Constitutive and inducible expression and regulation of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF), which was originally discovered as vascular permeability factor, is critical to human cancer angiogenesis through its potent functions as a stimulator of endothelial cell survival, mitogenesis, migration, differentiation and self-assembly, as well as vascular permeability, immunosuppression and mobilization of endothelial progenitor cells from the bone marrow into the peripheral circulation. Genetic alterations and a chaotic tumor microenvironment, such as hypoxia, acidosis, free radicals, and cytokines, are clearly attributed to numerous abnormalities in the expression and signaling of VEGF and its receptors. These perturbations confer a tremendous survival and growth advantage to vascular endothelial cells as manifested by exuberant tumor angiogenesis and a consequent malignant phenotype. Understanding the regulatory mechanisms of both inducible and constitutive VEGF expression will be crucial in designing effective therapeutic strategies targeting VEGF to control tumor growth and metastasis. In this review, molecular regulation of VEGF expression in tumor cells is discussed.     

Mathematical Modeling of Cellular Cross-Talk Between Endothelial and Tumor Cells Highlights Counterintuitive Effects of VEGF-Targeted Therapies

Tumor growth and progression are critically dependent on the establishment of a vascular support system. This is often accomplished via the expression of pro-angiogenic growth factors, including members of the vascular endothelial growth factor (VEGF) family of ligands. VEGF ligands are overexpressed in a wide variety of solid tumors and therefore have inspired optimism that inhibition of the different axes of the VEGF pathway—alone or in combination—would represent powerful anti-angiogenic therapies for most cancer types. When considering treatments that target VEGF and its receptors, it is difficult to tease out the differential anti-angiogenic and anti-tumor effects of all combinations experimentally because tumor cells and vascular endothelial cells are engaged in a dynamic cross-talk that impacts key aspects of tumorigenesis, independent of angiogenesis. Here we develop a mathematical model that connects intracellular signaling responsible for both endothelial and tumor cell proliferation and death to population-level cancer growth and angiogenesis. We use this model to investigate the effect of bidirectional communication between endothelial cells and tumor cells on treatments targeting VEGF and its receptors both in vitro and in vivo. Our results underscore the fact that in vitro therapeutic outcomes do not always translate to the in vivo situation. For example, our model predicts that certain therapeutic combinations result in antagonism in vivo that is not observed in vitro. Mathematical modeling in this direction can shed light on the mechanisms behind experimental observations that manipulating VEGF and its receptors is successful in some cases but disappointing in others.     

Bioactivity of anti-angiogenic ribozymes targeting Flt-1 and KDR mRNA.

Vascular endothelial growth factor (VEGF) and its receptors Flt-1 and KDR play important roles in physiological and pathological angiogenesis. Ribozymes that target the VEGF receptor mRNAs were developed and their biological activities in cell culture and an animal model were assessed. Ribozymes targeting Flt-1 or KDR mRNA sites reduced VEGF-induced proliferation of cultured human vascular endothelial cells and specifically lowered the level of Flt-1 or KDR mRNA present in the cells. Anti- Flt-1 and KDR ribozymes also exhibited anti-angiogenic activity in a rat corneal pocket assay of VEGF-induced angiogenesis. This report illustrates the anti-angiogenic potential of these ribozymes as well as their value in studying VEGF receptor function in normal and pathophysiologic states.     

Asiatic acid inhibits pro-angiogenic effects of VEGF and human gliomas in endothelial cell culture models

Malignant gliomas are one of the most devastating and incurable tumors. Sustained excessive angiogenesis by glioma cells is the major reason for their uncontrolled growth and resistance toward conventional therapies resulting in high mortality. Therefore, targeting angiogenesis should be a logical strategy to prevent or control glioma cell growth. Earlier studies have shown that Asiatic Acid (AsA), a pentacyclic triterpenoid, is effective against glioma and other cancer cells; however, its efficacy against angiogenesis remains unknown. In the present study, we examined the anti-angiogenic efficacy of AsA using human umbilical vein endothelial cells (HUVEC) and human brain microvascular endothelial cells (HBMEC). Our results showed that AsA (5-20 μM) inhibits HUVEC growth and induces apoptotic cell death by activating caspases (3 and 9) and modulating the expression of apoptosis regulators Bad, survivin and pAkt-ser473. Further, AsA showed a dose-dependent inhibition of HUVEC migration, invasion and capillary tube formation, and disintegrated preformed capillary network. AsA also inhibited the VEGF-stimulated growth and capillary tube formation by HUVEC and HBMEC. Next, we analyzed the angiogenic potential of conditioned media collected from human glioma LN18 and U87-MG cells treated with either DMSO (control conditioned media, CCM) or AsA 20 μM (AsA20 conditioned media, AsA20CM). CCM from glioma cells significantly enhanced the capillary tube formation in both HUVEC and HBMEC, while capillary tube formation in both endothelial cell lines was greatly compromised in the presence of AsA20CM. Consistent with these results, VEGF expression was lesser in AsA20CM compared to CCM, and indeed AsA strongly inhibited VEGF level (both cellular and secreted) in glioma cells. AsA also showed dose-dependent anti-angiogenic efficacy in Matrigel plug assay, and inhibited the glioma cells potential to attract HUVEC/HBMEC. Overall, the present study clearly showed the strong anti-angiogenic potential of AsA and suggests its usefulness against malignant gliomas.     

