Vascular endothelial growth factor (VEGF), a survival factor for tumour cells: implications for anti-angiogenic therapy

Angiogenesis is central to both the growth and metastasis of solid tumours. Anti-angiogenic strategies result in blood vessel regression accompanied by tumour cell apoptosis. Radiotherapy and many chemotherapeutic agents kill tumours by inducing apoptotic cell death. We propose that, in addition to its role as an angiogenic factor, vascular endothelial growth factor (VEGF) can act as a survival factor for tumour cells protecting them from apoptosis. Thus anti-angiogenics, in particular those directed against VEGF, have multiple anti-tumour effects. We suggest that anti-VEGF strategies prevent vessel growth and block a tumour cell survival factor, VEGF, rendering tumour cells more sensitive to chemotherapy and radiotherapy. In addition, as chemotherapy and radiotherapy have been shown to increase VEGF expression, anti-VEGF strategies may overcome therapy- induced tumour cell resistance.     

Vascular endothelial growth factor-dependent and -independent regulation of angiogenesis

Angiogenesis, the formation of blood vessels, is essential for preparing a closed circulatory system in the body, and for supplying oxygen and nutrition to tissues. Major diseases such as cancer, rheumatoid arthritis, and atherosclerosis include pathological angiogenesis in their malignant processes, suggesting anti-angiogenic therapy to be a new strategy for suppression of diseases. However, until the 1970s, the molecular basis of angiogenesis was largely unknown. In recent decades, extensive studies have revealed a variety of angiogenic factors and their receptors, including vascular endothelial growth factor (VEGF)-VEGFRs, Angiopoietin-Tie, Ephrin-EphRs and Delta-Notch to be the major regulators of angiogenesis in vertebrates. VEGF and its receptors play a central role in physiological as well as pathological angiogenesis, and functional inhibitors of VEGF and VEGFRs such as anti-VEGF neutralizing antibody and small molecules that block the tyrosine kinase activity of VEGFRs have recently been approved for use to treat patients with colorectal, lung, renal and liver cancers. These drugs have opened a novel field of cancer therapy, i.e. anti-angiogenesis therapy. However, as yet they cannot completely cure patients, and cancer cells could become resistant to these drugs. Thus, it is important to understand further the molecular mechanisms underlying not only VEGF-VEGFR signaling but also the VEGF-independent regulation of angiogenesis, and to learn how to improve anti-angiogenesis therapy.     

Mechanism of thrombin-induced angiogenesis

Clinical, laboratory, histopathological and pharmacological evidence support the notion that a systemic activation of blood coagulation is often present in cancer patients. Additionally, thrombin was shown to promote tumour progression and metastasis in animals, and epidemiological studies suggest an increased risk of cancer diagnosis after primary thromboembolism. We have proposed that the aforementioned results may be related to our finding that thrombin is a potent activator of angiogenesis. This is a thrombin receptor-mediated event (the receptor is referred to as protease-activate receptor) and is independent of fibrin formation. Many cellular effects of thrombin on endothelial cells can contribute to the angiogenic action of thrombin. (i) Exposure of endothelial cells to thrombin cause a time- and dose-dependent decrease in the attachment of these cells to basement membrane components, with a concomitant increase in matrix metalloproteinase 2 activation. (ii) Thrombin upregulates the expression of integrin alphavbeta3, the marker of the angiogenic phenotype of endothelial cells. (iii) Thrombin has chemotactic and aptotactic effects on endothelial cells and upregulates the expression of the vascular endothelial growth factor (VEGF) receptors (KDR and Flt1). Thus, thrombin synergizes with the key angiogenic factor VEGF in endothelial cell proliferation. Furthermore, thrombin enhances the secretion of VEGF and matrix metalloproteinase 9 of PC3 prostate cancer cells. These results can explain the angiogenic and tumour-promoting effect of thrombin and provide the basis for development of thrombin receptor mimetics or antagonists for therapeutic application.     

