氧化型低密度脂蛋白诱导巨噬细胞凋亡

氧化型低密度脂蛋白诱导巨噬细胞凋亡牛喜林，张修武，郭兆贵（湖南医科大学分子药理研究室，长沙４１００７８，中国）关键词ＤＮＡ片断；细胞凋亡；低密度脂蛋白；腹腔巨噬细胞目的：研究氧化型低密度脂蛋白（ｏｘＬＤＬ）诱导巨噬细胞凋亡．方法：超速离心法分离人血...     

氧化型低密度脂蛋白诱导主动脉和心内膜内皮细胞凋亡

目的：研究氧化型低密度脂蛋白（ｏｘ－ＬＤＬ）诱导血管和心内膜内皮细胞凋亡。方法：用超速离心法分离健康人血浆低密度脂蛋白（ＬＤＬ），以ＣｕＳＯ４１０μｍｏｌ．Ｌ＾－１氧化，观察ｏｘ－ＬＤＬ对培养新生小牛主动脉内皮细胞及心内膜细胞的损伤作用，琼脂糖凝胶电泳和Ｈｏｅｃｈｓｔ３３２５８荧光密度法定性与定量分析ＤＮＡ降解，结果：ｏｘ－ＬＤＬ诱导血管内皮细胞及以内膜细胞典型凋亡形态学改变，ＤＮＡ降解呈时间和剂     

雌激素对血管平滑肌细胞凋亡的影响

目的 :观察雌激素对血管平滑肌细胞 (SMC)凋亡的影响。方法 :以大鼠为对象 ,观察正常对照组、去卵巢组及去卵巢 +雌二醇治疗组大鼠血管平滑肌细胞凋亡的关系。结果 :雌二醇治疗组血管平滑肌细胞凋亡较去卵巢组相比明显减少(P>0 .0 5 ) ,与正常对照组比较差别无显著性意义 (P >0 .0 5 )。结论 :雌二醇对血管平滑肌细胞的凋亡有抑制作用 ,可能是其预防动脉粥样硬化的机制     

氧化甾醇抑制血管平滑肌细胞(VSMC)增殖及诱导其凋亡的作用

目的 :研究 3β ,5α ,6 β 胆甾烷三醇 (Triol)和2 5 羟胆固醇 (2 5OH)对VSMC增殖的影响 ,探讨氧化甾醇在动脉粥样硬化 (AS)发病中的作用和意义。方法 :培养和鉴定新西兰白兔主动脉VSMC ;以含不同浓度的Triol和 2 5OH的培养液作用于VSMC为实验组 ,不含氧化甾醇或含胆固醇的培养液作为对照组。VSMC增殖及其代谢活性以WST 1测定 ,细胞凋亡以光镜、透射电镜、脱氧核苷酸末端转移酶介导脱氧尿苷三磷酸缺口末端标记 (TUNEL)判断。结果 :在1× 10 - 9～ 1× 10 - 7mol·L- 1范围的Triol和 2 5OH对VSMC的WST 1代谢活性无明显变化 (P >0 .0 5 ) ,而≥ 1× 10 - 6 mol·L- 1的氧化甾醇则使其代谢活性显著下降 (P <0 .0 1) ;≥ 2 0× 10 - 6 mol·L- 1的Triol和2 5OH诱导VSMC胞体皱缩变小、染色质在核周边浓集、核碎裂、空泡和凋亡小体形成等超微结构变化 ;TUNEL呈阳性反应。结论 :小剂量氧化甾醇无促进VSMC增殖而较大剂量氧化甾醇可诱导VSMC凋亡。     

