Role of caspases in Ox-LDL-induced apoptotic cascade in human coronary artery endothelial cells

Oxidized low-density lipoprotein (ox-LDL) induces apoptosis in endothelial cells. However, steps leading to ox-LDL-induced apoptosis remain unclear. We examined the role of ox-LDL and its newly described receptor LOX-1 in the expression of intracellular pro- and antiapoptotic proteins and caspase pathways in human coronary artery endothelial cells (HCAECs). Cells were cultured and treated with different concentrations (10 to 80 microg/mL) of ox-LDL for different times (2 to 24 hours). Ox-LDL induced apoptosis in HCAECs in a concentration- and time-dependent manner. Ox-LDL also activated caspase-9 and caspase-3, but not caspase-8. After ox-LDL treatment, there was a significant release of activators of caspase-9, including cytochrome c and Smac from mitochondria to cytoplasmic compartment, and their release was not affected by treatment of cells with inhibitors of either caspase-8 or caspase-9. Ox-LDL also decreased expression of antiapoptotic proteins Bcl-2 and c-IAP (inhibitory apoptotic protein)-1, which are involved in the release of cytochrome c and Smac and activation of caspase-9, in a concentration- and time-dependent manner. On the other hand, ox-LDL did not change the expression of Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP) and proapoptotic protein Fas, which are required for the activation of caspase-8. Further, ox-LDL did not cause the truncation of Bid, which implies the activation of caspase-8. In other experiments, pretreatment of HCAECs with the caspase-9 inhibitor z-LEHD-fmk, but not the caspase-8 inhibitor z-IETD-fmk, blocked ox-LDL-induced activation of caspase-3 and apoptosis. As expected, pretreatment with the caspase-3 inhibitor DEVD-CHO inhibited ox-LDL-induced activation of caspase-3 and resultant apoptosis. The proapoptotic effects of ox-LDL were mediated by its receptor LOX-1, because pretreatment of HCAECs with antisense-LOX-1, but not sense-LOX-1, blocked these effects of ox-LDL. These findings suggest that ox-LDL through its receptor LOX-1 decreases the expression of antiapoptotic proteins Bcl-2 and c-IAP-1. This is followed by activation of apoptotic signaling pathway, involving release of cytochrome c and Smac and activation of caspase-9 and then caspase-3.     

消退素E1对氧化型低密度脂蛋白诱导的血管内皮细胞损伤的保护作用

目的 研究消退素E1是否对氧化型低密度脂蛋白诱导的人脐静脉内皮细胞损伤有保护作用,并探讨其分子机制.方法 将人脐静脉内皮细胞(HUVEC)随机分成6组,分别用生理盐水、消退素E1、PI3K抑制剂wortmanin、氧化型低密度脂蛋白、氧化型低密度脂蛋白+消退素E1、氧化型低密度脂蛋白+消退素E1+ PI3K抑制剂wortmanin处理人脐静脉内皮细胞48 h.随后用噻唑蓝法分析内皮细胞存活率、流式细胞仪检测细胞凋亡率、酶联免疫法分析细胞上清中肿瘤坏死因子-α( TNF-α)含量,酶标仪测量细胞内半胱天冬蛋白酶(caspase)3、9的活性,免疫印迹法检测激活的AKT和血凝素样氧化低密度脂蛋白受体-1( LOX-1)的表达.结果 氧化型低密度脂蛋白组细胞活力显著低于生理盐水组(P＜0.01),细胞凋亡率、TNF-α含量、caspase3和9活性,LOX-1蛋白的表达均显著高于生理盐水组(P均＜0.01).氧化型低密度脂蛋白+消退素E1组细胞凋亡率、TNF-α含量、caspase3和9活性,LOX-1蛋白的表达均显著低于氧化型低密度脂蛋白组(P＜0.01),细胞活力和激活的AKT均显著高于氧化型低密度脂蛋白组(P均＜0.01).氧化型低密度脂蛋白+消退素E1+ PI3K抑制剂wortmanin组的细胞活力和细胞凋亡率、TNF-α含量、caspase3和9活性,LOX-1蛋白的表达均显著高于氧化型低密度脂蛋白+消退素E1组(P＜0.05).结论 消退素E1能有效地抑制氧化型低密度脂蛋白诱导的内皮细胞凋亡,此作用可能通过PI3 K/Akt信号通路介导.     