Glioma angiogenesis: Towards novel RNA therapeutics

Brain tumors exhibit marked and aberrant blood vessel formation indicating angiogenic endothelial cells as a potential target for brain tumor treatment. The brain tumor blood vessels are used for nutrient delivery, and possibly for cancer cell migration. The process of angiogenesis is complex and involves multiple players. The current angiogenesis inhibitors used in clinical trials mostly target single angiogenic proteins and so far show limited effects on tumor growth. Besides the conventional angiogenesis inhibitors, RNA-based inhibitors such as small-interfering RNAs (siRNAs) are being analyzed for their capacity to silence the message of proteins involved in neovascularization. More recently, a new family of non-coding RNAs, named angiomirs [microRNAs (miRNAs) involved in angiogenesis] has emerged. These small RNAs have the advantage over siRNAs in that they have the potential of silencing multiple messages at the same time and therefore they might become therapeutically relevant in a “one-hit multiple-target” context against brain tumor angiogenesis. In this review we will discuss the emerging technologies in anti-angiogenesis emphasizing on RNA-based therapeutics.Key words: glioma, angiogenesis, anti-angiogenesis therapy, siRNA, miRNA, endothelial cells, blood vessels     

VEGF function in vascular pathogenesis

Vascular endothelial growth factor (VEGF) has proven to be the most critical angiogenic factor identified to date; its discovery has transformed the field of angiogenesis research. After two decades of intense studies by numerous laboratories, the complex biology of VEGF is slowly being elucidated. VEGF is a collection of several different isoforms with overlapping but also unique functions. Furthermore, there are at least four different VEGF receptors, adding to the complexity of the VEGF/receptor system. VEGF exerts its effects on endothelial cells, inflammatory cells and neuronal cells, and its expression may contribute to the pathologies of diseases ranging from retinopathies to cancer. This review focuses on the biochemistry and cell biology of VEGF, with particular emphasis on the different isoforms and receptors, thereby illustrating the multifunctional nature of this growth factor. Lastly, critical considerations based on the complex biology of VEGF for developing specific and more targeted anti-VEGF therapeutic agents will be discussed.     

Mesenchymal stem cells overexpressing PEDF decrease the angiogenesis of gliomas

The present study is an exploration of a novel strategy to target a therapeutic gene to brain tumour tissues. In the present study, we evaluated the feasibility of using hMSCs (human mesenchymal stem cells) to deliver PEDF (pigment epithelium-derived factor), a potent inhibitor of tumour angiogenesis, in a model of intracranial gliomas. To assess its potential of tracking gliomas, MSCs (mesenchymal stem cells) were injected into the cerebral hemisphere and it showed that MSCs infiltrated into the vessel beds and scattered throughout the tumour. In vitro migration assay showed that the VEGF (vascular endothelial growth factor) enhanced MSC migration. In contrast, the migratory activity of MSCs was significantly inhibited with the presence of PEDF. Systematic delivery of AAV (adeno-associated virus)–PEDF to established glioma xenografts resulted in increased apoptosis of gliomas. In addition, MSC–PEDF treatment prolonged the survival of mice bearing U87 gliomas. Taken together, these data validate that MSCs–PEDF can migrate and deliver PEDF to target glioma cells, which may be a novel and promising therapeutic approach for refractory brain tumour.     

Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis.

