Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis.

New blood vessel formation (angiogenesis) is a fundamental event in the process of tumor growth and metastatic dissemination. Hence, the molecular basis of tumor angiogenesis has been of keen interest in the field of cancer research. The vascular endothelial growth factor (VEGF) pathway is well established as one of the key regulators of this process. The VEGF/VEGF-receptor axis is composed of multiple ligands and receptors with overlapping and distinct ligand-receptor binding specificities, cell-type expression, and function. Activation of the VEGF-receptor pathway triggers a network of signaling processes that promote endothelial cell growth, migration, and survival from pre-existing vasculature. In addition, VEGF mediates vessel permeability, and has been associated with malignant effusions. More recently, an important role for VEGF has emerged in mobilization of endothelial progenitor cells from the bone marrow to distant sites of neovascularization. The well-established role of VEGF in promoting tumor angiogenesis and the pathogenesis of human cancers has led to the rational design and development of agents that selectively target this pathway. Studies with various anti-VEGF/VEGF-receptor therapies have shown that these agents can potently inhibit angiogenesis and tumor growth in preclinical models. Recently, an anti-VEGF antibody (bevacizumab), when used in combination with chemotherapy, was shown to significantly improve survival and response rates in patients with metastatic colorectal cancer and thus, validate VEGF pathway inhibitors as an important new treatment modality in cancer therapy.     

Interleukin-2 and histamine in combination inhibit tumour growth and angiogenesis in malignant glioma

Biotherapy including interleukin-2 (IL-2) treatment seems to be more effective outside the central nervous system when compared to the effects obtained when the same tumour is located intracerebrally. Recently published studies suggest that reduced activity of NK cells in tumour tissue can be increased by histamine. The present study was designed to determine whether IL-2 and histamine, alone or in combination, can induce anti-tumour effects in an orthotopic rat glioma model. One group of rats was treated with histamine alone (4 mg kg(-1)s.c. as daily injections from day 6 after intracranial tumour implantation), another group with IL-2 alone as a continuous subcutaneous infusion and a third group with both histamine and IL-2. The animals were sacrificed at day 24 after tumour implantation. IL-2 and histamine in combination significantly reduced tumour growth. The microvessel density was significantly reduced, an effect mainly affecting the small vessels. No obvious alteration in the pattern of VEGF mRNA expression was evident and no significant changes in apoptosis were observed. Neither IL-2 nor histamine alone caused any detectable effects on tumour growth. Histamine caused an early and pronounced decline in tumour blood flow compared to normal brain. The results indicate that the novel combination of IL-2 and histamine can be of value in reducing intracerebral tumour growth and, thus, it might be of interest to re-evaluate the therapeutic potential of biotherapy in malignant glioma.     

Hypoxia, angiogenesis, and colorectal cancer

The induction of angiogenesis is a nearly universal feature of solid malignancies, and therapeutic strategies that specifically inhibit this process have now entered the clinical arena. Colorectal cancers are highly angiogenic and were the first tumor type to exhibit significant responses to anti-angiogenic therapy. Multiple genetic and environmental stimuli induce the angiogenic phenotype. The characterization of these molecular pathways as well as their complex interactions is rapidly evolving, and a more comprehensive understanding is essential to optimize therapeutic approaches.     

Hypoxia correlates with angiogenesis in cervical cancers

Background Tissue hypoxia stimulates the induction of the angiogenic substances vascular endothelial growth factor and erythropoietin in the locus of the tissue. We have previously demonstrated that erythropoietin promotes angiogenesis by binding to its receptor in the endothelial cells of uterine and ovarian malignancies. In the present study, we examined whether malignant uterine cervix tissue showed hypoxia and whether hypoxia correlated with high vascular density through vascular endothelial growth factor.Methods To detect tissue hypoxia, we estimated the content of ATP in squamous cell carcinoma of the uterine cervix and in the normal cervix, using liquid chromatography columns. Surgically resected samples were fixed in Zamboni solution and processed for immunohistochemical microscopy to identify the endothelial cells and the location of vascular endothelial growth factor, with the use of anti-factor VIII and anti-vascular endothelial growth factor₁₆₅ antibody, respectively. The microvessels in a definite area were counted in sections of each specimen.Results Significantly lower ATP levels and significantly higher vascular density were seen in squamous cell carcinoma than in the controls (P < 0.05). The microvessel number in relation to ATP content was significantly higher in squamous cell carcinoma than in the controls (P < 0.001). Moreover vascular endothelial growth factor, the hyperplastic epithelium of the squamous cell carcinoma contained the immunoreactivity, with characteristic histopathological features suggesting retention of tissue fluid.Conclusion Squamous cell carcinoma of the uterine cervix showed hypoxia which correlated with abundant vascularity. Vascular endothelial growth factor expressed in the hyperplastic epithelium appears to promote angiogenesis in squamous cell carcinoma of the uterine cervix.     

