青蒿琥酯的抗血管生成作用

目的研究青蒿琥酯对血管生成的抑制作用。方法用人脐静脉内皮细胞(HUVEC)的生长、迁移及小管形成实验研究药物的体外抗血管生成作用;用人卵巢癌裸鼠移植瘤模型和免疫组化法研究药物的体内抗血管生成作用。结果青蒿琥酯浓度2.5 μmol·L^-1时,对HUVEC的增殖、迁移和小管形成均有显著的抑制作用;HUVEC 48 h的IC₅₀值为(21±3) μmol·L^-1。在整体实验中,青蒿琥酯50 mg·kg^-1·d^-1即可明显减少肿瘤的血管生成,抑制肿瘤生长;免疫组化结果表明,青蒿琥酯可以抑制VEGF和KDR/flk-1在肿瘤组织中的表达。结论青蒿琥酯有抗血管生成作用,提示该类药物在抗血管生成中有潜在的应用价值。     

Inhibition of human cancer cell line growth and human umbilical vein endothelial cell angiogenesis by artemisinin derivatives in vitro.

Artemisinin derivatives artesunate (ART) and dihydroartemisinin are remarkable anti-malarial drugs with low toxicity to humans. In the present investigation, we find they also inhibited tumor cell growth and suppressed angiogenesis in vitro. The anti-cancer activity was demonstrated by inhibition (IC(50)) of four human cancer cell lines: cervical cancer Hela, uterus chorion cancer JAR, embryo transversal cancer RD and ovarian cancer HO-8910 cell lines growth by the MTT assay. IC(50) values ranged from 15.4 to 49.7 microM or from 8.5 to 32.9 microM after treatment with ART or dihydroartemisinin for 48 h, indicating that dihydroartemisinin was more effective than ART in inhibiting cancer cell lines. The anti-angiogenic activities were tested on in vitro models of angiogenesis, namely, proliferation, migration and tube formation of human umbilical vein endothelial (HUVE) cells. We investigated the inhibitory effects of ART and dihydroartemisinin on HUVE cells proliferation by cell counting, migration into the scratch wounded area in HUVE cell monolayers and microvessel tube-like formation on collagen gel. The results showed ART and dihydroartemisinin significantly inhibited angiogenisis in a dose-dependent form in range of 12.5-50 microM and 2.5-50 microM, respectively. They indicated that dihydroartemisinin was more effective than ART in inhibiting angiogenesis either. These results and the known low toxicity are clues that ART and dihydroartemisinin may be promising novel candidates for cancer chemotherapy.     

Pseudolarix acid B inhibits angiogenesis by antagonizing the vascular endothelial growth factor-mediated anti-apoptotic effect

Angiogenesis is controlled by a number of growth factors, including vascular endothelial growth factor (VEGF). In this study, pseudolarix acid B, isolated from the traditional Chinese medicinal plant Pseudolarix kaempferi and originally identified as an early pregnancy-terminating agent, was evaluated for its potential as an angiogenesis inhibitor, using in vitro and in vivo models. After exposure to pseudolarix acid B 0.625-5 microM for 72 h, the proliferation of human umbilical vein endothelial cells was significantly inhibited. Pseudolarix acid B 0.313-2.5 microM for 24 h potently blocked the VEGF-induced tube formation of human umbilical vein endothelial cells in a dose-dependent manner. Matrigel plug assays disclosed that pseudolarix acid B reduced angiogenesis induced by VEGF in vivo. In addition, pseudolarix acid B antagonized VEGF-mediated anti-apoptotic effects on serum-deprived human umbilical vein endothelial cells and increased apoptosis of endothelial cells induced by VEGF in Matrigel plug assays. Moreover, pseudolarix acid B significantly inhibited VEGF-induced tyrosine phosphorylation of kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/flk-1), in correlation with a marked decrease in the phosphorylation of Akt and extracellular signal-regulated kinases (ERK). These findings collectively suggest that pseudolarix acid B possesses anti-angiogenic activity. One of the main anti-angiogenesis mechanisms of pseudolarix acid B may involve antagonism of the VEGF-mediated anti-apoptosis effect via inhibition of KDR/flk-1, ERK1/2, and Akt phosphorylation in endothelial cells.     

