新的血管抑素抗血管生成作用的实验研究

目的 观察一种新的血管抑素对新生务管的抑制作用，井初步探讨其作用靶点及机制。方法 采用人脐静脉内皮细胞增殖、迁移法。体内采用C57BL／CN纯系小鼠及鸡胚尿囊绒膜（CAM）模型，观察新生血管生成情况。结果 新的血管抑素在体外对血管内皮细胞增殖、迁移具有明显的抑制作用，在体内使用C57BL／CN纯系小鼠皮下移植瘤体积明显缩小，并对CAM新生血管也具有明显抑制作用，具有剂量依赖性。结论 新的血管抑素能明显抑制新生血管的形成，其机制可能通过抑制血管内皮细胞来阻断新生血管形成。     

新的杂合肽抗肿瘤血管生成的作用机制

目的探讨具有生物活性的小分子杂合肽对肿瘤血管生成的抑制作用机制。方法利用多肽仪合成新的杂合肽并对其合成产物进行体内外实验活性分析。结果在体外新的杂合肽在浓度为1.8μmol/L并在48 h对Lewis肿瘤细胞增殖及迁移具有明显抑制作用;在体内新的杂合肽在浓度为120μmol/L、并连续治疗21 d时能明显抑制碱烧伤后兔角膜新生血管及C57BL/6N肿瘤小鼠皮下移植瘤新生血管,使肿瘤体积明显减小,抑制程度具有一定的时-量依赖关系。并在细胞水平与整体水平上具有一定相关性。结论新的杂合肽能够明显抑制荷瘤鼠肿瘤的生长,其作用机制可能与抑制血管内皮细胞生成有关。     

熊果酸对膀胱癌T-24细胞生长的影响

目的 探讨三萜类化合物熊果酸(UA)对人膀胱癌T-24细胞增殖、迁移及裸鼠皮下移植瘤生长的抑制作用及其机制.方法 应用熊果酸处理体外培养的人膀胱癌T-24细胞,四甲基偶氮唑蓝(MTT)比色法、细胞迁移法检测UA对T-24细胞的增殖抑制效应;建立膀胱癌皮下移植瘤模型,观察UA对移植瘤的生长抑制作用.结果 体内﹑外实验结果显示:各治疗组UA能较明显抑制T-24细胞增殖、迁移,并在48 h抑制作用明显.在体内使裸鼠皮下移植瘤体积明显缩小,并表现具有一定的剂量-时间依赖关系.结论 UA能明显抑制肿瘤细胞生成,其作用靶点可能为肿瘤新生血管内皮细胞.     

Vasostatin与肿瘤血管生成

血管生成在肿瘤生长和转移的过程中具有重要作用,涉及一系列血管生成、抑制因子的表达和调控。内源性血管抑制因子vasostatin具有显著的抑制内皮细胞增殖和血管生成的功能,已有研究显示va-sostatin单独应用或联合其他治疗对多种恶性肿瘤有较好的治疗效果。其作用机制可能与vasostatin与层黏连蛋白(Laminin)结合,阻止由bFGF等引起的内皮细胞增生等作用有关。进一步研究vasostatin对肿瘤血管生成的抑制作用及其机制,有助于临床寻找新的有效分子靶向药物。     

海水浸泡降低血管内皮细胞迁移能力并抑制血管生成

目的:观察海水浸泡对血管内皮细胞迁移和血管生成的影响,为探讨海水致伤机制、制定科学救治原则提供实验依据.方法:建立ECV304细胞划痕创伤模型,分别给予生理盐水和人工海水浸泡处理,划痕创伤愈合修复分析观察细胞迁移能力;建立人食管鳞癌EC0156裸鼠皮下移植瘤模型,分别给予生理盐水和人工海水干预,监测肿瘤体积并绘制肿瘤生长曲线,观察肿瘤边缘血管的生成,免疫组织化学染色检测肿瘤组织内微血管密度.结果:海水浸泡后,ECV304细胞失去正常形态,并随浸泡时间的延长,呈明显"脱水"状态.在划痕后17 h,对照组划痕已基本愈合,而实验组仍可见清晰划痕,且以实验4 h组划痕尤其明显.与对照组相比,实验组细胞划痕愈合明显延迟,细胞迁移能力降低;与对照组相比实验组肿瘤边缘新生血管较少,前者肿瘤边缘新生血管较丰富,两者有显著性差异(微血管密度值:1.2±0.44 vs 3.2±0.83,P<0.05).实验组肿瘤生长缓慢,肿瘤体积较小(P<0.05).结论:海水浸泡能够降低血管内皮细胞迁移能力并抑制血管生成.     

