大鼠HO-1表达质粒构建、活力检测及抗HUVEC凋亡的作用研究

异种移植排斥反应的主要特征为内皮细胞发生Ⅱ型激活,引起黏附分子、细胞因子和前促凝分子等基因高表达,造成血管收缩、白细胞黏附、激活、聚集和血栓形成,最终导致内皮细胞凋亡。保护基因HO-1通过抑制前炎症反应及免疫调抑作用以保护异种移植器官。因此,通过构建含剪切的野生型大鼠HO-1 cDNA的表达型质粒,用DOTAP包裹转入HUVEC中表达,测定表达量及表达产物活性;采用TNF-α诱导细胞凋亡,以及Heme和SnPP分别刺激细胞,诱导和抑制细胞内HO-1表达量,流式细胞仪测定细胞凋亡率,明确HO-1的抗细胞凋亡作用。结果显示HO-1在HUVEC中高度表达,活力为对照组5倍;TNF-α诱导细胞凋亡,但Heme处理后细胞凋亡率下降至20%以下,而SnPP处理后细胞凋亡率显著上升,最高达到95%以上,并且HO-1基因表达抑制时细胞凋亡率是诱导时的5-20倍。本实验表明Heme处理后HO-1表达上调,具有显著抗细胞凋亡作用,细胞凋亡率与HO-1表达量呈负相关,提示HO-1通过抑制细胞凋亡,对细胞有保护作用。     

花生四烯酸细胞色素P450表氧化酶对凋亡内皮细胞Bc1-2表达、Caspase-3及MAPK活性的影响

花生四烯酸经细胞色素P450表氧化酶代谢产生的内皮来源超极化因子(EDHF)[表氧化二十烷烯酸(EETs)]对内皮细胞具有保护作用.研究了转染细胞色素P450表氧化酶基因CYPBM3·F87V、CYP2C110R及CYP2J2产生内源性EETs,通过检测内皮细胞中Bc1-2表达、caspase-3的活性及MAPK磷酸化水平探讨内源性EDHF的内皮细胞保护效应及其抗TNF-α诱导内皮细胞凋亡的作用机制.原代培养的牛主动脉血管内皮细胞转染CYP450表氧化酶基因24h后,加入TNF-α作用一定时间诱导内皮细胞凋亡,用Western印迹方法检测Bcl-2的表达,MAPK磷酸化水平,同时测定caspase-3的活性.结果显示转染表氧化酶基因能抑制TNF-α诱导的时间依赖性Bcl-2下调,抑制Caspase-3的激活.TNF-α使细胞内磷酸化MAPK水平呈时间依赖性减低,转染表氧化酶基因后细胞内的磷酸化MAPK水平较对照组升高.因此,转染表氧化酶基因CYPBM3·F87V、CYP2C11OR以及CYP2J2使内皮细胞产生内源性EETs(EDHF)通过激活MAPK(ERK1/2)途径,抑制抗凋亡基因Bcl-2的降解,抑制caspase-3的激活,从而抑制TNF-α诱导的内皮细胞凋亡,因而具有内皮保护效应.     