Hypoxia, angiogenesis, and lung cancer

Lung cancer is responsible for more deaths than any other cancer in America. As a result, novel ways to treat it are needed to improve patient outcomes. A tumor must form new blood vessels to grow and metastasize to distant sites; this angiogenesis is mediated by factors such as vascular endothelial growth factor (VEGF). Because it increases VEGF levels, hypoxia has been thought to be a primary trigger of angiogenesis. Tumor hypoxia and higher levels of serum markers of angiogenesis have been associated with poor prognosis in non-small cell lung cancer (NSCLC). In recent years, antiangiogenic compounds have been developed and tested in various solid malignancies, including NSCLC, for which bevacizumab, a monoclonal antibody against VEGF, was recently approved. Combinations of antiangiogenic drugs and conventional cytotoxic chemotherapy are currently under development.     

Survivin promotes glioma angiogenesis through vascular endothelial growth factor and basic fibroblast growth factor in vitro and in vivo

Survivin is involved in multiple signaling mechanisms in tumor maintenance, and accumulated studies elucidate that knockdown of survivin in endothelial cells could inhibit angiogenesis; however, the role of survivin in tumor cells to regulate tumor-derived angiogenesis remains largely unclear. In the present study 80 cases of brain glioma were chosen and protein expressions of survivin, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) in glioma cells were investigated by immunohistochemistry (IHC). Human umbilical vein endothelial cells (HUVEC) were cocultured with human glioma U251 wild-type cells, U251 cells survivin silenced, SHG44 wild-type cells, and SHG44 survivin-overexpressing cells, respectively. The proliferation and migration of HUVEC were evaluated by MTT assay and transwell chamber assay. The microvessels density (MVD) marked by CD31 expression in vascular endothelial cells in glioma xenografts in nude mice was detected by IHC. VEGF, bFGF, and PDGF in the aforementioned cells were detected by quantitive PCR (qPCR), Western blot, ELISA, and IHC in vitro and in vivo. The results showed that VEGF immunoreactivity score (IRS), bFGF IRS, and PDGF IRS were all positively correlated with survivin IRS in gliomas, respectively (P < 0.01). Survivin in human glioma cells could significantly promote the proliferation and migration of HUVEC and increase MVD, which could be contributed to survivin-dependent burst of VEGF and bFGF expression, followed by increase of tumor growth and proliferation. In summary, survivin, through upregulation of VEGF and bFGF, plays an essential role during glioma angiogenesis.     

Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor

Malignant gliomas are highly lethal cancers dependent on angiogenesis. Critical tumor subpopulations within gliomas share characteristics with neural stem cells. We examined the potential of stem cell-like glioma cells (SCLGC) to support tumor angiogenesis. SCLGC isolated from human glioblastoma biopsy specimens and xenografts potently generated tumors when implanted into the brains of immunocompromised mice, whereas non-SCLGC tumor cells isolated from only a few tumors formed secondary tumors when xenotransplanted. Tumors derived from SCLGC were morphologically distinguishable from non-SCLGC tumor populations by widespread tumor angiogenesis, necrosis, and hemorrhage. To determine a potential molecular mechanism for SCLGC in angiogenesis, we measured the expression of a panel of angiogenic factors secreted by SCLGC. In comparison with matched non-SCLGC populations, SCLGC consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, SCLGC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-SCLGC tumor cell-conditioned medium. The proangiogenic effects of glioma SCLGC on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from SCLGC but limited efficacy against xenografts derived from a matched non-SCLGC population. Together these data indicate that stem cell-like tumor cells can be a crucial source of key angiogenic factors in cancers and that targeting proangiogenic factors from stem cell-like tumor populations may be critical for patient therapy.     

