Roles for host and tumor angiotensin II type 1 receptor in tumor growth and tumor-associated angiogenesis

Angiotensin II (AII) is a multifunctional bioactive peptide, and host renin-angiotensin system (RAS) is closely associated with tumor growth. Recent reports have described that AII is a proangiogenic growth factor, and that Angiotensin II type 1 (AT1) receptor antagonists reduce tumor growth and tumor-associated angiogenesis. In this paper, we investigated the participation of AT1 receptor-signaling in cancer progression using murine Lewis lung carcinoma (LLC) cells, which express AT1 receptor, and AT1a receptor gene-deficient (AT1a-/-) mice. When LLC cells were implanted subcutaneously into wild-type (WT) mice, developed tumors showed intensive angiogenesis with an induction of vascular endothelial growth factor (VEGF) a. Compared with WT mice, tumor growth and tumor-associated angiogenesis was reduced in AT1a-/- mice with reduced expression of VEGFa. In AT1a-/- mice, administration of the AT1 receptor antagonist, TCV-116, showed further reductions of tumor growth, tumor-associated angiogenesis, and VEGFa expression. In vitro study, the expression of VEGFa mRNA and the production of VEGFa protein in LLC cells were significantly increased by AII, which were cancelled by AT1 receptor antagonist, CV-11974. Although the expression of other angiogenic factors, such as angiopoietin-1, angiopoietin-2, epidermal growth factor, and VEGF receptor 2 mRNA, was also investigated in tumor tissues, the expression of VEGFa was most correlated with tumor size among those other angiogenic factors. VEGFa induction by AT1 receptor-signaling in both host and tumor tissues is one of key regulators of tumor growth and tumor-associated angiogenesis. In conclusion, tumor tissue RAS as well as host tissue RAS were found to have an important role in tumor growth. AT1 receptor-signaling blockade may be a novel and effective target in the treatment of cancer.     

Cell Type-Specific Expression of Angiopoietin-1 and Angiopoietin-2 Suggests a Role in Glioblastoma Angiogenesis

Glioblastomas are highly vascular tumors which overexpress the angiogenesis factor vascular endothelial growth factor (VEGF). VEGF and its receptors, VEGF-R1 and VEGF-R2, have been shown to be necessary for embryonic angiogenesis as well as for tumor angiogenesis. Recently, the angiopoietin/Tie2 receptor system has been shown to exert functions in the cardiovascular system that are distinct from VEGF but are also critical for normal vascular development. To assess the potential role of Tie2 and its ligands angiopoietin-1 and angiopoietin-2 in tumor vascularization, we analyzed their expression pattern in human gliomas. Tie-2 was up-regulated in tumor endothelium compared to normal human brain tissue. We further observed cell type-specific up-regulation of the message for both angiopoietin-1 and angiopoietin-2 in gliomas. Whereas Ang-1 mRNA was expressed in tumor cells, Ang-2 mRNA was detected in endothelial cells of a subset of glioblastoma blood vessels. Small capillaries with few periendothelial support cells showed strong expression of Angiopoietin-2, whereas larger glioblastoma vessels with many periendothelial support cells showed little or no expression. Although the function of Tie2 and its ligands in tumor angiogenesis remains a subject of speculation, our findings are in agreement with a recently proposed hypothesis that in the presence of VEGF, local production of Ang-2 might promote angiogenesis.     

Infectious angiogenesis: Bartonella bacilliformis infection results in endothelial production of angiopoetin-2 and epidermal production of vascular endothelial growth factor

Pathological angiogenesis, the development of a microvasculature by neoplastic processes, is a critical component of the development of tumors. The role of oncogenes in the induction of angiogenesis has been extensively studied in benign and malignant tumors. However, the role of infection in inducing angiogenesis is not well understood. Verruga peruana is a clinical syndrome caused by the bacterium Bartonella bacilliformis, and is characterized by the development of hemangioma-like lesions, in which bacteria colonize endothelial cells. To gain insight into how this bacteria induces angiogenesis in vivo, we performed in situ hybridization of clinical specimens of verruga peruana for the angiogenesis factors vascular endothelial growth factor (VEGF), its receptors VEGFR1 and VEGFR2, and angiopoietin-2. High-level expression of angiopoietin-2 and VEGF receptors was observed in the endothelium of verruga peruana. Surprisingly, the major source of VEGF production in verruga peruana is the overlying epidermis. Infection of cultured endothelium with B. bacilliformis also resulted in induction of angiopoetin-2 in vitro. These findings imply a collaboration between infected endothelium and overlying epidermis to induce angiogenesis.     