花生四烯酸细胞色素P450表氧化酶对凋亡内皮细胞Bcl-2表达、Caspase-3及MAPK活性的影响

花生四烯酸经细胞色素P450表氧化酶代谢产生的内皮来源超极化因子(EDHF)[表氧化二十烷烯酸(EETs)]对内皮细胞具有保护作用。研究了转染细胞色素P450表氧化酶基因CYPBM3·F87V、CYP2C11OR及CYP2J2产生内源性EETs,通过检测内皮细胞中Bcl-2表达、caspase-3的活性及MAPK磷酸化水平探讨内源性EDHF的内皮细胞保护效应及其抗TNF-α诱导内皮细胞凋亡的作用机制。原代培养的牛主动脉血管内皮细胞转染CYP450表氧化酶基因24h后,加入TNF-α作用一定时间诱导内皮细胞凋亡,用Western印迹方法检测Bcl-2的表达,MAPK磷酸化水平,同时测定caspase-3的活性。结果显示转染表氧化酶基因能抑制TNF-α诱导的时间依赖性Bcl-2下调,抑制Caspase-3的激活。TNF-α使细胞内磷酸化MAPK水平呈时间依赖性减低,转染表氧化酶基因后细胞内的磷酸化MAPK水平较对照组升高。因此,转染表氧化酶基因CYPBM3·F87V、CYP2C11OR以及CYP2J2使内皮细胞产生内源性EETs(EDHF)通过激活MAPK(ERK1/2)途径,抑制抗凋亡基因Bcl-2的降解,抑制caspase-3的激活,从而抑制TNF-α诱导的内皮细胞凋亡,因而具有内皮保护效应。     

腺苷酸活化蛋白激酶活化对单核细胞-内皮细胞黏附的影响及其机制北大核心CSCD

本研究旨在探讨腺苷酸活化蛋白激酶（AMP-activated protein kinase,AMPK）活化对单核细胞与内皮细胞黏附的影响及其分子机制。用不同剂量的AMPK激动剂5-氨基咪唑-4-甲酰胺核糖核苷酸（AICAR,0~2 mmol/L）或AMPK抑制剂compound C（10 mmol/L）处理肿瘤坏死因子α（tumor necrosis factorα,TNFα,10 ng/m L）诱导的人主动脉内皮细胞（human aortic endothelial cells,HAECs）,用TNFα诱导过表达活性型或显性抑制型AMPK蛋白的HAECs。用荧光染色法观察AMPK对荧光标记的单核THP-1细胞与HAECs黏附的影响。用荧光定量PCR检测血管细胞黏附分子1（vascular cell adhesion molecule-1,VCAM-1）和细胞间黏附分子1（intercellular cell adhesion molecule-1,ICAM-1）m RNA表达水平,用ELISA法检测二者的蛋白分泌量;用Western blot检测核因子-kappa B（nuclear factor-kappa B,NF-κB）p65的211位点赖氨酸乙酰化水平,用ELISA法检测NF-κB p65DNA结合活性,并用试剂盒检测p300乙酰转移酶活性。通过小干扰RNA抑制HAECs组蛋白乙酰转移酶p300蛋白表达后,检测TNFα对NF-κB p65 DNA结合活性、黏附分子ICAM-1、VCAM-1的表达及单核细胞黏附率的影响。结果显示,AICAR显著抑制TNFα诱导的单核细胞与HAECs的黏附,在HAECs中下调TNFα诱导的ICAM-1、VCAM-1的m RNA水平上调和蛋白分泌。AICAR的效应可以被AMPK抑制剂compound C完全阻断。转染活性型AMPKα显著抑制TNFα诱导的ICAM-1、VCAM-1m RNA表达和分泌,以及单核细胞-内皮细胞黏附,而转染显性抑制型AMPKα则无明显影响。RNAi干预抑制p300活性显著抑制TNFα诱导的黏附分子表达和单核-内皮细胞黏附。AMPK激活可抑制TNFα诱导的p300乙酰转移酶活性,抑制NF-κB p65的211位赖氨酸的乙酰化,降低NF-κB p65 DNA结合活性。以上结果提示,AMPK激活抑制单核细胞-内皮细胞黏附,作用机制可能与其降低p300酶活性,下调NF-κB p65转录活性密切相关。     