Vasostatin基因对胰腺癌血管生成的抑制作用

目的研究 vasostatin 对体内、外血管生成的作用,探讨其对胰腺癌生长抑制的作用机制。方法应用小管形成实验研究 vasostatin 对体外血管生成的影响,鸡胚绒毛尿囊膜实验观察 vasostatin 对体内血管生成的作用。复制人胰腺癌裸鼠移植瘤模型,瘤内注射10~8pfu Ad-vasostatin、10~8pfu Ad-lacZ 及缓冲液,观察移植瘤生长曲线及瘤重。应用免疫组化法检测肿瘤微血管密度(MVD),研究 vasostatin 对胰腺癌移植瘤血管生成的影响。结果体外实验中,Ad-vasostatin 组细胞数较少,细胞排列连续性差,可见条索状细胞链,但中空闭合管状结构缺如。鸡胚绒毛尿囊膜实验提示 Ad-vasostatin 组的血管纹理欠清晰,小血管分布凌乱、稀疏,形态不规整,小血管分支少,多数在第二、第三级,极少数达到第四级。肿瘤生长3周后,Ad-vasostatin 组移植瘤瘤重(112mg±78mg)显著小于 Ad-laeZ 组(322mg±148mg)(P<0.05)及缓冲液组(468mg±160mg)(P<0.01)。免疫组化结果提示,Ad-vasostatin 组肿瘤组织 MVD[(76.2±16.8)个/mm~2]显著低于 Ad-lacZ 组[(144.0±16.6)个/mm~2]与缓冲液组[(147.0±22.7)个/mm~2)(P<0.05)。结论腺病毒介导的 vasostatin 基因可显著抑制体内、外血管生成,vasostatin 基因对人胰腺癌裸鼠移植瘤血管生成的抑制作用是其抑制人胰腺癌裸鼠移植瘤生长的重要原因。     

血管内皮细胞迁移与肿瘤血管生成

肿瘤生长和转移均依赖于肿瘤的血管生成,而血管内皮细胞迁移是肿瘤血管生成过程的一个重要环节,有着重要的研究意义.现从肿瘤血管生成过程中促血管生成因子、骨架蛋白解聚、细胞外基质的降解三个方面对血管内皮细胞迁移的影响进行综述.     

血管抑制素基因抑制结肠癌细胞增殖及肿瘤血管生成的研究

目的:研究血管抑制素基因体外对结肠癌细胞增殖的抑制及体内对肿瘤血管生成的抑制,探讨血管抑制素基因对结肠癌的抑制作用。方法:构建重组质粒pcDNA3.1( )／angio,用脂质体转染法将重组质粒、空白载体导入结肠癌细胞Colo205,培养细胞观察细胞生长,绘制细胞生长曲线,计算增殖抑制率,然后将pcDNA3.1( )／angio、pcDNA3.1( )／Colo205、Colo205分别接种至裸鼠右侧颈部皮下,观察肿瘤的生长,测量肿瘤体积,计算抑瘤率,免疫组化检测肿瘤组织中微血管密度(MVD)、血管内皮生长因子(VEGF)的表达。结果:体外pcDNA3.1( )／angio组细胞的生长速度略慢于pcDNA3.1( )／Colo205、Colo205组,但差异无统计学意义(P>0.05);体内pcDNA3.1( )／angio组细胞的致力显著降低,瘤体增长缓慢,抑瘤率较高(P<0.01),瘤体组织中MVD及VEGF的表达较低(P<0.05);pcDNA3.1( )／Colo205、Colo205两组之间的差异无统计学意义(P>0.05)。结论:在体外血管抑制素不直接影响结肠癌细胞Colo205的生物学特性,但在体内可强烈抑制肿瘤的生长,其机制可能是通过降低血管生成因子而抑制肿瘤的新生血管生成,发挥抑制肿瘤作用。     

胃癌细胞血管内皮生长因子RNA干扰对血管内皮细胞生长的影响

目前抗肿瘤血管生成的研究多在动物模型中进行，这样虽然可以模拟肿瘤生长的微环境，但由于受到体内环境诸多因素的影响，促血管生成因子人为表达降低后，血管内皮细胞的生物学行为发生了哪些变化难以明确。采用分泌促血管生成因子的肿瘤细胞与血管内皮细胞共培养可以初步明确这一问题。本研究采用RNA干扰（RNAi）技术观察其抑制肿瘤细胞血管内皮生长因子（VEGF）表达后对血管内皮细胞生长的影响。     