Angiogenesis in gliomas: biology and molecular pathophysiology

Glioblastoma multiforme (GBM) is characterized by exuberant angiogenesis, a key event in tumor growth and progression. The pathologic mechanisms driving this change and the biological behavior of gliomas remain unclear. One mechanism may involve cooption of native blood vessels by glioma cells inducing expression of angiopoietin-2 by endothelial cells. Subsequently, vascular apoptosis and involution leads to necrosis and hypoxia. This in turn induces angiogenesis that is associated with expression of hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF) in perinecrotic pseudopalisading glioma cells. Here we review the molecular and cellular mechanisms implicated in HIF-1-dependent and HIF-1-independent glioma-associated angiogenesis. In GBMs, both tumor hypoxia and genetic alterations commonly occur and act together to induce the expression of HIF-1. The angiogenic response of the tumor to HIF-1 is mediated by HIF-1-regulated target genes leading to the upregulation of several proangiogenic factors such as VEGF and other adaptive response molecules. Understanding the roles of these regulatory processes in tumor neovascularization, tumor growth and progression, and resistance to therapy will ultimately lead to the development of improved antiangiogenic therapies for GBMs.     

Molecular Mechanisms of Developmental and Tumor Angiogenesis

Angiogenesis, the sprouting of capillaries from preexisting vessels, is of fundamental importance during embryonic development and is the principal process by which the brain and certain other organs become vascularized. Angiogenesis occurs during embryonic development but is almost absent in adult tissues. Transient and tightly controlled (physiological) angiogenesis in adult tissues occurs during the female reproductive cycle and during wound healing. In contrast, pathological angiogenesis is characterized by the persistent proliferation of endothelial cells, and is a prominent feature of diseases such as proliferative retinopathy, rheumathoid arthritis, and psoriasis. In addition, many tumors are able to attract blood vessels from neighbouring tissues. Tumor-induced angiogenesis requires a constitutive activation of endothelial cells. These endothelial cells dissolve their surrounding extracellular matrix, migrate toward the tumor, proliferate, and form a new vascular network, thus supplying the tumor with nutrients and oxygen and removing waste products. The onset of angiogenesis in human gliomas is characterized by the expression of genes encoding angiogenic growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) in tumor cells, and coordinate induction of genes in endothelial cells which encode the respective growth factor receptors. Developmental and tumor angiogenesis appear to be regulated by a paracrine mechanism involving VEGF and VEGF receptor-1 and -2.     

Vascular Endothelial Growth Factors VEGF-B and VEGF-C Are Expressed in Human Tumors

The growth of solid tumors is dependent on angiogenesis, the formation of new blood vessels. Vascular endothelial growth factor (VEGF) is a secreted endothelial-cell-specific mitogen. We have recently characterized two novel endothelial growth factors with structural homology to VEGF and named them VEGF-B and VEGF-C. To further define the roles of VEGF-B and VEGF-C, we have studied their expression in a variety of human tumors, both malignant and benign. VEGF-B mRNA was detected in most of the tumor samples studied, and the mRNA and the protein product were localized to tumor cells. Endothelial cells of tumor vessels were also immunoreactive for VEGF-B, probably representing the binding sites of the VEGF-B polypeptide secreted by adjacent tumor cells. VEGF-C mRNA was detected in approximately one-half of the cancers analyzed. Via in situ hybridization, VEGF-C mRNA was also localized to tumor cells. All lymphomas studied contained low levels of VEGF-C mRNA, possibly reflecting the cell-specific pattern of expression of the VEGF-C gene in the corresponding normal cells. The expression of VEGF-C is associated with the development of lymphatic vessels, and VEGF-C could be an important factor regulating the mutual paracrine relationships between tumor cells and lymphatic endothelial cells. Furthermore, VEGF-C and VEGF-B can, similarly to VEGF, be involved in tumor angiogenesis.     

