Relationship between cardiomyocyte cell death and cardiac function during hypertensive cardiac remodelling in Dahl rats

The exact mechanisms responsible for the progression of heart failure remain unclear. We investigated the in vivo relationship between the incidence of apoptotic cell death and left ventricular function serially from the beginning of hypertension to decompensated heart failure in Dahl salt-sensitive rats. Dahl salt-resistant and Dahl salt-sensitive rats were fed on a high-salt diet from 6 weeks of age. Systolic blood pressure was recorded by the tail-cuff method every week. Cardiac function in vivo was evaluated by echocardiography and cardiac catheterization. Cardiomyocyte apoptosis was detected by the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) method. The gene expression of Bax, Bcl-2 and Bcl-xL was analysed by Northern blotting. The TUNEL method revealed that the incidence of cardiomyocyte apoptosis was significantly increased in the hearts of 18-week-old Dahl salt-sensitive rats (apoptotic index 1.3 +/- 0.1%). Northern blot analysis revealed that the Bcl-xL mRNA level increased gradually during the progression towards heart failure. In conclusion, these data suggest that cardiomyocyte apoptosis is a terminal event, and plays a role as an aggravating factor in the vicious cycle of heart failure.     

Involvement of cardiomyocyte survival-apoptosis balance in hypertensive cardiac remodeling

The balance between cell death and cell survival is a tightly controlled process, especially in terminally differentiated cells, such as the cardiomyocyte. Accumulating data support a role for cardiomyocyte apoptosis in the development of several cardiac diseases, including the transition from hypertensive compensatory hypertrophy to heart failure. This review briefly summarizes the status of the knowledge regarding the death-survival balance of cardiomyocytes in the context of hypertensive heart disease. Several molecular and cellular aspects as well as the most relevant pathophysiological implications are presented. Moreover, diagnosis tools under development and the possibilities for pharmacological intervention are also examined.     

Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of Bnip3 restrains postinfarction remodeling in mice

Following myocardial infarction, nonischemic myocyte death results in infarct expansion, myocardial loss, and ventricular dysfunction. Here, we demonstrate that a specific proapoptotic gene, Bnip3, minimizes ventricular remodeling in the mouse, despite having no effect on early or late infarct size. We evaluated the effects of ablating Bnip3 on cardiomyocyte death, infarct size, and ventricular remodeling after surgical ischemia/reperfusion (IR) injury in mice. Immediately following IR, no significant differences were observed between Bnip3(-/-) and WT mice. However, at 2 days after IR, apoptosis was diminished in Bnip3(-/-) periinfarct and remote myocardium, and at 3 weeks after IR, Bnip3(-/-) mice exhibited preserved LV systolic performance, diminished LV dilation, and decreased ventricular sphericalization. These results suggest myocardial salvage by inhibition of apoptosis. Forced cardiac expression of Bnip3 increased cardiomyocyte apoptosis in unstressed mice, causing progressive LV dilation and diminished systolic function. Conditional Bnip3 overexpression prior to coronary ligation increased apoptosis and infarct size. These studies identify postischemic apoptosis by myocardial Bnip3 as a major determinant of ventricular remodeling in the infarcted heart, suggesting that Bnip3 may be an attractive therapeutic target.     

Niclosamide enhances ROS-mediated cell death through c-Jun activation

Radiotherapy is an effective treatment modality in the clinical treatment of cancers, and has been combined with chemotherapy in order to improve therapeutic efficacy. Therefore, we aimed to develop small molecules that enhance the cytotoxic effects of radiotherapy. In this study, we provide evidence that niclosamide is an effective radiosensitizer in non-small cell lung cancer cells. Using a cell-based high-throughput viability screen of 1040 compounds in combination with γ-ionizing radiation (IR), we found niclosamide, an FDA-approved antihelminthic agent, had a radiosensitizing effect on H1299 human lung cancer cells. Pretreatment with niclosamide enhanced IR- induced cell death of H1299 in a dose-dependent manner via apoptosis compared with IR or niclosamide alone. The combined treatment induced significantly more phosphorylation of p38 MAPK and c-Jun in H1299 cells than IR or niclosamide alone. Since IR induces apoptosis through generation of reactive oxygen species (ROS), hydrogen peroxide (H₂O₂) was employed as another ROS generator and we found that niclosamide also sensitized cells to H₂O₂. Niclosamide pretreatment also induced c-Jun and its phosphorylation in the presence of H₂O₂, thereby enhancing apoptosis. N-acetyl-L-cysteine (NAC) treatment abolished both cell death and c-Jun activation induced by the combination treatments. Knockdown of c-Jun also decreased PARP cleavage and clonogenic cell survival in niclosamide- and IR-treated H1299 cells. Our findings suggest that niclosamide could be a promising radiosensitizer in lung cancer patients through activation of the p38 MAPK-c-Jun axis.     