Vasorin stimulates malignant progression and angiogenesis in glioma

Glioma, the most common human primary brain tumor, is characterized by invasive capabilities and angiogenesis. Vasorin (VASN), a transmembrane protein, is reported to be associated with vascular injury repair and is overexpressed in some human tumors. However, its role in tumor progression and angiogenesis in glioma is unknown. In this study, VASN was shown to be overexpressed in high‐grade gliomas, and the expression level correlated with tumor grade and microvessel density in glioma specimens. Glioma patients with high VASN expression had a shorter overall survival time. Knockdown of VASN in glioma cells by shRNA significantly inhibited the malignancy of glioma, including cell proliferation, colony formation, invasion, and sphere formation. Ectopic expression of VASN increased glioma progression in vitro. The expression of VASN correlated with the mesenchymal type of glioblastoma multiforme (GBM) subtyped by gene set enrichment analysis (GSEA). Our results showed that the concentration of VASN was increased in the conditioned medium (CM) from glioma cells with VASN overexpression, and the CM from glioma cells with knockdown or overexpressed VASN inhibited or promoted HUVEC migration and tubulogenesis in vitro, respectively. Glioma growth and angiogenesis were stimulated upon ectopic expression of VASN in vivo. The STAT3 and NOTCH pathways were found to be activated and inhibited by VASN overexpression. Our findings suggest that VASN stimulates tumor progression and angiogenesis in glioma, and, as such, represents a novel therapeutic target for glioma.     

Combination treatment with temozolomide and thalidomide inhibits tumor growth and angiogenesis in an orthotopic glioma model

The chemotherapeutic agent temozolomide (TMZ) and the anti-angiogenic agent thalidomide (THD) have both demonstrated anti-tumor activity in patients with recurrent malignant glioma. Combination treatment with TMZ and THD in patients with glioblastoma multiforme (GBM) appears to be more effective than treatment with either drug alone. To investigate the mechanism of this anti-tumor effect, we examined the combined effects of TMZ and THD in a rat glioma xenograft model. We found that combination treatment markedly inhibited the growth of tumors that were orthotopically implanted into rat brains. Using proliferating cell nuclear antigen (PCNA) staining, we observed a significant decrease in cell proliferation in these tumors. CD31 staining of the microvasculature revealed a significant decrease in angiogenesis. We also found increased apoptosis in treated tumors by terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. We further demonstrated that the expression of angiogenic factors, such as vascular endothelial cell growth factor (VEGF) and basic fibroblastic growth factor (bFGF), were inhibited by THD. THD also decreased the number of ED1-positive, activated macrophages or microglial cells, which produce pro-angiogenic molecules around the glioma. Taken together, these results suggest that combination treatment with TMZ and THD inhibits tumor growth via the induction of apoptosis and the inhibition of angiogenesis in a rat model and may be a promising therapy for malignant gliomas.     

Short hairpin RNA targeting survivin inhibits growth and angiogenesis of glioma U251 cells

Survivin is a novel tumor-associated gene, its overexpression mostly associates with carcinogenesis and development. Nevertheless, the precise role of survivin in initiation and progression of gliomas is still not completely clear. We constructed here three short hairpin RNA (shRNA) targeting survivin plasmid vectors and introduced them into glioma U251 cells. The three shRNAs were efficiently and specifically able to knockdown the survivin expression in transiently transfected U251 cells. The stable transfectants expressing the shRNA having the strongest inhibitory effect against survivin exhibited decreased cell growth, increased spontaneous apoptosis, mitotic catastrophe and cell cycle arrest. Furthermore, in nude mice xenografts, the stable transfectants presented decreased de novo glioma formation and reduced development of angiogenesis. Results from this study indicate that survivin plays an important role in malignant proliferation, antiapoptosis and angiogenesis of gliomas, which may become an attractive target for gene therapy of gliomas, while RNA interference (RNAi) mediated by shRNA may become a new promising strategy for cancer gene therapy.     

The miR-24-Bim pathway promotes tumor growth and angiogenesis in pancreatic carcinoma

miRNAs are a group of small RNAs that have been reported to play a key role at each stage of tumorigenesis and are believed to have future practical value. We now demonstrate that Bim, which stimulates cell apoptosis, is obviously down-regulated in pancreatic cancer (PaC) tissues and cell lines. And Bim-related miR-24 is significantly up-regulated in PaC. The repressed expression of Bim is proved to be a result of miR-24, thus promoting cell growth of both cancer and vascular cells, and accelerating vascular ring formation. By using mouse tumor model, we clearly showed that miR-24 promotes tumor growth and angiogenesis by suppressing Bim expression in vivo. Therefore, a new pathway comprising miR-24 and Bim can be used in the exploration of drug-target therapy of PaC.     