Porcine Circovirus 2 Deploys PERK Pathway and GRP78 for Its Enhanced Replication in PK-15 Cells

Porcine circovirus type 2 (PCV2) infection induces autophagy and apoptosis. These cellular responses could be connected with endoplasmic reticulum (ER) stress. It remains unknown if PCV2 induces ER stress and if autophagy or apoptosis is primary to PCV2 infection or secondary responses following ER stress. Here, we demonstrate that PCV2 triggered unfolded protein response (UPR) in PK-15 cells by activating the PERK/eIF2α pathway without concomitant activation of IRE1 or ATF6. Since ATF4 and CHOP were induced later than PERK/eIF2α, it is clear that persistent PCV2 infection could lead to selective activation of PERK via the PERK-eIF2α-ATF4-CHOP axis. Therefore, PERK activation could be part of the pro-apoptotic signaling via induced expression of CHOP by PCV2. Since PERK inhibition by GSK2606414 or RNA silencing or suppression of eIF2α dephosphorylation by salubrinal limited viral replication, we suppose that PCV2 deploys UPR to enhance its replication. Over-expression of GRP78 or treatment with tauroursodeoxycholic acid could enhance viral capsid expression and/or viral titers, indicating that these chaperones, endogenous or exogenous, could help correct folding of viral proteins. Our findings provide the first evidence that ER stress plays a role in the pathogenesis of PCV2 infection probably as part of autophagic and apoptotic responses.     

Upregulation of endothelial receptor for oxidized LDL (LOX-1) by oxidized LDL and implications in apoptosis of human coronary artery endothelial cells: evidence from use of antisense LOX-1 mRNA and chemical inhibitors

A specific lectin-like endothelial receptor for oxidized low density lipoprotein (LOX-1), distinct from the scavenger receptor in monocytes/macrophages, has been identified and cloned. In this study, we examined the regulation of LOX-1 by oxidized low density lipoprotein (ox-LDL) and determined the role of LOX-1 in ox-LDL-induced apoptosis of cultured human coronary artery endothelial cells (HCAECs). Incubation of HCAECs with ox-LDL (40 microg/mL), but not native LDL, for 24 hours markedly increased LOX-1 expression (mRNA and protein). After 48 hours of preincubation of HCAECs with a specific antisense to LOX-1 mRNA (antisense LOX-1), ox-LDL-mediated upregulation of LOX-1 was suppressed (P<0.01). In contrast, treatment of HCAECs with sense LOX-1 had no effect. Ox-LDL also induced apoptosis (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA laddering) of HCAECs in a concentration- and time-dependent fashion. LOX-1 played an important role in ox-LDL-mediated apoptosis of HCAECs because antisense LOX-1 inhibited this effect of ox-LDL. Polyinosinic acid and carrageenan, 2 different chemical inhibitors of LOX-1, also decreased ox-LDL-mediated apoptosis of HCAECs. Nuclear factor (NF)-kappaB was markedly activated in ox-LDL-treated HCAECs. The critical role of NF-kappaB activation became evident in experiments with antisense LOX-1, which abolished ox-LDL-mediated NF-kappaB activation. In this process, an NF-kappaB inhibitor, caffeic acid phenethyl ester, also inhibited ox-LDL-mediated apoptosis of HCAECs. These findings indicate that ox-LDL upregulates its own endothelial receptor. Ox-LDL-induced apoptosis is mediated by the action of LOX-1. In this process, NF-kappaB activation may play an important role as a signal transduction mechanism.     

Oxidized-LDL through LOX-1 increases the expression of angiotensin converting enzyme in human coronary artery endothelial cells

BACKGROUND AND OBJECTIVES: Our previous studies have shown that oxidized low-density lipoprotein (ox-LDL) and angiotensin II (Ang II) influence each other's action in endothelial cells. This study was designed to examine the regulation by ox-LDL of the expression of angiotensin converting enzyme (ACE) gene in human coronary artery endothelial cells (HCAECs). In addition, we studied the effect of the HMG CoA reductase inhibitor simvastatin on this interaction. METHODS AND RESULTS: Cultured HCAECs were incubated with ox-LDL (10-80 microg/ml) for 1-24 h. Ox-LDL increased the expression of ACE in a concentration- and time-dependent fashion. The upregulation of ACE expression in response to ox-LDL was mediated by its endothelial receptor LOX-1, since pretreatment of HCAECs with a blocking antibody to LOX-1 prevented the expression of ACE (P<0.01). Native-LDL had no significant effect on ACE expression. In this process, ox-LDL-induced activation of mitogen-activated protein kinase (MAPK p42/44) played an important role, since pretreatment of HCAECs with the MAPK p42/44 inhibitor (PD98059, 10 microM) inhibited MAPK activation and subsequently attenuated the expression of ACE (P<0.01 vs. ox-LDL alone). In other experiments, we pretreated HCAECs with simvastatin (10 microM) and then exposed the cells to ox-LDL. Simvastatin markedly attenuated ox-LDL-induced MAPK activation, and concurrently reduced ACE expression (P<0.01 vs. ox-LDL alone). CONCLUSIONS: Our observations provide direct evidence that ox-LDL via LOX-1 activation induces ACE gene expression in HCAECs, and MAPK activation plays a signal transduction role in this process. Simvastatin, which inhibits MAPK activation, also blocks ox-LDL-mediated upregulation of ACE.     

