High levels of oestrogen receptor-alpha in tumorigenesis: inhibition of cell growth and angiogenic factors

We previously found that the stable overexpression of oestrogen receptor-alpha in the human endothelial cell line ECV304* inhibits its growth in vitro, and that this inhibition is possibly mediated through a down-regulation of the vasoactive agents endothelin-1 and vascular endothelial growth factor. Here we show an in vivo growth-inhibitory effect of oestrogen receptor-alpha overexpression in tumours initiated in nude mice from the same clone of ECV304. In addition, we show that this growth inhibition is accompanied by an alphavbeta3-mediated inhibition of cell migration in vitro, and a down-regulation of the integrin alphavbeta3, vascular endothelial growth factor and vascularization in vivo. The levels of vascular endothelial growth factor and integrin alphavbeta3, through their effect on cell growth and migration, contribute to the process of angiogenesis and to the pathogenesis of atherosclerosis and cancer. The results shown here demonstrate that a higher level of oestrogen receptor-alpha in the cell, through its effect on certain angiogenic factors, may play a role in the control of angiogenesis.     

Angiogenesis and the role of the endothelial nicotinic acetylcholine receptor

An endothelial nicotinic acetycholine receptor (nAChR) mediates endothelial proliferation, survival, migration and tube formation in vitro, and angiogenesis in vivo. Exogenous nicotine stimulates this angiogenic pathway. This action of nicotine may contribute to tumor angiogenesis and tumor growth; atherosclerotic plaque neovascularization and progression; and other tobacco-related diseases. The endothelial nAChR mediates an angiogenic pathway that is interdependent with growth factor mediated pathways, as shown by pharmacological and molecular studies. The characterization of this new angiogenic pathway may provide a new therapeutic avenue for disorders of insufficient or pathological angiogenesis.     

TIE2-high cervical cancer cells promote tumor angiogenesis by upregulating TIE2 and VEGFR2 in endothelial cells

Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (TIE2), the receptor for angiopoietins, has been found highly expressed in cervical cancer and associated with poor prognosis. However, the potential role of tumoral TIE2 in cervical cancer angiogenesis and the underlying mechanisms remain unexplored. Here, based on multicolor immunofluorescence of 64 cervical cancer tissues, we found that TIE2 level in cervical cancer cells was positively related to shorter survival and higher microvessel density in tumor. In vitro and in vivo experiments verified that TIE2-high cervical cancer cells could promote tumor angiogenesis. TIE2-high tumor cells induced an amplified expression of TIE2 and vascular endothelial growth factor receptor 2(VEGFR2) in HUVECs to promote angiogenesis via TIE2 -AKT/MAPK signals, which could be reversed or partially reversed by TIE2, AKT or MAPK inhibitors and activated by angiopoietin-1 and angiopoietin-2. In conclusion, TIE2-high cervical cancer cells promote tumor angiogenesis by upregulating TIE2 and VEGFR2 in endothelial cells via TIE2-AKT/MAPK axis inside tumor cells.     

Regulation of angiogenesis-expression of VEGF receptors]

Angiogenesis, the formation of new blood vessels from pre-existing endothelium, is a crucial process for tumor growth, metastasis and inflammation. Therefore, it is focused on the anti-tumor therapy to prohibit angiogenesis in animal model and clinical studies. Eicosapentaenoic acid (EPA 20: 5,n-3) can restrain tumor growth and inflammation. In this paper, we examined the effects of EPA on tube formation. In EPA-pretreated endothelial cells angiogenesis was attenuated and also proliferation induced by VEGF, but not by b-FGF, was suppressed. The reason why EPA suppressed endothelial cell proliferation induced by VEGF was that EPA selectively inhibited the expression of KDR. As we mentioned, the regulation of angiogenesis in vivo may be involved in the expression of VEGF receptors.     

血管内皮生长因子受体信号转导通路与肿瘤血管生成

血管内皮生长因子是促进血管生成的重要调节因子.它能促进内皮细胞增殖、迁移,阻止内皮细胞凋亡、管腔网状结构退化,增加血管渗透性.所有这些作用都是通过血管内皮生长因子受体信号转导通路实现的.它们在肿瘤血管生成、肿瘤生长中起着重要的作用.以血管内皮生长因子受体信号转导通路为靶点是开发肿瘤血管生成抑制剂的理想策略.     

