Ribozyme approach to downregulate vascular endothelial growth factor (VEGF) 189 expression in non-small cell lung cancer (NSCLC)

The aim of this study was to further clarify the role of the cell-associated isoform of vascular endothelial growth factor (VEGF189) on tumour growth and vascularity. Five isoforms of VEGF have been identified with different biological activities. VEGF121, VEGF145, VEGF165, VEGF189, VEGF206 are generated by alternative splicing. We used a hammerhead-type ribozyme (V189Rz) to suppress VEGF189 mRNA. The V189Rz specifically cleaved exon 6 of VEGF189 mRNA, but showed no activity against the VEGF121 or VEGF165 isoforms. The V189Rz was introduced into the human non-small cell lung cancer (NSCLC) cell line (OZ-6/VR). The expression level of VEGF189 mRNA was decreased in the OZ-6/VR cells, while VEGF121 and 165 expression was unaltered. The OZ-6/VR cells xenotransplanted into nude mice showed markedly reduced vascularisation and growth, whereas the cell line did not show any decreased growth under tissue culture conditions. The OZ-6/VR cells (1×10⁵ cells/mouse) formed no tumours, whereas the parental OZ-6 cells formed large tumours within 8 weeks. The specific suppression of VEGF189 by the ribozyme decreased vascularity and xenotransplantability of the lung cancer cell line. Thus, the cell-associated isoform of VEGF, VEGF189, might have a key role in stromal vascularisation and the growth of NSCLC xenografts in vivo.     

血管内皮生长因子抗体对肿瘤转移的抑制作用

目的探讨阻断血管内皮生长因子（ｖａｓｃｕｌａｒｅｎｄｏｔｈｅｌｉａｌｇｒｏｗｔｈｆａｃｔｏｒ,ＶＥＧＦ）是否可以抑制肿瘤的转移。方法应用ＩＶＴＡ２ＭＡ－８９１津白Ⅱ小鼠自发乳腺癌模型进行抗肿瘤转移的研究,此模型伴有高自发肺转移。结果Ｎｏｒｔｈｅｒｎ杂交及免疫组化证实,该乳腺癌原发灶及肺转移灶均可表达ＶＥＧＦ,且以后者为高。接种肿瘤后第９天,以ＶＥＧＦ抗体处理荷瘤小鼠,可明显抑制原发肿瘤的生长（４４．０％,Ｐ＜０．０５）,而对肺转移灶数目及转移灶大小的抑制率分别达７３．０％和８３．７％。结论ＶＥＧＦ抗体对肿瘤转移的抑制有潜在的应用价值。     

Cell-retained isoforms of vascular endothelial growth factor (VEGF) are correlated with poor prognosis in osteosarcoma

Vascular endothelial growth factor (VEGF) is a major angiogenic factor. Osteosarcoma is characterised by hypervascularity and metastatic potential. We examined VEGF mRNA expression, VEGF isoform pattern and VEGF receptor (flt-1 and KDR) by RT-PCR analysis in 30 osteosarcomas. All 30 osteosarcomas expressed VEGF mRNA. 17 osteosarcomas (57%) expressed flt-1 mRNA, whilst 20 (67%) expressed KDR mRNA. 6/30 (20%) osteosarcomas were positive for VEGF121 only, 8 (27%) for VEGF121+VEGF165, and 16 (53%) for VEGF121+VEGF165+VEGF189. Patients with osteosarcomas with VEGF165 (n=24) had significantly poorer prognosis in comparison with those without VEGF165 (P=0.022, Wilcoxon's test). The osteosarcomas with VEGF165 had significantly increased vascularity assessed on sections immunostained for CD34 (P<0.001, Mann–Whitney U test). Although VEGF165 is a soluble isoform, it is also retained on the cellular surface. These results suggest that cell-retained VEGF isoforms (VEGF165, VEGF189) might be essential for neovascularisation in osteosarcoma, whilst the soluble VEGF121 isoform is not sufficient to stimulate neovascularisation in this type of neoplasm.     

Xenografts of human solid tumors frequently express cellular-associated isoform of vascular endothelial growth factor (VEGF) 189.