肺癌与抗血管生成治疗的新进展

肺癌与其它实体瘤一样 ,其发生、发展和转移均依赖于血管生成 ,以新生血管为靶点 ,抑制肿瘤血管生成、阻断肿瘤的营养来源和迁移通道 ,这已成为近年来癌症治疗的新策略。我们就近年来国外此领域的最新进展综述如下。一、肺癌组织中血管生成的调控血管内皮细胞增殖在肿瘤组织中比正常组织中快 30～40倍 ,当肿瘤生长至 1～ 2ｍｍ3 大小后 ,其继续长大必须依赖于新血管的生成 ,而血管生成是一个多步骤的复杂过程 ,并由多种血管生成因子和血管生成抑制因子共同调控所决定的。1 血管生成因子 :在多种血管生成因子中 ,血管内皮生长因子 (ＶＥＧＦ)…     

A peptide derived from the non-receptor-binding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis in vivo in combination with cisplatin

The urokinase plasminogen activator system is involved in angiogenesis and tumor growth of malignant gliomas, which are highly neovascularized and so may be amenable to antiangiogenic therapy. In this paper, we describe the activity of A6, an octamer capped peptide derived from the non-receptor-binding region of urokinase plasminogen activator. A6 inhibited human microvascular endothelial cell migration but had no effect on the proliferation of human microvascular endothelial cells or U87MG glioma cells in vitro. In contrast, A6 or cisplatin (CDDP) alone suppressed subcutaneous tumor growth in vivo by 48% and 53%, respectively, and, more strikingly, the combination of A6 plus CDDP inhibited tumor growth by 92%. Such combination treatment also greatly reduced the volume of intracranial tumor xenografts and increased survival of tumor-bearing animals when compared with CDDP or A6 alone. Tumors from the combination treatment group had significantly reduced neovascularization, suggesting a mechanism involving A6-mediated inhibition of endothelial cell motility, thereby eliciting vascular sensitivity to CDDP-mediated toxicity. These data suggest that the combination of an angiogenesis inhibitor that targets endothelial cells with a cytotoxic agent may be a useful therapeutic approach.     

Antibody against murine PECAM-1 inhibits tumor angiogenesis in mice

Platelet endothelial cell adhesion molecule (PECAM-1/CD31), a member of the immunoglobulin superfamily expressed at high levels on endothelial cells, has been recently implicated in angiogenesis. Although antagonism of PECAM-1 inhibited neovascularization in two different animal models of growth factor/chemokine-induced angiogenesis, its participation in tumor angiogenesis has not been established. We therefore investigated its involvement in models of tumor angiogenesis in mice. An antibody against murine PECAM-1 that was shown to block in vitro murine endothelial tube formation inhibited the subcutaneous growth and tumor vascularity of three tumors in mice: A549 human non-small cell lung cancer in SCID mice, B16 murine melanoma in C57BL/6 mice and AB12 murine mesothelioma in Balb/c mice. These studies suggest a possible role for PECAM-1 in the complex process of tumor angiogenesis and provide additional evidence of the importance of endothelial cell adhesion molecules to the formation of new vessels. This revised version was published online in June 2006 with corrections to the Cover Date.     

Notch signaling regulates tumor-induced angiogenesis in SPARC-overexpressed neuroblastoma

Despite existing aggressive treatment modalities, the prognosis for advanced stage neuroblastoma remains poor with significant long-term illness in disease survivors. Advance stage disease features are associated with tumor vascularity, and as such, angiogenesis inhibitors may prove useful along with current therapies. The matricellular protein, secreted protein acidic and rich in cysteine (SPARC), is known to inhibit proliferation and migration of endothelial cells stimulated by growth factors. Here, we sought to determine the effect of SPARC on neuroblastoma tumor cell-induced angiogenesis and to decipher the molecular mechanisms involved in angiogenesis inhibition. Conditioned medium from SPARC-overexpressed neuroblastoma cells (pSPARC-CM) inhibited endothelial tube formation, cell proliferation, induced programmed cell death and suppressed expression of pro-angiogenic molecules such as VEGF, FGF, PDGF, and MMP-9 in endothelial cells. Further analyses revealed that pSPARC-CM-suppressed expression of growth factors was mediated by inhibition of the Notch signaling pathway, and cells cultured on conditioned medium from tumor cells that overexpress both Notch intracellular domain (NICD-CM) and SPARC resumed the pSPARC-CM-suppressed capillary tube formation and growth factor expression in vitro. Further, SPARC overexpression in neuroblastoma cells inhibited neo-vascularization in vivo in a mouse dorsal air sac model. Furthermore, SPARC overexpression-induced endothelial cell death was observed by co-localization studies with TUNEL assay and an endothelial marker, CD31, in xenograft tumor sections from SPARC-overexpressed mice. Our data collectively suggest that SPARC overexpression induces endothelial cell apoptosis and inhibits angiogenesis both in vitro and in vivo.     

