Effect of manoalide on apoptosis induced by deprivation of growth factors in vascular endothelial cells.

AIM: To study effect of manoalide on apoptosis induced by deprivation of acidic fibroblast growth factor (aFGF) and serum in vascular endothelial cells (VEC). METHODS: Morphologic changes were observed by light microscopy. Viability was determined by counting the cells that attached to dishes after treatments. DNA fragmentation was analyzed by agarose gel electrophoresis and fluorescence microscopy. RESULTS: The cells deprived of aFGF and serum were exposed to manoalide 1-4 mumol. L-1 for 48 h, detachment and DNA fragmentation of these cells were suppressed. At 7 mumol. L-1, manoalide promoted detachment and DNA fragmentation of VEC. CONCLUSION: manoalide 2 mumol.L-1 inhibited, but 7 mumol.L-1 promoted, apoptosis of VEC.     

1-甲基-3-异丁基黄嘌呤延迟去除生长因子诱导的血管内皮细胞凋亡(英文)

目的:研究1-甲基-3-异丁基黄嘌呤(MIBX)对去除生长因子(aFGF和血清)诱导的血管内皮细胞凋亡的影响.方法:通过细胞存活率的分析,荧光显微技术和DNA凝胶电泳等方法,检测MIBX对细胞凋亡的影响.结果:用25-200μmol/L的MIBX处理培养在无aFGF和血清的培养液中的血管内皮细胞,50-200μmol/L的MIBX在处理6h明显抑制了凋亡小体的形成和DNA的片断化.但是同样浓度的MIBX处理细胞12h以后,处理组和对照组之间无明显差别.结论:MIBX延迟去除aFGF和血清诱导的血管内皮细胞凋亡.     

Integrin beta4 mAb inhibited apoptosis induced by deprivation of growth factors in vascular endothelial cells.

AIM: To understand the mechanism by which anti-beta4 integrin monoclonal antibody (mAb) inhibits apoptosis of vascular endothelial cells (VEC). METHODS: Viability was determined by counting the cells that attached to dishes after treatments. DNA fragmentation was analyzed by agarose gel electrophoresis and fluorescence microscopy. The intracellular content of cAMP was measured by radioimmunoassay (RIA). The levels of p53 and Ras expressions were analyzed by fluorescence microscopy combined with immunofluorescence under laser scanning confocal microscopy. RESULTS: After the cells were deprived of fibroblast growth factor (FGF) and serum were exposed to the mAb 5 mg/L for 24 h, the detachment and DNA fragmentation of these cells were suppressed. When cells were deprived of FGF and serum, the intracellular cAMP level and Ras protein content decreased (P<0.05), while the level of p53 protein expression increased (P<0.05). But in the presence of anti-beta4 integrin mAb, VEC apoptosis was inhibited, and at the same time, the changes mentioned above were obviously blocked (P<0.05). CONCLUSION: Anti-4 integrin mAb inhibited apoptosis by affecting the level of cAMP, and blocking down-regulation of Ras protein and up-regulation of p53 protein in VEC.     

黄樟素氧化物对去除成纤维细胞生长因子所诱导的血管内皮细胞凋亡及生长的影响

目的:研究黄樟素氧化物对去除成纤维细胞生长因子(FGF)诱导的血管内皮细胞凋亡及生长的影响.方法:光学显微镜观察细胞形态学变化;MTT法测定细胞生长;DNA电泳和荧光显微技术检测DNA断裂;流式细胞术测定细胞周期分布.结果:黄樟素氧化物5-25mg/L处理去除FGF的血管内皮细胞24h,细胞铺展和生长被促进,细胞脱壁和DNA片段化被抑制,黄樟素氧化物10mg/L对细胞周期分布无明显影响.黄樟素氧化物50-100 mg/L促进血管内皮细胞的脱壁和DNA片段化,黄樟素氧化物100mg/L将细胞周期阻断于G_2-M期.结论:黄樟素氧化物在10mg/L时抑制血管内皮细胞凋亡,而在100mg/L时促进其凋亡,该药物对血管内皮细胞生长和凋亡有重要影响.     