心肌梗死后心肌细胞凋亡及左室重塑过程中肝细胞生长因子的作用

目的:探讨外源性肝细胞生长因子(hepatocytegrowthfactor,HGF)对心肌梗死后心肌细胞凋亡及左室重塑的影响。方法:将56只新西兰兔随机分为4组:假手术组、假手术+HGF组、对照组和干预组。结扎左冠状动脉前降支复制急性心肌梗死模型。干预组静脉注射给予HGF2mg/(kg·12h),4周后测心功能、左室重塑指标,取出心脏,梗死区/缺血区重量比为梗死范围。TUNEL法染色检测细胞凋亡。Westernblot法测定凋亡蛋白Bcl-2的表达。结果:与假手术组相比,对照组的左室舒张末容积、左室相对重量、左室壁厚度均显著升高(t=3.598,2.348,2.324,P<0.05～0.01),左室缩短分数(leftventricularfractionofshortening,LVFS)、左室射血分数(leftventricularejectionfraction,LVEF)均显著降低(t=4.311,3.330,P<0.01)。HGF干预组LVESV,LVEDV显著低于对照组(t=2.810,2.449,P<0.01);LVFS及LVEF均显著升高。HGF干预组细胞凋亡率、心肌梗死范围均低于对照组(t=2.302,2.344,P<0.01)。左室腔直径与心肌细胞凋亡率呈正相关(r=0.801,P<0.01)。干预组梗死心肌周围bcl-2表达(灰度值1.37)显著高于对照组(灰度值0.17)(t=21.043,P<0.01)。结论:HGF能减少心肌梗死范围、降低心肌细胞凋亡率并能限制心肌梗死后的左室重塑,改善心功能,其作用机制可能与其抗凋     

Inhibition of phenylephrine-induced cardiomyocyte hypertrophy by activation of multiple adenosine receptor subtypes

Plasma adenosine levels are elevated in cardiovascular disease including hypertension and heart failure, and the nucleoside has been proposed to serve as an endogenous antimyocardial remodeling factor. We studied the modulation of phenylephrine-induced hypertrophy by adenosine receptor activation in isolated neonatal cultured ventricular myocytes. Phenylephrine (10 muM) increased cell size by 35% and significantly increased expression of atrial natriuretic peptide. These effects were reduced by the stable adenosine analog 2-chloroadenosine and were completely blocked by the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (1 microM), the A(2A) receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (100 nM), and the A(3) receptor agonist N(6)-(3-iodobenzyl)adenosine-5'-methyluronamide (100 nM). The antihypertrophic effects of all three agonists were completely reversed by their respective antagonists. Phenylephrine significantly up-regulated expression of the immediate early gene c-fos especially within the first 30 min of phenylephrine treatment. These effects were almost completely inhibited by all adenosine receptor agonists. Although phenylephrine also induced early stimulation of both p38 mitogen-activated protein kinase and extracellular signal-regulated kinase, these responses were unaffected by adenosine agonists. The expression of the G-protein regulatory factors RGS2 and RGS4 were increased by nearly 3-fold by phenylephrine treatment although this was completely prevented by adenosine receptor agonists. These agents also blocked the ability of phenylephrine to up-regulate Na/H exchange isoform 1 (NHE1) expression in hypertrophied myocytes. Thus, our results demonstrate an antihypertrophic effect of adenosine acting via multiple receptor subtypes through a mechanism involving down-regulation of NHE1 expression. The ability to prevent regulators of G-protein signaling (RGS) up-regulation further suggests that adenosine receptor activation minimizes signaling which leads to hypertrophic responses.     

