In vitro inhibition of angiogenesis by hydroalcoholic extract of oak (Quercus infectoria) acorn shell via suppressing VEGF,MMP-2, and MMP-9 secretion

Context: Angiogenesis is an essential factor for cancer progression. Although more attention is paid in angiogenesis on its role in cancer biology, many other non-neoplastic diseases are also angiogenic-dependent. Recently, there is motivation to control cancer via inhibition of angiogenesis.

Objective: Quercus infectoria Olivier var (Fagaceae) (oak) is a plant whose different parts, such as its fruit shell, have been used extensively as a traditional drug in the west part of Iran. Although some biological properties of oak are determined, its effects on angiogenesis are unclear. So, we investigated the antiangiogenic effects of oak acorn shell.

Materials and methods: Fresh oak acorns were collected, and after authentication; hydroalcoholic extract of acorn shells (5, 10, 20, 30, 40, 60, 80, and 100 μg/ml) was used for evaluation of its cytotoxicity, antiproliferative, and antiangiogenic effects in vitro. Also, effects of the extract on vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and MMP-9 secretion were assayed using enzyme-linked immunosorbent assay (ELISA) and gelatin zymography. Results: Treatment with hydroalcoholic extract in eight doses resulted in a significant decrease of endothelial cell proliferation and angiogenesis with an IC₅₀ value of ~20 μg/ml, without any toxic effect. At 40 μg/ml, the extract inhibited MMP-9 activity; however, a dose-dependent reduction (60–80 µg/ml) in MMP-2 activity was seen. VEGF secretion was decreased with increase in the concentration of the extract from 5 to 100 μg/ml.

Discussion and conclusion: This study indicated that hydroalcoholic extract of oak acorn shell acts as a potent antiangiogenic agent which exerts its inhibitory effect mainly through downregulation of essential mediators such as VEGF and MMPs.     

Antagonising the expression of VEGF in pathological angiogenesis

Vascular endothelial growth factor (VEGF) is the prototype of a subfamily of five growth factors sharing structural homologies with the platelet-derived growth factor (PDGF) protein superfamily. The VEGFs are potent mitogens specific for endothelial cells of the blood or lymphatic vasculature and are effective modulators of vessel permeability in the normal physiological processes of angiogenesis and vasculogenesis. VEGF appears to be a significant promoter of the inappropriate angiogenesis that sustains tumour growth and metastasis, inflammatory joint disease and diabetic retinopathy. Understanding the cell biology and control of VEGF expression has led to the development of several strategies for antagonising the pathological angiogenesis associated with these common diseases. This review describes these promising approaches in the context of current understanding of VEGF biology.     

脉络宁注射液对人血管内皮细胞缺氧损伤的保护作用

目的:研究脉络宁注射液对人脐静脉血管内皮细胞缺氧损伤的保护作用及其机制。方法:常规进行人血管内皮细胞(HUVECs)培养,将细胞随机分为正常对照组、缺氧组和脉络宁20 mg.L-1组。采用CCK-8法检测细胞存活率;Hochest33258荧光染料检测细胞凋亡率;RT-PCR法检测各组细胞中血管内皮生长因子(VEGF),血管生成素-2(Ang-2)mRNA表达水平。结果:脉络宁注射液可显著提高缺氧损伤细胞的存活率(P<0.05);与正常对照组比较,缺氧组细胞内VEGF和Ang-2 mRNA表达水平明显增高。与缺氧组比较,脉络宁组VEGF和Ang-2的表达进一步增强,各组间比较均有差异(P<0.05)。结论:脉络宁注射液可减轻血管内皮细胞缺氧损伤,上调缺氧血管内皮细胞中VEGF和Ang-2的表达,对血管内皮细胞缺氧损伤有一定的保护作用。     

LYG-202, a Newly Synthesized Flavonoid,Exhibits Potent Anti-angiogenic Activity In Vitro and In Vivo

LYG-202 (C₂₅H₃₀N₂O₅) is a newly synthesized flavonoid that has been confirmed to possess an antitumor effect, but the mechanism is unclear. Our present study was performed to identify the anti-angiogenic activity of this novel compound in vitro and in vivo. LYG-202 inhibited vascular endothelial growth factor (VEGF) stimulated migration and tube formation of human umbilical vein endothelial cells and arrested microvessel outgrowth from rat aortic rings in vitro. Meanwhile, LYG-202 suppressed the neovascularization of Chicken Chorioallantoic Membrane in vivo. Mechanistic studies revealed that LYG-202 suppressed the VEGF-induced tyrosine phosphorylation of KDR/Flk-1 (VEGFR-2) as well as its downstream protein kinases activation, by decreasing phosphorylated forms of serine/threonine kinase Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase. LYG-202 exerts anti-angiogenic activity both in vitro and in vivo, and these results suggest that it deserves further investigation as a promising anti–tumor angiogenesis compound.     