氧化型低密度脂蛋白诱导肝窦内皮细胞植物血凝素样氧化型低密度脂蛋白受体1的表达

目的：探讨植物血凝素样氧化型低密度脂蛋白（ox-LDL）受体1（LOX-1）在肝窦内皮细胞（HSECs）中的表达和ox-LDL对其表达的调控作用。方法使用实时定量聚合酶链反应（RT-PCR）和Western blotting法从基因和蛋白水平检测未经处理HSECs 中LOX-1的表达。应用不同浓度ox-LDL（0、20、40、60、80和100 mg/L）对HSECs作用24 h并应用80 mg/L ox-LDL对HSECs作用不同时间（0、12、24和48 h）,作用后实时定量PCR检测HSECs内LOX-1 mRNA的表达水平,Western blotting法检测细胞内LOX-1蛋白表达。给予80 mg/L ox-LDL干预组多聚肌苷酸250 mg/L作用24 h后,测定LOX-1 mRNA和蛋白的表达。采用单因素方差分析及t检验进行数据分析。结果 LOX-1 mRNA和蛋白在HSECs中均有表达。20～80 mg/L ox-LDL组HSECs中LOX-1 mRNA、蛋白表达水平随ox-LDL剂量增加而升高,与剂量有明显相关性（F＝38.7、3.43,均P＜0.05）。与80 mg/L ox-LDL组相比,100 mg/L ox-LDL 组 LOX-1 mRNA、蛋白表达下降,差异有统计学意义（ t ＝23.75、18.26, P ＜0.05）。80 mg/L ox-LDL对HSECs作用时间在0～24 h时,随着时间延长,LOX-1 mRNA、蛋白表达递增,与ox-LDL作用时间有明显相关性（F＝2.36、0.33,均P＜0.05）。与作用24 h相比,作用48 h组HSECs中LOX-1 mRNA、蛋白表达下降（t＝69.21、36.27,均P＜0.05）。与80 mg/L ox-LDL组相比,多聚肌苷酸组中LOX-1 mRNA和蛋白表达降低,两组差异均有统计学意义（ t＝54.93、28.19,均P＜0.05）。结论LOX-1存在于HSECs。在一定浓度和时间范围内,ox-LDL对HSECs LOX-1的调控作用具有时间和浓度依赖性,而多聚肌苷酸可部分抑制这种效应。     

阿托伐他汀减轻巨噬细胞内质网应激及其介导的细胞凋亡

目的 探讨阿托伐他汀对7-酮胆固醇（7-KC）诱导的巨噬细胞内质网应激及细胞凋亡的影响。方法 AopE-/-小鼠左肾动脉和左颈总动脉联合部分结扎建立颈动脉易损斑块模型。采用HE染色方法观察斑块病理学改变,用免疫荧光结合激光扫描共聚焦显微镜技术检测斑块中内质网应激(ER stress)相关蛋白CHOP及磷酸化PERK（p-PERK）的表达。体外培养小鼠巨噬细胞RAW264.7,给予7-KC、H2O2或联合阿托伐他汀处理后,蛋白质免疫印迹方法（Western blot）测定ER stress相关蛋白CHOP、p-PERK 、XBP-1s及凋亡相关蛋白cleaved caspase-3的表达。结果 AopE-/-小鼠颈动脉易损斑块局部ER stress相关蛋白CHOP的表达及PERK磷酸化水平明显上调;7-KC可诱导小鼠巨噬细胞ER stress,进而诱导细胞凋亡;同时,氧化应激诱导剂H2O2也可通过诱导小鼠巨噬细胞ER stress介导细胞凋亡;而阿托伐他汀可抑制7-KC和H2O2诱导的巨噬细胞ER stress及其介导的细胞凋亡。结论 ER stress可能参与AS易损斑块的形成;阿托伐他汀可通过减少细胞内氧化应激的水平,减轻巨噬细胞ER stress,从而抑制细胞凋亡。     

