Vascular smooth muscle cell growth-promoting factor/F-spondin inhibits angiogenesis via the blockade of integrin alphavbeta3 on vascular endothelial cells

Vascular smooth muscle cell growth-promoting factor (VSGP) was originally isolated from bovine ovarian follicular fluid as a stimulator of vascular smooth muscle cell proliferation. Homology searches indicate that bovine and human VSGPs are orthologs of rat F-spondin. Here, we examined whether recombinant human VSGP/F-spondin affected the biological activities of endothelial cells. VSGP/F-spondin did not affect the proliferation of human umbilical vein endothelial cells (HUVECs); however, it did inhibit VEGF- or bFGF-stimulated HUVEC migration. To clarify the mechanism of this inhibitory effect, we examined the adhesion of HUVECs to extracellular matrix proteins. VSGP/F-spondin specifically inhibited the spreading of HUVECs on vitronectin via the functional blockade of integrin alphavbeta3. As a result, VSGP/F-spondin inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) when HUVECs were plated on vitronectin. Moreover, VSGP/F-spondin inhibited the activation of Akt when HUVECs on vitronectin were stimulated with VEGF. VSGP/F-spondin inhibited tube formation by HUVECs in vitro and neovascularization in the rat cornea in vivo. These results indicate that VSGP/F-spondin inhibits angiogenesis at least in part by the blockade of endothelial integrin alphavbeta3.     

Thematic review series: sphingolipids. Ganglioside GM3 suppresses the proangiogenic effects of vascular endothelial growth factor and ganglioside GD1a

Gangliosides are sialic acid-containing glycosphingolipids that have long been associated with tumor malignancy and metastasis. Mounting evidence suggests that gangliosides also modulate tumor angiogenesis. Tumor cells shed gangliosides into the microenvironment, which produces both autocrine and paracrine effects on tumor cells and tumor-associated host cells. In this study, we show that the simple monosialoganglioside GM3 counteracts the proangiogenic effects of vascular endothelial growth factor (VEGF) and of the complex disialoganglioside GD1a. GM3 suppressed the action of VEGF and GD1a on the proliferation of human umbilical vein endothelial cells (HUVECs) and inhibited the migration of HUVECs toward VEGF as a chemoattractant. Enrichment of added GM3 in the HUVEC membrane also reduced the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) and downstream Akt. Moreover, GM3 reduced the proangiogenic effects of GD1a and growth factors in the in vivo Matrigel plug assay. Inhibition of GM3 biosynthesis with the glucosyl transferase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), increased HUVEC proliferation and the phosphorylation of VEGFR-2 and Akt. The effects of NB-DNJ on HUVECs were reversed with the addition of GM3. We conclude that GM3 has antiangiogenic action and may possess therapeutic potential for reducing tumor angiogenesis.     

AMP-activated protein kinase (AMPK) signaling in endothelial cells is essential for angiogenesis in response to hypoxic stress

AMP-activated protein kinase (AMPK) is a stress-activated protein kinase that is regulated by hypoxia and other cellular stresses that result in diminished cellular ATP levels. Here, we investigated whether AMPK signaling in endothelial cells has a role in regulating angiogenesis. Hypoxia induced the activating phosphorylation of AMPK in human umbilical vein endothelial cells (HUVECs), and AMPK activation was required for the maintenance of pro-angiogenic Akt signaling under these conditions. Suppression of AMPK signaling inhibited both HUVEC migration to VEGF and in vitro differentiation into tube-like structures in hypoxic, but not normoxic cultures. Dominant-negative AMPK also inhibited in vivo angiogenesis in Matrigel plugs that were implanted subcutaneously in mice. These data identify AMPK signaling as a new regulator of angiogenesis that is specifically required for endothelial cell migration and differentiation under conditions of hypoxia. As such, endothelial AMPK signaling may be a critical determinant of blood vessel recruitment to tissues that are subjected to ischemic stress.     