Angiogenic factors in normal endometrium and endometrial adenocarcinoma

In the endometrium, angiogenesis plays important roles not only in tumor growth but also in the menstrual cycle. The purpose of the present paper was to investigate immunohistochemically the correlation between angiogenic factor expression and angiogenic score in normal and neoplastic endometrium. Immunohistochemical staining for vascular endothelial growth factor (VEGF), angiopoietin (Ang)-1, Ang2, Tie2, CD34 and CD105 was performed on formalin-fixed and paraffin-embedded tissues from 31 normal endometrium and 85 endometrial adenocarcinoma. VEGF, Ang1, Ang2 and Tie2 expression was localized in the cytoplasm of glandular and tumor cells. The levels of each angiogenic factor were different in the phases of the menstrual cycle and each layer of normal endometrium. In general, VEGF and Tie2 expression was higher in adenocarcinoma than in normal epithelial cells. Conversely, Ang1 and Ang2 expression was higher in normal epithelium than in adenocarcinoma. The angiogenic score (CD105/CD34) tended to be higher in the adenocarcinoma than in the normal epithelium. It is suggested that the angiogenic pathway and the role of these factors seem to differ between normal tissue and carcinoma of the endometrium.     

CAPE suppresses VEGFR-2 activation, and tumor neovascularization and growth

The growth and metastasis of human solid tumors and the development of conditions such as diabetic retinopathy, rheumatoid arthritis, inflammatory psoriasis, and others are regulated by the balance between angiogenic stimulators and inhibitors released in the angiogenic–pathological microenvironment. Vascular endothelial growth factor (VEGF), an angiogenic factor, is a potent endothelial-specific mitogen that activates endothelial cells in pathological angiogenesis. Recently, we demonstrated that caffeic acid phenethyl ester (CAPE) inhibits tumor growth, invasion, and metastasis. However, the precise molecular mechanism underlying the inhibitory effect of CAPE on VEGF-mediated angiogenesis remains unknown. Here, we show that CAPE suppressed VEGF-induced proliferation, tube formation, migration, the formation of actin stress fibers and loss of VE-cadherin at cell–cell contacts in endothelial cells, indicating the inhibition of VEGF-mediated VEGF receptor-2 (VEGFR-2) and its downstream signal activation in vitro. CAPE blocked VEGF-stimulated neovascularization in the Matrigel plugs assay, and reduced vascular permeability in mouse skin capillaries in vivo. CAPE inhibited the growth and neovascularization of primary tumor cells in C57BL/6 and BALB/c mice inoculated with Lewis lung carcinoma, colon carcinoma, and melanoma cells. These results suggest that CAPE negatively modulates VEGF-induced angiogenesis by suppressing VEGFR-2 activation, and might be a therapeutic avenue for anti-angiogenesis.     

The expression of receptor for advanced glycation end products is associated with angiogenesis in human oral squamous cell carcinoma

The receptor for advanced glycation end products (RAGE) is associated with cancer progression in several human cancers. In this study, we examined the roles of RAGE in the angiogenesis of oral squamous cell carcinoma (OSCC). RAGE concentration was examined in 20 OSCC tumors by enzyme-linked immunosorbent assay (ELISA). The microvessel density (MVD) and lymph vessel density (LVD) were examined by immunostaining. Concentrations of vascular endothelial growth factor (VEGF) and VEGF-C were examined in tumor tissues by ELISA. Tumoral RAGE concentration was associated with higher tumor MVD (P = 0.0123) and tumor VEGF concentration (P = 0.0344), but not with LVD and VEGF-C concentration. Treatment with RAGE ligand, high-mobility group box (HMGB)-1 increased the secretion of VEGF but not that of VEGF-C in human OSCC cell lines, HSC-3 and HSC-4. The effect of HMGB-1 was abrogated by RAGE down-regulation by antisense S-oligodeoxynucleic acid. These results suggest that RAGE expression is closely associated to angiogenesis in OSCC.     

c-Myc is essential for vasculogenesis and angiogenesis during development and tumor progression

c-Myc promotes cell growth and transformation by ill-defined mechanisms. c-myc(-/-) mice die by embryonic day 10.5 (E10.5) with defects in growth and in cardiac and neural development. Here we report that the lethality of c-myc(-/-) embryos is also associated with profound defects in vasculogenesis and primitive erythropoiesis. Furthermore, c-myc(-/-) embryonic stem (ES) and yolk sac cells are compromised in their differentiative and growth potential. These defects are intrinsic to c-Myc, and are in part associated with a requirement for c-Myc for the expression of vascular endothelial growth factor (VEGF), as VEGF can partially rescue these defects. However, c-Myc is also required for the proper expression of other angiogenic factors in ES and yolk sac cells, including angiopoietin-2, and the angiogenic inhibitors thrombospondin-1 and angiopoietin-1. Finally, c-myc(-/-) ES cells are dramatically impaired in their ability to form tumors in immune-compromised mice, and the small tumors that sometimes develop are poorly vascularized. Therefore, c-Myc function is also necessary for the angiogenic switch that is indispensable for the progression and metastasis of tumors. These findings support the model wherein c-Myc promotes cell growth and transformation, as well as vascular and hematopoietic development, by functioning as a master regulator of angiogenic factors.     