Toxicity and anti-angiogenicity evaluation of Pak1 inhibitor IPA-3 using zebrafish embryo model

p21-activated kinase 1 (Pak1)—a key node protein kinase regulating various cellular process including angiogenesis—has been recognised to be a therapeutic target for multitude of diseases, and hence, various small molecule inhibitors targeting its activity have been tested. However, the direct toxic and anti-angiogenic effects of these pharmacologic agents have not been examined. In this study, we evaluate the translational efficacy of Pak1 inhibitor IPA-3 using zebrafish toxicity model system to stratify its anti-angiogenic potential and off-target effects to streamline the compound for further therapeutic usage. The morphometric analysis has shown explicit delay in hatching, tail bending, pericardial sac oedema and abnormal angiogenesis. We provide novel evidence that Pak1 inhibitor could act as anti-angiogenic agents by impeding the development of sub-intestinal vessel (SIV) and intersegmental vessels (ISVs) by suppressing the expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), neurophilin 1 (NRP1) and its downstream genes matrix metalloproteinase (MMP)-2 and MMP-9. Knockdown studies using 2-O-methylated oligoribonucleotides targeting Pak1 also revealed similar phenotypes with inhibition of angiogenesis accompanied with deregulation of major angiogenic factor and cardiac-specific genes. Taken together, our findings indicate that Pak1 signalling facilitates enhanced angiogenesis and also advocated the design and use of small molecule inhibitors of Pak1 as potent anti-angiogenic agents and suggest their utility in combinatorial therapeutic approaches targeting anomalous angiogenesis.     

VEGF 家族及其在肿瘤生长中作用的研究

血管内皮生长因子(Vascular Endothelial Growth Factor,VEGF)家族是一类多功能的细胞因子,在血管生成和淋巴管生成中具有直接和间接的调控作用,可促进内皮细胞增殖、促进血管生成以及增加血管的通透性。VEGF/VEGFR轴由多重配基和受体质量叠加交错组成,并且受体与配基结合具有专一性,在不同的细胞中具有不同的细胞类型表达和功能.启动VEGF信号通路,触发了一个网状的信号过程,从而促进血管内皮细胞生长、转移和存活。进来研究发现,VEGF的一个重要作用表现为可动员内皮祖细胞从骨髓向远处转移从而形成新生血管,因而有必要设计和发展针对这一途径的抑制因子。随着研究的深入,VEGF促进肿瘤血管生成的作用和与人类癌症的发病机制的关系是确定的,因此,抑制VEGF途径被确认为是一种重要的有效的抗癌模式     

Autocrine functions of VEGF in breast tumor cells

Vascular endothelial growth factor A (VEGF-A) is well known for its key roles in blood vessel growth. Although most studies on VEGF and VEGF receptors have been focused on their functions in angiogenesis and in endothelial cells, the role of VEGF in cancer biology appears as an emerging area of importance. In this context, the presence of VEGF receptors in tumor cells strongly suggests that VEGF-A also promotes a wide range of functions, both in vitro and in vivo, all autocrine functions on tumor cells, including adhesion, survival, migration and invasion. Ultimately, refining our knowledge of VEGF signaling pathways in tumor cells should help us to understand why the current used treatments targeting the VEGF pathway in cancer are not universally effective in inhibiting metastasis tumors, and it should also provide new avenues for future therapies.     

Autocrine functions of VEGF in breast tumor cells: Adhesion,survival, migration and invasion

Vascular endothelial growth factor A (VEGF-A) is well known for its key roles in blood vessel growth. Although most studies on VEGF and VEGF receptors have been focused on their functions in angiogenesis and in endothelial cells, the role of VEGF in cancer biology appears as an emerging area of importance. In this context, the presence of VEGF receptors in tumor cells strongly suggests that VEGF-A also promotes a wide range of functions, both in vitro and in vivo, all autocrine functions on tumor cells, including adhesion, survival, migration and invasion. Ultimately, refining our knowledge of VEGF signaling pathways in tumor cells should help us to understand why the current used treatments targeting the VEGF pathway in cancer are not universally effective in inhibiting metastasis tumors, and it should also provide new avenues for future therapies.     