Regulation of cardiac myocyte cell death

Cardiac myocyte death, whether through necrotic or apoptotic mechanisms, is a contributing factor to many cardiac pathologies. Although necrosis and apoptosis are the widely accepted forms of cell death, they may utilize the same cell death machinery. The environment within the cell probably dictates the final outcome, producing a spectrum of response between the two extremes. This review examines the probable mechanisms involved in myocyte death. Caspases, the generally accepted executioners of apoptosis, are significant in executing cardiac myocyte death, but other proteases (e.g., calpains, cathepsins) also promote cell death, and these are discussed. The two principal cell death pathways (death receptor- and mitochondrial-mediated) are described in relation to the emerging structural information for the principal proteins, and they are discussed relative to current understanding of myocyte cell death mechanisms. Whereas the mitochondrial pathway is probably a significant factor in myocyte death in both acute and chronic phases of myocardial diseases, the death receptor pathway may prove significant in the longer term. The Bcl-2 family of proteins are key regulators of the mitochondrial death pathway. These proteins are described and their possible functions are discussed. The commitment to cell death is also influenced by protein kinase cascades that are activated in the cell. Whereas certain pathways are cytoprotective (e.g., phosphatidylinositol 3'-kinase), the roles of other kinases are less clear. Since myocyte death is implicated in a number of cardiac pathologies, attenuation of the death pathways may prove important in ameliorating such disease states, and possible therapeutic strategies are explored.     

N-乙酰半胱氨酸对心力衰竭兔核因子NF-κB活化及心肌凋亡的干预作用

目的 探讨慢性心衰兔心肌核因子-κB(NF-κB)活化与心肌凋亡的关系以及N-乙酰半胱氨酸(NAC)的作用机制.方法 健康日本大耳白兔共50只,雌雄不限(武汉大学医学院实验动物中心提供),随机选取10只设为对照组.余40只兔采用盐酸阿霉素复制心衰模型(阿霉素1mg/kg,每周2次,连续8周),造模成功25只,随机分为心衰组(n=12)和NAC干预组(n=13),NAC组给予NAC注射液(300 mg/kg)耳缘静脉注射,每天1次共4周.对照组与心衰组均注射等体积生理盐水.观测血流动力学指标、心肌凋亡指数、血清和心肌总抗氧化能力、bcl-2、Bax蛋白在心肌细胞中表达的水平,以及NF-κB p65的表达和核结合活性的改变.通过t检验、方差分析和直线相关分析等统计学方法,观察NAC对上述指标的影响.结果 对照组、心衰绀和NAC组心肌细胞凋亡指数(AI)分别为1.57%、55.62%和21.71%,NAC组较心衰组心肌细胞凋亡率显著降低(P＜0.001).心衰组Bax蛋白、NF-κBp65的核内表达增高,bcl-2表达下调,bcl-2/Bax比值和总抗氧化能力[血清中(8.86±2.21)U/ml,心肌中(1.26±0.30)U/mgprot]下降.NAC可不同程度地改善心功能指标,降低心肌凋亡指数和Bax的表达,显著降低NF-κB p65核内表达及升高总抗氧化能力[血清中(13.23±2.92)u/ml,心肌中(1.58±0.19)U/mgprot]、bcl-2/Bax比值和bcl-2的表达.结论 氧化应激可激活NF-κB,启动bcl-2和Bax的转录调节相应蛋白的合成,介导心肌凋亡,促进慢性心衰的发生发展.NAC具有抗氧化作用,通过降低心肌NF-κB活性,减少心肌细胞凋亡,改善心功能.     

Novel camphor-based pyrimidine derivatives induced cancer cell death through a ROS-mediated mitochondrial apoptosis pathway

A series of novel camphor-based pyrimidine derivatives (3a–3x) have been synthesized; their structures were determined by using conventional methods and compound 3f was further confirmed through single crystal XRD analysis. The cytotoxic activity of the target compounds against a panel of human normal (GES-1) and cancer cell lines (MDA-MB-231, RPMI-8226, A549) was evaluated by MTS assay. Here we found that compound 3f exhibited the strongest anti-tumor activity, comparable to that of etoposide, and had much lower cytotoxicity to normal GES-1 cells (IC₅₀ > 50 μM) than the reference drug (IC₅₀ = 8.89 μM). Subsequent mechanism studies in MDA-MB-231 cells revealed that compound 3f caused G0/G1 phase arrest and apoptosis in a dose dependent manner. Moreover, the loss of mitochondrial membrane potential and enhancement of cellular ROS levels were also observed upon 3f treatment, which indicated that 3f exerted cytotoxic activity by a ROS-mediated mitochondrial apoptosis pathway. This result was confirmed by a significant increase in the expression of pro-apoptotic proteins Bax, cytochrome C and caspase-3, and downregulation of anti-apoptosis protein Bcl-2. Overall, 3f can be adopted for further investigation in the development of antitumor agents based on natural products.