Stage-dependent expression of an angiogenic agent and vascular organization in experimental skin tumor development

Increased angiogenesis and expression of antibodies to vascular endothelial growth factor (VEGF), an angiogenic agent, have been shown in the tumor development of many tissues. Areas of skin expressing VEGF and total volume of vessels expressing laminin in the wall were measured in chemical carcinogen-exposed mice using CAS-200 morphometry apparatus having a sensitivity exceeding 99% and reproducibility exceeding 99%. The area of VEGF expression was increased in carcinogen-exposed skin, dysplasia and in well-differentiated squamous cell carcinomas, but decreased in squamous cell carcinomas with decreased degree of differentiation. The vessel volume increased prior to the formation of tumors in carcinogen-exposed skin as well as in highly malignant neoplasms. In well-differentiated squamous cell carcinomas with an expansive growth pattern, the vessels were parallel to the basal membrane, in moderately differentiated tumors the vessels were in the direction of tumor invasion, and in poorly differentiated tumors, active angiogenesis consisted of numerous, enlarged vessels within the tumor. This study showed increased VEGF expression and number of vessels occurring in early stages of skin tumor development, pointing to a role of angiogenesis in chemical risk assessment and in cancer prevention. Altered vessel structure and vessel arrangement were distinct in later stages of tumor growth and in malignant neoplasms, pointing to the utility of detailed vessel analysis in neoplasm characterization.     

LHT7, a chemically modified heparin,inhibits multiple stages of angiogenesis by blocking VEGF,FGF2 and PDGF-B signaling pathways

Despite the therapeutic benefits of the angiogenesis inhibitors shown in the clinics, they have encountered an unexpected limitation by the occurrence of acquired resistance. Although the mechanism of the resistance is not clear so far, the upregulation of alternative angiogenic pathways and stabilization of endothelium by mural cells were reported to be responsible. Therefore, blocking multiple angiogenic pathways that are crucial in tumor angiogenesis has been highlighted to overcome such limitations. To develop an angiogenesis inhibitor that could block multiple angiogenic factors, heparin is an excellent lead compound since wide array of angiogenic factors are heparin-binding proteins. In previous study, we reported a heparin-derived angiogenesis inhibitor, LHT7, as a potent angiogenesis inhibitor and showed that it blocked VEGF signaling pathway. Here we show that LHT7 could block the fibroblast growth factor 2 (FGF2) and platelet-derived growth factor B (PDGF-B) in addition to VEGF. Simultaneous blockade of these angiogenic factors resulted in inhibition of multiple stages of the angiogenic process, including initial angiogenic response to maturation of the endothelium by pericyte coverage in vitro. In addition, the treatment of LHT7 in vivo did not show any sign of vascular normalization and directly led to decreased blood perfusion throughout the tumor. Our findings show that LHT7 could effectively inhibit tumor angiogenesis by blocking multiple stages of the angiogenesis, and could potentially be used to overcome the resistance.     

CD40 ligation induces tissue factor expression in human vascular smooth muscle cells

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are important regulators of blood and lymphatic vessel growth and vascular permeability. Both blood and lymphatic vessels of the upper respiratory tract play important roles in pathological conditions, such as infections and tumors. Here we have studied the expression of VEGF-C and its receptor VEGFR-3 in the upper respiratory system by Northern blot analysis and immunohistochemistry of human tissues, and in situ mRNA hybridization of developing mouse embryos and β-galactosidase staining of mouse embryos having a LacZ marker gene in the VEGFR-3 gene locus. The results demonstrate expression of VEGF-C and VEGFR-3 in the developing and adult nasal respiratory epithelium and in the nasal vascular plexus, respectively. Unlike in most other tissues, in the nasal mucosa VEGFR-3 is expressed in both blood and lymphatic vessels. Expression of VEGF-C was also detected in nasal and nasopharyngeal tumor islands, which were surrounded by VEGFR-3-positive angiogenic blood vessels. These results suggest that VEGF-C and VEGFR-3 have a role in the development of the nasal submucosal vascular plexus and in its normal function and that they are associated with angiogenesis in nasal and nasopharyngeal tumors.     