Vascular endothelial growth factor (VEGF), a major factor mediating tumor stromal angiogenesis, is expressed as five splice variants encoded by a single gene (VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206). Recently, we demonstrated that the cell-associated isoform, VEGF189, plays important roles in establishment of human colon and esophageal cancer xenografts. We have established 228 xenografts originating from various human solid tumors. In this study, we investigated the expression patterns of VEGF isoforms in those tumor xenografts by RT-PCR. The isoform patterns were VEGF121/VEGF165 in 27 xenografts (11.8%) and VEGF121/VEGF165/VEGF189 in 201 (88.2%). All human solid tumor xenografts expressed VEGF189 more frequently than primary tumors reported previously. These results suggest that VEGF189 contributes to the successful xenotransplantability of various human solid tumors via augmentation of stromal vascularization.     

Use of soluble recombinant decoy receptor vascular endothelial growth factor trap (VEGF Trap) to inhibit vascular endothelial growth factor activity

Primary tumors and metastases require blood vessel formation to support their continued growth and eventual metastasis. They use existing vasculature during initial growth but eventually must orchestrate the development and maintenance of new vessels--a process termed angiogenesis--to grow beyond a small size and spread. Angiogenesis is regulated by a number of soluble factors, the relative proportions of which can exacerbate or inhibit the process. Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, produced by the majority of human solid tumors. Inhibitors of VEGF might have an impact on the growth and metastasis of these cancers. The relevance of this strategy to the treatment of colorectal cancer was first successfully demonstrated in human clinical trials using a monoclonal antibody against VEGF. A potent antiangiogenic soluble recombinant decoy, VEGF Trap is a protein constructed from VEGF receptor-binding domains linked to an immunoglobulin G(1) constant region. It possesses an affinity for VEGF that is significantly higher than that of the monoclonal antibody. VEGF Trap has demonstrated marked efficacy in halting angiogenesis and shrinking tumors in preclinical animal models and is currently being studied in phase I clinical trials in humans with advanced solid malignancies.     

Tumor-derived vascular endothelial growth factor (VEGF)-a facilitates tumor metastasis through the VEGF-VEGFR1 signaling pathway

Vascular endothelial growth factor (VEGF) and its receptors are involved in carcinogenesis, invasion and tumor angiogenesis, but the underlying mechanism by which VEGF promotes tumor metastasis is poorly understood. In this study, we show that in cancer patients high expression of VEGF is correlated with metastasis, and anti-VEGF treatment (bevacizumab) has clinical effects on tumor metastasis. Two human lung carcinoma cell lines (A549 and SPCA1 cells) with distinct VEGF expression were injected intravenously through the lateral tail vein of SCID mice and a murine model was developed. We investigated the association between the expression of VEGF and tumor metastasis by microvessel density, immunohistochemistry and whole mount staining. At sacrifice, in the high VEGF expression A549 cell line group, the induced tumor was distinctively larger in size and multiple metastatic lesions were found in lung tissues. Two specific neutralizing anti-mouse VEGFR1 and VEGFR2 antibodies were administered to the tumor-bearing mice; anti-VEGFR1, but not anti-VEGFR2 treatment produced inhibitive effects on VEGF-induced tumor metastasis. These findings demonstrate that the VEGF-VEGFR1 signaling pathway is crucial for tumor metastasis and the blockade of VEGF-VEGFR1-induced metastasis may provide a novel approach for the prevention and treatment of tumor metastasis.     

Blockage of vascular endothelial growth factor (VEGF) reduces experimental pleurodesis

Background and objective

Chemical pleurodesis controls recurrent malignant pleural effusion. The mechanism that determines pleural symphysis involves the action of vascular endothelial growth factor (VEGF). We assessed the influence of the anti-VEGF antibody (bevacizumab) on pleurodesis induced by talc or silver nitrate and analyzed the temporal development of pleural angiogenesis.

Methods

Sixty New Zealand rabbits received intrapleural injection (2 mL) of talc (400 mg/kg) or 0.5% silver nitrate. In each group, half of the animals received an intravenous injection of bevacizumab 30 min before the sclerosing agent. Five animals from each group were euthanized 7, 14, or 28 days after the procedure. Adhesions and inflammation (scores: 0-4), thickness (μm), vascular density (vessels/field), and collagen fibers (μm²) were evaluated in the visceral pleura.

Results

Antibody anti-VEGF interferes in pleurodesis induced by talc or silver nitrate. Pleural inflammation was discreet with no difference between the groups, regardless the anti-VEGF treatment. Concerning the vascular density of the visceral pleura, a smaller number of neoformed vessels was noted in the animals that received bevacizumab. In the animals receiving silver nitrate, the decrement in adhesions and vascular density was associated with reduced thick and thin collagen fibers, resulting in less pleural thickness.