A series of novel camphor-based pyrimidine derivatives were synthesized and characterized. We found the compound 3f exhibited strongest anti-tumor activity via ROS-mediated mitochondrial apoptosis pathway.     

Oligodendrocyte cell death in pathogenesis of multiple sclerosis: Protection of oligodendrocytes from apoptosis by complement

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. It is mediated by activated lymphocytes, macrophages, microglia, and complement. In MS, myelin-forming oligodendrocytes (OLGs) are the targets of inflammatory and immune attacks. OLG death by apoptosis or necrosis causes the cell loss seen in MS plaques. Studies of experimental allergic encephalomyelitis (EAE) in caspase 11-deficient mice show that caspase-mediated death of OLGs is critical to demyelination. Complement activation may affect MS pathogenesis through activated terminal complex C5b-9, which promotes demyelination, and through sublytic C5b-9, which protects OLGs from apoptosis. By inducing EAE in C5-deficient mice, we showed that complement C5 promotes axon preservation and new myelin formation, which protect OLGs from apoptosis. These findings indicate that activated complement C5b-9 plays a proinflammatory role in acute MS but may also protect OLGs from death in chronic MS.     

Tumor necrosis factor alpha-induced apoptosis in cardiac myocytes. Involvement of the sphingolipid signaling cascade in cardiac cell death.

In the present study, it was shown that physiologically relevant levels of the proinflammatory cytokine TNFalpha induced apoptosis in rat cardiomyocytes in vitro, as quantified by single cell microgel electrophoresis of nuclei ("cardiac comets") as well as by morphological and biochemical criteria. It was also shown that TNFalpha stimulated production of the endogenous second messenger, sphingosine, suggesting sphingolipid involvement in TNFalpha-mediated cardiomyocyte apoptosis. Consistent with this hypothesis, sphingosine strongly induced cardiomyocyte apoptosis. The ability of the appropriate stimulus to drive cardiomyocytes into apoptosis indicated that these cells were primed for apoptosis and were susceptible to clinically relevant apoptotic triggers, such as TNFalpha. These findings suggest that the elevated TNFalpha levels seen in a variety of clinical conditions, including sepsis and ischemic myocardial disorders, may contribute to TNFalpha-induced cardiac cell death. Cardiomyocyte apoptosis is also discussed in terms of its potential beneficial role in limiting the area of cardiac cell involvement as a consequence of myocardial infarction, viral infection, and primary cardiac tumors.     

Relationship between myocardial remodeling and neurohumoral activation in patients with cardiac failure

50 healthy subjects and 685 patients with heart failure (NYHA functional class I-IV) due to coronary heart disease (CHD) aged 34-74 years (mean age 48.1 +/- 8.22 years) were examined. Cardio-hemodynamics was assessed by M- and B-echocardiography from parasternal, subcostal and apical view on a short and long axis with transmitral Doppler. Plasma levels of epinephrine, norepinephrine, serotonin, aldosterone, STH, hydrocortisone were measured in the patients. Progression of cardiac failure is characterized by evolution of the left ventricular ellipse form into a ball shape primarily due to an increase in cross sectional cavity size. The changes of the left ventricular geometry in heart failure patients are combined with progressive reduction of the relative wall thickness index, intensification of the myocardial stress and impairment of diastolic function. The clearest intercoupling between myocardial remodeling and neurohumoral activation was registered in patients with asymptomatic heart failure. With growing severity of left ventricular dysfunction, plasm activity of serotonin, hydrocortisone and aldosterone increase more than levels of norepinephrine, epinephrine and STH.     

Bacterial programmed cell death of cerebral endothelial cells involves dual death pathways

Major barriers separating the blood from tissue compartments in the body are composed of endothelial cells. Interaction of bacteria with such barriers defines the course of invasive infections, and meningitis has served as a model system to study endothelial cell injury. Here we report the impressive ability of Streptococcus pneumoniae, clinically one of the most important pathogens, to induce 2 morphologically distinct forms of programmed cell death (PCD) in brain-derived endothelial cells. Pneumococci and the major cytotoxins H2O2 and pneumolysin induce apoptosis-like PCD independent of TLR2 and TLR4. On the other hand, pneumococcal cell wall, a major proinflammatory component, causes caspase-driven classical apoptosis that is mediated through TLR2. These findings broaden the scope of bacterial-induced PCD, link these effects to innate immune TLRs, and provide insight into the acute and persistent phases of damage during meningitis.     