Effect of vascular endothelial growth factor and epidermal growth factor on iatrogenic apoptosis in human endothelial cells

To study the effect of growth factors on iatrogenic apoptosis, we examined the influence of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) on staurosporine-induced apoptosis in primary cultures of human umbilical vein endothelial cells (HUVEC). Apoptosis was evaluated by a cell viability test, the TUNEL-POD assay and the activation of the pro-apoptotic caspase-3. Staurosporine (10-100nM) caused the activation of caspase-3. This effect was manifest after 2hr of incubation and reached its maximum after 5hr. Severe loss of viability followed within 18hr. VEGF or EGF (10-100ng/mL) added together with staurosporine decreased the activation of caspase-3. The loss of viability was 24hr delayed. The action of growth factors was observed at 1% serum concentration but also at concentration optimal for HUVEC survival (10%, v/v). Furthermore, the inhibition of PI-3 kinase (PI-3K) by wortmannin or LY294002 as well as the inhibition of MEK by PD098059 or U0126 prevented the protective effect of VEGF and EGF. Western blotting analysis showed that after 3hr of incubation with staurosporine the level of the anti-apoptotic protein Mcl-1 decreased and this effect was reverted by VEGF. It is concluded that VEGF and EGF antagonize the pro-apoptotic action of staurosporine by the combined signalling of PI-3K and ERKs pathways.     

Levamisole induced apoptosis in cultured vascular endothelial cells

To better understand the anticancer activity of Levamisole (LMS), which serves as an adjuvant in colon cancer therapy in combination with 5-Fluorouracil, this study analyses LMS' ability to induce apoptosis and growth arrest in cultured human micro- and macrovascular endothelial cells (ECs) and fibroblasts. Cells exposed (24 h) to Levamisole (range: 0.5 - 2 mmol l(-1)) alone or in combination with antioxidants (10 mmol l(-1) glutathione or 5 mmol l(-1) N-Acetylcysteine or 0.1 mmol l(-1) Tocopherol) were evaluated for apoptosis ((3)H-thymidine assays, in situ staining), mRNA/protein expression (Northern/Western blot), and proliferation ((3)H-thymidine incorporation). Levamisole dose-dependently increased apoptosis in ECs to 230% (HUVECs-human umbilical vein ECs), 525% (adult human venous ECs) and 600% (human uterine microvascular ECs) but not in fibroblasts compared to control cells (set as 100%). Levamisole increased in ECs integrin-dependent matrix adhesion, inhibited proliferation (-70%), reduced expression of survival factors such as clusterin (-30%), endothelin-1 (-43%), bcl-2 (-34%), endothelial NO-synthase (-32%) and pRb (Retinoblastoma protein: -89%), and increased that of growth arrest/death signals such as p21 (+73%) and bak (+50%). LMS (2 mmol l(-1))-induced apoptosis was inhibited by glutathione (-50%) and N-Acetylcysteine (-36%), which also counteracted reduction by Levamisole of pRb expression, suggesting reactive oxygen species and pRb play a role in these processes. The ability of LMS to selectively induce apoptosis and growth arrest in endothelial cells potentially hints at vascular targeting to contribute to Levamisole's anticancer activity.     

乙酰葛根素对血管紧张素Ⅱ致血管内皮细胞凋亡的抑制作用

目的探讨血管紧张素Ⅱ(AngⅡ)在不同浓度和不同作用时间下,对血管内皮细胞凋亡及凋亡调控基因的作用及乙酰葛根素对血管内皮细胞凋亡的抑制作用。方法采用流式细胞学技术,观察乙酰葛根素对AngⅡ致血管内皮细胞凋亡、Fas和Bcl-2表达的影响。结果AngⅡ可明显促进血管内皮细胞凋亡及Fas和Bcl-2的表达,且其作用呈剂量依赖性和时间依赖性;乙酰葛根素抑制AngⅡ引起的血管内皮细胞凋亡及Fas和Bcl-2的表达。结论乙酰葛根素明显抑制AngⅡ诱导的内皮细胞凋亡和凋亡调控基因表达。     

西红花苷对氧化甾醇诱导血管内皮细胞凋亡的影响

目的:研究西红花苷对培养的牛血管内皮细胞凋亡的影响。方法:采用3β、5α、6β三羟胆固(烷)醇(Triol)诱导内皮细胞凋亡的模型,用比色法测定丙二醛含量,光学显微镜、电子显微镜检测形态和超微结构的变化,DNA电泳检测DNAladder及流式细胞仪分析法检测凋亡率,采用逆转录聚合酶链式反应(RTPCR)法检测BaxmRNA表达的变化。结果:Triol处理后,培养液中MDA含量增加,为1.761nmol·L-1(P<0.01),细胞收缩,核浓缩,深染,线粒体肿胀空化,出现凋亡小体,出现凋亡典型的“DNAladder”,和亚二倍体峰,凋亡率为30.62%,BaxmRNA表达量增加;西红花苷(10-7,10-6mol·L-1) Triol组,MDA含量减少,内皮细胞形态结构的完整,凋亡率分别为24.4%,6.3%,BaxmRNA表达量降低。结论:西红花苷可能是抗脂质过氧化并通过调节BaxmRNA表达,减少细胞异常凋亡。     

