Novel approach to analysis of in vitro tumor angiogenesis with a variable-pressure scanning electron microscope: suppression by matrix metalloproteinase inhibitor SI-27

Degradation of basement membrane by metallo-proteinases (MMP) is a critical step in tumor angiogenesis. To evaluate in vitro angiogenesis, several models have been employed, including bovine cornea, fenestrated rat brain, Matrigel, and others. These models did not provide quantitative analysis of capillary formation. The current study aimed for a novel approach to in vitro assay of angiogenesis with a “wet scanning electron microscope (SEM)” to investigate suppression of tumor angiogenesis by the MMP inhibitor, SI-27. The effects of noncytotoxic concentrations of SI-27 (1–100μM) were determined on nonmitogenic vascular endothelial growth factor (VEGF) (10ng/ml)-mediated cell motility and in vitro angiogenesis of human umbilical vein endothelial cells (HUVECs). Activities of MMP and tissue inhibitor of metalloproteinase (TIMP) were determined by enzyme-linked immunosorbent assay (ELISA). Subsequently, the inhibitory effect of SI-27 was examined on in vitro angiogenesis stimulated by supernatants of human glioma cell lines (U87MG, U251MG, or U373MG). In vitro angiogenesis was quantitatively analyzed with a variable-pressure SEM. Cell motility and in vitro angiogenesis by HUVECs were significantly increased by VEGF along with elevated MMP-1 and-2 activity, whereas SI-27 significantly suppressed VEGF-mediated in vitro angiogenesis and inactivated both MMP-1 and MMP-2, but not inhibited cell motility. The angiogenesis promoted by glioma supernatants showed a significant reduction in the presence of SI-27. SI-27, a novel MMP inhibitor, inhibited tumor angiogenesis in vitro. It can be anticipated to prevent tumor growth through its angiosuppressive effect. Quantitative analysis with a variable-pressure SEM is a novel approach to in vitro angiogenesis assay.     

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.     

Vascular endothelial growth factor is an in vivo survival factor for tumor endothelium in a murine model of colorectal carcinoma liver metastases

BACKGROUND: Recent studies have suggested that vascular endothelial growth factor (VEGF), in addition to its proangiogenic properties, also functions as a survival factor for endothelial cells. The authors hypothesized that inhibition of VEGF activity by blockade of VEGF receptor-2 (R-2) function prevents angiogenesis and decreases tumor growth in colon carcinoma liver metastases. METHODS: Spleens of mice were injected with human colon carcinoma cells producing liver metastases. After 7 days of tumor growth, groups of mice received either antibody to VEGFR-2 (DC101) or phosphate-buffered saline (control). In a follow-up experiment, a similar treatment regimen was followed except that mice were sacrificed at 1-week intervals to assess the time course of endothelial cell and tumor cell apoptosis. RESULTS: After 21 days of therapy, the authors observed a significant decrease in vessel counts in liver metastases from human colon carcinoma in nude mice after therapy with VEGFR-2 antibody. Tumor cell apoptosis was increased significantly in the tumors of mice receiving DC101. Temporal studies with immunofluorescent double staining for the microvasculature and apoptotic cells revealed an increase in endothelial cell apoptosis that preceded an increase in tumor cell apoptosis. In vitro, treatment of human umbilical vein endothelial cells with antibody to VEGFR-2 produced a > 2.5-fold increase in endothelial cell apoptosis. CONCLUSIONS: Therapy targeting the VEGFR-2 inhibited tumor growth in a murine model of colon carcinoma liver metastasis. Surprisingly, this therapy did not only inhibit angiogenesis but also led to endothelial cell death. These findings suggest that VEGF, via VEGFR-2 signaling, functions as a survival factor for tumor endothelial cells in liver metastases from colon carcinoma.     

Membrane ganglioside enrichment lowers the threshold for vascular endothelial cell angiogenic signaling

Malignant tumor progression depends on angiogenesis, requiring vascular endothelial cell migration, and proliferation, triggered by tumor-derived vascular endothelial cell growth factor (VEGF). We show that gangliosides, which are actively shed by tumor cells and bind to normal cells in the tumor microenvironment, have the potential to sensitize vascular endothelial cells to respond to subthreshold levels of VEGF: Ganglioside enrichment of human umbilical vein vascular endothelial cells (HUVEC) caused very low, normally barely stimulatory, VEGF concentrations to trigger robust VEGF receptor dimerization and autophosphorylation, as well as activation of downstream signaling pathways, and cell proliferation and migration. Thus, by dramatically lowering the threshold for growth factor activation of contiguous normal stromal cells, shed tumor gangliosides may promote tumor progression by causing these normal cells to become increasingly autonomous from growth factor requirements by a process that we term tumor-induced progression of the microenvironment.     

