PPARdelta modulates lipopolysaccharide-induced TNFalpha inflammation signaling in cultured cardiomyocytes

Peroxisome proliferator activated receptors (PPARs: PPARalpha, gamma and delta) regulate fatty acid metabolism, glucose homeostasis, cell proliferation, differentiation and inflammation. Tumor necrosis factor alpha (TNFalpha) is one of the important pathological factors in inflammatory responses during the pathological progression of myocardial ischemic/reperfusion and hypertrophy. Accumulating evidence shows that synthetic ligands of PPARalpha and PPARgamma suppress myocardial inflammatory responses, such as the production of TNFalpha, thus exerting beneficial effects in animals who had undergone ischemia/reperfusion injury or cardiac hypertrophy. However, it remains obscured if PPARdelta and its ligands exert any effect on the production of TNFalpha, thus influencing cardiac inflammatory responses. In this study, we investigated the effects of PPARdelta and its synthetic ligand GW0742 on TNFalpha production in cultured cardiomyocytes. Our studies indicate that a PPARdelta-selective ligand inhibited lipopolysaccharide (LPS)-induced TNFalpha production from cardiomyocytes. Adenoviral-mediated overexpression of PPARdelta substantially inhibited TNFalpha expression in cultured cardiomyocytes compared to controls, whereas overexpression of a PPARdelta mutant with ablated ligand binding domain did not show similar effect. Conversely, absence of PPARdelta in cardiomyocytes further exaggerated LPS-induced TNFalpha production. Moreover, activation of PPARdelta abrogated LPS-induced degradation of IkappaBs, thus suppressing LPS-induced nuclear factor kappaB (NF-kappaB) activities. Therefore, PPARdelta is an important determinant of TNFalpha expression via the NF-kappaB signaling pathway, thus serving as therapeutic targets to attenuate inflammation that are involved in cardiac pathological progression.     

New insights into cellular mechanisms during sepsis

Despite intensive ongoing research efforts, the mortality of patients with sepsis remains unacceptably high. Clinical trials emerging from promising results in animal models have mostly failed to deliver sufficient treatment strategies so far. Many studies investigating the underlying mechanisms of sepsis have focused on deterioration of the humoral and cellular components of the immune system. However, in addition to septic shock, the main cause of death in septic patients is multiorgan failure. So far, not much is known about the effects of a dysregulated immune system as seen in sepsis on parenchymal cells of end organs. Studies on the interaction of the complement system and kidney as well as liver cells resulted in interesting yet still inconclusive data. In this review, we provide new insights into mechanisms during sepsis based on recent findings.     

人脐带间充质干细胞向心肌细胞诱导分化并体内移植的研究

目的探讨人脐带间充质干细胞(MSCs)经5-氮杂胞苷(5aza)诱导后能否向心肌样细胞分化及诱导后细胞移植到心肌梗死大鼠心脏后能否存活,为心肌细胞的移植探索新的细胞来源。方法从人脐带华尔通氏胶培养出MSCs,检测人脐带来源的MSCs的细胞表面标记。经5aza诱导,采用免疫组化方法及RT-PCR方法检测诱导后细胞能否表达心肌细胞标记物,诱导后细胞经5-溴-2-脱氧尿苷(BrdU)标记后移植到梗死心肌,观察移植后细胞能否存活。结果人脐带间充质干细胞经5aza诱导后能表达心肌细胞标记物,诱导后细胞移植到心肌梗死模型大鼠心肌后细胞能存活。结论人脐带间充质干细胞经5aza诱导能向心肌样细胞分化,诱导后细胞移植至体内能存活,人脐带间充质干细胞可以作为心肌细胞移植的来源。     

Human induced pluripotent stem cells and their use in drug discovery for toxicity testing

Predicting human safety risks of novel xenobiotics remains a major challenge, partly due to the limited availability of human cells to evaluate tissue-specific toxicity. Recent progress in the production of human induced pluripotent stem cells (hiPSCs) may fill this gap. hiPSCs can be continuously expanded in culture in an undifferentiated state and then differentiated to form most cell types. Thus, it is becoming technically feasible to generate large quantities of human cell types and, in combination with relatively new detection methods, to develop higher-throughput in vitro assays that quantify tissue-specific biological properties. Indeed, the first wave of large scale hiSC-differentiated cell types including patient-derived hiPSCS are now commercially available. However, significant improvements in hiPSC production and differentiation processes are required before cell-based toxicity assays that accurately reflect mature tissue phenotypes can be delivered and implemented in a cost-effective manner. In this review, we discuss the promising alignment of hiPSCs and recently emerging technologies to quantify tissue-specific functions. We emphasize liver, cardiovascular, and CNS safety risks and highlight limitations that must be overcome before routine screening for toxicity pathways in hiSC-derived cells can be established.     

Release kinetics of intact and degraded troponin I and T after irreversible cell damage

Purpose

We characterized the release kinetics of cardiac troponin I and T in relation to lactate dehydrogenase (LDH) from cardiomyocytes before and after the transition from reversible to irreversible cell damage.

Methods

Cardiomyocytes were exposed to mild metabolic inhibition (1 mmol/L sodium azide) to induce a necrotic cell death process that is characterized by a reversible (0-12 h) and irreversible phase (12-30 h). At various time intervals cells and media were collected and analyzed for LDH activity, intact cTnI and cTnT, and their degradation products.