The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40ng/ml in the culture medium, but decreased at 80ng/ml. Under CoCl(2)-induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or anti-angiogenic activities of BMSCs.     

Molecular pathways regulating mobilization of marrow-derived stem cells for tissue revascularization

Adult bone marrow is a rich reservoir of hematopoietic and vascular stem and progenitor cells. Mobilization and recruitment of these cells are essential for tissue revascularization. Physiological stress, secondary to tissue injury or tumor growth, results in the release of angiogenic factors, including vascular endothelial growth factor (VEGF), which promotes mobilization of stem cells to the circulation, contributing to the formation of functional vasculature. VEGF interacts with its receptors, VEGFR2 and VEGFR1, expressed on endothelial and hematopoietic stem cells, and thereby promotes recruitment of these cells to neo-angiogenic sites, accelerating the revascularization process. The mobilization of stem cells from marrow is a dynamic process, regulated by shear stress imparted by blood flow, and the activation of metalloproteinases that induce the release of 'Kit ligand', facilitating egress from the marrow to the circulation. Identification of the molecular pathways that support the proliferation and differentiation of vascular stem and progenitor cells will open up new avenues for the design of clinical trials to accelerate tissue vascularization and organogenesis.     

Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells

Endothelial nitric oxide synthase (eNOS) is essential for neovascularization. Here we show that the impaired neovascularization in mice lacking eNOS is related to a defect in progenitor cell mobilization. Mice deficient in eNOS (Nos3(-/-)) show reduced vascular endothelial growth factor (VEGF)-induced mobilization of endothelial progenitor cells (EPCs) and increased mortality after myelosuppression. Intravenous infusion of wild-type progenitor cells, but not bone marrow transplantation, rescued the defective neovascularization of Nos3(-/-) mice in a model of hind-limb ischemia, suggesting that progenitor mobilization from the bone marrow is impaired in Nos3(-/-) mice. Mechanistically, matrix metalloproteinase-9 (MMP-9), which is required for stem cell mobilization, was reduced in the bone marrow of Nos3(-/-) mice. These findings indicate that eNOS expressed by bone marrow stromal cells influences recruitment of stem and progenitor cells. This may contribute to impaired regeneration processes in ischemic heart disease patients, who are characterized by a reduced systemic NO bioactivity.     

Role of NOD- like Receptors in Glioma Angiogenesis: Insights into future therapeutic interventions

Gliomas are the most common solid tumors among central nervous system tumors. Most glioma patients succumb to their disease within two years of the initial diagnosis. The median survival of gliomas is only 14.6 months, even after aggressive therapy with surgery, radiation, and chemotherapy. Gliomas are heavily infiltrated with myeloid- derived cells and endothelial cells. Increasing evidence suggests that these myeloid- derived cells interact with tumor cells promoting their growth and migration. NLRs (nucleotide-binding oligomerization domain (NOD)-containing protein like receptors) are a class of pattern recognition receptors that are critical to sensing pathogen and danger associated molecular patterns. Mutations in some NLRs lead to autoinflammatory diseases in humans. Moreover, dysregulated NLR signaling is central to the pathogenesis of several cancers, autoimmune and neurodegenerative diseases. Our review explores the role of angiogenic factors that contribute to upstream or downstream signaling pathways leading to NLRs. Angiogenesis plays a significant role in the pathogenesis of variety of tumors including gliomas. Though NLRs have been detected in several cancers including gliomas and NLR signaling contributes to angiogenesis, the exact role and mechanism of involvement of NLRs in glioma angiogenesis remain largely unexplored. We discuss cellular, molecular and genetic studies of NLR signaling and convergence of NLR signaling pathways with angiogenesis signaling in gliomas. This may lead to re-appropriation of existing anti-angiogenic therapies or development of future strategies for targeted therapeutics in gliomas.     

An In Vitro Cord Formation Assay Identifies Unique Vascular Phenotypes Associated with Angiogenic Growth Factors

Vascular endothelial growth factor (VEGF) plays a dominant role in angiogenesis. While inhibitors of the VEGF pathway are approved for the treatment of a number of tumor types, the effectiveness is limited and evasive resistance is common. One mechanism of evasive resistance to inhibition of the VEGF pathway is upregulation of other pro-angiogenic factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). Numerous in vitro assays examine angiogenesis, but many of these assays are performed in media or matrix with multiple growth factors or are driven by VEGF. In order to study angiogenesis driven by other growth factors, we developed a basal medium to use on a co-culture cord formation system of adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs). We found that cord formation driven by different angiogenic factors led to unique phenotypes that could be differentiated and combination studies indicate dominant phenotypes elicited by some growth factors. VEGF-driven cords were highly covered by smooth muscle actin, and bFGF-driven cords had thicker nodes, while EGF-driven cords were highly branched. Multiparametric analysis indicated that when combined EGF has a dominant phenotype. In addition, because this assay system is run in minimal medium, potential proangiogenic molecules can be screened. Using this assay we identified an inhibitor that promoted cord formation, which was translated into in vivo tumor models. Together this study illustrates the unique roles of multiple anti-angiogenic agents, which may lead to improvements in therapeutic angiogenesis efforts and better rational for anti-angiogenic therapy.     