Effects of adrenomedullin on endothelial cells in the multistep process of angiogenesis: involvement of CRLR/RAMP2 and CRLR/RAMP3 receptors

Recently, we demonstrated that U87 glioblastoma xenograft tumors treated with anti-adrenomedullin (AM) antibody were less vascularized than control tumors, suggesting that AM might be involved in neovascularization and/or vessel stabilization. Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, is a multistep process that involves migration and proliferation of endothelial cells, remodeling of the extracellular matrix and functional maturation of the newly assembled vessels. In our study, we analyzed the role of AM on human umbilical vein endothelial cell (HUVEC) phenotype related to different stages of angiogenesis. Here we report evidence that AM promoted HUVEC migration and invasion in a dose-dependent manner. The action of AM is specific and is mediated by the calcitonin receptor-like receptor/receptor activity-modifying protein-2 and -3 (CRLR/RAMP2; CRLR/RAMP3) receptors. Furthermore, AM was able to induce HUVEC differentiation into cord-like structures on Matrigel. Suboptimal concentrations of vascular endothelial growth factor (VEGF) and AM acted synergistically to induce angiogenic-related effects on endothelial cells in vitro. Blocking antibodies to VEGF did not significantly inhibit AM-induced capillary tube formation by human endothelial cells, indicating that AM does not function indirectly through upregulation of VEGF. These findings suggest that the proangiogenic action of AM on cultured endothelial cells via CRLR/RAMP2 and CRLR/RAMP3 receptors may translate in vivo into enhanced neovascularization and therefore identify AM and its receptors acting as potential new targets for antiangiogenic therapies.     

Radiation induced-tubulogenesis in endothelial cells is antagonized by the antiangiogenic properties of green tea polyphenol (-) epigallocatechin-3-gallate

Radiation therapy is a widely-used option for the treatment of a variety of solid tumors. Although effective, ionizing radiation (IR) may give rise to various side effects, including secondary tumors. In agreement with this, recent reports have demonstrated increased invasive potential in different tumor-derived cell lines following radiation treatment. Many of the molecular effects of IR specifically on the endothelial cells involved in tumor neo-vascularization remain unknown. In this study, we found that low sublethal single doses of IR applied to human umbilical vein endothelial cells stimulated cell migration and in vitro tubulogenesis. This correlated with an increase in membrane type-1 matrix metalloproteinase (MT1-MMP) protein expression, a crucial enzyme that promotes endothelial cell migration and tube formation, and of caveolin-1, a protein that regulates tube formation. Cell adhesion was also promoted by IR, reflected in increased gene expression levels of cell surface beta(3) integrin. Pretreatment of the cells with epigallocatechin-3-gallate (EGCg), a green tea catechin that possesses anti-angiogenic properties, prevented most of the IR-induced cellular and molecular events. These observations suggest that current protocols involving radiation therapy for the treatment of cancer can paradoxically promote angiogenesis, but can be improved by combination with anti-angiogenic molecules such as EGCg to target those tumor-derived endothelial cells that escaped IR-induced apoptosis.     

Human chondrosarcoma secretes vascular endothelial growth factor to induce tumor angiogenesis and stores basic fibroblast growth factor for regulation of its own growth.

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are well-known factors that induce neovascularization in many tumors. The molecular mechanisms that regulate tumor angiogenesis in human chondrosarcoma are not clear. We assessed in this work the angiogenic activities of a human chondrosarcoma cell line (OUMS-27) in vivo and determined the efficacies of angiogenic factors derived from OUMS-27 cells on human umbilical vein endothelial cells (HUVECs) in vitro. Tumor xenografts induced an increase in the formation of neovessels, but the distributions of Ki-67 antigen, VEGF and bFGF were unaffected. We also demonstrated that OUMS-27 cells secreted VEGF(165) into the culture medium and that it was the maximal angiogenic factor to stimulate endothelial proliferation and migration in chondrosarcoma. Anti-VEGF antibodies induced an approximately 70% inhibition of these responses of HUVECs, but did not have any effect on OUMS-27 cells. Anti-bFGF antibodies suppressed not only the activities of HUVECs but also the growth of tumor cells in vitro. We indicate that angiogenesis is principally elicited by VEGF(165) and that tumorigenesis is mainly regulated by bFGF stored in the extracellular matrix of OUMS-27 cells. The present study may offer the availability of combination therapies for inhibition of VEGF and bFGF action on vascular endothelial cells and chondrosarcoma cells, respectively.     