Tumor necrosis factor alpha (TNF-alpha) is a macrophage/monocyte-derived polypeptide which modulates the expression of various genes in vascular endothelial cells and induces angiogenesis. However, the underlying mechanism by which TNF-alpha mediates angiogenesis is not completely understood. In this study, we assessed whether TNF-alpha-induced angiogenesis is mediated through TNF-alpha itself or indirectly through other TNF-alpha-induced angiogenesis-promoting factors. Cellular mRNA levels of interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors were increased after the treatment of human microvascular endothelial cells with TNF-alpha (100 U/ml). TNF-alpha-dependent tubular morphogenesis in vascular endothelial cells was inhibited by the administration of anti-IL-8, anti-VEGF, and anti-bFGF antibodies, and coadministration of all three antibodies almost completely abrogated tubular formation. Moreover, treatment with Sp1, NF-kappaB, and c-Jun antisense oligonucleotides inhibited TNF-alpha-dependent tubular morphogenesis by microvascular endothelial cells. Administration of a NF-kappaB antisense oligonucleotide almost completely inhibited TNF-alpha-dependent IL-8 production and partially abrogated TNF-alpha-dependent VEGF production, and an Sp1 antisense sequence partially inhibited TNF-alpha-dependent production of VEGF. A c-Jun antisense oligonucleotide significantly inhibited TNF-alpha-dependent bFGF production but did not affect the production of IL-8 and VEGF. Administration of an anti-IL-8 or anti-VEGF antibody also blocked TNF-alpha-induced neovascularization in the rabbit cornea in vivo. Thus, angiogenesis by TNF-alpha appears to be modulated through various angiogenic factors, both in vitro and in vivo, and this pathway is controlled through paracrine and/or autocrine mechanisms.     

Arginine-rich anti-vascular endothelial growth factor peptides inhibit tumor growth and metastasis by blocking angiogenesis

Tumor angiogenesis is a critical step for the growth and metastasis of solid tumors. Vascular endothelial growth factor (VEGF) is a specific and potent angiogenic factor and contributes to the development of solid tumors by promoting tumor angiogenesis. Therefore, it is a prime therapeutic target for the development of antagonists for treatment of cancer. We identified from peptide libraries arginine-rich hexapeptides that inhibit the interaction of VEGF(165) with VEGF receptor (IC(50) = 2-4 micrometer). They have no effect on binding of basic fibroblast growth factor to cellular receptor. The hexapeptides inhibit the proliferation of human umbilical vein endothelial cells induced by VEGF(165) without toxicity. The peptides bind to VEGF and inhibit binding of both VEGF(165) and VEGF(121), suggesting that the peptides interact with the main body of VEGF but not the heparin-binding domain that is absent in VEGF(121). The identified peptides block the angiogenesis induced by VEGF(165) in vivo in the chick chorioallantoic membrane and the rabbit cornea. Furthermore, one of the hexapeptides, RRKRRR, blocks the growth and metastasis of VEGF-secreting HM7 human colon carcinoma cells in nude mice. Based on our results, the arginine-rich hexapeptides may be effective for the treatment of various human tumors and other angiogenesis-dependent diseases that are related to the action of VEGF and could also serve as leads for development of more effective drugs.     

Fungal polysaccharopeptide inhibits tumor angiogenesis and tumor growth in mice

Angiogenesis is crucial to tumor growth and metastasis, and interruption of this process is a prime avenue for therapeutic intervention of tumor proliferation. The present study has made use of the S180 tumor-bearing mouse model to investigate the polysaccharopeptide, PSP, isolated from the edible mushroom Coriolus versicolor, a herbal medicine known for its anti-angiogenesis properties. Quantitative analysis of microcorrosion casting of the tumor tissue showed more angiogenic features such as dense sinusoids and hot spots, in control (untreated) than in PSP-treated animals. Immunostaining of tumor tissues with antibody against the endothelial cell marker (Factor VIII) demonstrated a positive correlation in that both the vascular density and tumor weight were lower in mice treated with PSP. Morphometric analysis of corrosion casts revealed that, even though the total amount of new vessel production was reduced, the basic tumor type-specific vascular architecture was retained. However, the expression of vascular endothelial cell growth factor (VEGF) in these tumors was suppressed. In conclusion, anti-angiogenesis should be one of the pathways through which PSP mediated its anti-tumor activity.     