氧化甾醇诱导血管平滑肌细胞凋亡及其作用特点的研究

目的研究３β,５α,６β－胆甾烷二醇（Ｔｒｉｏｌ）诱导血管平滑肌细胞（ＶＳＭＣｓ）凋亡及其与２５－羟胆固醇（２５－ＯＨ）比较的特点。方法体外培养ＶＳＭＣｓ、光镜、透射电镜及ＴＵＮＥＬ技术。结果ＶＳＭＣｓ经Ｔｒｉｏｌ处理后,贴壁层细胞呈现核染色质浓集,核碎裂和凋亡小体形成等凋亡的超微结构变化;ＴＵＮＥＬ显示ＶＳＭＣｓ凋亡细胞数随Ｔｒｉｏｌ浓度的增加而增多;剂量为 ３０ μｍｏｌ·Ｌ－１的 Ｔｒｉｏｌ和 ２５－ＯＨ诱导 ＶＳＭＣｓ的凋亡作用,前者不能而后者能被 ５０ μｍｏｌ·Ｌ－１的胆固醇所抑制。结论Ｔｒｉｏｌ能诱导ＶＳＭＣｓ凋亡,不同氧化甾醇可能有不同的作用通道和机制;氧化甾醇诱导ＶＳＭＣｓ凋亡可能是引起动脉粥样硬化斑块破溃,导致急性心血管事件发生的机制之一。     

他汀类药物与血管平滑肌细胞凋亡

血管平滑肌细胞（VSMC）的异常增殖和迁移在粥样斑块形成和冠状动脉介入治疗（PCI）后再狭窄中起非常重要的作用。他汀类药物不仅是一种有效的调脂药，同时还有多种调脂以外的作用。如他汀类药物可以抑制VSMC的增殖和迁移，并诱导其凋亡。临近病变部位的中层VSMC的凋亡可能造成斑块纤维帽容易破裂，进而引起斑块不稳定和临床事件。但研究证实他汀类药物有稳定斑块的作用，且新生内膜VSMC对他汀类药物诱导的凋亡作用比普通的血管中层VSMC更敏感。因此，他汀类药物的促新生内膜VSMC凋亡作用可能对预防PCI术后再狭窄起有益作用。具体作用机制以及临床上如何合理发挥此类作用尚待进一步研究。     

葛根通脉饮诱导血管平滑肌细胞凋亡的实验研究

目的研究葛根通脉饮对同型半胱氨酸(homocysteinemia,HCY)诱导增殖大鼠血管平滑肌细胞(vascular smooth muscle cell,VSMC)的促凋亡作用。方法制备葛根通脉饮含药血清,大鼠VSMC原代培养、鉴定,分为空白组、HCY组、葛根通脉饮组、瑞舒伐他汀(可定)组、葛根通脉饮联合可定组。药物干预后,Annexin/PI双染法检测细胞凋亡率。结果与空白组比较,HCY组细胞凋亡率显著降低(P<0.01);与HCY组比较,葛根通脉饮组、可定组、葛根通脉饮联合可定组细胞凋亡率显著升高(P<0.05,P<0.01)。结论葛根通脉饮可促进HCY诱导增殖的大鼠VSMC凋亡,可能是其抗动脉粥样硬化(atherosclerosis,AS)作用机制之一。     

普罗布考抑制过氧化氢诱导血管平滑肌细胞的凋亡

目的研究抗氧化剂普罗布考(probucol)对过氧化氢(H2O2)诱导血管平滑肌细胞(VSMCs)凋亡的影响。方法以1mmol·L-1H2O2为VSMC凋亡诱导剂,probucol浓度为100、10和1μmol·L-1,孵育6h,采用Annexin V-FITC染色、TUNEL法和Hoechst33258染色法观察细胞凋亡;Western blot检测细胞中ASK-1和Trx-1蛋白的表达。结果H2O2可促使VSMCs凋亡,细胞中ASK-1蛋白表达增加,Trx-1蛋白表达降低。Probucol可减轻H2O2所致的细胞凋亡,呈浓度依赖性;同时细胞中ASK-1蛋白表达降低,Trx-1蛋白表达增加。结论probucol能拮抗H2O2诱导的血管平滑肌细胞凋亡,其机制可能与降低细胞中ASK-1蛋白表达,升高Trx-1蛋白表达有关。     