Adiponectin stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in endothelial cells

Adiponectin is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Here, we investigated whether adiponectin regulates angiogenic processes in vitro and in vivo. Adiponectin stimulated the differentiation of human umbilical vein endothelium cells (HUVECs) into capillary-like structures in vitro and functioned as a chemoattractant in migration assays. Adiponectin promoted the phosphorylation of AMP-activated protein kinase (AMPK), protein kinase Akt/protein kinase B, and endothelial nitric oxide synthesis (eNOS) in HUVECs. Transduction with either dominant-negative AMPK or dominant-negative Akt abolished adiponectin-induced eNOS phosphorylation as well as adiponectin-stimulated HUVEC migration and differentiation. Dominant-negative AMPK also inhibited adiponectin-induced Akt phosphorylation, suggesting that AMPK is upstream of Akt. Dominant-negative Akt or the phosphatidylinositol 3-kinase inhibitor LY294002 blocked adiponectin-stimulated Akt and eNOS phosphorylation, migration, and differentiation without altering AMPK phosphorylation. Finally, adiponectin stimulated blood vessel growth in vivo in mouse Matrigel plug implantation and rabbit corneal models of angiogenesis. These data indicate that adiponectin can function to stimulate the new blood vessel growth by promoting cross-talk between AMP-activated protein kinase and Akt signaling within endothelial cells.     

Anti-Cancer Activity of an Osthole Derivative,NBM-T-BMX-OS01: Targeting Vascular Endothelial Growth Factor Receptor Signaling and Angiogenesis

Angiogenesis occurs during tissue growth, development and wound healing. It is also required for tumor progression and represents a rational target for therapeutic intervention. NBM-T-BMX-OS01 (BMX), derived from the semisynthesis of osthole, an active ingredient isolated from Chinese herb Cnidium monnieri (L.) Cuss., was recently shown to enhance learning and memory in rats. In this study, we characterized the anti-angiogenic activities of NBM-T-BMX-OS01 (BMX) in an effort to develop novel inhibitors to suppress angiogenesis and tumor growth. BMX inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration and endothelial tube formation in human umbilical endothelial cells (HUVECs). BMX also attenuated VEGF-induced microvessel sprouting from aortic rings ex vivo and reduced HCT116 colorectal cancer cells-induced angiogenesis in vivo. Moreover, BMX inhibited the phosphorylation of VEGFR2, FAK, Akt and ERK in HUVECs exposed to VEGF. BMX was also shown to inhibit HCT116 cell proliferation and to suppress the growth of subcutaneous xenografts of HCT116 cells in vivo. Taken together, this study provides evidence that BMX modulates vascular endothelial cell remodeling and leads to the inhibition of tumor angiogenesis. These results also support the role of BMX as a potential drug candidate and warrant the clinical development in the treatment of cancer.     

Pharmacoproteomic analysis of a novel cell-permeable peptide inhibitor of tumor-induced angiogenesis

P11, a novel peptide ligand containing a PDZ-binding motif (Ser-Asp-Val) with high affinity to integrin α(v)β(3) was identified from a hexapeptide library (PS-SPCL) using a protein microarray chip-based screening system. Here, we investigated the inhibitory mechanism of P11 (HSDVHK) on tumor-induced angiogenesis via a pharmacoproteomic approach. P11 was rapidly internalized by, human umbilical vein endothelial cells (HUVECs) via an integrin α(v)β(3)-mediated event. Caveolin and clathrin appeared to be involved in the P11 uptake process. The cell-penetrating P11 resulted in suppression of bFGF-induced HUVEC proliferation in a dose-dependent manner. Phosphorylation of extracellular-signal regulated kinase (ERK1/2) and mitogen-activated protein kinase kinase (MEK) in bFGF-stimulated HUVECs was inhibited by cell-permeable P11. Proteomic analysis via antibody microarray showed up-regulation of p53 in P11-treated HUVECs, resulting in induction of apoptosis via activation of caspases-3, -8, and -9. Several lines of experimental evidence strongly suggest that the molecular mechanism of P11, a novel anti-angiogenic agent, inhibits bFGF-induced HUVEC proliferation via mitogen-activated protein kinase kinase and extracellular-signal regulated kinase inhibition as well as p53-mediated apoptosis related with activation of caspases.     

MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor

Although the significance of vascular endothelial growth factor (VEGF) and its receptors in angiogenesis is well established, the signal transduction cascades activated by VEGF and their involvement in mediating the mitogenic response of endothelial cells to VEGF are incompletely characterized. Here we demonstrate that VEGF activates mitogen-activated protein (MAP) kinases, including the extracellular signal-regulated protein kinase (ERK) and p38 MAP kinase, phosphatidylinositol 3-kinase (PI 3-kinase), and p70 S6 kinase in human umbilical vein endothelial cells (HUVEC). The activation of these enzymes was assayed by kinase phosphorylation and by kinase activity towards substrates. Studies with PI 3-kinase inhibitors revealed that activation of p70 S6 kinase was mediated by PI 3-kinase. Selective inhibition of ERK, PI 3-kinase, and p70 S6 kinase with the inhibitors PD098059, LY294002, and rapamycin, respectively, inhibited VEGF-stimulated HUVEC proliferation. In marked contrast, the p38 MAP kinase inhibitor SB203580 not only failed to inhibit but actually enhanced HUVEC proliferation; this effect was associated with the phosphorylation of Rb protein. Rb phosphorylation resulted from a decrease in the level of the cdk inhibitor p27KiP1. These results indicate that the activities of ERK, PI 3-kinase, and p70 S6 kinase are essential for VEGF-induced HUVEC proliferation. p38 MAP kinase suppresses endothelial cell proliferation by regulating cell-cycle progression.     

Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis.     

Helicobacter pylori-derived Heat shock protein 60 enhances angiogenesis via a CXCR2-mediated signaling pathway

Helicobacter pylori is a potent carcinogen associated with gastric cancer malignancy. Recently, H. pylori Heat shock protein 60 (HpHSP60) has been reported to promote cancer development by inducing chronic inflammation and promoting tumor cell migration. This study demonstrates a role for HpHSP60 in angiogenesis, a necessary precursor to tumor growth. We showed that HpHSP60 enhanced cell migration and tube formation, but not cell proliferation, in human umbilical vein endothelial cells (HUVECs). HpHSP60 also indirectly promoted HUVEC proliferation when HUVECs were co-cultured with supernatants collected from HpHSP60-treated AGS or THP-1 cells. The angiogenic array showed that HpHSP60 dramatically induced THP-1 cells and HUVECs to produce the chemotactic factors IL-8 and GRO. Inhibition of CXCR2, the receptor for IL-8 and GRO, or downstream PLCβ2/Ca2+-mediated signaling, significantly abolished HpHSP60-induced tube formation. In contrast, suppression of MAP K or PI3 K signaling did not affect HpHSP60-mediated tubulogenesis. These data suggest that HpHSP60 enhances angiogenesis via CXCR2/PLCβ2/Ca2+ signal transduction in endothelial cells.     

Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.     

Diallyl trisulfide inhibits migration,invasion and angiogenesis of human colon cancer HT‐29 cells and umbilical vein endothelial cells,and suppresses murine xenograft tumour growth

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis‐dependent diseases including cancer. We examined the cytotoxic, anti‐metastatic, anti‐cancer and anti‐angiogenic effects of diallyl trisulfide (DATS). In HT29 cells, DATS inhibited migration and invasion through the inhibition of focal adhesion kinase (FAK), extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38 which was associated with inhibition of matrix metalloproteinases‐2, ‐7 and ‐9 and VEGF. In human umbilical vein endothelial cells (HUVEC), DATS inhibited the migration and angiogenesis through FAK, Src and Ras. DATS also inhibited the secretion of VEGF. The capillary‐like tube structure formation and migration by HUVEC was inhibited by DATS. The chicken egg chorioallantoic membrane (CAM) assay indicated that DATS treatment inhibited ex‐vivo angiogenesis. We investigated the anti‐tumour effects of DATS against human colon cancer xenografts in BALB/c^nu/nu mice and its anti‐angiogenic activity in vivo. In this in‐vivo study, DATS also inhibited the tumour growth, tumour weight and angiogenesis (decreased the levels of haemoglobin) in HT29 cells. In conclusion, the present results suggest that the inhibition of angiogenesis may be an important mechanism in colon cancer chemotherapy by DATS.     

Antiangiogenic activity of 11,11'-dideoxyverticillin, a natural product isolated from the fungus Shiraia bambusicola