Regulation of vascular permeability by vascular endothelial growth factors

Increased vascular permeability is one of the first stages in both physiological and pathological angiogenesis-the generation of new blood vessels from preexisting vasculature. Although this has been hypothesised to be true in physiological angiogenesis, it is clearly a mark of blood vessel growth in disease. Normal, healthy blood vessel growth (physiological angiogenesis) occurs throughout development as well as during tissue repair and growth in adult tissues. Angiogenesis is also seen in a wide variety of diseases, which include all the major causes of mortality in the West-heart disease, cancer, stroke, vascular disease, and diabetes. Much of this angiogenesis is significantly different from normal blood vessel growth and is termed pathological angiogenesis. Angiogenesis is regulated by vascular growth factors, the most notable being the vascular endothelial growth factor family of proteins (VEGF). These act on specific receptors in the vascular system to stimulate new vessel growth by a number of mechanisms. VEGFs also directly stimulate increased vascular permeability to water and large molecular weight proteins and vasodilatation. These two effects result in a large flux of water and macromolecules from the vasculature to the interstitium, often resulting in oedema. This review will outline the mechanisms by which VEGFs do this and discuss some of the difficulties in interpreting data from VEGF studies due to the conflicting and synergistic effects of these actions.     

程序性细胞死亡因子 4基因对川崎病血清诱导血管内皮细胞凋亡的影响

目的研究程序性细胞死亡因子 4(PDCD4)对川崎病病人血清诱导的血管内皮细胞凋亡的作用。方法研究时间为 2017年 3月至 2018年 4月。采集邵阳学院附属第一医院川崎病病人血清,处理血管内皮细胞,实时荧光定量 PCR(realtime PCR)和蛋白质印迹法检测细胞中 PDCD4 mRNA和蛋白表达水平。在血管内皮细胞中转染 PDCD4 siRNA,用川崎病血清培养,以 Realtime PCR和蛋白质印迹法检测细胞中 PDCD4表达水平。 MTT方法检测细胞增殖,流式细胞术检测细胞凋亡,蛋白质印迹法检测细胞中活化型半胱氨酸天冬氨酸蛋白酶 3(C?Caspase?3)、活化型 Caspase?9(C?Caspase?9)蛋白水平。结果川崎病病人血清处理后的血管内皮细胞中 PDCD4表达上调［mRNA:(1.00±0.01)比( 3.51±0.36);蛋白:(0.26±0.08)比(0.57±0.06)］。 PDCD4 siRNA可以下调川崎病病人血清诱导的血管内皮细胞中 PDCD4表达水平［mRNA:(0.91±0.09)比( 0.41±0.06);蛋白:     

诺帝对血管内皮生长因子诱导的人脐血源性内皮祖细胞功能的影响

探讨手性化合物诺帝(Nordy)对血管内皮生长因子(VEGF)诱导的人脐血源性内皮祖细胞(EPCs)功能的影响及其意义。应用密度梯度离心法分离新鲜人脐血的单个核细胞，接种于EGM-2培养液中培养7～10 d获得内皮祖细胞(EPCs)。分别采用MTT法、Millicell-PCF培养小室系统和Matrigel内小管形成试验检测诺帝对VEGF刺激下EPCs增殖活性、迁移能力和体外形成小管样结构能力的影响。结果表明，100 μmol·L^-1诺帝作用24 h明显抑制EPCs增殖活性(P<0.05)，诺帝(25～50 μmol·L^-1)作用48～72 h也明显抑制EPCs增殖活性(P<0.05)。诺帝(25～100 μmol·L^-1)显著抑制VEGF诱导的EPCs迁移活性和体外形成小管样结构的能力(P<0.05)。诺帝能抑制体外VEGF诱导的人脐血源性EPCs增殖、迁移和体外小管形成能力，提示其具有抗EPCs效应。     

血管内皮生长因子165诱导人血管内皮细胞多药耐药表型

目的：研究血管内皮生长因子165（VEGF165）对血管内皮细胞药物敏感性的影响。方法：人真皮微血管内皮细胞（HDMEC）与抗癌药物和VEGF165混合培养,MTT法分析药敏变化,琼脂糖电泳检测细胞凋亡,RT-PCR、免疫印迹法分析HDMEC产生多药耐药表型的机制。结果：VEGF165体外诱导HDMEC对多种抗癌药物耐药,如表柔比星、顺铂、足叶乙甙、丝裂霉素C、长春新碱、CPT-11、泰素等,其机制与VEGF165诱导HDMEC上调表达多药耐药相关蛋白和肺耐药相关蛋以及下调表达Bax蛋白有关。结论：VEGF165诱导血管内皮细胞的多药耐药表型,提示化疗时抗癌药物的抗血管生成活性可能取决于肿瘤微环境。     