Epo is involved in angiogenesis in human glioma

In this study, the extent of angiogenesis, evaluated as microvascular density, and the immunoreactivity of tumor cells to erythropoietin (Epo) and of endothelial cells to Epo receptor (EpoR) have been correlated in human glioma specimens, and the effect of anti-Epo antibody on glioma-induced angiogenesis in vivo in the chick embryo chorioallantoic membrane (CAM) has been investigated. Results show that: (1) Epo/EpoR expression correlates with angiogenesis, (2) in the CAM assay, tumor bioptic specimens induce a strong angiogenic response, comparable to that induced by VEGF, and (3) an anti-Epo antibody co-administered with tumor bioptic specimens significantly inhibits the angiogenic response. These findings suggest the presence of a loop in the Epo/EpoR system, i.e. Epo is secreted by glioma tumor cells and it affects glioma vascular endothelial cells via its receptor and promotes angiogenesis in a paracrine manner. Moreover, as demonstrated by in vivo experiments, Epo is responsible for the strong angiogenic response induced by human glioma bioptic specimens, because an anti-Epo antibody is able to significantly inhibit this response.     

Gene expression of angiogenesis related factors in glioma

Angiogenesis plays an important role in growth and proliferation of cancer. Various angiogenic and angiostatic factors regulate angiogenesis. In this study, we examined gene expression of the angiopoietin family including angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2) in 39 gliomas and 5 glioma-xenografts by RT-PCR. Ang1 and Ang2 genes were expressed in 54%, and 77% of gliomas, respectively. The expression of Ang1 was significantly correlated with the expression of Ang2. Both Ang1 and Ang2 were shown to be expressed in the glioma cells. Ang2 gene expression was correlated with VEGF gene expression. Angiopoietin molecules may synergistically cooperate in growth and vascularization in glioma.     

Up-regulation of vascular endothelial growth factor by membrane-type 1 matrix metalloproteinase stimulates human glioma xenograft growth and angiogenesis.

Membrane-type (MT) 1 matrix metalloproteinase (MMP) is up-regulated in many tumor types and has been implicated in tumor progression and metastasis. MT1-MMP is critical for pericellular degradation of the extracellular matrix, thereby promoting tumor cell invasion and dissemination. To grow efficiently in vivo, tumor cells induce angiogenesis in both primary solid tumors and metastatic foci. The present study describes a functional link between the expression of MT1-MMP and vascular endothelial growth factor (VEGF) production in human glioma U251 xenografts in athymic mice. To investigate the effects of MT1-MMP on VEGF expression, U251 cells were stably transfected with MT1-MMP to generate the U-MT cell line overexpressing the enzyme. In vitro, the U-MT cells had an increased rate of proliferation and migration as well as the ability to activate the MMP-2 proenzyme and directionally remodel a three-dimensional collagen matrix. These findings suggested higher tumorigenicity of U-MT cells relative to the vector-control U-neo cells. In agreement with the in vitro data, U-MT xenografts in BALB/c nu/nu mice displayed markedly increased growth rates and elevated levels of angiogenesis. In contrast, U-neo cells formed small, minimally vascularized tumors. The elevated angiogenesis in U-MT xenografts was associated with an up-regulation of VEGF expression in tumor cells. In addition, U-MT cells in vitro secreted twice as much VEGF as the control cells. GM6001, a hydroxamate inhibitor of MMP activity, down-regulated the production of VEGF in U-MT cells to the levels observed in the U-neo control. Our results demonstrate that the enhanced tumorigenicity of glioma cells overexpressing MT1-MMP involves stimulation of angiogenesis through the up-regulation of VEGF production.     

Gene variants in the angiogenesis pathway and prostate cancer

Although the causes of prostate cancer are still unknown, numerous studies support the role of genetic factors in the development and progression of this disease. Single nucleotide polymorphisms (SNPs) in key angiogenesis genes have been studied in prostate cancer. In this review, we provide an overview of the current knowledge of the role of genetic variants in the angiogenesis pathway in prostate cancer risk and progression. Of the 17 prostate cancer genome-wide association studies (GWAS) conducted to date, only one identified disease-associated SNPs in a region of an angiogenesis pathway gene. An association was observed between aggressive disease and three intergenic SNPs (rs11199874, rs10749408 and rs10788165) in a region on chromosome 10q26 that encompasses FGFR2. The majority (27/32, 84.4%) of primary candidate gene studies reviewed had a small (n < 800, 20/32, 62.5%) to medium sample size (n = 800-2000, 7/32, 21.9%), whereas only five (15.6%) had a large sample size (n ≥ 2000). Results from the large studies revealed associations with risk and aggressive disease for SNPs in NOS2A, NOS3 and MMP-2 and risk for HIF1-α. Meta-analyses have so far been conducted on FGFR2, TGF-β, TNF-α, HIF1-α and IL10 and the results reveal an association with risk for SNPs in FGFR2 and TGF-β and aggressive disease for SNPs in IL-10. Thus, existing evidence from GWAS and large candidate gene studies indicates that SNPs from a limited number of angiogenesis pathway genes are associated with prostate cancer risk and progression.