Apolipoprotein E: A potent inhibitor of endothelial and tumor cell proliferation

Recombinant human apolipoprotein E3 (apoE), purified from E. coli, inhibited the proliferation of several cell types, including endothelial cells and tumor cells in a dose- and time-dependent manner. ApoE inhibited both de novo DNA synthesis and proliferation as assessed by an increase in cell number. Maximal inhibition of cell growth by apoE was achieved under conditions where proliferation was dependent on heparin-binding growth factors. Thus, at low serum concentrations (0–2.5%) basic fibroblast growth factor (bFGF) stimulated the proliferation of bovine aortic endothelial (BAE) cells severalfold. The bFGF-dependent proliferation was dramatically inhibited by apoE with an IC₅₀ ≈ 50 nM. Under conditions where cell proliferation was mainly serum-dependent, apoE also suppressed growth but required higher concentrations to be effective (IC₅₀ ≈ 500 nM). ApoE also inhibited growth of bovine corneal endothelial cells, human melanoma cells, and human breast carcinoma cells. The IC₅₀ values obtained with these cells were generally 3–5 times higher than with BAE cells. Inhibition of cell proliferation by apoE was reversible and dependent on the time of apoE addition to the culture. In addition, apoE inhibited the chemotactic response of endothelial cells that were induced to migrate by a gradient of soluble bFGF. Inhibition of cell proliferation by apoE may be mediated both by competition for growth factor binding to proteoglycans and by an antiadhesive activity of apoE. The present results demonstrate that apoE is a potent inhibitor of proliferation of several cell types and suggest that apoE may be effective in modulating angiogenesis, tumor cell growth, and metastasis.     

几种扩血管多肽对bFGF促血管平滑肌细胞增殖作用的影响

目的和方法：研究肾上腺髓质素（Ａｄｍ）、降钙素基因相关肽（ＣＧＲＰ）及Ｃ－型心房利太（ＣＮＰ）对碱性成纤维细胞生长因子（ｂＦＧＦ）促血管平滑细胞（ＶＳＭＣ）增殖作用的影响及其机制。结果：孵育２４ｈ后,ｂＦＧＦ刺激ＶＳＭＣ增殖较对照组增加２．１倍（Ｐ〈０．０１）,细胞内蛋白磷酸化程度增加１．４倍（Ｐ〈０．０１）,ＰＫＣ及ＭＡＰＫ活性分别增加１．５和２．５倍（Ｐ〈０．０１０;Ａｄｍ．ＣＧＲＰｔ ＣＮＰ     

Intra- and extracellular signaling by endothelial neuregulin-1

Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.     

Targeted inhibition of alphavbeta3 integrin with an RNA aptamer impairs endothelial cell growth and survival

Alphavbeta3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. Alphavbeta3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of alphavbeta3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-alphavbeta3 that binds recombinant alphavbeta3 integrin, for its ability to bind endogenous alphavbeta3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-alphavbeta3 binds alphavbeta3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-alphavbeta3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-alphavbeta3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-alphavbeta3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.     

Upregulation of neuropilin-1 by basic fibroblast growth factor enhances vascular smooth muscle cell migration in response to VEGF