Nmp4促进TNF-α诱导的成骨细胞凋亡

核基质蛋白4(nuclear matrix protein4,Nmp4)是一种具有核质穿梭功能的结构性转录因子.主要通过负调控调节成骨细胞分化和增殖,抑制骨密度及骨量增加,而Nmp4是否调节成骨细胞凋亡,还未有相关报道.本课题通过分离Nmp4基因敲除(Nmp4-KO)和野生型(WT)小鼠原代成骨细胞,以肿瘤坏死因子(TNF-α)为凋亡诱导手段,研究了Nmp4对成骨细胞凋亡的影响及其作用机制.体外细胞实验发现,Nmp4-KO显著抑制TNF-α诱导的成骨细胞内caspase-3激活.Nmp4-KO促进细胞外信号调节激酶(Erk)和丝氨酸/苏氨酸蛋白激酶(Akt)信号途径的激活,抑制c-Jun氨基末端激酶(JNK)磷酸化,从而对抗成骨细胞凋亡.TNF-α诱导处理可增强成骨细胞核因子NFκB磷酸化及其核转位,但Nmp4基因缺失无进一步促进作用.未经诱导处理的Nmp4-KO细胞内NFκB磷酸化水平显著高于WT对照.此外,TNF-α诱导处理促使线粒体途径信号分子Bad磷酸化及Bcl-xl表达水平适当升高,但在两种细胞表型间无显著差异.这些结果证实,Nmp4基因敲除可促进相关抗凋亡信号分子的激活和表达,抑制促凋亡信号的激活,进而抑制成骨细胞凋亡的发生.     

Apelin 通过调节ROS 水平抑制TNF-α 诱导的HepG2 细胞凋亡

目的:探讨apelin在肿瘤坏死因子(tumor necrosis factor-α,TNF-α)诱导的肝细胞凋亡中的作用及可能机制。方法:PCR检测HepG2细胞和原代小鼠肝细胞中APJ受体的表达;采用Hoechst 33342染色检测TNF-α诱导的HepG2细胞凋亡;用活性氧(ROS)检测试剂盒结合流式细胞术测定细胞内ROS水平;通过Western blot检测信号分子JNK的磷酸化水平;比较给予apelin处理对上述指标的影响。结果:HepG2细胞和原代小鼠肝细胞均表达APJ受体;apelin可抑制TNF-α导致的细胞内ROS生成增多和JNK磷酸化水平升高并减少TNF-α诱导的HepG2细胞凋亡。结论:Apelin可能通过拮抗TNF-α诱导的细胞内ROS水平升高,使JNK信号失活,从而抑制HepG2细胞凋亡。     

葡萄球菌肠毒素B诱导脐静脉内皮细胞凋亡机制的探讨

目的探讨金黄色葡萄球菌(金葡菌)肠毒素B诱导脐静脉内皮细胞凋亡的机制。方法将不同浓度金葡菌肠毒素B感染脐静脉内皮细胞8 h后,用流式细胞术检测细胞凋亡率,同时用比色法检测TNF-α、caspase-3及caspase-8的产生量,并检测加入TNF-α抗体、caspase-3和caspase-8抑制剂后的细胞凋亡率。结果不同浓度肠毒素B作用脐静脉内皮细胞8 h后均可诱导细胞凋亡,且TNF-α、caspase-3和caspase-8的产生量均高于对照组(P0.01);而加入TNF-α抗体、caspase-3和caspase-8抑制剂后凋亡率明显降低。结论金葡菌肠毒素B可以诱导脐静脉内皮细胞凋亡,其凋亡机制可能是通过TNF-α介导的caspase-8及caspase-3激活的外源性死亡因子受体途径。     

球状脂联素在波动性高血糖诱导人脐静脉内皮细胞凋亡中的作用

目的球状脂联素在波动性高血糖诱导人脐静脉内皮细胞凋亡中的作用。方法在不同条件下体外培养人脐静脉内皮细胞,分别或联合加入球状脂联素(gAD)、单磷酸腺苷激活蛋白激酶(AMPK)的激活剂AICAR和AMPK的阻滞剂araA。采用MTT比色法测定细胞活性,流式细胞仪检测细胞凋亡率,Western blot检测AMPKα和磷酸化AMPKα蛋白表达。结果分别与对照组和恒定高血糖组比较,波动性高血糖显著抑制细胞活性和增加细胞凋亡率。gAD明显抑制波动性高血糖诱导的细胞凋亡。AICAR和gAD可明显激活AMPK的表达。araA可明显抑制gAD诱导的AMPK蛋白表达。结论波动性高血糖比恒定性高血糖更易促进内皮细胞凋亡,gAD明显抑制波动性高血糖诱导内皮细胞凋亡,其机制可能与激活AMPK有关。     