Inhibition of angiogenesis by vitamin D-binding protein: characterization of anti-endothelial activity of DBP-maf

Angiogenesis is a complex process involving coordinated steps of endothelial cell activation, proliferation, migration, tube formation and capillary sprouting with participation of intracellular signaling pathways. Regulation of angiogenesis carries tremendous potential for cancer therapy. Our earlier studies showed that vitamin D-binding protein-macrophage activating factor (DBP-maf) acts as a potent anti-angiogenic factor and inhibits tumor growth in vivo. The goal of this investigation was to understand the effect of DBP-maf on human endothelial cell (HEC) and the mechanism of angiogenesis inhibition. DBP-maf inhibited human endothelial cell (HEC) proliferation by inhibiting DNA synthesis (IC(50) = 7.8 +/- 0.15 microg/ml). DBP-maf significantly induced S- and G0/G1-phase arrest in HEC in 72 h. DBP-maf potently blocked VEGF-induced migration, tube-formation of HEC in a dose dependent manner. In addition, DBP-maf inhibited growth factor-induced microvessel sprouting in rat aortic ring assay. Moreover, DBP-maf inhibited VEGF signaling by decreasing VEGF-mediated phosphorylation of VEGFR-2 and ERK1/2, a downstream target of VEGF signaling cascade. However, Akt activation was not affected. These studies collectively demonstrate that DBP-maf inhibits angiogenesis by blocking critical steps such as HEC proliferation, migration, tube formation and microvessel sprouting. DBP-maf exerts its effect by inhibiting VEGR-2 and ERK1/2 signaling cascades. Understanding the cellular and molecular mechanisms of anti-endothelial activity of DBP-maf will allow us to develop it as an angiogenesis targeting novel drug for tumor therapy.     

IFN-gamma-mediated inhibition of tumor angiogenesis by natural killer T-cell ligand,alpha-galactosylceramide

Alpha-galactosylceramide (alpha-GalCer), which is a specific ligand for CD1d-restricted variable-alpha14 chain (V(alpha)14) natural killer T (NKT) cells, exerts a potent antitumor effect. We recently demonstrated that interferon-gamma (IFN-gamma) secreted by both NKT cells and NK cells plays a critical role in mediating the antimetastatic effect of alpha-GalCer; however, the IFN-gamma-dependent antitumor mechanisms remain poorly defined. In the present study, we demonstrate IFN-gamma-dependent inhibition of tumor angiogenesis by alpha-GalCer. In alpha-GalCer-treated mice, subcutaneous tumor growth and tumor-induced angiogenesis were inhibited in an IFN-gamma-dependent manner. The alpha-GalCer-activated splenic or hepatic mononuclear cells inhibited murine endothelial cell proliferation in vitro, and this inhibitory effect was mediated mostly by IFN-gamma produced by NKT cells and NK cells. NK cell depletion resulted in significant but partial inhibition of tumor growth and angiogenesis in vivo. These results suggest that the IFN-gamma-mediated inhibition of tumor angiogenesis is critically involved in the effector mechanisms of antitumor effects evoked by alpha-GalCer.     

Treatment of metastatic colorectal carcinomas by systemic inhibition of vascular endothelial growth factor signaling in mice

AIM: Tumor angiogenesis has been shown to be promoted by vascular endothelial growth factor (VEGF) via stimulating endothelial cell proliferation, migration, and survival. Blockade of VEGF signaling by different means has been demonstrated to result in reduced tumor growth and suppression of tumor angiogenesis in distinct tumor entities. Here, we tested a recombinant adenovirus, AdsFIt1-3, that encodes an antagonistically acting fragment of the VEGF receptor 1 (Flt-1), for systemic antitumor effects in pre-established subcutaneous CRC tumors in mice. METHODS: Murine colorectal carcinoma cells (CT26) were inoculated subcutaneously into Balb/c mice for in vivo studies. Tumor size and survival were determined. 293 cell line was used for propagation of the adenoviral vectors. Human lung cancer line A549 and human umbilical vein endothelial cells were transfected for in vitro experiments. RESULTS: Infection of tumor cells with AdsFlt1-3 resulted in protein secretion into cell supernatant, demonstrating correct vector function. As expected, the secreted sFlt1-3 protein had no direct effect on CT26 tumor cell proliferation in vitro, but endothelial cell function was inhibited by about 46% as compared to the AdLacZ control in a tube formation assay. When AdsFlt1-3 (5×109 PFU/animal) was applied to tumor bearing mice, we found a tumor inhibition by 72% at d 12 after treatment initiation. In spite of these antitumoral effects, the survival time was not improved. According to reduced intratumoral microvessel density in AdsFIt1-3-treated mice, the antitumor mechanism can be attributed to angiostatic vector effects. We did not detect increased systemic VEGF levels after AdsFlt1-3 treatment and liver toxicity was low as judged by serum alanine aminotransferase determination. CONCLUSION: In this study we confirmed the value of a systemic administration of AdsFIt1-3 to block VEGF signaling as antitumor therapy in an experimental metastatic colorectal carcinoma model in mice.     