红花黄色素B对AngⅡ诱导内皮细胞线粒体损伤的保护作用

血管内皮细胞(vascular endothelial cell,VEC)作为血液与组织之间的屏障,参与多种生理和病理过程,其功能异常与动脉粥样硬化、高血压、糖尿病等有紧密的联系[1?4]。血管紧张素Ⅱ(angiotensin-Ⅱ,Ang-Ⅱ)可促进内皮细胞内活性氧自由基(reactiveoxygen species,ROS)的生成,改变胞内Ca2+     

Stevioside enhances apoptosis induced by serum deprivation in PC12 cells

In the laboratory, using a PC12 cell system, studies have been conducted on the effects of various chemicals on apoptosis, as it is considered to be an essential part of normal development, maintenance, and defense in organisms. Stevioside is a natural sweetener extracted from the leaves of Stevia rebaudiana. Since it is widely used as a sugar replacement, it was decided to evaluate the toxicological effects of low concentrations of stevioside on apoptosis induced by serum deprivation using the PC12 cell system. It was found that based on data from DNA electrophoresis and TUNEL signal assays stevioside enhanced apoptosis induced by serum deprivation. This enhancement was caused by increased expression of Bax and of cytochrome c released into the cytosol. These findings suggest that stevioside affects the regulation of the normal apoptotic condition. Further investigation will be needed to clarify the detailed mechanism of the enhancement due to the treatment with stevioside.     

Stevioside enhances apoptosis induced by serum deprivation in PC12 cells

In the laboratory, using a PC12 cell system, studies have been conducted on the effects of various chemicals on apoptosis, as it is considered to be an essential part of normal development, maintenance, and defense in organisms. Stevioside is a natural sweetener extracted from the leaves of Stevia rebaudiana. Since it is widely used as a sugar replacement, it was decided to evaluate the toxicological effects of low concentrations of stevioside on apoptosis induced by serum deprivation using the PC12 cell system. It was found that based on data from DNA electrophoresis and TUNEL signal assays stevioside enhanced apoptosis induced by serum deprivation. This enhancement was caused by increased expression of Bax and of cytochrome c released into the cytosol. These findings suggest that stevioside affects the regulation of the normal apoptotic condition. Further investigation will be needed to clarify the detailed mechanism of the enhancement due to the treatment with stevioside.     

外源性羟自由基诱导血管内皮细胞凋亡及卡托普利的干预作用

目的 观察外源性氧自由基（OFR）衍生的羟自由基（OH^-)诱导培养的VECs凋亡及卡托普利（Captopril,Cap)的干预作用，探讨血管内皮细胞（VECs)凋亡（Apoptosis)与心血管疾病的关系。方法 （1）采用改良的Jaffe法，进行人脐静脉血管内皮细胞（HUVECs)原代、传代培养；（2）取4－6代HUVECs，无血清条件下分别为10^-5、10^-4、10^-3、10^-2、10^-1mol/L羟自由基（OH^-)共孵育，观察细胞存活率、贴壁率、形态学、DNA凝胶电泳、DNA片段率及培养液一氧化氮（NO）含量变化；（3）观察10^-3mol/L OH^-诱导HUVECs凋亡及10^-7、10^-6、10^-5mol/L Cap干预后上述参数的变化。结果 （1）正常培养HUVECs存活率＞95％，贴壁率＞90％；（2）外源性OH－明显降HUVECs存活率、贴壁率，且呈剂量依赖性；10^-5、10^-4、10^-3mol/L OH^-诱导HUVECs凋亡，DNA“梯形”以10^-3mol/L OH^-作用8－24h显著，DNA片段率、凋亡细胞百分率明显升高；而10^-2、10^-1mol/L OH^-可诱导HUVECs坏死；（3）10^-7、10^-6、10^-5mol/L Cap干预明显提高OH^-处理的HUVECs存活率、贴壁率，降低其凋亡细胞百分率、DNA片段率，使DNA“梯形”渐消失，NO分泌减少渐逆转。结论 （1）采用改良的Jaffe法，可以建立HUVECs体外模型；（2）外源性OH^-在较低浓度时可诱导培养HUVECs的典型凋亡；（3）Cap能减轻或抑制OH^-诱导培养HUVECs的凋亡，恢复其NO分泌。     