血红素加氧酶／一氧化碳系统在1，6-二磷酸果糖抗IL-1β致胰岛细胞凋亡中的作用

目的：探讨1,6-二磷酸果糖（FDP）对白介素-1β（IL-1β）致胰岛细胞凋亡的保护作用及其机制与血红素加氧酶／一氧化碳（HO-1／CO）系统的关系。方法：应用离体培养乳鼠的胰岛细胞,分别检测IL-1β、FDP作用后细胞形态、细胞活性、细胞凋亡率、细胞HO-1的活性和细胞培养上清液中胰岛素的基础和高糖刺激分泌量以及CO的含量的变化,同时设立正常对照组。结果：正常胰岛细胞HO-1活性较低,CO含量少,凋亡细胞率为4．71±0．62。IL-1β作用20h后,与正常组比较胰岛细胞活性明显降低,基础和高糖刺激胰岛素分泌减少,胰岛细胞凋亡率明显增加（P〈0．01）;细胞HO-1活性有所增加,上清液中CO生成增多（P〈0．05）,损伤后与FDP共同孵育细胞活性显著升高,胰岛素基础和高糖分泌量增多,胰岛凋亡率明显降低,HO-1活性和CO生成显著提高（P〈0．01）,具有统计学意义。结论：FDP能降低IL-1β诱导胰岛细胞的凋亡,改善细胞活性,促进细胞的分泌功能,机制可能与FDP增加HO-1活性,从而CO生成增多有关。     

缺氧诱导因子-1α在缺氧预处理预防心肌细胞损伤中的作用

本工作旨在研究缺氧预处理(hypoxic preconditioning,HPC)对于心肌细胞外信号调节激酶(extracellular signal-regulated proteinkinases,ERK)活性、缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响,及其在缺氧复氧诱导心肌细胞损伤中的作用。通过在培养的SD乳鼠心肌细胞缺氧／复氧(H／R)模型上,观察HPC对于24h后H／R诱导心肌细胞损伤的影响,以台盼蓝排斥实验检测心肌细胞存活率、以TUNEL法检测细胞凋亡、并用荧光素染料Hoechst33258测定心肌细胞凋亡率：制备心肌细胞蛋白提取物,以磷酸化的ERK1／2抗体测定ERK1／2活性,以抗HIF-1α抗体检测HIF-1α的表达,并观察ERKs的上游激酶(MEK1／2)抑制剂PD98059对于HPC诱导的ERKs磷酸化、HIF-1α表达以及心肌细胞保护作用的影响,并分析细胞损伤与ERK1／2活性、HIF-1α表达量之间的相互关系。结果 显示缺氧复氧造成心肌细胞损伤,HPC可以增加心肌细胞H／R后存活率,降低凋亡率,并激活ERKll2,诱导HIF-1α表达：细胞凋亡与ERKs活性、HIF-1α表达量之间存在负相关,即ERKs活化、HIF-1α表达与预防细胞损伤有关：而ERKs活性与HIF-1α表达量之间存在正相关,ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的ERKs磷酸化、HIF-1α表达和心肌细胞保护作用。由此得出的结论是HPC可以提高乳鼠心肌细胞对于H／R的耐受性,其机制涉及ERKs介导的HIF-1α表达。     

Inhibition of heme oxygenase-1 interferes with the transforming activity of the Kaposi sarcoma herpesvirus-encoded G protein-coupled receptor

Heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in the heme catabolism, is expressed in AIDS-Kaposi sarcoma (KS) lesions. Its expression is up-regulated by the Kaposi sarcoma-associated herpesvirus (KSHV) in endothelial cells, but the mechanisms underlying KSHV-induced HO-1 expression are still unknown. In this study we investigated whether the oncogenic G protein-coupled receptor (KSHV-GPCR or vGPCR), one of the key KSHV genes involved in KS development, activated HO-1 expression. Here we show that vGPCR induces HO-1 mRNA and protein levels in fibroblasts and endothelial cells. Moreover, targeted knock-down gene expression of HO-1 by small hairpin RNA and chemical inhibition of HO-1 enzymatic activity by tin protoporphyrin IX (SnPP), impaired vGPCR-induced survival, proliferation, transformation, and vascular endothelial growth factor (VEGF)-A expression. vGPCR-expressing cells implanted in the dorsal flank of nude mice developed tumors with elevated HO-1 expression and activity. Chronic administration of SnPP to the implanted mice, under conditions that effectively blocked HO-1 activity and VEGF-A expression in the transplanted cells, strikingly reduced tumor growth, without apparent side effects. On the contrary, administration of the HO-1 inducer cobalt protoporphyrin (CoPP) further enhanced vGPCR-induced tumor growth. These data postulate HO-1 as an important mediator of vGPCR-induced tumor growth and suggest that inhibition of intratumoral HO-1 activity by SnPP may be a potential therapeutic strategy.     

Heme oxygenase 1 regulates apoptosis induced by heat stress in bovine ovarian granulosa cells via the ERK1/2 pathway

Heat stress can inhibit follicular development in dairy cows, and thus can affect their reproductive performance. Follicular granulosa cells can synthesize estrogen, that affects the development and differentiation of follicles by apoptosis. Heme oxygenase 1 (HO-1/heat shock protein 32) plays an antiapoptotic and cytoprotective role in various cells during stress-induced apoptosis, but little is known about its definitive function in bovine (ovarian) granulosa cells (bGCs). In our study, the roles and mechanism of HO-1 on the heat stress-induced apoptosis of bGCs were studied. Our results show that the expression of HO-1 was significantly increased under heat stress. Moreover, HO-1 silencing increased apoptosis, whereas its overexpression dampened apoptosis by regulating the expression of Bax/Bcl-2 and the levels of cleaved caspase-3. In addition, HO-1 can also play a cytoprotective role by affecting estrogen levels and decomposing heme to produce biologically active metabolite carbon monoxide (CO). Meanwhile, CO significantly increased the level of HO-1, decreased Bax/Bcl-2 levels, and inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The apoptosis of ovarian GCs can affect the secretion of estrogen and lead to disorder of the ovarian microenvironment, thus affecting the normal function of the ovary. Our results indicate that HO-1 acts as a cytoprotective enzyme and plays a protective role in heat-induced apoptosis of bGCs. In conclusion, HO-1 and its metabolite CO inhibit the apoptosis of bGCs induced by heat stress through the ERK1/2 pathway. The results of this study provide a valuable clue for improving the fertility of heat stressed cows in summer.     

Protection from abortion by heme oxygenase-1 up-regulation is associated with increased levels of Bag-1 and neuropilin-1 at the fetal-maternal interface

Tolerance mechanisms allowing pregnancy success resemble those involved in allograft acceptance. Heme oxygenase (HO) is a tissue-protective molecule, which allows graft acceptance and is known to have antiapoptotic effects on several cell types. We previously reported down-regulated levels of HO-1 and HO-2 in placenta from allopregnant mice undergoing abortion. In this study, we analyzed whether the up-regulation of HO-1 by cobalt-protoporphyrin (Co-PP) during implantation window can rescue mice from abortion. Induction of HO-1 by Co-PP treatment prevented fetal rejection, whereas the down-regulation of HOs by zinc-protoporphyrin application boosted abortion. The beneficial effect of HO-1 induction was not related to a local shift to Th2-profile or to a change in the NO system. Interestingly, the expression of the antiapoptotic/cytoprotective molecule Bag-1 as well as the levels of neuropilin-1, a novel marker for T regulatory cells, were up-regulated after Co-PP treatment. Our data strongly support a very important role for HO-1 in fetal allotolerance and suggest that HO-1 might be protective by up-regulating tissue protective molecules, i.e., Bag-1, and by activating T regulatory cells rather than by changing the local cytokine profile.     