Effect of heparin affin regulatory peptide on the expression of vascular endothelial growth factor receptors in endothelial cells

BACKGROUND: Heparin affin regulatory peptide (HARP) is an 18-kDa secreted protein that has been implicated in tumor growth and angiogenesis, although the mechanisms involved remain largely unknown. In the present work, the effect of human recombinant HARP on the expression of the vascular endothelial growth factor (VEGF) receptors KDR, Flt-1 and neuropilin-1 was studied in cultured human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: The mRNA and protein levels of VEGF receptors were estimated by semi-quantitative RT-PCR and Western blot, respectively. Cell proliferation and migration were measured by MTT, direct counting of the cells and modified Boyden chamber assays. RESULTS: HARP decreased the expression of KDR but increased the expression of Flt-1 and neuropilin-1 at both the mRNA and protein level. The effect reached a maximum 4 h after the addition of HARP into the cell culture medium and was reversed at later time-points. When HARP was added to the culture medium 4 h before the addition of VEGF165, it inhibited VEGF165-induced proliferation and migration of HUVEC. CONCLUSION: These data suggest that HARP affects the expression of VEGF receptors and inhibits VEGF165-induced activation of HUVEC.     

青蒿琥酯抑制新生血管生成的作用

目的　为研究青蒿琥酯成为抗血管生成药物的可能性 ,观察其对新生血管的影响 ,并初步探讨其机制。方法　采用鸡胚绒毛尿囊膜、大鼠主动脉环无血清培养、人脐静脉内皮细胞损伤迁移等实验 ,检测青蒿琥酯对新生血管增殖与迁移的抑制作用。结果　鸡胚绒毛尿囊膜实验表明 ,青蒿琥酯有较强的血管增殖抑制作用 ,对微血管作用强于大血管 ;大鼠主动脉环无血清培养实验表明 ,青蒿琥酯能明显推迟血管新生 ,减少新生血管数量 ;人脐静脉内皮细胞损伤迁移实验表明青蒿琥酯对内皮细胞具有增殖和迁移抑制作用。在这些体内外实验中 ,青蒿琥酯抑制血管生成作用呈剂量依赖性。结论　青蒿琥酯具有抑制新生血管生成作用 ,此作用可能与抑制血管内皮细胞迁移有关。     

Chrysin inhibits lipopolysaccharide-induced angiogenesis via down-regulation of VEGF/VEGFR-2(KDR) and IL-6/IL-6R pathways

The relationship between chrysin and inflammation-induced angiogenesis remains unclear. The aim of this study was to evaluate the suppressive effects of chrysin on lipopolysaccharide (LPS)-induced angiogenesis in chicken chorioallantoic membrane (CAM) as well as in human umbilical endothelial cells (HUVEC). The IN VIVO CAM model was applied to evaluate the percentage of new vessels formation, followed by measuring endothelial migration and tube formation in HUVEC cultures. The mechanisms of the suppressive effect of chrysin on LPS-induced angiogenesis, in terms of VEGF, VEGF receptors (VEGFR), interleukin 6 (IL-6) and IL-6 receptor gene expressions, were analyzed by Western blot, ELISA cytokine assay, and quantitative real time PCR. The results showed that chrysin (10(-8) - 10(-5) M) inhibited LPS-induced CAM neovascular density. There was a significant down-regulation of VEGF and VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) gene expression by chrysin in LPS-treated HUVEC cultures. Besides, chrysin concentration-dependently inhibited the auto-regulation loop of IL-6/IL-6R in LPS-treated HUVEC cells. We conclude that chrysin suppresses both IN VITRO and IN VIVO LPS-induced angiogenesis.     