Growth-regulated oncogene is pivotal in thrombin-induced angiogenesis

The mechanism of thrombin-induced angiogenesis is poorly understood. Using a gene chip array to investigate the pro-malignant phenotype of thrombin-stimulated cells, we observed that thrombin markedly up-regulates growth-regulated oncogene-alpha (GRO-alpha) in several tumor cell lines as well as endothelial cells by mRNA and protein analysis. Thrombin enhanced the secretion of GRO-alpha from tumor cells 25- to 64-fold. GRO-alpha is a CXC chemokine with tumor-associated angiogenic as well as oncogenic activation following ligation of its CXCR2 receptor. GRO-alpha enhanced angiogenesis in the chick chorioallantoic membrane assay 2.2-fold, providing direct evidence for GRO-alpha as an angiogenic growth factor. Anti-GRO-alpha antibody completely inhibited the 2.7-fold thrombin-induced up-regulation of angiogenesis, as well as the 1.5-fold thrombin-induced up-regulation of both endothelial cell cord formation in Matrigel and growth in vitro. Thrombin as well as its PAR-1 receptor activation peptide [thrombin receptor activation peptide (TRAP)] as well as GRO-alpha all markedly increased vascular regulatory proteins and growth factors: matrix metalloproteinase (MMP)-1, MMP-2, vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), CD31, and receptors KDR and CXCR2 in human umbilical vein endothelial cells. All of the thrombin/TRAP gene up-regulations were completely inhibited by anti-GRO-alpha antibody and unaffected by irrelevant antibody. Similar inhibition of gene up-regulation as well as thrombin-induced chemotaxis was noted with small interfering RNA (shRNA) GRO-alpha KD 4T1 breast tumor and B16F10 melanoma cells. In vivo tumor growth studies in wild-type mice with shRNA GRO-alpha KD cells revealed 2- to 4-fold impaired tumor growth, metastasis, and angiogenesis, which was not affected by endogenous thrombin. Thus, thrombin-induced angiogenesis requires the up-regulation of GRO-alpha. Thrombin up-regulation of GRO-alpha in tumor cells as well as endothelial cells contributes to tumor angiogenesis.     

Selection of high affinity human neutralizing antibodies to VEGFR2 from a large antibody phage display library for antiangiogenesis therapy.

Compelling evidence suggests that vascular endothelial growth factor (VEGF) and its receptors play an important role in angiogenesis associated with tumor growth and metastasis. VEGF exerts its biologic activities through 2 transmembrane tyrosine kinase receptors: the fms-like tyrosine kinase receptor (Flt-1, or VEGFR1) and kinase insert domain-containing receptor (KDR or VEGFR2). We have previously produced a panel of antibodies directed against KDR from mice immunized with the recombinant form receptor. These antibodies efficiently neutralized VEGF-induced KDR activation and mitogenesis of human umbilical vascular endothelial cells (HUVEC). Murine antibodies, however, may not be suitable candidates for human therapy because of their propensity to elicit human anti-mouse antibody response. Here we isolated several high-affinity human Fab antibody fragments directed against KDR from an antibody phage display library constructed from the pooled B lymphocytes of nonimmunized healthy human donors. These human Fab fragments bind specifically to KDR with nanomolar affinity and block KDR/VEGF interaction with IC(50) of approximately 2-20 nM. Further, they effectively inhibit VEGF-stimulated mitogenesis of HUVEC and migration of human leukemia cells. Epitope mapping studies demonstrated that all neutralizing human antibodies bound the epitope(s) located within the first 3 N-terminal immunoglobulin-like domains of KDR, the same region that encompasses the binding site of VEGF. Our results suggest that these human anti-KDR antibodies may have potential application in the treatment of cancer and other diseases in which pathologic angiogenesis occurs.     