Therapeutic myocardial angiogenesis with vascular endothelial growth factors

Emerging evidence has shown that administration of angiogenic growth factors, either as recombinant protein or by gene transfer, can augment tissue perfusion through neovascularization in animal models of myocardial and hindlimb ischemia. Many cytokines have angiogenic activity; one of those that have been best studied in animal models and clinical trials is vascular endothelial growth factor (VEGF). VEGF has been known to be a key regulator of physiologic and pathologic angiogenesis associated with tumor. Recently the effect of VEGF is not restricted to the direct angiogenic effect in vivo but includes mobilization of bone-marrow-derived endothelial progenitor cells and augmentation of postnatal vasculogenesis in situ. Clinical trials of therapeutic angiogenesis with VEGF in patients with end-stage coronary artery disease have shown increases in exercise time and reductions in anginal symptoms and have provided objective evidence of improved perfusion and left ventricular function. Larger scale placebo-controlled trials with recombinant protein (rhVEGF165) have been limited to intracoronary and intravenous administration and have shown favorable trends in exercise time and angina frequency. Small-scale, placebo-controlled, randomized clinical trials of gene transfer (phVEGF-2) via thoracotomy or percutaneous intramyocardial delivery demonstrated significant improvement of both subjective symptoms and objective measures of myocardial ischemia. Both therapeutic modalities appear to be safe and well tolerated. Further studies are required to determine the optimal dose, formulation, route of administration, and combinations of growth factors and the utility of adjunctive endothelial progenitor cell or other stem cell supplementation, to provide safe and effective therapeutic myocardial neovascularization.     

Activation of chemokine receptor CXCR4 in malignant glioma cells promotes the production of vascular endothelial growth factor

Numerous studies have showed that chemokine receptors, such as CXCR4, contribute to the growth and metastasis of a variety of malignant tumors. In this study, we investigated the role of CXCR4 in the production of angiogenic factor, vascular endothelial growth factor (VEGF), in various human glioma cells from astrocytic origin. The expression of CXCR4 mRNA and protein in three glioma cell lines, U87-MG, SHG-44, and CHG-5, was determined by RT-PCR and immunocytochemistry, respectively. The malignancies of three gliomas were evaluated by expression of glial fibrillary acidic protein and vimentin, the differentiation markers of astrocytic cells. The role of functional CXCR4 in tumor cell migration was studied with chemotaxis assay. Ca2+ mobilization and VEGF production were measured in the cells after stimulation with CXCR4 ligand, SDF1beta. The results showed that the levels of functional CXCR4 expression at both mRNA and protein levels by several human glioma cell lines were correlated with the degree of differentiation of the tumor cells. Activation of CXCR4 induced glioma cell chemotaxis and could trigger the increase of intracellular [Ca2+]i. Such an activation could result in the increased production of VEGF by the stimulated tumor cells. Our results suggest that CXCR4 may contribute to the high level of VEGF produced by malignant glioma cells and thus constitute a therapeutic target for antiangiogenesis strategy.     

The neurotransmitter dopamine inhibits angiogenesis induced by vascular permeability factor/vascular endothelial growth factor

Angiogenesis has an essential role in many important pathological and physiological settings. It has been shown that vascular permeability factor/vascular endothelial growth factor (VPF/VEGF), a potent cytokine expressed by most malignant tumors, has critical roles in vasculogenesis and both physiological and pathological angiogenesis. We report here that at non-toxic levels, the neurotransmitter dopamine strongly and selectively inhibited the vascular permeabilizing and angiogenic activities of VPF/VEGF. Dopamine acted through D2 dopamine receptors to induce endocytosis of VEGF receptor 2, which is critical for promoting angiogenesis, thereby preventing VPF/VEGF binding, receptor phosphorylation and subsequent signaling steps. The action of dopamine was specific for VPF/VEGF and did not affect other mediators of microvascular permeability or endothelial-cell proliferation or migration. These results reveal a new link between the nervous system and angiogenesis and indicate that dopamine and other D2 receptors, already in clinical use for other purposes, might have value in anti-angiogenesis therapy.     