槲皮素抑制血管生成作用的实验研究

目的　研究槲皮素 (Quercetin)对血管生成和培养的人脐静脉内皮细胞 (HUVEC)的影响。方法　采用生长因子 (血管内皮细胞生长因子VEGF、碱性成纤维细胞生长因子bFGF)诱导的鸡胚绒毛尿囊膜 (CAM)血管增生模型观察槲皮素对血管生成的影响 ;利用培养的HUVEC ,用MTT法观察槲皮素抑制内皮细胞增殖的作用 ;流式细胞仪观察槲皮素对HUVEC细胞周期的影响。结果　槲皮素 (0 1、0 0 5和 0 0 2 5mmol·L-1)能明显抑制VEGF诱导的CAM小血管生成 ;槲皮素 (0 1和 0 0 5mmol·L-1)能明显抑制bFGF诱导的CAM小血管生成 ;槲皮素 (2 4 0、12 0 μmol·L-1和 6 0 μmol·L-1)对内皮细胞增殖有抑制作用 ,抑制率分别为 6 7 0 %、5 8 1%和39 7% ;槲皮素 (2 4 0、12 0 μmol·L-1)能显著导致HUVEC的S、G2 期阻滞。结论　槲皮素能抑制VEGF和bFGF诱导的血管生成 ,且对内皮细胞增殖具有抑制作用。     

A protein tyrosine phosphatase inhibitor accelerates angiogenesis in a rat model of hindlimb ischemia

Vascular endothelial growth factor (VEGF) receptor-2 (KDR/flk-1) has a tyrosine kinase domain and, once activated, induces the autophosphorylation of the tyrosine residues. The phosphorylated KDR/flk-1 can be a substrate for intracellular protein tyrosine phosphatases (PTPs). In the present study, we have examined whether the PTP inhibitor sodium orthovanadate (SOV) activates KDR/flk-1 and accelerates angiogenesis in a rat model of hindlimb ischemia. The left femoral artery was exposed and excised to induce limb ischemia. The PTP activity in ischemic adductors increased, whereas SOV significantly suppressed the increase in the activity. Tyrosine phosphorylation of KDR/flk-1 and Akt phosphorylation significantly increased in the muscles injected with SOV compared with those injected with saline. The amount of VEGF increased in both the muscles injected with SOV and those injected with the saline but did not differ significantly. At 21 days after the induction of ischemia, immunohistochemical studies demonstrated that muscles injected with SOV showed significantly increased capillary density compared with those injected with saline. In a rat model of hindlimb ischemia, not only VEGF but also PTP, which might impair angiogenesis, increased. SOV activated KDR/flk-1 and accelerated angiogenesis. Thus, a PTP inhibitor can be a new drug for therapeutic angiogenesis in peripheral ischemic diseases.     

松萝酸抑制小鼠H22肿瘤生长及作用机制研究

摘 要 目的： 探讨松萝酸对小鼠H22肿瘤生长的影响及其作用机制。方法: 昆明种小鼠前腋下皮下接种H22细胞,第2天腹腔注射松萝酸,每3 d测量1次肿瘤体积。用免疫组化法检测微血管密度（MVD）;ELISA 法检测小鼠血清和人脐静脉血管内皮细胞（HUVEC）细胞培养基上清中血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）水平;MTT法检测松萝酸对人脐静脉血管内皮细胞（HUVEC）体外增殖的影响。结果: 松萝酸组小鼠H22肿瘤体积、重量增长明显较模型组缓慢(P<0.05),同时松萝酸组的肿瘤微血管密度显著减少(P<0.05),松萝酸对HUVEC体外增殖有显著抑制作用。松萝酸可显著降低HUVEC细胞和小鼠血清VEGF和bFGF水平(P<0.01)。结论:松萝酸可显著抑制小鼠H22肿瘤生长及血管生成,且能抑制VEGF和bFGF表达和HUVEC体外增殖。松萝酸对VEGF和bFGF分泌的抑制作用可能是其抗H22肿瘤及抗血管生成的一个重要机制。     

半边旗提取物5F对高转移卵巢癌细胞HO-8910PM侵袭转移的影响

目的研究半边旗提取物5F(以下简称5F)对人高转移卵巢癌细胞HO8910PM转移相关能力的影响及其作用的机制。方法以MTT法检测5F对高转移卵巢癌细胞HO8910PM细胞增殖的影响;以细胞粘附人工重组基底膜实验检测5F对HO8910PM细胞粘附能力的影响;用Transwell小室法检测5F对HO8910PM细胞的侵袭能力和趋化运动能力的影响;Westernblot法检测5F对HO8910PM细胞NFκB(P65)和VEGF蛋白表达的影响。结果50μmol·L-1的5F作用细胞6h后能抑制HO8910PM细胞体外侵袭人工基底膜、趋化运动和粘附能力,抑制率分别为(37.57±0.62)%,(28.42±0.67)%,(46.07±4.49)%;5F能明显下调HO8910PM细胞中NFκB(P65)和VEGF蛋白的表达。结论5F能抑制高转移卵巢癌细胞HO8910PM的侵袭、运动和粘附能力。5F抗肿瘤侵袭转移的作用机制与NFκB(P65)和VEGF蛋白的表达下调有关。     