Neuropilin-1 (NRP-1) is a co-receptor for vascular endothelial growth factor (VEGF). During neovascularization, vascular smooth muscle cells (VSMCs) and pericytes modulate the function of endothelial cells. Factors that mediate NRP-1 in human VSMCs (hVSMCs) remain to be elucidated. We studied various angiogenic cytokines to identify factors that increase NRP-1 expression in hVSMCs. Treatment of hVSMCs with basic fibroblast growth factor (b-FGF) induced expressions of NRP-1 mRNA and protein whereas epidermal growth factor, insulin-like growth factor-1, and interleukin-1beta did not. b-FGF induced phosphorylation of Erk-1/2 and JNK. MEK1/2 and nuclear factor kappa B (NF-kappaB) inhibitors (U0126 and TLCK, respectively) blocked the ability of b-FGF to induce NRP-1 mRNA expression, but inhibition of JNK (SP600125) or PI3-kinase activity (wortmannin) did not. Further, the increase in NRP-1 expression by b-FGF enhanced hVSMCs migration in response to VEGF(165). This effect was dependent on the binding of VEGF(165) to VEGFR-2, as blocking antibodies to VEGFR-2, but not VEGFR-1, inhibited VEGF(165)-induced migration. In conclusion, b-FGF increased NRP-1 expression in hVSMCs that in turn enhance the effect of VEGF(165) on cell migration. The enhanced migration of hVSMCs was mediated through binding of VEGF(165) to both NRP-1 and VEGFR-2, as inhibition of VEGFR-2 on these cells blocked the effect of VEGF-mediated cell migration.     

Identification of an active site on the laminin alpha4 chain globular domain that binds to alphavbeta3 integrin and promotes angiogenesis

Angiogenesis is important for wound healing, tumor growth, and metastasis. The laminin alpha4 chain, a component of laminin-8 and -9, is expressed in endothelial cell basement membranes. It mediates endothelial cell adhesion by binding with its receptors such as alphavbeta3 integrin and participates in new blood vessel formation. In this study, we found the recombinant laminin alpha4LG modules (rLG1-3, rLG1, and rLG2) mediate HUVECs adhesion. The attachment of HUVECs to the rLG2 was specifically inhibited by a function-blocking monoclonal antibody LM609 specific for alphavbeta3 integrin. Using deletion mutants of the alpha4LG2 revealed the HUVECs-adhesion site is located in amino acids 1121-1139. A synthetic G(1121-1139) peptide could be attached by HUVECs at same efficiency with the rLG2 and promoted angiogenesis in CAM. In conclusion, we have identified a new alphavbeta3 integrin-interacting peptide within laminin alpha4 G domain. This suggests that G(1121-1139) peptide-containing proteins may perform their biological functions by interacting with alphavbeta3 integrin.     

Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis.

We provide anatomic and functional evidence that nicotine induces angiogenesis. We also show that nicotine accelerates the growth of tumor and atheroma in association with increased neovascularization. Nicotine increased endothelial-cell growth and tube formation in vitro, and accelerated fibrovascular growth in vivo. In a mouse model of hind-limb ischemia, nicotine increased capillary and collateral growth, and enhanced tissue perfusion. In mouse models of lung cancer and atherosclerosis, we found that nicotine enhanced lesion growth in association with an increase in lesion vascularity. These effects of nicotine were mediated through nicotinic acetylcholine receptors at nicotine concentrations that are pathophysiologically relevant. The endothelial production of nitric oxide, prostacyclin and vascular endothelial growth factor might have a role in these effects.     

Antiangiogenesis efficacy of nitric oxide donors

Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis‐mediated disorders. Because nitric oxide (NO) is known to play a key role in various vascular diseases, the present study was undertaken to determine the role of NO in angiogenesis‐mediated processes using the NO donor, S‐nitroso N‐acetyl penicillamine (SNAP) and S‐nitroso N‐acetyl glutathione (SNAG). The antiangiogenic efficacy of these NO donors was examined using in vivo and in vitro model systems. The in vitro studies demonstrated the ability of SNAP to inhibit cytokine fibroblast growth factor (FGF2)‐stimulated tube formation and serum‐induced cell proliferation. The inhibitory effect on cell proliferation by SNAP concentrations above the millimolar range was associated with significant shifts in the concentration of NO metabolites. Furthermore, using the mouse Matrigel implant model and the chick chorioallantoic membrane (CAM) models, SNAP demonstrated maximal inhibitory efficacy (85–95% inhibition) of cytokine (FGF2)‐induced neovascularization in both in vivo models. SNAP and SNAG resulted in 85% inhibition of FGF2‐induced neovascularization in the mouse Matrigel model when given at 5 mg/kg/day infusion in minipumps during 14 days and 87% inhibition of angiogenesis induced by FGF2 in the CAM when administered a single dose of 50 μg. Thus, NO donors might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or neovascular, ocular, and inflammatory diseases. J. Cell. Biochem. 80:104–114, 2000. © 2000 Wiley‐Liss, Inc.     

