＜0.05）；生存分析显示：VEGF阳性表达与生存期呈明显负相关（P＜0.01）；COX风险比例模型分析显示：VEGF表达可作为判断NSCLC患者预后的独立指标（P＜0.01）。结论　VEGF与NSCLC的临床病理特征、预后密切相关，是促进肺癌生长的重要因子。     

Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases

Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF-induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process.     

人星形细胞瘤血管内皮生长因子表达

目的:探讨VEGF在星形细胞瘤中表达与星形细胞瘤组织学分级和患者年龄的关系.方法:应用免疫组化方法检测52例星形细胞瘤石蜡包埋病理切片.结果:VEGF在不同病理级别星形细胞瘤中表达程度不同,随组织级别增加,表达增强.VEGF高表达组患者平均年龄高于低表达组.结论:VEGF与星形细胞瘤生长及恶性进展关系密切,VEGF高表达与高龄有关,预示预后不良.     

Preclinical data targeting vascular endothelial growth factor in colorectal cancer

Vascular endothelial growth factor (VEGF) is the driving force behind angiogenesis in most solid malignancies. This also holds true for colorectal cancer (CRC), where increased levels of VEGF in primary cancers are associated with increased microvessel density and poor prognosis. These findings have led to preclinical studies evaluating the efficacy of anti-VEGF therapy in inhibiting the growth of CRC in ectopic and orthotopic locations. In preclinical models, numerous approaches to inhibit VEGF activity led to decreased tumor growth and angiogenesis. These studies led to clinical trials in which, unfortunately, single-agent anti-VEGF therapy was relatively ineffective for patients with metastatic CRC. However, combinations of anti-VEGF therapies with chemotherapy have clearly demonstrated clinical benefit. Understanding the mechanisms of the role of VEGF in CRC angiogenesis and the effect of anti-VEGF therapy on the tumor vasculature will allow oncologists to optimize therapeutic regimens targeting VEGF and its receptors.     

血管内皮生长因子在人脑胶质瘤侵袭中的作用

 目的　研究血管内皮生长因子 (Vascularendothelialgrowthfactor ,VEGF)在脑胶质细胞瘤中的表达情况 ,探讨其表达与肿瘤的恶性程度、肿瘤侵袭及预后的关系。方法　采用免疫组织化学染色检测 5 2例脑胶质瘤标本 ,4例垂体腺瘤 ,5例正常脑组织的VEGF表达水平。结果　在正常脑组织中未见VEGF免疫组化染色阳性细胞 ,垂体腺瘤偶有表达 ,脑胶质瘤组织中均有VEGF表达 (P <0 .0 5 )。随着脑胶质瘤恶性程度的增加 ,VEGF的表达增高 (P <0 .0 1)。生存期 <3年者VEGF的表达显著高于生存期 >3年者 (P <0 .0 1)。结论　VEGF的表达与胶质瘤的恶性程度和侵袭性等有关 ,并对其预后有一定的意义     

Regulation of vascular endothelial growth factor receptor-1 expression by specificity proteins 1, 3, and 4 in pancreatic cancer cells

Vascular endothelial growth factor receptor-1 (VEGFR1) is expressed in cancer cell lines and tumors and, in pancreatic and colon cancer cells, activation of VEGFR1 is linked to increased tumor migration and invasiveness. Tolfenamic acid, a nonsteroidal anti-inflammatory drug, decreases Sp protein expression in Panc-1 and L3.6pl pancreatic cancer cells, and this was accompanied by decreased VEGFR1 protein and mRNA and decreased luciferase activity on cells transfected with constructs (pVEGFR1) containing VEGFR1 promoter inserts. Comparable results were obtained in pancreatic cancer cells transfected with small inhibitory RNAs for Sp1, Sp3, and Sp4 and all three proteins bound to GC-rich elements in the VEGFR1 promoter. These results show that VEGFR1 is regulated by Sp proteins and that treatment with tolfenamic acid decreases expression of this critical angiogenic factor. Moreover, in vitro studies in Panc-1 cells show that activation of VEGFR1 by VEGFB to increase mitogen-activated protein kinase 1/2 phosphorylation and cell migration on collagen-coated plates is also inhibited by tolfenamic acid. Thus, targeted degradation of Sp proteins is highly effective for inhibiting VEGFR1 and associated angiogenic responses in pancreatic cancer.     

血管内皮生长因子受体-1与肿瘤

血管内皮生长因子受体-1（VEGFR-1）通过其酪氨酸激酶活性调节肿瘤血管的新生从而影响肿瘤的发生、发展和转移。近年研究发现一些肿瘤细胞本身也可以表达VEGFR-1,抗VEGFR-1抗体不仅可以对表达VEGFR-1的肿瘤血管内皮发生直接的抗血管生成活性,也可以通过旁分泌的配体活化VEGFR-1阳性肿瘤细胞而直接影响肿瘤的发生、发展。     

Dendritic cells and vascular endothelial growth factor in colorectal cancer: correlations with clinicobiological findings

OBJECTIVE: Dendritic cells (DC) are central to the development of immune system responses. In a cohort of 54 patients affected by colorectal cancer, we prospectively investigated the number of peripheral blood (PB) DC type 1 (DC1) and type 2 (DC2) and correlated their counts and functionality to the stage of the disease and to vascular endothelial growth factor (VEGF) levels. RESULTS: At diagnosis, compared with healthy controls, patients presented reduced PBDC1 and PBDC2 numbers (p < 0.001). Moreover, in cancer patients, PBDC showed low levels of DC-associated antigens (HLA DR, p = 0.004; CD11c, p < 0.001; CD83, p = 0.01; CD86, p = 0.007 and Mannose receptor, p = 0.029), an upregulation of CXCR4 (p = 0.017) and a reduced T cell stimulation capability (p < 0.001). DC1 and DC2 loss was higher in stage D versus stage ABC patients (p = 0.003 and p = 0.002, respectively); surgery and chemotherapy appeared to attenuate a DC defect, although the restoration of normal PBDC levels is completed only at 6 and 12 months after diagnosis, respectively. In this series of patients, PBDC1 and PBDC2 numbers inversely correlated with VEGF serum levels (p < 0.001), suggesting a possible effect of this cytokine on DC compartment. In culture, the exposure of monocyte-derived DC to VEGF produced a dramatic alteration of DC differentiation by (1) induction of apoptosis, (2) alteration of DC immunophenotypic profile and (3) increased CXCR4 expression. Exposure to anti-VEGF blocking antibodies reversed VEGF inhibitory effects in all cases. CONCLUSIONS: These findings suggest that in colorectal cancer patients there is a numerical and functional impairment of PBDC compartment possibly related to the stage of the disease and to VEGF levels.