血管内皮生长因子受体在胶质瘤中的表达

目的 研究人脑胶质瘤组织中血管内皮生长因子受体(KDR、Flt-1)mRNA的表达，同时分析肿瘤间质血管增生的情况，并探讨两之间的关系。方法 采用半定量逆转录-聚合酶链反应(RT-PCR)方法，对49例人脑胶质瘤手术切除标本，U251等3株胶质瘤细胞、人类脐静脉内皮细胞(HUVEC)以及12例脑外伤内减压术中得到的正常脑组织检测了KDR、Flt-1 mRNA表达水平。另外，用免疫组化SP法对胶质瘤及正常脑组织的微血管密度(MVD)进行了分析。结果 胶质瘤组织中的KDR、Flt-1 mRNA表达水平明显增高，分别达59．2％、63．2％，随着病理分级的提高其表达水平有升高趋势，HUVEC及3株胶质瘤细胞亦表达水平较高。胶质瘤中的微血管密度(MVD)与胶质瘤级别有关，KDR、Flt-1 mRNA的表达同MVD呈正相关。结论 KDR、Flt-1、MVD对胶质瘤的恶性生物学行为评估有重要意义，血管内皮生长因子受体的高表达与肿瘤组织血管增生有关，在胶质瘤侵袭过程中发挥重要作用。     

Sanguiin H-6 blocks endothelial cell growth through inhibition of VEGF binding to VEGF receptor

The vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, which is a process where new blood vessels develop from the endothelium of a pre-existing vasculature. VEGF exerts its activity by binding to its receptor tyrosine kinase, KDR/Flk-1, which is expressed on the surface of endothelial cells. A methanol extract and organic solvent (n-hexane, ethyl acetate, n-butanol, aqueous) fractions from Rubus coreanus were examined for their inhibitory effects on VEGF binding to the VEGF receptor. The methanol extract from the crude drug were found to significantly inhibit VEGF binding to the VEGF receptor (IC50 approximately = 27 microg/mL). Among the fractions examined, the aqueous fraction from the medicinal plant showed potent inhibitory effects against the binding of KDR/Flk-1-Fc to immobilized VEGF165 in a dose-dependent manner (IC50 approximately = 11 microg/mL). Sanguiin H-6 was isolated as an active principle from the aqueous fraction, and inhibited the binding of KDR/Flk-1-Fc to immobilized VEGF165 in a dose-dependent manner (IC50 approximately = 0.3 microg/mL). In addition, sanguiin H-6 efficiently blocked the VEGF-induced HUVEC proliferation in a dose-dependent manner (IC50 approximately = 7.4 microg/mL) but had no effect on the growth of HT1080 human fibrosarcoma cells. This suggests that sanguiin H-6 might be a potential anti-angiogenic agent.     

Novel 2,3-dihydro-1,4-benzoxazines as potent and orally bioavailable inhibitors of tumor-driven angiogenesis

Angiogenesis is vital for solid tumor growth, and its prevention is a proven strategy for the treatment of disease states such as cancer. The vascular endothelial growth factor (VEGF) pathway provides several opportunities by which small molecules can act as inhibitors of endothelial proliferation and migration. Critical to these processes is signaling through VEGFR-2 or the kinase insert domain receptor (KDR) upon stimulation by its ligand VEGF. Herein, we report the discovery of 2,3-dihydro-1,4-benzoxazines as inhibitors of intrinsic KDR activity (IC 50 < 0.1 microM) and human umbilical vein endothelial cell (HUVEC) proliferation with IC 50 < 0.1 microM. More specifically, compound 16 was identified as a potent (KDR: < 1 nM and HUVEC: 4 nM) and selective inhibitor that exhibited efficacy in angiogenic in vivo models. In addition, this series of molecules is typically well-absorbed orally, further demonstrating the 2,3-dihydro-1,4-benzoxazine moiety as a promising platform for generating kinase-based antiangiogenic therapeutic agents.     