Differential activation of myocardial ER stress response: A possible role in hypoxic tolerance

Background

Low oxygen availability in the high altitude milieu causes adverse physiological and pathological consequences to the cardiopulmonary system. A key role is played by proteins in maintaining optimal cardiac function under stress. Differential response to hypoxia may be linked to the susceptibility of proteins to free radical induced modifications. The present study was designed to understand the significance of protein oxidation and ER stress in the myocardial response to hostile environments.

Methods

Sprague–Dawley rats were exposed to simulated hypoxia equivalent to 223 mm Hg pressure, screened on the basis of time taken for onset of a characteristic hyperventilatory response and categorized as susceptible (< 10 min), normal (10–25 min) or tolerant (> 25 min). Protein modifications and activity of cellular proteolytic enzymes were assayed in myocardial tissue extracts to identify alterations in protein homeostasis. To evaluate the ER stress response, expression of various ER marker chaperones was investigated.

Results

Susceptible animals displayed a distinct increase in protein oxidation and intracellular thiol content. They showed higher expression of ER stress hallmarks, GRP78, PDI and ERO1α, and exhibited a greater activation of the proteasome and calpain proteolytic systems, associated with elevated oxidized proteins. While a marked upregulation in the prosurvival signaling cascade PI3K/Akt/mTOR was observed in tolerant animals, the expression of pro-apoptotic caspase-3 and CHOP remained unaltered.

Conclusion

Thus, higher susceptibility to hypoxia is linked to a disruption in the proteostasis and activation of the ER stress response. Enhanced tolerance to hostile environments may be contributed by better maintenance of protein folding homeostasis.     

β1-AR持久兴奋通过CaMKIIδ-内质网应激凋亡通路导致心力衰竭的机制

目的研究β1-AR持久兴奋通过CaMKIIδ内质网应激（ERS）凋亡通路导致心力衰竭的机制。方法30只SD大鼠随机分成三组，正常对照组（Control）、异丙肾上腺组（Iso）和、异丙肾上腺＋美托洛尔组（Iso＋Meto），每组10只，所有动物均自由进食进水。（1）Iso组大鼠背部皮下注射Iso5mg／（kg·d），连续10d；Control组背部皮下注射相同体积的生理盐水；Iso＋Meto组大鼠背部皮下注射Iso5mg／（kg·d），连续10d，在背部皮下注射Iso前一天开始Meto 10mg／（kg·d）灌胃，连续4周；（2）所有大鼠饲养4周后，采用美国Millar公司P—V Loop导管经颈动脉插管至左心室，使用Pow—erlab生理记录系统测量血流动力学相关指标；统计各组大鼠心脏重量和心脏重量／体重比值；（3）TUNEL法和Caspase-3活性检测心肌细胞凋亡；（4）Western blot分析CaMKIIδERS相关基因（GRP78、CHOP和caspase-12）和凋亡相关基因Bcl-2／Bax的表达水平。结果30只SD大鼠实验过程精神状态好，进食进水正常。（1）与Control组比较，Iso组SD大鼠心脏重塑和血流动力学指标有显著性差异（P〈0．05）；而Iso＋Meto组心脏重塑和血流动力学指标与Iso组相比明显改善（P〈0．05）；（2）TUNEL法原位检测各组大鼠心肌细胞凋亡示与Control组比较，Iso组TUNEL阳性细胞数明显增高（P〈0．05）；而Iso＋Met组明显低于Iso组（P〈0．05）。心肌细胞Caspase-3活性和TUNEL凋亡阳性细胞核指数各组变化一致。Western blot检测心肌细胞凋亡相关基因bcl-2／Bax蛋白表达。与Control组相比，Iso组bcl-2蛋白表达明显降低和Bax蛋白表达明显增加（P〈0．05）；而Iso＋Met组与Iso组相比,bcl-2明显增高和Bax明显降低（P〈0．05）；（3）Western blot分析CaMKIIδp-CaMKIIδ白表达显示，与Control组比较，Iso组CAMKII活性和p-CaMKIIδ蛋白表达明显增加；而Iso＋Meto组与Iso组相比，CAMKII活性和p-     

京尼平抑制饱和脂肪酸诱导的HepG2细胞凋亡和内质网应激

目的 探讨京尼平减轻棕榈酸对HepG2细胞毒性的作用及机制.方法 将HepG2细胞分为4组,分别用牛血清白蛋白、棕榈酸(1 mmol/L)、京尼平(20 μmol/L)或京尼平(20 μmol/L)预处理30 min后棕榈酸(1 mmol/L)孵育24 h,检测细胞活力及乳酸脱氧酶(LDH)释放;孵育16 h后经流式细...     