Enhanced pathological angiogenesis in mice lacking beta3 integrin or beta3 and beta5 integrins.

Inhibition of alphavbeta3 or alphavbeta5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking beta3 integrins or both beta3 and beta5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither beta3 nor beta5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of beta3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the beta3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in beta3-null endothelial cells. These data indicate that alphavbeta3 and alphavbeta5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of alphav-integrin antagonists in anti-angiogenic therapeutics.     

8-prenylnaringenin, a novel phytoestrogen, inhibits angiogenesis in vitro and in vivo

8-Prenylnaringenin is a recently discovered phytoestrogen. Using an in vitro model of angiogenesis in which endothelial cells can be induced to invade a three-dimensional collagen gel within which they form capillary-like tubes, we demonstrate that 8-prenylnaringenin inhibits angiogenesis induced by basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), or the synergistic effect of the two cytokines in combination, with an IC(50) of between 3 and 10 microM. This effect was seen with bovine microvascular endothelial cells derived from the adrenal cortex (BME cells) and with endothelial cells from the bovine thoracic aorta (BAE cells). The inhibitory effects of 8-prenylnaringenin were found to be roughly equipotent to those of genistein that has previously been shown to inhibit angiogenesis in vitro. Early chorioallantoic membrane (CAM) assay results showed reductions in both vessel lengths and vein diameters, with similar potency in the 8-prenylnaringenin and genistein groups. Similar effects on the CAM vessels were seen when the two substances were co-added. These findings suggest that 8-prenylnaringenin has potential therapeutic applications for diseases in which angiogenesis is an important component.     

Neprilysin inhibits angiogenesis via proteolysis of fibroblast growth factor-2

Neprilysin is a cell surface peptidase that catalytically inactivates neuropeptide substrates and functions as a tumor suppressor via its enzymatic function and multiple protein-protein interactions. We investigated whether neutral endopeptidase could inhibit angiogenesis in vivo utilizing a murine corneal pocket angiogenesis model and found that it reduced fibroblast growth factor-2-induced angiogenesis by 85% (p < 0.01) but had no effect on that of vascular endothelial growth factor. Treatment with recombinant neprilysin, but not enzymatically inactive neprilysin, resulted in a slight increase in basic fibroblast growth factor electrophoretic mobility from proteolytic cleavage between amino acids Leu-135 and Gly-136, which was inhibited by the neutral endopeptidase inhibitor CGS24592 and heparin. Cleavage kinetics were rapid, comparable with that of other known neprilysin substrates. Functional studies involving neprilysin-expressing vascular endothelial cells demonstrated that neutral endopeptidase inhibition significantly enhanced fibroblast growth factor-mediated endothelial cell growth, capillary array formation, and signaling, whereas exogenous recombinant neprilysin inhibited signaling. Recombinant constructs confirmed that cleavage products neither promoted capillary array formation nor induced signaling. Moreover, mutation of the cleavage site resulted in concomitant loss of cleavage and increased the potency of fibroblast growth factor-2 to induce capillary array formation. These data indicate that neprilysin proteolytically inactivates fibroblast growth factor-2, resulting in negative regulation of angiogenesis.     

血管形成在双歧杆菌预防大肠癌生长中的作用

目的 从血管形成角度探索青春型双歧杆菌预防大肠癌生长的途径。方法 建立大肠癌裸鼠移植瘤模型,以免疫组化法检测大肠癌组织血管内皮生长因子（VEGF）的蛋白表达水平及其微血管密度（MVD）。结果 双歧杆菌预防组大肠癌VEGF的阳性细胞密度及MVD的数量均明显低于肿瘤对照组（P〈0．01）。结论 青春型双歧杆菌能下调大肠癌VEGF的表达,进而抑制其血管形成,这可能是它预防大肠癌生长的途径之一。