Glioma apoptosis induced by macrophages involves both death receptor-dependent and independent pathways

Apoptosis of glioma may represent a promising intervention for tumor treatment. Macrophages are able to induce apoptosis in a number of tumor cells, including glioma. It is known that apoptosis of cells is executed on either a death receptor-dependent or independent pathway. Whether and how apoptosis of glioma cells induced by activated macrophages is involved in these two pathways simultaneously are not known. Using in vitro and in vivo experimental models, we investigated Bcl-2 system and Fas/FasL channel, representing the death receptor-dependent and independent pathways, respectively, in glioma cells treated with the supernatant from the activated macrophages, which was rich in tumor necrosis factor-alpha and interferon-gamma. We found that levels of Fas and FasL were up-regulated both in vitro and in vivo, accompanying an increase in the expression of caspase-8. The number of apoptotic cells was also increased significantly, although the percentage of death cells exceeded the number of tumor cells positive for Fas or FasL. It was also evident that the expression of Bax was increased, whereas the level of Bcl-2 was decreased, in glioma cells treated with the supernatant from the activated macrophages. The alteration of molecules related to both death pathways led to apoptosis of glioma and the inhibition of xenograft glioma growth in mice. Apoptosis of glioma induced by the activated macrophage is executed by way of both death receptor-dependent and independent pathways, and such an apoptosis-induced approach can effectively inhibit the growth of glioma in vivo.     

Transforming growth factor beta induces mesangial cell apoptosis through NO- and p53-dependent and -independent pathways.

BACKGROUND: Because transforming growth factor beta (TGF-beta) has been shown to have a bimodal effect on mesangial cell (MC) proliferation, we studied its effect on MC apoptosis. METHODS: Cultured mouse MCs were used to evaluate the effect of TGF-beta. Morphologic evaluation of MC apoptosis was performed by staining cells with H-33342 and propidium iodide. To confirm the effect of TGF-beta on MC apoptosis, DNA was extracted from control and TGF-beta-treated MCs and run on gel electrophoresis. We evaluated the effect of NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, on TGF-beta-induced MC apoptosis to determine the role of NO and studied the effect of sodium nitroprusside (SNP) and SNAP (S-nitroso-N-acetyl-penicillamine) on MC apoptosis to confirm the effect of NO. We examined the role of p53 by studying the effect of TGF-beta on MCs derived from p53 knockout mice (p53KO-MC) as well as a normogenic strain (N-MC). We also examined the effect of TGF-beta, SNP, and SNAP on apoptosis of p53 mutant (MDAMB-231) and wild-type p53 (MCF-7) breast cancer cell lines. In addition, Western blots were generated from control, TGF-beta-treated, and SNAP-treated MCs and probed for the expression of p53. RESULTS: TGF-beta promoted MC apoptosis. Moreover, TGF-beta-treated MCs displayed integer multiples of 180 base pairs (ladder pattern). L-NAME inhibited TGF-beta-induced MC apoptosis. Furthermore, SNP and SNAP, NO donors, promoted MC apoptosis. TGF-beta also enhanced the MC expression of p53. TGF-beta induced only a moderate degree of apoptosis in MCs derived from p53KO-MC when compared with N-MCs. Similarly, the TGF-beta-induced apoptosis of MDAMB-231 was of a moderate degree when compared with MCF-7 cells. CONCLUSIONS: We hypothesize that TGF-beta promotes MC apoptosis through NO generation and p53-dependent and -independent pathways.     

Role of mesenchymal cell death in lung remodeling after injury.

Repair after acute lung injury requires elimination of granulation tissue from the alveolar airspace. We hypothesized that during lung repair, signals capable of inducing the death of the two principal cellular elements of granulation tissue, fibroblasts and endothelial cells, would be present at the air-lung interface. Bronchoalveolar lavage fluid obtained from patients during lung repair induced both fibroblast and endothelial cell death, while fluid obtained at the time of injury or from patient controls did not. The mode of cell death for endothelial cells was apoptosis. Fibroblast death, while morphologically distinct from necrosis, also differed from typical apoptosis. Only proliferating cells were susceptible to the bioactivities in lavage fluid, which were trypsin sensitive and lipid insoluble. Histological examination of lung tissue from patients after lung injury revealed evidence of apoptotic cells within airspace granulation tissue. Our results suggest that cell death induced by peptide(s) present at the air-lung interface may participate in the remodeling process that accompanies tissue repair after injury.