3,4,5,6-Tetrahydroxyxanthone prevents vascular endothelial cell apoptosis induced by high glucose

Apoptosis of endothelial cells may be an important risk factor contributing to the incidence of vascular complications in diabetes. In the present study, we tested the effect of 3,4,5,6-tetrahydroxyxanthone, a synthetic xanthone derivative, on apoptosis induced in human umbilical vein endothelial cells (HUVEC) by a high glucose concentration. Cell apoptosis was detected using DNA ladder formation and flow cytometric techniques. The expression of Bcl-2 protein was analysed using flow cytometric techniques. Lactate dehydrogenase (LDH) activity and malonyldialdehyde (MDA) content in the medium were measured. Cell viability was assayed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. Exposure of HUVEC to a high glucose concentration (30 mM) for 48 h markedly increased LDH release and MDA content in the medium and induced apoptosis and Bcl-2 protein expression in HUVEC. Pretreatment with 3,4,5,6-tetrahydroxyxanthone (1, 3 or 10 M) or probucol (10 M) significantly decreased the level of LDH and MDA in the medium, reduced apoptosis and increased the expression of Bcl-2 protein in HUVEC. These results suggest that 3,4,5,6-tetrahydroxyxanthone inhibits high-glucose-induced endothelial cell apoptosis by increasing Bcl-2 protein expression in HUVEC.     

Diethyl phthalate enhances apoptosis induced by serum deprivation in PC12 cells

In this study, to examine the mechanism of diethyl phthalate toxicity to cells, the effects of diethyl phthalate on apoptosis in a PC12 cell system were investigated by assaying apoptotic factors such as caspase-3, Bax, cytochrome c and DNA damage. Diethyl phthalate was shown to enhance the apoptosis induced by serum deprivation according to the results of DNA electrophoresis and TUNEL signal assays, although it could not induce apoptosis itself in the cells. This enhancement was thought to be because of an increase in caspase-3-like activity. In addition, the expression of bax and contents of cytochrome c in the cytosol showed a tendency to increase the cells exposed to diethyl phthalate. These results indicated that diethyl phthalate, a potential endocrine disrupter, affects the apoptotic system in PC12 cells. Diethyl phthalate may enhance oxidative stress such as that induced by reactive oxygen species in PC12 cells.     

乙酰葛根素对氧糖剥离诱导海马神经元凋亡的影响

目的研究新型异黄酮类化合物乙酰葛根素对氧糖剥离神经元的保护作用及其机制。方法采用氧糖剥离建立细胞模型。DAPI法观察神经元凋亡率,RT-PCR法检测NF-κBp65、HIF-1α及p53mRNA的表达,Western blot法测定Hsp70蛋白的含量及乙酰葛根素对其的影响。结果与模型组相比较,乙酰葛根素可以减少凋亡细胞数;降低NF-κBp65、HIF-1α及p53mRNA表达;乙酰葛根素(低,中和高浓度组)增加Hsp70蛋白的表达。结论乙酰葛根素降低NF-κBp65、HIF-1α、p53的表达和升高Hsp70蛋白表达。     

Effect of an oversulfated exopolysaccharide on angiogenesis induced by fibroblast growth factor-2 or vascular endothelial growth factor in vitro

The aim of this study was to determine the angiogenic properties of an oversulfated exopolysaccharide (OS-EPS) derived from a polysaccharide secreted by the mesophilic bacterium Alteromonas infernus. We compared the effect of this OS-EPS with that of a non-oversulfated exopolysaccharide (EPS) on human umbilical vein endothelial cell (HUVEC) proliferation, migration and differentiation induced by basic fibroblast growth factor (FGF-2) or vascular endothelial growth factor (VEGF). OS-EPS enhanced HUVEC proliferation by 58% when used alone, and by respectively 30% and 70% in the presence of FGF-2 and VEGF. OS-EPS also increased the density of tubular structures on Matrigel in the presence of FGF-2 or VEGF. Vascular tube formation was related to alpha(6) integrin subunit expression, which was enhanced by 50% in the presence of the growth factors. Indeed, a monoclonal anti-alpha(6) blocking antibody abolished this vascular tube formation. EPS had no effect in any of the experimental conditions, underlying the importance of sulfation in the angiogenic effects of exopolysaccharide. By potentiating the angiogenic activity of FGF-2 and/or VEGF, OS-EPS, which possesses low anticoagulant activity and thus a low hemorrhagic risk, could potentially be used to accelerate vascular wound healing or to promote the growth of collateral blood vessels in ischemic tissues.