Angiopoietin/Tie receptor system: emerging targets for therapeutic angiogenesis and anti-angiogenic therapy

Balanced regulation of endothelial cell function and co-ordination of endothelial cells and periendothelial support cells by angiogenic growth factors and cell type-specific receptor tyrosine kinases is crucially involved in physiological angiogenesis. Disturbance of this fine-tuned balance is associated with disease-related neoangiogenesis, such as in tumour angiogenesis or in retinopathy, or in insufficient angiogenesis in occlusive vascular disease. In addition to the well known function of vascular endothelial growth factor (VEGF) as an endothelial cell-specific angiogenesis inducer and survival factor, recent studies on angiopoietins and their receptors have provided insight into the interplay of these endothelium-specific ligand-receptor systems in formation, maintenance and remodelling of the vasculature. Knowledge of the mechanisms by which these ligand-receptor systems are involved in regulation of the interaction of endothelial cells and their periendothelial support cells opens new opportunities for therapeutic angiogenesis to induce formation of functional blood vessels in occlusive disease, as well as to complement and/or enhance current anti-VEGF-based strategies for anti-angiogenic therapy.     

血管内皮生长因子受体及其小分子抑制剂的研究进展

目前抗血管生成治疗是抗肿瘤研究的热点.血管内皮生长因子(VEGF)是刺激血管生成的重要因子之一,血管内皮生长因子受体(VEGFR)在肿瘤新生血管中高表达,因此成为肿瘤靶向治疗的理想靶点.以VEGF、VEGFR为靶点的抗肿瘤药物除了常见的单克隆抗体药物阿伐斯汀外,小分子抑制剂舒尼替尼、索拉非尼等也已经广泛使用;另外,一些...     

Arsenite suppresses angiogenesis of vascular endothelial cells mediated by Platelet Derived Growth Factor Receptor-beta

The present study aimed to investigate the effects of sodium arsenite (NaAsO₂) on the angiogenesis of human umbilical vein endothelial cells (HUVECs) and the mechanism involved. Firstly, a Matrigel-based in vitro angiogenesis assay demonstrated that arsenite suppressed the angiogenesis of HUVECs in a dose-dependent manner. Then by using a global inhibitor for multiple growth factor receptors (E7080) and a specific inhibitor of PDGFR-beta (CP-673451), we found that E7080 completely prevented and CP-673451 significantly decreased the angiogenesis of HUVECs. This suggested that angiogenesis of HUVECs depends on the signal pathway mediated by tyrosine kinase receptors and that among them, PDGFR-beta has an important regulatory function. Finally by using porcine aortic endothelial cells which stably express human PDGFR-beta, we found that arsenite suppressed the angiogenesis mediated by PDGFR-beta. Based on these results, we conclude that arsenite suppressed the angiogenesis of the vascular endothelial cells, that this effect is mediated by PDGFR-beta, and postulate that it might contribute to the injuries of blood vessel in arsenism.     

The endoplasmic reticulum stress inducer thapsigargin enhances the toxicity of ZnO nanoparticles to macrophages and macrophage-endothelial co-culture

It was recently shown that exposure to ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress both in vivo and in vitro, but the role of ER stress in ZnO NP induced toxicity remains unclear. Because macrophages are sensitive to ER stress, we hypothesized that stressing macrophages with ER stress inducer could enhance the toxicity of ZnO NPs. In this study, the effects of ER stress inducer thapsigargin (TG) on the toxicity of ZnO NPs to THP-1 macrophages were investigated. The results showed that TG enhanced ZnO NP induced cytotoxicity as revealed by water soluble tetrazolium-1 (WST-1) and neutral red uptake assays, but not lactate dehydrogenase (LDH) assay. ZnO NPs dose-dependently enhanced the accumulation of intracellular Zn ions without the induction of reactive oxygen species (ROS), and the presence of TG did not significantly affect these effects. In the co-culture, exposure of THP-1 macrophages in the upper chamber to ZnO NPs and TG significantly reduced the viability of human umbilical vein endothelial cells (HUVECs) in the lower chamber, but the release of tumor necrosis factor α (TNFα) was not induced. In summary, our data showed that stressing THP-1 macrophages with TG enhanced the cytotoxicity of ZnO NPs to macrophages and macrophage-endothelial co-cultures.     