维拉帕米诱导血管平滑肌细胞凋亡及其在再狭窄机制中的作用

目的探讨维拉帕米能否诱导血管平滑肌细胞凋亡及细胞凋亡在再狭窄机制中的作用。方法分别取正常兔髂动脉,动脉粥样硬化及再狭窄髂动脉血管中膜组织进行平滑肌细胞培养,［３Ｈ］ ＴｄＲ参入法测定细胞增殖活性,维拉帕米诱导平滑肌细胞凋亡,通过电镜观察,ＤＮＡ凝胶电泳及流式细胞仪了解各组平滑肌细胞凋亡情况。结果维拉帕米诱导下,平滑肌细胞的变化具有凋亡的典型特征。血管成形术后,细胞增殖及凋亡系统均被激活,再狭窄组细胞增殖程度增加２６％,细胞凋亡激活程度增加１９％,二者比例失衡。结论维拉帕米能够引起血管平滑肌细胞凋亡,而平滑肌细胞凋亡的相对减少在再狭窄的发生机制中发挥一定的作用。     

3种药物诱导兔血管平滑肌细胞凋亡实验研究

目的 研究阿克拉霉素(ACR)、雷公藤甲素(TP)、羟基喜树碱(HCPT)是否具有抑制血管平滑肌细胞(VSMCs)增殖并诱导其发生凋亡的作用，为临床开发新型药物涂层支架提供实验依据。方法 体外主动脉SMCs培养和流式细胞仪检测，DNA凝胶电泳。结果TP组与阳性对照地塞米松组相比．凋亡率有显著增高，ACR组凋亡率与正常细胞组相比基本无变化；HCPT组细胞凋亡率高于正常细胞组，但相比阳性对照地塞米松组无显著性差异。3种药物干预组凝胶电泳谱型呈凋亡特征性梯状条带。结论 ACR、TP、HCPT可抑制指数生长期SMCs增殖并诱导其凋亡。     

Oxidized low-density lipoprotein sensitizes human vascular smooth muscle cells to FAS (CD95)-mediated apoptosis

1. It was investigated in the present study whether oxidized low-density lipoprotein (oxLDL) was implicated in the susceptibility of human vascular smooth muscle cells (VSMC) to Fas-mediated death. Human fetal aorta smooth muscle cells were treated with agonistic anti-Fas antibody (CH11) and oxLDL and cell death was then determined by viability and DNA fragmentation. 2. The results of the present study show that cross-linking of Fas receptor with anti-Fas antibody in the presence of oxLDL induced death and DNA fragmentation in human VSMC, which were blocked by the caspase inhibitor z-VAD.fmk, followed by the upregulation of cell surface Fas. 3. The data indicate that oxLDL is implicated in death in VSMC and provide evidence that oxLDL is involved in Fas signal transduction. The present study proposes a novel mechanism(s) by which VSMC become susceptible to Fas ligand. 4. One of the mechanisms proposed by which oxLDL upregulates cell surface Fas is by inhibiting the degradation of Fas through the ubiquitin-proteasome pathway.     

Oxidized low-density lipoproteins induce apoptosis in aortic and endocardial endothelial cells

目的：研究氧化型低密度脂蛋白（ｏｘＬＤＬ）诱导血管和心内膜内皮细胞凋亡．方法：用超速离心法分离健康人血浆低密度脂蛋白（ＬＤＬ），以ＣｕＳＯ４１０μｍｏｌ·Ｌ－１氧化．观察ｏｘＬＤＬ对培养新生小牛主动脉内皮细胞及心内膜细胞的损伤作用．琼脂糖凝胶电泳和Ｈｏｅｃｈｓｔ３３２５８荧光密度法定性与定量分析ＤＮＡ降解．结果：ｏｘＬＤＬ诱导血管内皮细胞及心内膜细胞典型凋亡形态学改变，ＤＮＡ降解呈时间和剂量依赖性．环己米特和硫酸葡聚糖对此作用无影响．ＢＨＴ２０μｍｏｌ·Ｌ－１可取消ＤＮＡ降解．溶血性磷脂酰胆碱５０μｍｏｌ·Ｌ－１无诱导凋亡作用．ｏｘＬＤＬ诱导的ＤＮＡ降解可被依他酸取消．结论：ｏｘＬＤＬ诱导血管内皮细胞及心内膜细胞凋亡．     

Fluvastatin enhances apoptosis in cytokine-stimulated vascular smooth muscle cells.

Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) have been shown to attenuate proliferation of vascular smooth muscle cells (VSMCs) by mechanisms independent of lipid reduction. In the current study, we investigated the effect of lipophilic and hydrophilic statins (fluvastatin and pravastatin) on apoptosis in unstimulated or cytokine-stimulated VSMCs. The presence of apoptosis in rat VSMCs was evaluated by electrophoresis of DNA fragments and 4'6'-diamidine-2'-phenylindole staining and quantified by flow cytometry. Fluvastatin but not pravastatin enhanced apoptosis in interleukin-1beta-stimulated VSMCs. The proapoptotic effect of fluvastatin was fully reversed by mevalonate and geranylgeranyl-pyrophosphate, and partially by farnesyl-pyrophosphate, but not by squalene. Inhibition of the extracellular signal-regulated protein kinase (ERK1/2) pathway significantly increased fluvastatin-enhanced apoptosis, whereas inhibition of the p38-mitogen-activated protein kinase (MAPK) pathway significantly prevented this increase. However, fluvastatin showed no effect on the activity of ERK1/2 and p38-MAPK. Furthermore, fluvastatin-induced apoptosis was inhibited by YVAD-FMK (a caspase-1/interleukin-1beta-converting enzyme-like protease inhibitor) and DEVD-FMK (a caspase-3/CPP32 inhibitor), indicating involvement of an important segment in the apoptosis signaling pathway. These findings suggest that fluvastatin enhances apoptosis in cytokine-stimulated VSMCs and that protein prenylation, MAPK (ERK1/2 and p38-MAPK), and caspases are critically involved in the pathways of fluvastatin-enhanced apoptosis.     

Troglitazone and rosiglitazone induce apoptosis of vascular smooth muscle cells through an extracellular signal-regulated kinase-independent pathway

Recent evidence suggests that apoptosis may be involved in the control of vascular smooth muscle cell (VSMC) number in atherosclerotic lesions. The peroxisome proliferator-activated receptor gamma (PPARgamma) ligands thiazolidinediones have been reported to induce apoptosis in macrophages and in a variety of tumor cell lines. To evaluate whether these agents also induce apoptosis in VSMC, cultured rat VSMC were treated with increasing doses of the thiazolidinedione analogues troglitazone (TRO) and rosiglitazone (RSG). Both ligands induced cell death in a concentration-dependent manner (EC50 12.1+/-3.3 microM and 1.43+/-0.39 microM, respectively), causing almost complete cell death at the highest concentrations (100 microM and 10 microM for TRO and RSG, respectively), along with an expected parallel decrease in [3H]thymidine uptake into cell DNA (EC50 6.7+/-2.4 microM and 0.75+/-0.19 microM, respectively). The cell count was determined by the coulter counter principle. Furthermore two apoptotic markers were measured, the caspase 3 activity and the cytoplasmic histone-associated DNA fragments, both of which were significantly increased when the aforementioned high concentrations were used. This indicates that apoptosis is involved in the TRO- and RSG-induced VSMC growth suppression. The same concentrations of TRO and RSG caused an unexpected stimulation of the extracellular signal-regulated response kinases 1 and 2 (ERK1/2) and stimulated the p38 mitogenic-activated protein (MAP) kinase as determined by Western blotting. In order to establish whether the proapoptotic effects of TRO and RSG are mediated through ERK1/2 activation, we used the selective MAP kinase kinase (MEK) inhibitor PD98059 (20 microM), which suppressed the TRO- and RSG-induced ERK1/2 activation but did not abolish their proapoptotic effects. We conclude that the thiazolidinedione analogues TRO and RSG induce cell death due to apoptosis in VSMC through an ERK1/2-independent pathway.     