VEGF-null cells require PDGFR alpha signaling-mediated stromal fibroblast recruitment for tumorigenesis

We generated VEGF-null fibrosarcomas from VEGF-loxP mouse embryonic fibroblasts to investigate the mechanisms of tumor escape after VEGF inactivation. These cells were found to be tumorigenic and angiogenic in vivo in spite of the absence of tumor-derived VEGF. However, VEGF derived from host stroma was readily detected in the tumor mass and treatment with a newly developed anti-VEGF monoclonal antibody substantially inhibited tumor growth. The functional significance of stroma-derived VEGF indicates that the recruitment of stromal cells is critical for the angiogenic and tumorigenic properties of these cells. Here we identified PDGF AA as the major stromal fibroblast chemotactic factor produced by tumor cells, and demonstrated that disrupting the paracrine PDGFR alpha signaling between tumor cells and stromal fibroblasts by soluble PDGFR alpha-IgG significantly reduced tumor growth. Thus, PDGFR alpha signaling is required for the recruitment of VEGF-producing stromal fibroblasts for tumor angiogenesis and growth. Our findings highlight a novel aspect of PDGFR alpha signaling in tumorigenesis.     

Histidine-Rich Glycoprotein Can Prevent Development of Mouse Experimental Glioblastoma

Extensive angiogenesis, formation of new capillaries from pre-existing blood vessels, is an important feature of malignant glioma. Several antiangiogenic drugs targeting vascular endothelial growth factor (VEGF) or its receptors are currently in clinical trials as therapy for high-grade glioma and bevacizumab was recently approved by the FDA for treatment of recurrent glioblastoma. However, the modest efficacy of these drugs and emerging problems with anti-VEGF treatment resistance welcome the development of alternative antiangiogenic therapies. One potential candidate is histidine-rich glycoprotein (HRG), a plasma protein with antiangiogenic properties that can inhibit endothelial cell adhesion and migration. We have used the RCAS/TV-A mouse model for gliomas to investigate the effect of HRG on brain tumor development. Tumors were induced with platelet-derived growth factor-B (PDGF-B), in the presence or absence of HRG. We found that HRG had little effect on tumor incidence but could significantly inhibit the development of malignant glioma and completely prevent the occurrence of grade IV tumors (glioblastoma).     

Therapeutic myocardial angiogenesis with vascular endothelial growth factors

Emerging evidence has shown that administration of angiogenic growth factors, either as recombinant protein or by gene transfer, can augment tissue perfusion through neovascularization in animal models of myocardial and hindlimb ischemia. Many cytokines have angiogenic activity; one of those that have been best studied in animal models and clinical trials is vascular endothelial growth factor (VEGF). VEGF has been known to be a key regulator of physiologic and pathologic angiogenesis associated with tumor. Recently the effect of VEGF is not restricted to the direct angiogenic effect in vivo but includes mobilization of bone-marrow-derived endothelial progenitor cells and augmentation of postnatal vasculogenesis in situ. Clinical trials of therapeutic angiogenesis with VEGF in patients with end-stage coronary artery disease have shown increases in exercise time and reductions in anginal symptoms and have provided objective evidence of improved perfusion and left ventricular function. Larger scale placebo-controlled trials with recombinant protein (rhVEGF₁₆₅) have been limited to intracoronary and intravenous administration and have shown favorable trends in exercise time and angina frequency. Small-scale, placebo-controlled, randomized clinical trials of gene transfer (phVEGF-2) via thoracotomy or percutaneous intramyocardial delivery demonstrated significant improvement of both subjective symptoms and objective measures of myocardial ischemia. Both therapeutic modalities appear to be safe and well tolerated. Further studies are required to determine the optimal dose, formulation, route of administration, and combinations of growth factors and the utility of adjunctive endothelial progenitor cell or other stem cell supplementation, to provide safe and effective therapeutic myocardial neovascularization. (Mol Cell Biochem 264: 63–74, 2004)     