Conclusion

The anti-VEGF antibody inhibits adhesions between pleural layers. Despite being an experimental study in animals with normal pleura, the results call attention to a likely lack of success in pleurodesis when VEGF blockers are used.     

Predictive value of vascular endothelial growth factor (VEGF) in metastasis and prognosis of human colorectal cancer.

Vascular endothelial growth factor (VEGF) may affect the phenotype of cancer cells, such as growth velocity and metastatic potential, due to its probable multifunctional property including a mitogenic activity for vascular endothelial cells. The present study was designed to investigate the association of VEGF mRNA expression with progression and metastasis of human colorectal cancer. The level of VEGF mRNA expression was quantified by Northern blot hybridization in tumorous and non-tumorous tissues obtained from 60 primary colorectal cancer patients. The ratio of the former to the latter was defined as the VEGF T/N ratio, and the prognostic significance of this ratio, following surgery, in addition to the relationship to progression and metastatic potential, was evaluated. The value of the VEGF T/N ratio was significantly correlated with the depth of tumour infiltration (P=0.046), the incidence of liver metastasis (P < 0.0001) and lymph node metastasis (P=0.036). Patient prognosis was estimated by the Kaplan-Meier method and the log-rank test. When the VEGF T/N ratio was higher than 4.8 for which the chi2 value of the log-rank test was maximal, the tumour was defined as showing overexpression of VEGF mRNA. Patients with overexpression of VEGF mRNA demonstrated poorer survival than patients without overexpression of VEGF mRNA (P < 0.001). The overall estimated hazard ratio for death in patients with overexpression of VEGF mRNA was 1.94 according to a multivariate analysis (P=0.005). Thus, VEGF is associated with the progression, invasion and metastasis of colorectal cancer, and overexpression of VEGF mRNA in the primary tumour is assumed to be closely correlated with poor prognosis in colorectal cancer patients. Moreover, the VEGF T/N ratio may be used as an independent prognostic marker in colorectal cancer patients.     

Overexpression of vascular endothelial growth factor-A165 enhances tumor angiogenesis but not metastasis during beta-cell carcinogenesis.

The pivotal role of vascular endothelial growth factor (VEGF-A) in the regulation of angiogenesis, in particular in the onset and maintenance of tumor angiogenesis, has been demonstrated repeatedly in experimental model systems and, more recently, in clinical trials. Experimental evidence has also suggested that up-regulated expression of VEGF-A may cooperate with other genetic or epigenetic changes to induce or accelerate tumor progression to invasive and metastatic cancers. Here we report the generation of transgenic mouse lines that express human VEGF-A165 under the control of the rat insulin promoter in the beta cells of pancreatic islets of Langerhans (Rip1VEGF-A). These mice do not exhibit detectable changes in islet development, vascularization, or physiology. Intercrosses of these mice with a transgenic mouse model of pancreatic beta cell carcinogenesis (Rip1Tag2) result in an earlier onset of tumor angiogenesis and with it accelerated tumor growth and mortality. The transition from benign tumors (adenoma) to malignant tumors (carcinoma) is modestly accelerated; however, tumor metastases are not observed. Our findings indicate that in beta-cell tumorigenesis, overexpression of VEGF-A165 accelerates the onset of tumor angiogenesis and with it tumor progression but is not sufficient to induce tumor metastasis.     

Immunohistochemical evaluation of vascular endothelial growth factor (VEGF) in colorectal carcinoma

BACKGROUND: Vascular endothelial growth factor (VEGF) is one of the most important proangiogenic factors over-expressed in many human cancers and is usually associated with an unfavorable outcome. This work was planned to assess VEGF and microvessel density (MVD) in colorectal carcinoma (CRC) and to correlate them with the available clinicopathological parameters. MATERIAL AND METHODS: Ten normal colonic mucosa, 21 adenoma and 70 CRC cases were examined by immunohistochemical staining using anti-VEGF antibody. RESULTS: Eighty-one percent (81%) of adenoma and 78.6% of CRC cases showed VEGF immunoreactivity; however, the intensity of staining was significantly in favour of malignant cases (p=0.032). VEGF immunostaining in CRC was correlated with low grade (p=0.009), early stage either by Dukes' system (p=0.03) or TNM stage (p=0.03), negative nodal status (p=0.04) and high mast cell count (p=0.04). MVD assessed by Haematoxylin and Eosin staining was associated with dense inflammatory response (p=0.003) and high mast cell count (p=0.006). CONCLUSIONS: VEGF could be an early carcinogenic factor in CRC that declines with its progression. Inflammatory cells including mast cells could play a role in CRC angiogenesis.     