The fungus Shiraia bambusicola yields the phytochemical 11,11'-dideoxyverticillin, which has been shown to possess potent anticancer activity both in vitro and in vivo. In this study, we reveal that 11,11'-dideoxyverticillin has anti-angiogenic activities and explore the potential mechanisms for this effect. Treatment with 11,11'-dideoxyverticillin inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) with IC(50) values of 0.17+/-0.05muM for VEGF-stimulated cells and 0.39+/-0.08muM for serum-stimulated cells. 11,11'-Dideoxyverticillin also antagonized the antiapoptotic effects of VEGF on serum-deprived HUVECs, inhibited VEGF-induced HUVEC migration in vitro, and blocked serum-induced HUVEC tube formation. Moreover, 11,11'-dideoxyverticillin completely blocked VEGF-induced microvessel sprouting from Matrigel-embedded rat aortic rings and vessel growth in Matrigel plugs in mice. In addition, 11,11'-dideoxyverticillin decreased VEGF secretion by MDA-MB-468 breast cancer cells, and significantly suppressed VEGF-induced tyrosine phosphorylation of Flt-1 and KDR/Flk-1. This inhibition of receptor phosphorylation was correlated with a marked decrease in VEGF-triggered pERK activation and a dramatic increase in pP38 MAPK, but no apparent change in pAkt. Together, these findings strongly suggest that 11,11'-dideoxyverticillin is a structurally novel angiogenesis inhibitor.     

Dietary compound glycyrrhetinic acid suppresses tumor angiogenesis and growth by modulating antiangiogenic and proapoptotic pathways in vitro and in vivo

Glycyrrhetinic acid (GA) is a major bioactive compound of licorice. The objective of this study was to investigate the effects of GA on ovarian cancer, particularly those related to angiogenesis and apoptosis, and to elucidate the underlying mechanisms of action. In vitro studies showed that GA significantly inhibited proliferation, migration, invasion and tube formation in human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. GA inhibited the phosphorylation of major receptors and enzymes involved in angiogenesis, such as VEGFR2, mTOR, Akt, ERK1/2, MEK1/2, p38 and JNK1/2 in HUVECs. In addition, GA induced apoptosis, loss of mitochondrial membrane potential and cell cycle arrest in G1 phase in A2780 ovarian cancer cells. The proapoptotic effect of GA involved the increased phosphorylation of p38 and JNK1/2; increased cleavage of caspase 3, caspase 9 and PARP; reduced phosphorylation of mTOR, Akt and ERK1/2; and reduced expressions of survivin and cyclin D1. Ex vivo studies showed that GA significantly inhibited microvessel sprouting in rat aortic ring model. In vivo studies showed that GA inhibited the formation of new blood vessels in zebrafish and mouse Matrigel plug. GA also significantly reduced the size of ovarian cancer xenograft tumors in nude mice. Taken together, GA possesses potential antitumor effects, and the underlying mechanisms may involve the inhibition of signaling pathways related to angiogenesis and the activation of apoptotic pathways in cancer cells. Our findings suggest that GA could serve as an effective regimen in the prevention or treatment of cancer.     

Targeting integrins and PI3K/Akt-mediated signal transduction pathways enhances radiation-induced anti-angiogenesis

The integrins and PI3K/Akt are important mediators of the signal transduction pathways involved in tumor angiogenesis and cell survival after exposure to ionizing radiation. Selective targeting of either integrins or PI3K/Akt can radiosensitize tumors. In this study, we tested the hypothesis that the combined inhibition of integrin alphanubeta3 by cRGD and PI3K/Akt by LY294002 would significantly enhance radiation-induced inhibition of angiogenesis by vascular endothelial cells. Treatment with cRGD inhibited the adhesion and tube formation of human umbilical vein endothelial cells (HUVECs). The inhibitory effect was further increased when cRGD and LY294002 were applied simultaneously. Both radiation and cRGD induced Akt phosphorylation, up-regulated COX2 expression, and increased PGE2 production in HUVECs. Treatment with LY294002 effectively inhibited radiation- and cRGD-induced Akt phosphorylation and up-regulation of COX2 and increased apoptosis of HUVECs. The combined use of cRGD and LY294002 enhanced radiation-induced cell killing. The clonogenic survival of HUVECs was decreased from 34% with 2 Gy radiation to 4% with these agents combined. These results demonstrate that combined use of ionizing radiation, cRGD and LY294002 inhibited multiple signaling transduction pathways involved in tumor angiogenesis and enhanced radiation-induced effects on vascular endothelial cells.     

Cxc chemokine receptor expression on human endothelial cells.