消癌解毒方通过内质网应激诱导肝癌HepG2细胞凋亡的研究

[目的]探讨内质网应激在消癌解毒方在人肝癌HepG2细胞凋亡中的作用。[方法]CCK-8法检测不同浓度的消癌解毒方处理HepG2细胞12、24、48 h后细胞的增殖情况;采用流式细胞术检测细胞消癌解毒方处理HepG2细胞24 h凋亡情况;应用qRT-PCR检测内质网应激上游分子标记GRP78、PERK、ATF-6、IRE-1的mRNA水平;采用Western blot方法分析消癌解毒方对PERK、ATF4、CHOP和TRB3蛋白的表达情况;并用CCK-8法检测内质网抑制剂4-苯基丁酸(4-PBA)对HepG2细胞凋亡率的影响。[结果]消癌解毒方抑制HepG2细胞的增殖,并且这种效应呈现时间和浓度依赖;ATF-6和IRE-1 mRNA水平无明显变化,GRP78和PERK mRNA水平较阴性对照组均显著增加;消癌解毒方可上调内质网应激通路的标志蛋白质PERK、ATF4、CHOP水平,增加下游TRB3蛋白的表达。4-PBA与消癌解毒方联合作用组的细胞凋亡率显著减少。[结论]消癌解毒方通过内质网应激诱导人肝癌HepG2细胞的凋亡,其可能是通过PERK通路上调内质网应激相关凋亡蛋白CHOP的表达。     

Potential pathological roles for oxidized low-density lipoprotein and scavenger receptors SR-AI,CD36, and LOX-1 in aortic valve stenosis

Objective

To clarify the potential mechanisms by which oxidized low-density lipoprotein (oxLDL) could contribute to the progression of aortic valve stenosis (AVS).

Methods

We investigated a total of 46 stenotic and 20 control human aortic valves. The mRNA expression levels of scavenger receptor class A type 1 (SR-A1), CD36, Lectin-like oxidized LDL receptor-1 (LOX-1), and scavenger receptor class B type 1 (SR-B1) were studied using qPCR. Their cellular distribution in the valves was assessed by immunohistochemistry, and the potential effects of oxLDL were studied in cultured myofibroblasts isolated from the aortic valves.

Results

In AVS, the proinflammatory SR-A1 and the angiogenic LOX-1 were upregulated (p = 0.003 and p = 0.002), whereas the antiangiogenic CD36 was downregulated (p = 0.02). The expression of the atheroprotective SR-B1 remained unchanged. Immunohistochemistry revealed that SR-A1 was expressed by macrophages, whereas the expression of CD36 and LOX-1 was confined to myofibroblasts and endothelial cells in the diseased valves. In cultured valvular myofibroblasts, mast cell-derived components and TNF-α induced LOX-1 expression (p = 0.05 and p < 0.001), whereas oxLDL promoted the expression of proinflammatory cytokines. Furthermore, the expression of osteoprotegerin, an inhibitor of valvular calcification, decreased in response to oxLDL. Finally, myofibroblasts derived from stenotic valves accumulated more DiI-labeled oxLDL than myofibroblasts derived from macroscopically healthy valves (p = 0.035), so revealing enhanced foam cell-forming potential of myofibroblasts in the diseased valves.

Conclusion

This study unveils the presence of SR-A1, CD36, and LOX-1 in aortic valves and suggests potential mechanisms by which they may contribute to the pathological angiogenesis, inflammation, calcification, and lipid accumulation in AVS.     

沉默MAP4K4通过调控PPARγ/ABCA1通路缓解ox-LDL诱导的血管内皮细胞损伤

目的 探讨丝裂原活化蛋白激酶(MAP4K4)在氧化型低密度脂蛋白(ox-LDL)诱导的血管内皮细胞损伤中的作用及其机制.方法 采用100mg/Lox-LDL诱导人脐静脉内皮细胞(HUVEC)建立细胞损伤模型.RT-PCR检测HUVEC 中MAP4K4的mRNA表达水平.Western blot 法测定MAP4K4、Ba...