Verapamil stereoisomers induce antiproliferative effects in vascular smooth muscle cells via autophagy

Calcium channel blockers (CCBs) are important in the management of hypertension and limit restenosis. Although CCB efficacy could derive from decreased blood pressure, other mechanisms independent of CCB activity also can contribute to antiproliferative action. To understand mechanisms of CCB-mediated antiproliferation, we studied two structurally dissimilar CCBs, diltiazem and verapamil, in cultured rat vascular smooth muscle cells (VSMC). To elucidate CCB-independent effects, pure stereoisomers of verapamil (R-verapamil, inactive VR; S-verapamil, active, VS) were used. The effects of CCB exposure on cell viability (MTT reduction), cell proliferation (³H-thymidine incorporation), VSMC morphology by light and transmission electron microscopy (TEM) and autophagy (LC3I/II, ATG5) were measured. In general, verapamil, VR or VS treatment alone (80 μM) appreciably enhanced MTT absorbance although higher concentrations (VR or VS) slightly decreased MTT absorbance. Diltiazem (140 μM) markedly decreased MTT absorbance (40%) at 120 h. VR or VS treatment inhibited ³H-thymidine incorporation (24 h) and induced cytological alterations (i.e., karyokinesis, enhanced perinuclear MTT deposition, accumulated perinuclear “vacuoles”). TEM revealed perinuclear “vacuoles” to be aggregates of highly laminated and electron-dense vesicles resembling autophagosomes and lysosomes, respectively. Increased autophagosome activity was confirmed by a concentration-dependent increase in LC3-II formation by Western blotting and by increased perinuclear LC3-GFP⁺ puncta in verapamil-treated VSMC. Verapamil stereoisomers appeared to decrease perinuclear mitochondrial density. These observations indicate that antiproliferative effects of verapamil stereoisomers are produced by enhanced mitochondrial damage and upregulated autophagy in VSMC. These effects are independent of CCB activity indicating a distinct mechanism of action that could be targeted for more efficacious anti-atherosclerotic and anti-restenosis therapy.     

福辛普利对平滑肌细胞凋亡及凋亡调控基因的影响

目的：探讨血管紧张素Ⅱ在不同浓度和不同作用时间下对血管平滑肌细胞凋亡及凋亡调控基因表达的作用，并研究福辛普利对其的影响。方法：采用流式细胞技术测定在不同浓度和不同作用时间血管紧张素Ⅱ作用下血管平滑肌细胞凋亡率及凋亡调控基因Fas、Bcl-2表达的变化和福辛普利对其的影响。结果：血管紧张素Ⅱ对血管平滑肌细胞凋亡和凋亡调控基因表达有较明显的诱导作用。福辛普利可抑制AngⅡ作用，对细胞凋亡有一定的影响，同时可影响凋亡调控基因的表达。结论：血管紧张素Ⅱ具有一定的的促进血管平滑肌细胞凋亡作用和调节凋亡调控基因表达的作用，尤其大剂量作用较明显。而福辛普利可明显抑制AngⅡ对血管平滑肌细胞的作用。     

Glucocorticoids induce endothelin release from vascular smooth muscle cells but not endothelial cells

Vascular smooth muscle cells in culture are capable of secreting endothelin which is a vasoconstrictor and mitogenic peptide. The effect of glucocorticoids on endothelin release from vascular smooth muscle cells of the rat and rabbit aortas was investigated. Micromolar concentrations of dexamethasone and cortisol caused a 2 to 5-fold increase in endothelin release from the two smooth muscle cell types but no such response was observed in endothelial cells of the bovine aorta. Glucocorticoids appear to selectively induce endothelin release from vascular smooth muscle cells and this may be relevant to glucocorticoid-induced hypertension.