CXC chemokines play a important role in the process of leukocyte recruitment and activation at sites of inflammation. However, recent evidence suggests that these molecules can also regulate endothelial cell functions such as migration, angiogenesis and proliferation. In this study we have investigated CXC chemokine receptor expression in both primary cultures of human umbilical vein endothelial cells (HUVEC) and the spontaneously transformed HUVEC cell line, ECV304. We found that both cell types express mRNA for chemokine receptors CXCR1, CXCR2 and CXCR4, but not CXCR3. Flow cytometric analysis revealed low levels of CXCR1 but higher levels of CXCR4 cell surface expression. HUVECs responded to SDF-1alpha with a rapid and robust calcium flux, however no calcium flux was seen with either IL-8 or Gro-alpha. HUVECs and ECV304 cells did not proliferate in response to CXC chemokines, although ECV304 cells did migrate towards SDF-1alpha and IL-8. These data demonstrate that HUVECs and the endothelial cell line, ECV304 express functional CXC chemokine receptors.     

BRN-103, a novel nicotinamide derivative, inhibits VEGF-induced angiogenesis and proliferation in human umbilical vein endothelial cells

Anti-angiogenesis is regarded as an effective strategy for cancer treatment, and vascular endothelial growth factor (VEGF) plays a key role in the regulations of angiogenesis and vasculogenesis. In the present study, the authors synthesized five novel nicotinamide derivatives which structurally mimic the receptor tyrosine kinase inhibitor sunitinib and evaluated their anti-angiogenic effects. Transwell migration assays revealed that 2-(1-benzylpiperidin-4-yl) amino-N-(3-chlorophenyl) nicotinamide (BRN-103), among the five derivatives most potently inhibited VEGF-induced human umbilical vein endothelial cells (HUVECs). In addition, BRN-103 dose-dependently inhibited VEGF-induced migration, proliferation, and capillary-like tube formation of HUVECs and vessel sprouting from mouse aortic rings. To understand the molecular mechanisms responsible for these activities, the authors examined the effect of BRN-103 on VEGF signaling pathways in HUVECs. BRN-103 was found to suppress the VEGF-induced phosphorylation of VEGF receptor 2 (VEGR2) and the activations of AKT and eNOS. Taken together, these results suggest that BRN-103 inhibits VEGF-mediated angiogenesis signaling in human endothelial cells.     

Inhibition of P38 MAPK Reduces Tumor Conditioned Medium-Induced Angiogenesis in Co-Cultured Human Umbilical Vein Endothelial Cells and Fibroblasts

Tumor conditioned medium (CM) has been widely used to stimulate endothelial cells to form capillary-like structures in in vitro angiogenesis models. We report herein the effect of HT1080 and A549 CM after they were mixed with microvascular endothelial cells medium-2 (EGM-2) on angiogenesis in human umbilical vein endothelial cells (HUVECs). Both HT1080 and A549 CM decreased HUVEC proliferation, to different extents. While A549 CM significantly increased capillary-like structure formation in a co-culture system, no effect of HT1080 was apparent. Inhibition of p38 mitogen-activated protein kinase (MAPK) blocked both basal and A549 CM induced capillary-like structure formation, but inhibition of extracellular signal-regulated kinases (ERK) and that of c-Jun N-terminal protein kinases (JNK) MAPK had no such effect. Activation of ERK MAPK was inhibited by both CMs, whereas p38 MAPK was inactivated by HT1080 and activated by A549 CM and a control. Neither CM had an effect on JNK MAPK. The results suggest that p38 MAPK played a critical role in capillary-like structure formation in the co-culture, partly via promotion of apoptosis in HUVECs.     

Green tea extract inhibits angiogenesis of human umbilical vein endothelial cells through reduction of expression of VEGF receptors

Epidemiological and animal studies have indicated that consumption of green tea is associated with a reduced risk of developing certain forms of cancer. However, the inhibitory mechanism of green tea in angiogenesis, an important process in tumor growth, has not been well established. In the present study, green tea extract (GTE) was tested for its ability to inhibit cell viability, cell proliferation, cell cycle dynamics, vascular endothelial growth factor (VEGF) and expression of VEGF receptors fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/Kinase insert domain containing receptor (Flk-1/KDR) in vitro using human umbilical vein endothelial cells (HUVECs). GTE in culture media did not affect cell viability but significantly reduced cell proliferation dose-dependently and caused a dose-dependent accumulation of cells in the G1 phase. The decrease of the expression of Flt-1 and KDR/Flk-1 in HUVEC by GTE was detected with immunohistochemical and Western blotting methods. These results suggest that GTE may have preventive effects on tumor angiogenesis and metastasis through reduction of expression of VEGF receptors.