Over-expression of HO-1 on mesenchymal stem cells promotes angiogenesis and improves myocardial function in infarcted myocardium

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with diverse cytoprotective effects, and reported to have an important role in angiogenesis recently. Here we investigated whether HO-1 transduced by mesenchymal stem cells (MSCs) can induce angiogenic effects in infarcted myocardium. HO-1 was transfected into cultured MSCs using an adenoviral vector. 1 × 10⁶ Ad-HO-1-transfected MSCs (HO-1-MSCs) or Ad-Null-transfected MSCs (Null-MSCs) or PBS was respectively injected into rat hearts intramyocardially at 1 h post-myocardial infarction. The results showed that HO-1-MSCs were able to induce stable expression of HO-1 in vitro and in vivo. The capillary density and expression of angiogenic growth factors, VEGF and FGF2 were significantly enhanced in HO-1-MSCs-treated hearts compared with Null-MSCs-treated and PBS-treated hearts. However, the angiogenic effects of HO-1 were abolished by treating the animals with HO inhibitor, zinc protoporphyrin. The myocardial apoptosis was marked reduced with significantly reduced fibrotic area in HO-1-MSCs-treated hearts; Furthermore, the cardiac function and remodeling were also significantly improved in HO-1-MSCs-treated hearts. Our current findings support the premise that HO-1 transduced by MSCs can induce angiogenic effects and improve heart function after acute myocardial infarction.     

Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis

Heme oxygenase-1 (HO-1) is emerging as an important cytoprotective enzyme system in a variety of injury models. To optimize future therapeutic applications of HO-1, it is necessary to delineate the precise functions and mechanisms as well as modes of externally regulating HO-1 expression. Investigations have been limited by difficulties with the generation of HO-1 null mice and the lack of specific HO-1 inhibitors. Lung ischemia-reperfusion (I-R) injury is the inciting event in acute lung failure following transplantation, surgery, and shock. To study the function of HO-1 in I-R-induced lung injury, we designed small interfering RNA (siRNA) sequences that effectively suppress HO-1 expression both in vitro and in vivo in an organ-specific manner. In this study we show that there is enhanced apoptosis, via increased Fas expression and caspase 3 activity, in the presence of HO-1 siRNA in endothelial cells and mouse lung during I-R injury, whereas HO-1 overexpression attenuates apoptosis. To the best of our knowledge, we are the first to demonstrate that lung-specific siRNA delivery can be achieved by intranasal administration without the need for viral vectors or transfection agents in vivo, thereby obviating potential concerns for toxicity if siRNA technology is to have clinical application in the future.     

Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection

In cerebral circulation, epileptic seizures associated with excessive release of the excitatory neurotransmitter glutamate cause endothelial injury. Heme oxygenase (HO), which metabolizes heme to a vasodilator, carbon monoxide (CO), and antioxidants, biliverdin/bilirubin, is highly expressed in cerebral microvessels as a constitutive isoform, HO-2, whereas the inducible form, HO-1, is not detectable. Using cerebral vascular endothelial cells from newborn pigs and HO-2-knockout mice, we addressed the hypotheses that 1) glutamate induces oxidative stress-related endothelial death by apoptosis, and 2) HO-1 and HO-2 are protective against glutamate cytotoxicity. In cerebral endothelial cells, glutamate (0.1–2.0 mM) increased formation of reactive oxygen species, including superoxide radicals, and induced major keystone events of apoptosis, such as NF-B nuclear translocation, caspase-3 activation, DNA fragmentation, and cell detachment. Glutamate-induced apoptosis was greatly exacerbated in HO-2 gene-deleted murine cerebrovascular endothelial cells and in porcine cells with pharmacologically inhibited HO-2 activity. Glutamate toxicity was prevented by superoxide dismutase, suggesting apoptotic changes are oxidative stress related. When HO-1 was pharmacologically upregulated by cobalt protoporphyrin, apoptotic effects of glutamate in cerebral endothelial cells were completely prevented. Glutamate-induced reactive oxygen species production and apoptosis were blocked by a CO-releasing compound, CORM-A1 (50 µM), and by bilirubin (1 µM), consistent with the antioxidant and cytoprotective roles of the end products of HO activity. We conclude that both HO-1 and HO-2 have anti-apoptotic effects against oxidative stress-related glutamate toxicity in cerebral vascular endothelium. Although HO-1, when induced, provides powerful protection, HO-2 is an essential endogenous anti-apoptotic factor against glutamate toxicity in the cerebral vascular endothelium. endothelium; carbon monoxide; bilirubin; injury; reactive oxygen species; heme oxygenase     

Endoplasmic reticulum stress stimulates heme oxygenase-1 gene expression in vascular smooth muscle. Role in cell survival

Heme oxygenase-1 (HO-1) is a cytoprotective protein that catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide (CO). In the present study, we found that endoplasmic reticulum (ER) stress induced by a variety of experimental agents stimulated a time- and concentration-dependent increase in HO-1 mRNA and protein in vascular smooth muscle cells (SMC). The induction of HO-1 by ER stress was blocked by actinomycin D or cycloheximide and was independent of any changes in HO-1 mRNA stability. Luciferase reporter assays indicated that ER stress stimulated HO-1 promoter activity via the antioxidant response element. Moreover, ER stress induced the nuclear import of Nrf2 and the binding of Nrf2 to the HO-1 antioxidant response element. Interestingly, ER stress stimulated SMC apoptosis, as demonstrated by annexin V binding, caspase-3 activation, and DNA laddering. The induction of apoptosis by ER stress was potentiated by HO inhibition, whereas it was prevented by addition of HO substrate. In addition, exposure of SMC to exogenously administered CO inhibited ER stress-mediated apoptosis, and this was associated with a decrease in the expression of the proapoptotic protein, GADD153. In contrast, the other HO-1 products failed to block apoptosis or GADD153 expression during ER stress. These results demonstrated that ER stress is an inducer of HO-1 gene expression in vascular SMC and that HO-1-derived CO acts in an autocrine fashion to inhibit SMC apoptosis. The capacity of ER stress to stimulate the HO-1/CO system provides a novel mechanism by which this organelle regulates cell survival.     

Neutrophil gelatinase-associated lipocalin: a new antioxidant that exerts its cytoprotective effect independent on Heme Oxygenase-1

Neutrophil Gelatinase-Associated Lipocalin (NGAL/Lcn2), a member of the lipocalin family, has a variety of functions. There are extensive studies examining the expression of NGAL under harmful conditions. However, its precise function remains poorly understood. Heme Oxygenase 1 (HO-1) is an enzyme with well-established cytoprotective effects. Previous work showed that NGAL induces expression of HO-1. Interestingly, the same stimuli induced the expression of both NGAL and HO-1. The current study was designed to (1) determine whether NGAL exerts its cytoprotective effect through HO-1 and (2) compare NGAL and HO-1 with each other in terms of their protective role against oxidative stress. The current data indicate that NGAL exerts its cytoprotective effect independent of HO-1 and protects cells against oxidative stress more efficiently than HO-1. The data also strongly suggest that induction of NGAL under harmful conditions is a compensatory response to ameliorate oxidative stress-mediated toxicity. These findings may suggest new applications of NGAL, particularly when oxidative stress is a major factor.