Examining the role of Rac1 in tumor angiogenesis and growth: a clinically relevant RNAi-mediated approach

Angiogenesis, the sprouting of new blood vessels from the pre-existing vasculature, is a well established target in anti-cancer therapy. It is thought that the Rho GTPase Rac1 is required during vascular endothelial growth factor (VEGF)-mediated angiogenesis. In the present study, we have used a clinically relevant RNA interference approach to silence Rac1 expression. Human umbilical vein endothelial cells were transiently transfected with non-specific control siRNA (siNS) or Rac1 siRNA (siRac1) using electroporation or Lipofectamine 2000. Functional assays with transfected endothelial cells were performed to determine the effect of Rac1 knockdown on angiogenesis in vitro. Silencing of Rac1 inhibited VEGF-mediated tube formation, cell migration, invasion and proliferation. In addition, treatment with Rac1 siRNA inhibited angiogenesis in an in vivo Matrigel plug assay. Intratumoral injections of siRac1 almost completely inhibited the growth of grafted Neuro2a tumors and reduced tumor angiogenesis. Together, these data indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis. Knockdown of Rac1 may represent an attractive approach to inhibit tumor angiogenesis and growth.     

Platelet-endothelial interaction in tumor angiogenesis and microcirculation

Activated platelets release angiogenic growth factors and have therefore been proposed to contribute to tumor angiogenesis within a potentially prothrombotic tumor microcirculation. The aim of the study was to investigate interactions of platelets with the angiogenic microvascular endothelium of highly vascularized solid tumors during growth and in response to endothelial stimulation in comparison with normal subcutaneous tissue. Experiments were performed in the dorsal skinfold chamber preparation of C57BL/6J mice bearing the Lewis lung carcinoma (LLC-1) or methylcholanthrene-induced fibrosarcoma (BFS-1). Fluorescently labeled rolling and adherent platelets, red blood cell velocity, and vessel diameters were assessed by intravital fluorescence microscopy on days 1, 3, 8, and 14 after tumor cell implantation. Slightly elevated numbers of rolling platelets were observed in the early stages of tumor angiogenesis at day 1 (control, 1.7 +/- 0.6; LLC-1, 3.4 +/- 1.8; BFS-1, 3.0 +/- 0.7 [1/mm/s], P <.05) and day 3 (control, 1.6 +/- 0.6; LLC-1, 4.1 +/- 1.7, P <.05; BFS-1, 2.3 +/- 0.5 [1/mm/s]) after tumor cell implantation. Endothelial stimulation with calcium ionophore A23187 at day 14 after tumor cell implantation resulted in a minor increase to 2.1 +/- 0.4 (LLC-1) and 1.8 +/- 0.8 (BFS-1) rolling platelets (1/mm/s) in tumor microvessels compared with 4.9 +/- 0.9 in controls (P <.05). Platelet adherence was not observed. We therefore conclude that in the 2 experimental tumors under study, (1) slightly increased platelet rolling is a transient phenomenon after tumor cell implantation, and (2) platelet-endothelial interaction in response to endothelial stimulation is reduced in tumor microvessels.     

Interaction of endostatin with integrins implicated in angiogenesis

Endostatin, a fragment of collagen XVIII, is a potent antagonist of angiogenesis and inhibitor of tumor growth in mouse models. At present, the mechanism of action of endostatin is unknown. We show here that recombinantly produced human endostatin interacts with alpha(5)- and alpha(v)-integrins on the surface of human endothelial cells. We further demonstrate that the endostatin-integrin interaction is of functional significance in vitro, as we found that immobilized endostatin supports endothelial cell survival and migration in an integrin-dependent manner. Soluble endostatin in turn inhibits integrin-dependent endothelial cell functions, such as cell migration. Taken together, these results implicate integrins as potential targets for endostatin function and support the importance of integrins in endothelial cell biology and angiogenesis.