Antibody against vascular endothelial growth factor (VEGF) inhibits angiogenic switch and liver metastasis in orthotopic xenograft model with site-dependent expression of VEGF

We previously reported that upregulation of angiogenesis, i.e. angiogenic switch (AS), may occur simultaneously to initiation of invasion in the early development of human colon cancer. We also showed that mRNA upregulation of the gene of vascular endothelial growth factor (VEGF) occurs immediately prior to metastasis in human colon cancer in an orthotopic nude mouse model of colon cancer liver metastasis. In this paper, we studied whether the antibody against VEGF inhibits AS and liver metastasis in an orthotopic xenograft model with site-dependent expression of VEGF. We examined levels of vessel density, VEGF mRNA by in situ hybridization (ISH) method and liver metastasis in pre-AS (on days 8 and 11) and post-AS (on days 15 and 18) treatment groups. The mean vessel density and the intensity of VEGF mRNA by ISH in the pre-AS treatment group were significantly lower than those for the post-AS treatment and the control group. Liver metastases were completely inhibited (0/10) in the pre-AS treatment, while they occurred in 4 out of 10 and 5 out of 10 mice in the post-AS treatment and the control groups, respectively (p<0.01). These results suggest that VEGF antibody treatment performed before AS could efficiently inhibit AS and liver metastasis, which may indicate that VEGF antibody has another potential as a drug for chemoprevention of colon cancer.     

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.     

Prognostic impact of vascular endothelial growth factor-A expression in resected gallbladder carcinoma

The purpose of this study was to evaluate the value of vascular endothelial growth factor-A (VEGF-A) expression and other confirmed prognostic factors in predicting clinical outcomes after the resection of gallbladder carcinoma (GBC). Between January 1999 and January 2006, a total of 84 consecutive and non-selected patients who underwent resection for GBC were retrospectively reviewed. Of the 84 patients studied, 45 cases (53.6%) exhibited high expression of VEGF-A and were placed into the high expression group. The 14 cases (16.7%) that showed no VEGF expression and the 25 cases (29.7%) that had lower VEGF-A levels were pooled into the low expression group (46.4%). There was a relationship between VEGF-A status and pM stage (P = 0.027) as well as histologic differentiation (P < 0.001). In univariate analysis by log-rank test, ECOG performance status, CA 19-9, pN stage, pM stage, histologic differentiation, and VEGF-A expression were significant prognostic factors (P = 0.015, 0.001, 0.020, <0.001, 0.040, and <0.001, respectively). Multivariate analysis revealed that pN status and VEGF-A expression maintained independent prognostic influence on overall survival (P < 0.001 and P = 0.013, respectively). VEGF-A expression has a positive correlation with pM stage and histologic differentiation. pN status and VEGF-A expression were independent prognostic factors of overall survival in patients with resected GBC.     

The role of vascular endothelial growth factor (VEGF) in AIDS-related Kaposi's sarcoma

Kaposi's sarcoma (KS) is the most common neoplasm associated with human immunodeficiency virus-1 (HIV-1) infection. KS involves the skin and mucous membranes as well as other organs and can lead to tumor-associated edema and ulcerations. Despite therapy with highly active antiviral agents, most patients with HIV-1-related KS eventually develop disseminated disease. In the treatment of KS, a strong rationale exists for the use of agents that inhibit vascular endothelial growth factor (VEGF). Angiogenesis appears to be an important feature of this disease, and recent experimental studies have demonstrated the role of VEGF and its receptors in the pathogenesis of KS. Thus, therapeutic agents that target the VEGF pathway may be an effective strategy in reducing the tumor growth and edema associated with KS. Phase I study results with SU5416, a synthetic low molecular-weight inhibitor of the VEGF-Flk-1/KDR receptor tyrosine kinase, demonstrate that this agent is well tolerated. Preliminary results show that in a majority of patients with autoimmune deficiency syndrome (AIDS)-related disease, SU5416 clearly has biological activity (it flattens, shrinks, or dissolves lesions and reduces or resolves edema) or stabilizes the disease. Angiogenesis inhibition with SU5416 is a promising therapeutic approach in treating patients with KS, and further clinical evaluation is currently under way.     

The clinical toxicity profile of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors; a review

Clinical experience with vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor (VEGFR) targeting angiogenesis inhibitors is rapidly increasing, and some compounds have already been approved for regular anticancer treatment.Apart from their activity, much attention has been focussed on the clinical toxicity profile of these compounds.This review describes the most frequently occurring side-effects of both antibodies and tyrosine kinase inhibitors and discusses some of the underlying mechanisms. Some practical guidelines for treatment of the side-effects are given.     