魟鱼软骨多糖对人脐静脉内皮细胞形成新生血管的影响

目的探讨缸鱼软骨多糖(RCG)对人脐静脉内皮细胞形成新生血管的作用.方法原代培养人脐静脉内皮细胞,实验分为生理氯化钠溶液(NS)对照组、RCG 100,50,25,10,2 g·L-1组和阳性对照鲨鱼软骨多糖(SCG,50 g·L-1)组.采用MTT法检测RCG对HUVEC增殖的影响,流式细胞仪检测RCG对HUVEC细胞周期的影响;并观察RCG对HUVEC迁移及小管形成的作用.结果10～100 g·L-1RCG可明显抑制HUVEC的体外增殖,IC50为62.93 g·L-1.流式细胞仪检测表明,RCG阻止HUVEC在G2/M期.10～100g·L-1 RCG明显抑制HUVEC迁移和小管形成.结论RCG具有良好的体外抗血管生成活性.     

感染Ad-HGF对高糖诱导的人脐静脉内皮细胞Bax和Bcl-2蛋白表达的影响

目的 探讨高糖条件下感染携带肝细胞生长因子的重组腺病毒(Ad-HGF)对人脐静脉内皮细胞(humanumbilical vein endothelial cells,HUVECs)凋亡相关蛋白Bax、Bcl-2表达的影响.方法 将HUVECs分为低糖组(LG组,5.5 mmol/L)、高糖组(HG组,35 mmol/L)、腺病毒对照组(HG+ Ad-GFP组)和实验组(HG+ Ad-HGF组).检测4组HUVECs增殖情况、细胞内活性氧(ROS)水平及凋亡相关蛋白Bax、Bcl-2的表达.结果 HG组、HG+ Ad-GFP组HUVECs存活率低于LG组,HG+ Ad-HGF组高于HG组(P＜0.05,P＜0.01).HG组、HG+ Ad-HGF组HUVECs内ROS水平高于LG组,但HG+ Ad-HGF组低于HG组(P＜0.05).HG组、HG+ Ad-GFP组Bax、Bax/Bcl-2高于LG组,Bcl-2低于LG组,但HG+ Ad-HGF组Bax、Bax/Bcl-2低于HG组,Bcl-2高于HG组(P＜0.05).结论 感染Ad-HGF可通过降低细胞内ROS水平和Bax/Bcl-2减少细胞凋亡,进而对高糖诱导的HUVECs起到保护作用.     

新藤黄酸干预肺腺癌血管生成的细胞学研究

目的 通过对人脐静脉内皮细胞HUVEC和人肺腺癌A549的培养,检测含新藤黄酸(GNA)条件培养基对血管内皮细胞存活率、成管和生长的影响.方法 采用甲基噻唑基四唑(MTT)法和平板克隆实验法研究GNA对HUVEC存活率和克隆形成率的影响;应用薄层胶原建立血管内皮细胞的二维培养模型,观察GN A对于血管内皮细胞成管现象的影响;采用细胞划痕愈合和小室迁移实验考察GNA对HUVEC的迁移能力影响;Westernblot检测血管内皮生长因子(VEGF)和缺氧诱导因子(HIF-1α)蛋白的表达.结果 MTT检测结果显示,HUVEC细胞存活率和克隆形成率随GNA剂量增加而降低.GNA可抑制HUVEC细胞的迁移.还可抑制HUVEC管腔样结构形成.此外,GNA可下调HUVEC中VEGF和HIF-1α蛋白的表达.结论GNA可在体外抑制血管生成,其作用机制可能与抑制肿瘤细胞分泌的HIF-1α和VEGF有关.     

