Differential Gene Expression In Aging-related Embryonic Development

老化的心肌表达许多在胚胎和胎儿发育过程中正常表达的基因转录本。使用基于转录组和基于启动子的技术，我们已经努力了解在发育模型和衰老模型中这种基因表达控制的潜在机制。基因表达序列分析(SAGE)提供了一个定量的、具有代表性的和全面的差异基因表达谱。我们使用SAGE分析来生成定量的成绩单评估，这已被证明比其他技术更快速、更经济。我们使用基于RT-PCR的技术来确定一些中胚层和心脏限制性基因产物在分化的EC来源的心肌细胞中表达的时间点，以及最近普遍用于维持胚胎干细胞的饲养层。这些后一项研究旨在确定可能对细胞进行重新编程以变得更具多能性的因素。 我们利用定量聚合酶链式反应和原位杂交技术分析了SAGE鉴定的一些差异调控基因的时间和空间分布，其中一些基因在胎儿或成人心脏中显示出心脏优势。这项研究中确定的新基因产物为分析人类和小鼠胚胎干细胞的心脏前期和早期发育过程提供了一个框架，是积极研究的对象。在这些进行后续分析的基因转录本中，包括Cripto和多营养素。 自从最初的发现以来，我们已经确定多营养素(PTN)是一种发育调节的细胞因子生长因子，它促进血管生成、细胞增殖和分化，在发育中的心肌中显著表达，但在成人心脏中表达很低。相反，在心肌梗死的大鼠模型和人类扩张型心肌病中，多营养素显著上调，这表明它可能在损伤修复中具有功能作用。为了阐明PTN对收缩细胞的影响，我们采用了原代培养的新生大鼠(NN)和成年(A)心肌细胞(CM)。我们发现，PTN以剂量和时间依赖的方式促进caspase介导的基因组DNA片段化。更重要的是，它显著增强了NNCM对低氧应激和紫外线(UV)照射的凋亡反应，以及ACM对低氧复氧的反应。我们进一步发现，在siRNA介导的内源性PTN蛋白被敲除后，PTN增强了紫外线诱导的NNCM细胞的凋亡。机制上，PTN拮抗IGF-1相关的AKT/PKB的Ser-473磷酸化，同时降低BAD和GSK3β的磷酸化。腺病毒表达AKT和氯化锂介导的GSK3β抑制作用也减少了PTN增强的细胞程序性死亡。这些数据表明，PTN至少部分地通过抑制AKT信号通路促进CM细胞死亡。总之，我们发现了PTN在CMS中的一个新的作用。这种细胞因子，除了它在心脏中的促血管生成作用外，还加强了CM对促凋亡应激反应的程序性细胞死亡。

The aging myocardium expresses many gene transcripts that are normally expressed during embryonic and fetal development. Using transcriptome-based and promoter-based techniques, we have worked towards understanding the mechanisms underlying this control of gene expression both in models of development and in aging. Serial analysis of gene expression (SAGE) yields a quantitative, representative and comprehensive differential gene expression profile. We have employed SAGE analysis to generate a quantitative transcript assessment that has proven to be much more rapid and economical than other techniques. We used an RT-PCR based technique to determine the time points where a number of mesodermal and cardiac-restricted gene products are expressed in differentiated EC derived cardiomyocytes, and more recently feeder layers general employed to maintain embryonic stem cells. These latter studies were designed to identify factors that could possibly reprogram cells to become more multipotent. We have utilized quantitative PCR and in situ hybridizations to analyze the temporal and spatial distribution of a number of the most differentially regulated transcripts identified by SAGE, several of which have shown a cardiac predominance in either fetal or adult heart. The novel gene products identified in this study provide a framework for the analysis of pre- and early cardiac developmental processes in human and mouse embryonic stem cells and are the subject of active investigation. Included among these gene transcripts that have been subjected to follow-up analysis are Cripto and pleiotrophin. Since the original identification, we have determined that pleiotrophin (PTN), a development-regulated cytokine growth factor that promotes angiogenesis, cell proliferation and differentiation, is prominently expressed in developing myocardium but it is poorly expressed in adult heart. Conversely, in a rat model of myocardial infarction and in human dilated cardiomyopathy, pleiotrophin is markedly up-regulated, suggesting that it may have a functional role in injury repair. To elucidate the effects of PTN on contractile cells, we employed primary cultures of rat neonatal (NN) and adult (A) cardiomyocytes (CM). We found that PTN promotes caspase-mediated genomic DNA fragmentation in a dose- and time-dependent manner. More importantly, it significantly potentiates the apoptotic response of NNCMs to hypoxic stress and to ultraviolet (UV) irradiation, and of ACMs to hypoxia-reoxygenation. We furthermore found that PTN potentiated UV-induced apoptosis is abolished in NNCMs following siRNA-mediated knockdown of endogenous PTN proteins. Mechanistically, PTN antagonizes IGF-1 associated Ser-473 phosphorylation of AKT/PKB, and it concomitantly decreases both BAD and GSK3beta; phosphorylation. Adenoviral expression of constitutively active AKT and lithium chloride mediated inhibition of GSK3beta; also reduced the programmed cell death potentiated by PTN. These latter data indicate that PTN promotes CM cell death, at least partially, through inhibition of AKT signaling. In conclusion, we have uncovered a novel role for PTN in CMs. This cytokine, which in addition to its pro-angiogenic effects in heart, potentiates CM programmed cell death in response to pro-apoptotic stress.