DMBA-induced mammary pathologies are angiogenic in vivo and in vitro

We have previously shown that human pre-invasive diseases of the breast are angiogenic. In addition, normal epithelium from women with coincident or subsequent invasive breast cancer is more vascular than normal epithelium from women with no breast cancer. To develop a model in which to study the regulation of angiogenesis in pre-invasive mammary pathologies, we examined 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tissues for the presence of neovascularization in pre-invasive histopathologies. These studies included morphometric analysis of tissue vascularity in pre-invasive lesions. In addition, we isolated fresh tumors and histologically normal epithelium (organoids) from DMBA or vehicle-treated control rats to test their ability to induce endothelial cell tubule formation in vitro. Finally, we examined tumors for their ability to produce vascular endothelial cell growth factor. The morphometric studies documented that with epithelial progression, the ability of individual cells to elicit angiogenesis increases. The in vitro studies showed that isolated tumors from these animals stimulate angiogenesis. Furthermore, normal epithelium from DMBA-treated rats is more angiogenic than epithelium from control animals. Finally, DMBA-induced tumors produce vascular endothelial growth factor (VEGF) mRNA, therefore, DMBA-induced mammary tumorigenesis is one model in which to test the dependency of progression on angiogenesis.     

Transgenic induction of vascular endothelial growth factor-C is strongly angiogenic in mouse embryos but leads to persistent lymphatic hyperplasia in adult tissues

Vascular endothelial growth factor-C (VEGF-C) is the quintessential lymphangiogenic growth factor that is required for the development of the lymphatic system and is capable of stimulating lymphangiogenesis in adults by activating its receptor, VEGFR-3. Although VEGF-C is a major candidate molecule for the development of prolymphangiogenic therapy for defective lymphatic vessels in lymphedema, the stability of lymph vessels generated by exogenous VEGF-C administration is not currently known. We studied VEGF-C-stimulated lymphangiogenesis in inducible transgenic mouse models in which growth factor expression can be spatially and temporally controlled without side effects, such as inflammation. VEGF-C induction in adult mouse skin for 1 to 2 weeks caused robust lymphatic hyperplasia that persisted for at least 6 months. VEGF-C induced lymphangiogenesis in numerous tissues and organs when expressed in the vascular endothelium in either neonates or adult mice. Very few or no effects were observed in either blood vessels or collecting lymph vessels. Additionally, VEGF-C stimulated lymphangiogenesis in embryos after the onset of lymphatic vessel development. Strikingly, a strong angiogenic effect was observed after VEGF-C induction in vascular endothelium at any point before embryonic day 16.5. Our results indicate that blood vessels can undergo VEGF-C-induced angiogenesis even after down-regulation of VEGFR-3 in embryos; however, transient VEGF-C expression in adults can induce long-lasting lymphatic hyperplasia with no obvious side effects on the blood vasculature.     

Co-expression of VEGF, c-Met and HGF/SF in secondary pleural tumors

Tumor angiogenesis is influenced by a large number of angiogenic factors among which vascular endothelial growth factor (VEGF) is one of the most important cytokines. Together with hepatocyte growth factor/scatter factor (HGF/SF), c-Met receptor forms a paracrine signaling system. The aim was to study the characterization of the proteins, VEGF, c-Met and HGF/SF with expression pattern and possible co-expression in secondary pleural tumors. Biopsy specimens of the pleural region from 70 patients were chosen and analyzed using immunohistochemistry and in situ hybridization. In the investigated tumors, a marked intracytoplasmic expression, sometimes over-expression of VEGF, c-Met and HGF/SF was detected. This expression was not connected to certain tumor types or a certain histogenetic origin of the tumor. These results indicate a role of these factors in angiogenesis. The synthesis of VEGF and c-Met within the tumor cells was established by in situ hybridization. There was a significant co-expression of VEGF and c-Met/HGF. Thus, autocrine stimulation of these angio-genetically effective systems may be present here. Importantly, the autocrine mechanism between over-expressed c-Met and HGF/SF in malignant tumors, already preferred by other authors, with demonstration of the proteins in the same tumor cells, has to be assumed in the process of pleural metastatic spread. Simultaneous synthesis of these three different proteins is also possible via the plasminogen-urokinase system. VEGF is reported to increase vascular permeability, which in turn causes pleural effusions. The results presented here may be the basis for possible future palliative therapeutical strategies in malignant pleural effusions.