阿托伐他汀对血管新生的促进作用

目的探讨阿托伐他汀对血管新生的促进作用及其作用机制。方法建立野生型C3H／He小鼠下肢缺血模型,观察用药后肢体血流、毛细血管数、血管内皮生长因子（VEGF）蛋白表达。并行离体实验（血管新生共同培养）,计数血管样结构物管腔形成数。结果阿托伐他汀使实验小鼠术后缺血肢血流明显改善,缺血肢与非缺血肢血流面积比明显增加;缺血肢毛细血管密度明显增加,VEGF蛋白表达增强。血管新生共同培养,血管样结构物管腔数明显高于对照组和加入血管新生抑制剂组,但与阳性对照组比较无明显差异。结论阿托伐他汀有促进血管新生的作用。     

Cytotoxicity,oxidative stress and inflammation induced by ZnO nanoparticles in endothelial cells: interaction with palmitate or lipopolysaccharide

Recent studies showed that ZnO nanoparticles (NPs) might induce the toxicity to human endothelial cells. However, little is known about the interaction between ZnO NPs and circulatory components, which is likely to occur when NPs enter the blood. In this study, we evaluated ZnO NP‐induced cytotoxicity, oxidative stress and inflammation in human umbilical vein endothelial cells (HUVECs), with the emphasis on the interaction with palmitate (PA) or lipopolysaccharide (LPS), because PA and LPS are normal components in human blood that increase in metabolic diseases. Overall, ZnO NPs induced cytotoxicity and intracellular reactive oxygen species (ROS) at a concentration of 32 μg ml⁻¹, but did not significantly affect the release of inflammatory cytokines or adhesion of THP‐1 monocytes to HUVECs. In addition, exposure to ZnO NPs dose‐dependently promoted intracellular Zn ions in HUVECs. PA and LPS have different effects. Two hundred μm PA significantly induced cytotoxicity and THP‐1 monocyte adhesion, but did not affect ROS or release of inflammatory cytokines. In contrast, 1 μg ml⁻¹ LPS significantly induced ROS, release of inflammatory cytokines and THP‐1 monocyte adhesion, but not cytotoxicity. The presence of ZnO NPs did not significantly affect the toxicity induced by PA or LPS. In addition, the accumulation of Zn ions after ZnO NP exposure was not significantly affected by the presence of PA or LPS. We concluded that there was no interaction between ZnO NPs and PA or LPS on toxicity to HUVECs in vitro . Copyright © 2016 John Wiley & Sons, Ltd.     

Comparison of P25 and nanobelts on Kruppel-like factor-mediated nitric oxide pathways in human umbilical vein endothelial cells

Recently, we reported that titanium dioxide (TiO₂) materials activated endothelial cells via Kruppel-like factor (KLF)-mediated nitric oxide (NO) dysfunction, but the roles of physical properties of materials are not clear. In this study, we prepared nanobelts from P25 particles and compared their adverse effects to human umbilical vein endothelial cells (HUVECs). TiO₂ nanobelts had belt-like morphology but comparable surface areas as P25 particles. When applied to HUVECs, P25 particles or nanobelts did not induce cytotoxicity, although nanobelts were much more effective to increase intracellular Ti element concentrations compared the same amounts of P25 particles. Only nanobelts significantly induced THP-1 adhesion onto HUVECs. Consistently, nanobelts were more significant to induce the expression of intracellular adhesion molecule-1 (ICAM1) and the release of soluble ICAM-1 (sICAM-1), indicating that nanobelts were more potent to induce endothelial activation in vitro. As the mechanisms for endothelial activation, both P25 and nanobelts reduced the generation of intracellular NO as well as the expression of NO regulators KLF2 and KLF4. Combined, the results from this study indicated that the different morphologies of P25 particles and nanobelts only changed their internalization into HUVECs but showed minimal impact on KLF-mediated NO signaling pathways.     

N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide promotes vascular endothelial cell angiogenesis and migration in the absence of serum and FGF-2

Aim:

To investigate the effect of N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide (BPC) on angiogenesis in human umbilical vein endothelial cells (HUVECs).