Hepatoma-derived growth factor induces tumorigenesis in vivo through both direct angiogenic activity and induction of vascular endothelial growth factor

Hepatoma-derived growth factor (HDGF) is highly expressed in tumor cells, and stimulates their proliferation. In the present study, we investigated the role of HDGF in tumorigenesis and elucidated the mechanism of action. Stable transfectants of NIH3T3 cells overexpressing HDGF did not show significant anchorage-independent growth in soft agar assay. However, these stable transfectants overexpressing HDGF generated sarcomatous tumors in nude mice. These tumors were red-colored macroscopically, and histologically showed a rich vascularity. Immunohistochemical analysis using CD31 antibody showed new vessel formation. Recombinant HDGF stimulated proliferation of human umbilical vein endothelial cells in a dose-dependent manner, and stimulated tubule formation. Furthermore, vascular endothelial growth factor (VEGF) was detected immunohistochemically in the tumor tissues. Transient expression of HDGF induced both VEGF gene and protein expression as demonstrated by a reporter assay using VEGF gene promoter. The administration of anti-VEGF neutralizing antibody significantly suppressed, but did not block, the tumor growth of HDGF-overexpressing cells in nude mice. Thus, these findings suggested that HDGF-induced tumor formation in vivo involves induction of VEGF as well as direct angiogenic activity.     

20（S）-人参皂苷Rg3对血管内皮细胞增殖和迁移的抑制作用

 目的 探讨20（S）-人参皂苷Rg3（SPG-Rg3）抗肿瘤诱导新生血管生成的作用机制。方法 采用MTT法、PCNA免疫荧光染色和Boyden小室迁移实验,观察SPG-Rg3对B16黑色素瘤细胞条件培养液（Conditioned Medium of B16 Melanoma Cells, BMCM）诱导的人脐静脉内皮细胞增殖和迁移的影响;并通过免疫细胞化学染色法观察了SPG-Rg3对B16黑色素瘤细胞血管内皮细胞生长因子（Vascular Endothelial Growth Factor, VEGF）及金属基质蛋白酶-9（Matrix Metalloproteinase-9, MMP-9）表达的影响。结果SPG-Rg3组BMCM的促内皮细胞增殖和迁移作用均明显弱于对照组,且SPG-Rg3浓度为5μg/ml时能够降低B16黑色素瘤细胞VEGF及MMP-9的表达。结论SPG-Rg3通过减少肿瘤细胞分泌细胞因子VEGF和MMP-9,抑制肿瘤细胞对血管内皮细胞增殖和迁移的促进作用,从而间接发挥其抗肿瘤新生血管生成的作用。     

Inhibition of VEGF receptors significantly impairs mammary cancer growth in C3(1)/Tag transgenic mice through antiangiogenic and non-antiangiogenic mechanisms

Cancer growth and progression is often critically influenced by the production of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis. VEGF produced by tumor cells stimulates endothelial cell growth through the binding and activation of the KDR/Flk-1 receptor (VEGFR-2) on endothelial cells. Recently, some human breast cancer epithelial cells have been shown to express VEGF receptors, suggesting a potential autocrine-mediated growth stimulation of a subset of cancers by VEGF. We demonstrate that mammary tumors in the C3(1)/Tag transgenic model express VEGF and VEGF receptors and tumor growth is stimulated by this autocrine mechanism. GW654652, an indazolylpyrimidine, is a VEGFRs tyrosine kinase inhibitor that dramatically reduces both angiogenesis and tumor cell growth in this model, as demonstrated using both in vitro and in vivo assays. GW654652 significantly decreased cell proliferation and induced apoptosis in human umbilical vein endothelial cells and M6 mammary tumor cells derived from C3(1)/Tag (Tag: simian virus 40 T-antigen) transgenic mice. A 75% reduction in VEGF-induced angiogenesis was observed with GW654652 using the chick chorioallantoic membrane assay, whereas GW654652 produced an approximately 85% reduction in angiogenesis as assessed by the Matrigel plug assay. A profound inhibitory effect on tumor growth in the C3(1)/Tag transgenic model of human breast cancer was observed with oral administration of GW654652 as measured by delayed tumor onset, decreased multiplicity, reduced tumor volume, and extended animal survival. The antitumor effects of GW654652 were associated with reduced tumor vascularization and no apparent toxicity. Tumor growth, however, rapidly advanced following cessation of treatment. This is the first demonstration that a VEGF receptor inhibitor, GW654652, has a strong inhibitory effect on angiogenesis and tumor progression in a transgenic model of mammary cancer, suggesting that this is a useful approach for preclinical testing of such agents.     