Perlecan regulates developmental angiogenesis by modulating the VEGF-VEGFR2 axis

Using the zebrafish, we previously identified a central function for perlecan during angiogenic blood vessel development. Here, we explored the nature of perlecan function during developmental angiogenesis. A close examination of individual endothelial cell behavior revealed that perlecan is required for proper endothelial cell migration and proliferation. Because these events are largely mediated by VEGF-VEGFR2 signaling, we investigated the relationship between perlecan and the VEGF pathway. We discovered that perlecan knockdown caused an abnormal increase and redistribution of total VEGF-A protein suggesting that perlecan is required for the appropriate localization of VEGF-A. Importantly, we linked perlecan function to the VEGF pathway by efficiently rescuing the perlecan morphant phenotype by microinjecting VEGF-A₁₆₅ protein or mRNA. Combining the strategic localization of perlecan throughout the vascular basement membrane along with its growth factor-binding ability, we hypothesized a major role for perlecan during the establishment of the VEGF gradient which provides the instructive cues to endothelial cells during angiogenesis. In support of this hypothesis we demonstrated that human perlecan bound in a heparan sulfate-dependent fashion to VEGF-A₁₆₅. Moreover, perlecan enhanced VEGF mediated VEGFR2 activation of human endothelial cells. Collectively, our results indicate that perlecan coordinates developmental angiogenesis through modulation of VEGF-VEGFR2 signaling events. The identification of angiogenic factors, such as perlecan, and their role in vertebrate development will not only enhance overall understanding of the molecular basis of angiogenesis, but may also provide new insight into angiogenesis-based therapeutic approaches.     

FtMt promotes glioma tumorigenesis and angiogenesis via lncRNA SNHG1/miR-9-5p axis

ObjectiveThis study is to investigate the effects and the mechanisms of mitochondrial ferritin (FtMt) on the glioma tumorigenesis and angiogenesis.MethodsFtMt expression was detected in glioma tissues and cells as well as in nude mouse tissues. Cell proliferation and apoptosis rate were observed following transfection of LV-FtMt or sh-FtMt in glioma cell line. Moreover, glioma cells with FtMt over-expression/knockdown were co-cultured with human umbilical vein endothelial cells (HUVECs) to observe its function on HUVEC proliferation, angiogenic ability and the vascular endothelial growth factor (VEGF) content. Gain and loss of function of small nucleolar RNA host gene 1 (SNHG1) and miR-9-5p were performed in glioma cells and GBM nude mice to observe its effect on glioma cell proliferation and HUVEC angiogenic ability. Luciferase reporter gene and RIP assay were employed to inspect the interactions among SNHG1, FtMt and miR-9-5p. Additionally, a xenograft mouse model was applied to determine the role of FtMt in glioma.ResultsIn this work, FtMt was strongly expressed in glioma tissues and cells as well as in nude mouse tumor tissues. The employment of the loss-of and gain-of functions assays illustrated that FtMt enhanced glioma tumorigenesis and angiogenesis. Mechanistically, our findings showed that FtMt positively related to SNHG1 while negatively correlated with miR-9-5p, and both SNHG1 and FtMt can competitively bind with miR-9-5p. Besides, the inhibition effects of sh-FtMt on glioma were surveyed in vivo experiments.ConclusionEvidence in this study suggested that FtMt promotes glioma tumorigenesis and angiogenesis via SNHG1 mediated miR-9-5p expression, which may provide a theoretical basis for glioma treatment.     

Glioma angiogenesis

Brain tumors exhibit marked and aberrant blood vessel formation indicating angiogenic endothelial cells as a potential target for brain tumor treatment. The brain tumor blood vessels are used for nutrient delivery, and possibly for cancer cell migration. The process of angiogenesis is complex and involves multiple players. The current angiogenesis inhibitors used in clinical trials mostly target single angiogenic proteins and so far show limited effects on tumor growth. Besides the conventional angiogenesis inhibitors, RNA-based inhibitors such as small-interfering RNAs (siRNAs) are being analyzed for their capacity to silence the message of proteins involved in neovascularization. More recently, a new family of non-coding RNAs, named angiomirs [microRNAs (miRNAs) involved in angiogenesis] has emerged. These small RNAs have the advantage over siRNAs in that they have the potential of silencing multiple messages at the same time and therefore they might become therapeutically relevant in a “one-hit multiple-target” context against brain tumor angiogenesis. In this review we will discuss the emerging technologies in anti-angiogenesis emphasizing on RNA-based therapeutics.