Molecular mechanisms of phenotypic modulation and growth of smooth muscle cells

本研究的目的是阐明血管平滑肌细胞表型调节和生长的分子机制。为此，我们的目标是确定影响血管平滑肌细胞中分子标记物基因表达的内在和外在因素，并将这些知识应用于血管疾病如动脉粥样硬化的新疗法的开发。研究分为三个主要研究领域; 1）平滑肌细胞中特异性基因表达和转录的调节和表型调节的潜在机制的阐明，2）平滑肌细胞生长和表型调节的潜在生长因子和细胞信号传导途径，和3）血管和动脉粥样硬化的有效基因治疗技术，特别关注平滑肌基因。四年的研究期如下：1）转录因子的功能鉴定 ...更多信息 在去分化的平滑肌细胞中受压：分析平滑肌肌球蛋白重链SMemb基因的转录调节，该基因是去分化的平滑肌细胞的分子标记，其导致转录因子BTEB 2的分离。对BTEB 2的分析进一步阐明了其激活机制和转录诱导机制，以及在人冠状动脉再狭窄病变中的相关作用; 2）阐明了平滑肌细胞中的转录机制：参与平滑肌转录的转录机制，即染色质重塑因子（CIA）和组蛋白乙酰转移酶（Tip 60，p300），并阐明了它们在转录激活机制中的作用; 3）HB-EGF对平滑肌细胞生长的调控机制：HB-EGF上调平滑肌细胞生长和趋化性刺激的基因表达、蛋白质产生和脱落。分离HB-EGF脱落抑制剂KB-R7785。此外，分析了作为脱落酶候选者的ADAM家族成员的作用。4)衰老抑制基因Klotho的分子生物学特性Klotho基因主要表达于对钙稳态有影响的组织中。Klotho在调节电解质代谢方面起着关键作用。klotho基因的缺陷导致细胞和组织被钙蛋白酶激活而降解。重要的是，在老年小鼠的组织中，□钙蛋白酶的激活增加与klotho基因表达的下调一起发生。5)钙调蛋白hl功能作用的基因敲除分析在小鼠中敲除钙调蛋白hl基因，表明钙调蛋白参与肌肉收缩装置。6)通过基因转移调节平滑肌细胞表型调节：针对参与平滑肌生长的转录因子（E2 F，NF κ B）的诱饵方法已经完成，并扩展到人类临床研究。新的基因转移方法也被开发出来。少

The purpose of the present research was to elucidate the molecular mechanisms underlying phenotypic modulation and growth of vascular smooth muscle cells. To this end, our objective was to determine the intrinsic and external factors which affect gene expression of molecular markers in vascular smooth muscle cells, and to apply this knowledge to the development of new therapies for vascular diseases such as atherosclerosis. Studies were grouped into three main research fields ; 1) regulation of specific gene expression and transcription in smooth muscle cells and elucidation of mechanisms underlying phenotypic modulation, 2) growth factors and cell signaling pathways underlying smooth muscle cell growth and phenotypic modulation, and 3) efficient gene therapy techniques for the vasculature and atherosclerosis with a particular focus on smooth muscle genes.Research achievements during the four year research term are as follows : 1) functional characterization of transcription factors ex … More pressed in dedifferentiated smooth muscle cells : the transcriptional regulation of the smooth muscle myosin heavy chain SMemb gene which is a molecular marker of dedifferentiated smooth muscle cells was analyzed which resulted in the isolation of the transcription factor BTEB2. Analysis of BTEB2 further resulted in elucidation of its activation mechanism and mechanisms of transcriptional induction in addition to a correlative role in human coronary restenotic lesions ; 2) elucidation of transcriptional mechanisms in smooth muscle cells : transcriptional machinery involved in smooth muscle transcription, namely chromatin remodeling factors (CIA) and histone acetyltransferases (Tip60, p300) were analyzed and their roles in transcriptional activation mechanisms were elucidated ; 3) regulatory mechanisms of HB-EGF on smooth muscle cell growth ; HB-EGF was shown to be upregulated for gene expression, protein production and shedding on smooth muscle growth and chemotaxic stimuli. The HB-EGF shedding inhibitor KB-R7785 was isolated. Additionally, the role of the ADAM faimily members which are candidates for shedding enzymes were analyzed. 4) Molecular characterization of the aging suppressor gene Klotho ; The klotho gene is expressed principally in t**npotant tissues for calcium homeostasis. Klotho plays a critical role for the regulation of electrolyte metabolism. The deficiency of the klotho gene results in degradation of cells and tissues by the actibation □calpain. Importantly, the increased activation of □calpain occurs in tissues of old mice together with the downregulation of klotho gene expression. 5) Genetic knockout analysis of the functional role of calponin hl ; the calponin hl gene was knocked out in mice which showed that calponin is involved in the muscle contractile apparatus. 6) Regulation of smooth muscle cell phenotypic modulation by gene transfer : Decoy approaches against transcription factors involved in smooth muscle growth (E2F, NFKB) were done and were extended to human clinical studies. New gene transfer methods were also developed. Less