Vascular endothelial growth factor (VEGF) directly enhances osteoclastic bone resorption and survival of mature osteoclasts

In bone development and regeneration, angiogenesis and bone/cartilage resorption are essential processes and are closely associated with each other, suggesting a common mediator for these two biological events. To address this interrelationship, we examined the effect of vascular endothelial growth factor (VEGF), the most critical growth factor for angiogenesis, on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. VEGF caused a dose- and time-dependent increase in the area of bone resorption pits excavated by the isolated osteoclasts, partially by enhancing the survival of the cells. Two distinct VEGF receptors, KDR/Flk-1 and Flt-1, were detectable in osteoclasts at the gene and protein levels, and VEGF induced tyrosine phosphorylation of proteins in osteoclasts. Thus, osteoclastic function and angiogenesis are up-regulated by a common mediator such as VEGF.     

Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth.

The role of bone marrow (BM)-derived precursor cells in tumor angiogenesis is not known. We demonstrate here that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells (CEPs). We used the angiogenic defective, tumor resistant Id-mutant mice to show that transplantation of wild-type BM or vascular endothelial growth factor (VEGF)-mobilized stem cells restore tumor angiogenesis and growth. We detected donor-derived CEPs throughout the neovessels of tumors and Matrigel-plugs in an Id1+/-Id3-/- host, which were associated with VEGF-receptor-1-positive (VEGFR1+) myeloid cells. The angiogenic defect in Id-mutant mice was due to impaired VEGF-driven mobilization of VEGFR2+ CEPs and impaired proliferation and incorporation of VEGFR1+ cells. Although targeting of either VEGFR1 or VEGFR2 alone partially blocks the growth of tumors, inhibition of both VEGFR1 and VEGFR2 was necessary to completely ablate tumor growth. These data demonstrate that recruitment of VEGF-responsive BM-derived precursors is necessary and sufficient for tumor angiogenesis and suggest new clinical strategies to block tumor growth.     

Quantification and cell-to-cell variation of vascular endothelial growth factor receptors

The vascular endothelial growth factor receptors (VEGFR) play a significant role in angiogenesis, the formation of new blood vessels from existing vasculature. Systems biology offers promising approaches to better understand angiogenesis by computational modeling the key molecular interactions in this process. Such modeling requires quantitative knowledge of cell surface density of pro-angiogenic receptors versus anti-angiogenic receptors, their regulation, and their cell-to-cell variability. Using quantitative fluorescence, we systematically characterized the endothelial surface density of VEGFRs and neuropilin-1 (NRP1). We also determined the role of VEGF in regulating the surface density of these receptors. Applying cell-by-cell analysis revealed heterogeneity in receptor surface density and VEGF tuning of this heterogeneity. Altogether, we determine inherent differences in the surface expression levels of these receptors and the role of VEGF in regulating the balance of anti-angiogenic or modulatory (VEGFR1) and pro-angiogenic (VEGFR2) receptors.     

Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1(-/-), or VEGFR2(-/-) embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.     

Gene Expression of Neuropilin-1 and Its Receptors,VEGF/Semaphorin 3a,in Normal and Cancer Cells

Extracellular domains of the transmembrane glycoprotein, neuropilin-1 (Np1), specifically bind an array of factors and co-receptors including class-3 semaphorins (Sema3a), vascular endothelial growth factor (VEGF), hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor-β 1 (TGF-β1), and fibroblast growth factor2 (FGF2). Np1 may have a role in immune response, tumor cell growth, and angiogenesis, but its relative expression in comparison to its co-primary receptors, VEGF and Sema3a, is not known. In this study we determined the mRNA expression of Np1 and its co-receptors, VEGF and Sema3a, and the ratio of VEGF/Sema3a in different human and rodent cell lines. Expression of Np1, VEGF and Sema3a is very low in cells derived from normal tissues, but these proteins are highly expressed in tumor-derived cells. Furthermore, the ratio of VEGF/Sema3a is highly variable in different tumor cells. The elevated mRNA expression of Np1 and its putative receptors in tumor cells suggests a role for these proteins in tumor cell migration and angiogenesis. As different tumor cells exhibit varying VEGF/Sema3a ratios, it appears that cancer cells show differential response to angiogenic factors. These results bring to light the individual variation among the cancer-related genes, Np1, VEGF, and Sema3a, and provide an important impetus for the possible personalized therapeutic approaches for cancer patients.