Methods:

Capillary-like tube formation on matrigel and cell migration analyses were performed in the absence of serum and fibroblast growth factor (FGF-2). Reactive oxygen species (ROS) were measured using a fluorescent probe, 2′, 7′- dichlorodihydrofluorescein (DCHF). The nitric oxide (NO) production of HUVECs was examined using a NO detection kit. Morphological observation under a phase contrast microscope, a viability assay using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium (MTT) and a lactate dehydrogenase (LDH) activity analysis by a detection kit were performed to evaluate the toxicity of BPC on HUVECs in the presence of serum and FGF-2. The level of hypoxia-inducible factor 1α (HIF-1α) and the release of vascular endothelial growth factor (VEGF) were measured by Western blot and ELISA, respectively.

Results:

In the absence of serum and FGF-2, cells treated with BPC (5-20 μmol/L) rapidly aligned with one another and formed tube-like structures within 12 h. In the presence of serum and FGF-2, cells treated with BPC for 24, 48 and 72 h had no changes in morphology, viability or LDH release compared with the control group. Cell migration in the BPC-treated group was significantly increased compared with the control group. During this process, NO production and ROS level were elevated dramatically, and the levels of HIF-1α and VEGF were increased dependent on the generation of ROS.

Conclusion:

BPC most effectively promoted angiogenesis and migration in HUVECs in the absence of FGF-2 and serum.     

Effects of Cordyceps militaris extract on angiogenesis and tumor growth

INTRODUCTIONAngiogenesis is the process of new blood vesselformation from existing blood vessels and a natural re-sponse of tissues to ischemia[1]. The steps of angio-genesis involve proteolytic degradation of extracelluarmatrix, endothelial cell-matrix adhesion, migration,proliferation, and differentiation[2]. This biological re-action is often associated with some diseases, such assolid tumor[3], diabetic retinopathy[4], and rheumatoidarthritis[5].Tumor formation requires the developm…     

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。     

力达霉素通过下调VEGF表达抑制斑马鱼胚胎血管生成

本研究采用斑马鱼胚胎模型研究力达霉素在整体动物水平对血管生成的影响。力达霉素处理胚胎后, 利用形态学观察、血管染色法、转基因斑马鱼检测其对胚胎血管生成影响, 以荧光定量PCR和蛋白免疫印迹法检测VEGF基因的表达情况。结果显示: 力达霉素处理后, 胚胎出现心包水肿、血流速度减缓等症状; 血管生长率降低, 肠下静脉生成受到抑制。荧光定量PCR和蛋白免疫印迹检测表明, 力达霉素对胚胎的VEGF mRNA表达水平没有影响, 但VEGF蛋白的表达受到显著抑制。研究结果表明, 力达霉素可以下调VEGF蛋白表达, 从而抑制斑马鱼胚胎血管生成。     

巴戟天醇提取物促大鼠缺血心肌治疗性血管生成的实验研究

目的探讨巴戟天糖链(MOO)对急性心肌梗死(AMI)大鼠缺血心肌治疗性血管生成的影响及其机制。方法♂Wistar大鼠,结扎冠状动脉左前降支,成功制成AMI模型40只,随机分为MOO小、中、大剂量组、麝香保心丸组及模型组,每组8只。另取10只建立假手术组。药物治疗组分别灌胃给予巴戟天醇提物水溶性部分(0.7、1.4、2.8mg.kg-1.d-1)及麝香保心丸悬浊液(30mg.kg-1.d-1),其余两组灌胃给予等量蒸馏水。连续灌胃6wk后处死大鼠,心肌取材,应用免疫组织化学法检测大鼠缺血心肌Ⅷ因子及血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)、碱性成纤维细胞生长因子(basic fibroblast growthfactor,bFGF)蛋白表达情况;计算微血管密度(microvessecdensity,MVD),用图像分析软件测定VEGF及bFGF表达灰度值,并进行半定量分析。结果与模型组相比,MOO中、大剂量组能增加缺血心肌MVD及VEGF、bFGF灰度值(P<0.05),但作用弱于麝香保心丸组(P<0.05);MOO3个剂量组之间MVD差异均有显著性(P<0.05);MOO3个剂量组之间VEGF灰度值差异均有显著性(P<0.05);MOO中、大剂量组bFGF灰度值与小剂量组相比差异有显著性(P<0.05)。结论MOO可促进AMI后大鼠缺血心肌的血管生成,其机制可能与上调缺血心肌VEGF、bFGF蛋白的表达有关。