Inhibition of vascular endothelial growth factor (VEGF)-induced endothelial proliferation, arterial relaxation, vascular permeability and angiogenesis by dobesilate

Vascular endothelial growth factor (VEGF) is a key factor in angiogenesis and vascular permeability which is associated with many pathological processes. 2,5-hydroxybenzene sulfonate (DHBS; dobesilate) is a small molecule with anti-angiogenic activity that has been described as an inhibitor of fibroblast growth factors (FGF). The aim of the present study was to evaluate the effects of DHBS on VEGF-induced actions. The effects of DHBS were evaluated on VEGF-induced proliferation in human umbilical vein endothelial cells (HUVEC) and rat aorta relaxation, as well as on in vivo VEGF-induced skin vascular permeability and neovascularization in rats. DHBS at 50 and 100 μM concentration significantly inhibited the proliferation of HUVEC induced by VEGF (10 ng/ml), without significantly affecting HUVEC proliferation in the absence of VEGF. Rapid VEGF-induced activation of Akt in HUVEC was also prevented by DHBS (100 μM). Additionally, DHBS (2 μM) specifically inhibited the relaxation of rat aorta induced by VEGF (0.1 to 30 ng/ml), but not endothelium-dependent relaxation to acetylcholine (1 nM to 10 μM). The in vivo enhancement of vascular permeability caused by VEGF injection (50 μl at 10 ng/ml) in rat skin was also inhibited by DHBS co-administration (200 μM) (74.8±3.8% inhibition of dye extravasation). Administration of DHBS (200 mg/kg/day; i.p.) also reduced VEGF-induced angiogenesis in vivo. DHBS inhibits main responses elicited in vitro and in vivo by VEGF. As a dual antagonist of VEGF and FGF activities, DHBS could be of therapeutic interest in the treatment of diseases related to VEGF/FGF overproduction and excessive angiogenesis.     

芸香宁碱抑制血管生成作用的研究

目的：研究芸香宁碱对血管生成的抑制作用,并研究其初步的作用机制。方法用人脐静脉内皮细胞（HUVEC）的生长、迁移实验及体内动物模型－鸡胚绒毛尿囊膜法（CAM 法）研究化合物的体内外抗血管生成作用;用酶联免疫吸附法检测芸香宁碱在非凋亡剂量时对肿瘤细胞培养上清液中 VEGF 蛋白分泌量的影响。结果芸香宁碱对血管内皮细胞具有优先抑制作用,对 HUVEC 的半数抑制剂量为（33．2±0．4）μmol·L －1,而对其他肿瘤细胞的 IC50值均大于这一数值;芸香宁碱在体外能够明显抑制内皮细胞的迁移和对细胞外基质黏附作用,并呈剂量依赖性,体内实验显示30μmol·L －1剂量浓度时显著抑制 CAM新生血管形成;芸香宁碱在15μmol·L －1和30μmol·L －1的浓度剂量时显著抑制人肝癌 HepG2细胞培养上清液中VEGF 蛋白的分泌,具有显著性差异。结论芸香宁碱在非凋亡浓度剂量具有抑制新生血管形成作用,其机制与抑制血管内皮细胞增殖以及抑制肿瘤细胞表达 VEGF 有关。     

Inhibition of angiogenesis involves in anticancer activity of riccardin D, a macrocyclic bisbibenzyl, in human lung carcinoma