Results

During the first 12 h of metabolic inhibition, cell viability was unchanged with no release of intact cTnI and cTnT nor their degradation products. Between 12 and 30 h of azide treatment, cardiomyocytes showed progressive cell death accompanied by release of intact cTnI (29 kDa), intact cTnT (39 kDa), four cTnI degradation products of 26, 20, 17 and 12 kDa, and three cTnT degradation products of 37, 27 and 14 kDa. Possibly due to degradation, there is progressive loss of cTnI and cTnT protein that is obviously undetected by the antibodies used.

Conclusions

Metabolic inhibition of cardiomyocytes induces a parallel release of intact cTnI and cTnT and their degradation products, starting only after onset of irreversible cardiomyocyte damage.     

氧化低密度脂蛋白诱导心肌细胞LOX-1表达的研究

目的探讨氧化低密度脂蛋白（ox-LDL）对心肌细胞血凝素样氧化低密度脂蛋白受体1（LOX-1）表达的影响。方法原代培养乳鼠心肌细胞,分别用不同浓度的ox-LDL（0,10,20,50,100μg/ml）与心肌细胞作用不同时间（0,6,12,24,36h）。用RT-PCR测定LOX-1mRNA表达水平,Western blot测定LOX-1蛋白表达水平。结果不同浓度的ox-LDL均可诱导心肌细胞LOX-1mRNA和蛋白表达,与对照组比较有显著性差异（P〈0.05）,在10-50μg/ml的剂量范围内存在明显的剂量-效应关系,各组间比较有显著性差异（P〈0.05）。随着ox-LDL与心肌细胞作用时间的延长,心肌细胞LOX-1mRNA和蛋白表达增加,与对照组比较差异具有显著性（P〈0.05）,在6～24h内呈明显的时间-效应关系,各组间比较有显著性差异（P〈0.05）。结论 ox-LDL可诱导心肌细胞LOX-1表达,并在一定范围内随ox-LDL浓度增加和作用时间延长而表达增强。     

骨髓间充质干细胞分化为心肌细胞的研究进展

骨髓中至少存在两种干细胞：造血干细胞（Hemopoieticstemcells,HSCs）ffx＇f~充质干细胞（Mesenchymalstemcells,MSCs）。以前认为MSCs的重要作用是支持造血,近来的研究发现MSCs具有自我更新和多向分化的潜能。缺血性心脏病因其功能性心肌细胞减少而发生心室重构,最终导致心力衰竭,严重威胁着人类的身体健康。MSCs在体外或体内可诱导分化为心肌样细胞,并应用于缺血性心脏病的治疗,通过减少梗死面积,改善心肌功能,为心脏病的治疗提供了新途径。骨髓间充质干细胞的来源广泛,取材方便,较易培养已成为研究的热点。     

小鼠胚胎干细胞分化心肌细胞过程中基因表达情况和电生理特性研究

目的： 探讨小鼠胚胎干细胞在体外分化心肌样细胞过程中αMHC、Anf、MLC2v基因的表达情况以及分化的心肌样细胞的电生理特点。 方法： 将转染PαMHC-EGFP小鼠D3胚胎干细胞系,体外诱导分化心肌样细胞，应用RT-PCR检测0 d、3 d、5 d、7 d、10 d、14 d拟胚体（EB）细胞中αMHC、Anf、 MLC2v基因的表达情况，同时应用膜片钳技术，观察心肌样细胞的电生理特性。 结果： 在EB形成的7d和8d可见EB内出现有绿色荧光蛋白表达的心肌样细胞群；RT-PCR结果显示αMHC基因表达在EB体形成第7 d，而在10 d、14 d跳动的心肌样细胞中该基因表达增强（P<0.05），而Anf、 MLC2v基因的表达仅出现在心肌样细胞；膜片钳记录的心肌样细胞动作电位显示,73.5%（n=34）的细胞记录到典型心房肌细胞动作电位［APD90=（78.4±3.1）ms］；20.5%细胞表现为起搏细胞动作电位［APD90=（112.6±5.5）ms］；1个细胞显示了典型的心室肌细胞动作电位的特征（APD90=200 ms）。 结论： 在心肌样细胞分化的早期可见αMHC、Anf、 MLC2v基因的表达，这3种基因可为心肌发育过程中的特征性基因并对心肌发育起调节作用。而此干细胞系分化的心肌样细胞主要为心房肌细胞和起搏细胞。     

人骨髓间充质干细胞体外培养扩增及向心肌细胞诱导分化的实验研究

目的:培养人骨髓间充质干细胞(Human mesenchymal stem cell,hMSCs),探讨hMSCs体外向心肌细胞(Cardio-myocytes,CM)定向诱导分化的实验研究。方法:取人的骨髓血,用Percoll(1.073g/ml)密度梯度离心及贴壁筛选结合的方法体外培养扩增hMSCs,并进行流式细胞仪分析鉴定其免疫学表型,以未加一抗只加二抗的hMSCs作为平行对照组。选用生长良好、纯度达到95%的P5代hMSCs,用不同诱导浓度的5-氮杂胞苷(5-Azacytidine)1、5、10和20μmol/L进行诱导,对诱导后的细胞进行心肌特异性标志TroponinⅠ及Desmin的免疫组化鉴定。结果:体外分离纯化培养扩增出hMSCs,其CD44阳性率平均为93.26%±2.48%,与平行对照组(3.42%±1.09%)相比有明显差异(P<0.01)。经5和10μmol/L5-Aza诱导分化的hMSCs表达心肌特异性标记TroponinⅠ、Desmin;10μmol/L5-Aza诱导分化的hMSCs阳性率明显高于5μmol/L组;在20μmol/L组中,诱导后超过50%的细胞脱落死亡。结论:hMSCs可体外分离培养扩增,并具有向心肌细胞分化的潜能,5-Aza最佳诱导浓度为10μmol/L。