Differential Gene Expression In Aging-related Embryonic Development

衰老心肌表达许多基因转录本，这些基因转录本在胚胎和胎儿发育期间正常表达。使用基于转录组和基于启动子的技术，我们一直致力于了解在发育和衰老模型中基因表达控制的机制。基因表达系列分析（SAGE）产生定量的、代表性的和全面的差异基因表达谱。我们采用SAGE分析来生成定量转录本评估，该评估已被证明比其他技术更快速和经济。我们使用了RT-PCR为基础的技术，以确定时间点，在分化的EC衍生的心肌细胞，和最近饲养层一般用于维持胚胎干细胞中表达的中胚层和心脏限制性基因产物的数量。后面的这些研究旨在确定可能重新编程细胞使其变得更具多能性的因素。 我们已经利用定量PCR和原位杂交分析的时间和空间分布的一些最差异调节的转录鉴定SAGE，其中几个已显示出心脏优势，无论是胎儿或成人的心脏。在这项研究中发现的新基因产物提供了一个框架，用于分析人类和小鼠胚胎干细胞中的前和早期心脏发育过程，并且是积极研究的主题。在这些基因转录本中，已经进行了后续分析的是Cripto和多效生长因子。 自从最初的鉴定以来，我们已经确定了多效生长因子（PTN），一种促进血管生成、细胞增殖和分化的发育调节细胞因子生长因子，在发育中的心肌中显著表达，但在成人心脏中表达很差。相反，在大鼠心肌梗死模型和人扩张型心肌病中，多效生长因子显著上调，表明它可能在损伤修复中发挥功能性作用。为了阐明PTN对收缩细胞的影响，我们采用了大鼠新生（NN）和成年（A）心肌细胞（CM）的原代培养物。我们发现PTN以剂量和时间依赖性方式促进半胱天冬酶介导的基因组DNA片段化。更重要的是，它显着增强NNCM的细胞凋亡反应，缺氧应激和紫外线（UV）照射，和ACMs缺氧-复氧。我们还发现，PTN增强的UV诱导的细胞凋亡在siRNA介导的内源性PTN蛋白敲低后的NNCM中被消除。从机制上讲，PTN拮抗IGF-1相关的AKT/PKB的Ser-473磷酸化，并同时降低BAD和GSK 3 β磷酸化。腺病毒表达的组成型活性AKT和氯化锂介导的抑制GSK 3 β;也减少了PTN增强的程序性细胞死亡。这些后面的数据表明，PTN促进CM细胞死亡，至少部分地，通过抑制AKT信号传导。 总之，我们发现了PTN在CM中的新作用。这种细胞因子，除了其在心脏中的促血管生成作用外，还增强CM程序性细胞死亡以响应促凋亡应激。最近，我们已经开始分析转录因子B-MYB在心肌细胞中的作用。

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 summary, 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. More recently, we have begun analyzing the effects of the transcription factor B-MYB in cardiomyocytes.