Molecular mechanisms of phenotypic modulation and growth of smooth muscle cells

平滑肌细胞表型调节和生长的分子机制

• 批准号: 09281102
• 负责人: NAGAI Ryozo
• 金额: $ 127.87万
• 依托单位: University of Tokyo (1999-2001)Gunma University (1997-1998)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas (A)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09281102/
• 关键词: blood vessels; smooth muscle; gene therapy; gene expression; phenotypic modulation; atherosclerosis; aging; cardiovascular remodeling; 転写因子; klotho; クロマチン転写; HB-EGF; カルポニン; Smooth muscle; Klotho; 血管平滑筋; BTEB2; プロモーター; INOS; Znフィンガー; 組織

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

本研究的目的是阐明血管平滑肌细胞表型改变和生长的分子机制。为此，我们的目标是确定影响血管平滑肌细胞分子标志物基因表达的内在和外部因素，并将这些知识应用于动脉粥样硬化等血管疾病的新疗法的开发。这些研究分为三个主要研究领域：1)特定基因在平滑肌细胞中的表达和转录的调控及表型调控机制的阐明；2)生长因子和细胞信号通路对平滑肌细胞生长和表型调控的影响；3)血管和动脉粥样硬化的有效基因治疗技术，特别是对平滑肌基因的研究。在四年的研究期间取得的研究成果如下：1)转录因子EX…的功能表征在去分化的平滑肌细胞中受到更多的压力：分析去分化的平滑肌细胞的分子标志--平滑肌肌球蛋白重链SMemb基因的转录调控，从而分离到转录因子BTEB2。通过对BTEB2的分析，进一步阐明了其激活机制和转录诱导机制，以及在人类冠状动脉再狭窄病变中的相关作用；2)阐明了平滑肌细胞的转录机制：分析了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(Tip60，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被证明上调了基因表达、蛋白合成，并释放了对平滑肌生长和趋化刺激的作用。分离得到HB-EGF脱落剂KB-R7785。此外，还分析了作为脱落酶候选成员的ADAM FIMELY成员的作用。4)衰老抑制基因Klotho的分子特征；Klotho基因主要在钙稳态的潜在组织中表达。Klotho在调节电解质代谢中起着至关重要的作用。Klotho基因的缺失会导致细胞和组织的降解。重要的是，在老年小鼠的组织中，-calain的活性增加，同时klotho基因的表达下调。5)Calponin H1功能作用的基因敲除分析在小鼠中，Calponin H1基因被敲除，这表明Calponin参与了肌肉收缩装置。6)基因转移对平滑肌细胞表型调控的研究：针对参与血管平滑肌生长的转录因子(E2F、NFKB)进行诱骗实验，并将其推广到人类临床研究中。新的基因转移方法也被开发出来。较少

项目成果

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