Elevated vascular endothelial growth factor (VEGF) serum levels in idiopathic myelofibrosis.

An increase of angiogenesis has been shown in idiopathic myelofibrosis with myeloid metaplasia (MMM) by microvessel density count method but evaluation of circulating angiogenic factors is still incomplete. In 31 patients affected by MMM and in 12 healthy subjects we evaluated the serum levels of VEGF (vascular endothelial growth factor) and correlated VEGF with clinical and laboratory features of disease. We found that MMM patients had circulating VEGF concentrations much higher than controls (Median 1208 ng/ml vs 138 ng/ml, P < 0.0001). No correlation was found between VEGF and Hb, WBC, PLT, LDH, creatinine, bone marrow cellularity, fibrosis, splenomegaly, hepatomegaly, and therapy. However, in the subgroup of patients with a normal or low VEGF concentration, a direct correlation between VEGF and platelet count (r = 0.90, P = 0.002) was detected. Moreover, patients with a platelet count < 300 x 10(9)/l had VEGF serum levels lower than patients with a higher PLT count (median VEGF 864 vs 1557 pg/ml, P = 0.001). In six patients and in eight controls we also had the opportunity to measure VEGF in the plasma and we calculated that VEGF concentration was much higher in platelet-rich than in platelet-poor plasma and that platetets of MMM patients contained four times more VEGF than those of healthy controls. These results indicate that VEGF is overproduced in MMM, thus confirming an increased angiogenic activity. Platelets are probably a major source of VEGF in MMM but not the only one.     

抗VEGF发卡状核酶抑制肝癌VEGF表达和移植瘤生长

目的:采用抗人血管内皮生长因子(vascular endothelial growth factor,VEGF)发卡状核酶基因转染肝癌细胞,观察核酶对VEGF表达及肿瘤生长的影响。方法:采用脂质体介导的方法,将人工合成的抗VEGF发卡状核酶基因转染肝癌细胞SMMC-7721,同时制备空载体和细胞对照,经G418筛选获得阳性克隆,基因组水平鉴定核酶在细胞中的表达,半定量RT-PCR法和免疫组化法观察核酶对SMMC-7721细胞VEGF表达的影响。接种各组细胞于裸鼠皮下,观察瘤体体积大小和质量,免疫组化法观测各组肿瘤微血管变化和VEGF的表达。结果:核酶基因成功导入到肿瘤细胞中,转基因细胞的增殖速率明显下降(P<0.01),转染核酶组细胞VEGF水平明显下降(P<0.01)。移植瘤成瘤率和生长速度减慢(P<0.01),肿瘤组织VEGF表达和血管形成减少(P<0.01)。结论:抗人VEGF发卡状核酶基因在体内、体外均可抑制肝癌VEGF表达,通过减少血管形成抑制肿瘤生长,为进一步开展肝癌血管靶向基因治疗提供实验依据。     

Vascular endothelial growth factor (VEGF) in leptomeningeal metastasis: diagnostic and prognostic value

This study examined the diagnostic and prognostic value of vascular endothelial growth factor (VEGF) levels in the cerebrospinal fluid (CSF) of 39 patients with leptomeningeal metastasis (LM). Vascular endothelial growth factor levels at diagnosis were significantly higher in patients with LM (median 359 pg ml(-1)) than in patients with other neurological diseases (median <25 pg ml(-1)). The specificity of VEGF levels above 250 pg ml(-1) for LM was high (98.3%), while the sensitivity was low (51.4%; 73% for VEGF values above 100 pg ml(-1)). In 49% of the LM patients, particularly with lymphoma or medulloblastoma, VEGF levels were below 250 pg ml(-1) and thus in the range of VEGF levels in other neurological diseases. Vascular endothelial growth factor levels correlated significantly with CSF lactate and albumin. Vascular endothelial growth factor levels mirrored the clinical course with a marked reduction in response to therapy and an increase at relapse in some patients who had serial CSF samples available. Multivariate Cox regression analysis showed VEGF below 100 pg ml(-1) (relative risk (RR)=4.24, P=0.0002) and age below 60 years (RR=2.5, P=0.004) to be associated with longer survival in LM. In conclusion, CSF VEGF levels in LM vary considerably. High VEGF levels have a very high specificity for LM and may help to establish the diagnosis. The role of VEGF as a predictor of outcome should be substantiated in prospective studies.