NM-3, an isocoumarin, increases the antitumor effects of radiotherapy without toxicity

We examined the effects of a new antiangiogenic isocoumarin, NM-3, as a radiation modifier in vitro and in vivo. The present studies demonstrate that NM-3 is cytotoxic to human umbilical vein endothelial cells (HUVECs) but not to Lewis lung carcinoma (LLC) cells nor Seg-1, esophageal adenocarcinoma cells, in clonogenic survival assays. When HUVEC cultures are treated with NM-3 combined with ionizing radiation (IR), additive cytotoxicity is observed. In addition, the combination of NM-3 and IR inhibits HUVEC migration to a greater extent than either treatment alone. The effects of treatment with NM-3 and IR were also evaluated in tumor model systems. C57BL/6 female mice bearing LLC tumors were given injections for 4 consecutive days with NM-3 (25 mg/kg/day) and treated with IR (20 Gy) for 2 consecutive days. Combined treatment with NM-3 and IR significantly reduced mean tumor volume compared with either treatment alone. An increase in local tumor control was also observed in LLC tumors in mice receiving NM-3/IR therapy. When athymic nude mice bearing Seg-1 tumor xenografts were treated with NM-3 (100 mg/kg/day for 4 days) and 20 Gy (four 5 Gy fractions), significant tumor regression was observed after combined treatment (NM-3 and IR) compared with IR alone. Importantly, no increase in systemic or local tissue toxicity was observed after combined treatment (NM-3 and IR) when compared with IR alone. The bioavailability and nontoxic profile of NM-3 suggests that the efficacy of this agent should be tested in clinical radiotherapy.     

Head and neck squamous cell carcinoma lines produce biologically active angiogenic factors

Angiogenesis is the formation of capillaries from pre-existing blood vessels. Angiogenesis occurs in many developmental, physiologic, and pathologic processes including tumor growth. Previous studies have shown that angiogenesis is required for growth and metastasis of solid tumors. Fibroblast growth factors (FGF-1, -2) and vascular endothelial growth factor (VEGF) are extremely potent angiogenesis inducers by stimulating the proliferation and migration of capillary endothelial cells. Expression of these factors is upregulated in many solid tumors and correlates with high vascularity, lymph node metastasis, and poor clinical prognosis. Few studies have examined whether established head and neck squamous cell carcinoma (SCC) lines produce biologically active angiogenic factors. By immunoprecipitation, we detected FGF and VEGF proteins in cellular lysates of SCC lines. We also detected FGF-1 and -2 proteins in serum-free-conditioned medium from these lines. Conditioned medium from SCC lines significantly increased proliferation of human umbilical vein endothelial cells (HUVEC). This increased proliferation was abrogated by pre-incubation of conditioned medium with neutralizing antibodies to FGFs and VEGF. Conditioned medium from SCC lines also significantly stimulated HUVEC invasion across a reconstituted basement membrane. We concluded that head and neck SCC lines secrete biologically active angiogenic factors.     

Induction of tube formation by angiopoietin-1 in endothelial cell/fibroblast co-culture is dependent on endogenous VEGF

The angiopoietin-1 (Ang1)/Tie2 receptor system is known to be important for angiogenesis and vascular remodeling. However, its contribution to the survival and morphogenesis of endothelial cells is still not well elucidated. In this study, we analyzed the role of the Ang1/Tie2 pathway in cell survival and tube formation using a human umbilical vein endothelial (HUVE) cell and fibroblast co-culture system. In this system, which mimics angiogenesis in vivo , fibroblasts secrete a basal level of vascular endothelial growth factor (VEGF), and Ang1 stimulated tube formation. However, anti-VEGF or anti-VEGF receptor-2 neutralizing antibody blocked the Ang1-induced tube formation. Furthermore, other angiogenic factors such as hepatic growth factor (HGF) and basic fibroblast growth factor (bFGF) showed the same pheno-type as Ang1, i.e., a stimulatory effect only in the presence of endogenous VEGF. The Ang1-promoted tube formation was mainly due to suppression of HUVE cell apoptosis in a PI3-kinase-dependent manner. These findings suggest that Ang1 stimulates tube formation in vivo via the PI3-kinase/Akt pathway, but this effect takes place only in a VEGF-dependent manner.     

Characterization and purification of angiogenic factor derived from highly liver metastatic colon cancer cells

We investigated the effects of serum-free conditioned medium (SFCM) of colon cancer cells on human umbilical vein endothelial cells (HUVECs). SFCM of highly liver metastatic cell line (LM-HS) strongly enhanced proliferation, migration and tube formation of HUVECs. Moreover, it was suggested that a main angiogenic factor derived from LM-H5 may be VEGF based on HPLC, SDS-PAGE analysis and neutralizing experiment using anti-growth factor antibodies. These findings suggest that angiogenesis is related to the processes of metastasis of colon cancer, and that VEGF synthesized by colon cancer cells acts as a key factor inducing angiogenesis.