Riccardin D is a novel macrocyclic bisbibenzyl compound extracted from Chinese liverwort plant Dumortiera hirsuta. Our previous studies showed that riccardin D is a DNA topo II inhibitor and has therapeutic potential for treatment of cancers. In this combined in vitro and in vivo study, we examined the inhibitory effects of riccardin D on tumor angiogenesis and the subsequent effect of anticancer activity was evaluated. Incubation with riccardin D weakly inhibited the proliferation of human umbilical vascular endothelial cells (HUVEC) as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The scratch wound experiment showed that riccardin D effectively decreased the motility and migration of HUVEC cells. Riccardin D inhibited the formation of capillary tube as demonstrated by decrease of branch points formed by HUVEC cells on 3-D Matrigel. We examined the levels of angiogenic factors including vascular endothelial growth factor (VEGF), VEGF receptor 2, epidermal growth factor receptor (EGF receptor), and matrix metalloproteinase (MMPs) in HUVEC cells. The expressions of VEGF, phospho-VEGF receptor 2, EGF receptor and MMP-2 were significantly reduced by riccardin D as estimated by Western blot assay and real-time quantitative PCR analysis. The decrease of VEGF was also detected in riccardin D-treated human lung cancer H460 cells. The anticancer activity of riccardin D was then evaluated in a mouse model in which riccardin D delayed the growth of H460 xenografts without obvious toxicity to animals after three weeks injection. To evaluate the role of antiangiogenesis of riccardin D in mice, CD34 immunohistochemical staining was employed to analyze the mean vascular density in H460 xenograft tissues. The number of blood vessels was significantly decreased after riccardin D treatment. These results suggest that riccardin D display the inhibitory effect on growth of human lung carcinoma cells and that the inhibition of angiogenesis may involve in anticancer activity of riccardin D.     

卡立泊来德对K562细胞诱导的血管生成的影响

目的研究卡立泊来德(cariporide)处理对K562细胞诱导的血管生成能力的影响。方法应用MTT检测K562细胞上清液对脐静脉内皮细胞增殖能力的影响;transwell检测K562细胞上清液对脐静脉内皮细胞迁移能力的影响;基质胶血管形成法检测K562细胞上清液对脐静脉内皮细胞体外血管形成能力的影响;激光扫描共聚焦显微镜测定K562细胞的细胞内的pH;酶联免疫吸附实验(ELISA)检测K562细胞上清中血管内皮生长因子的表达水平。结果cariporide处理可以明显降低K562细胞上清液对脐静脉内皮细胞增殖,迁移和体外成管能力的诱导;cariporide处理后K562细胞的细胞内pH明显下降,分泌VEGF能力也受到抑制。结论 cariporide能抑制K562细胞的血管生成诱导能力,这种抑制是通过细胞内pH下降以及VEGF分泌减少引起的。     

Uncaria rhynchophylla induces angiogenesis in vitro and in vivo

Angiogenesis consists of the proliferation, migration, and differentiation of endothelial cells, and angiogenic factors and matrix protein interactions modulate this process. The aim of this study was to determine the angiogenic properties of Uncaria rhynchophylla. Uncaria rhynchophylla significantly enhanced human umbilical vein endothelial cells (HUVECs) proliferation in a dose-dependent manner. Neutralization of vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) by monoclonal antibody suppressed the Uncaria rhynchophylla stimulatory effect on proliferation. In addition, Uncaria rhynchophylla significantly increased chemotactic-migration on gelatin and tubular structures on Matrigel of HUVECs in a dose-dependent manner. Interestingly, Uncaria rhynchophylla dose-dependently increased VEGF, and bFGF gene expression and protein secretion of HUVEC. The angiogenic activity of Uncaria rhynchophylla was confirmed using an in vivo Matrigel angiogenesis model, showing promotion of blood vessel formation. These results suggest that Uncaria rhynchophylla could potentially used to accelerate vascular wound healing or to promote the growth of collateral blood vessel in ischemic tissues.     

番茄红素异构体对人卵巢癌HO-8910细胞增殖和凋亡的影响效果初探

目的研究番茄红素异构体对体外培养的人卵巢癌HO-8910细胞增殖和凋亡的影响。方法观察含不同比例的顺、反式异构体番茄红素对人卵巢癌HO-8910细胞的增殖抑制作用;采用PI流式细胞仪检测细胞凋亡率。结果番茄红素异构体对人卵巢癌HO-8910细胞有一定程度的抑制增殖和诱导凋亡的作用,且呈浓度依赖性。结论对人卵巢癌HO-8910细胞,含较高比例顺式番茄红素的Ⅱ号药组抑制增殖与诱导凋亡的作用比全反式番茄红素的I号药组具更强的生物活性。