The role of TCF21 in coronary heart disease

 描述（由申请人提供）：虽然大约40%的冠心病（CHD）风险可归因于遗传变异，但我们目前仅了解一小部分导致这种风险的遗传变异。最近的全基因组关联研究（GWAS）指出，CHD相关基因座可能通过完全意想不到的机制对CHD病理生物学做出贡献，从而提高了新治疗策略的可能性。我们的研究小组最近发现，这些CHD相关的多态性之一直接影响TCF 21基因的表达，暗示它在CHD病理生物学。TCF 21是一种基本的螺旋-环-螺旋转录因子，存在于发育中的冠状动脉血管系统中，在决定前体平滑肌细胞（SMC）的细胞命运决定中起关键作用。表达tcf 21的细胞存在于人和鼠冠状动脉的外膜和主动脉中膜。在ApoE-/-小鼠模型中对动脉粥样硬化疾病的反应中，这些Tcf 21表达细胞迁移到正在形成的病变中，并在纤维帽处对齐，在那里它们显示出SMC分化的证据。这是特别有趣的，因为SMC被认为可以加强纤维帽结构，从而防止斑块破裂和心肌梗死。然而，Tcf 21在这些前体SMC中的作用是未知的。本文所述的一系列实验旨在确定Tcf 21如何在体内调节前体SMC的表型和病变组成，并阐明介导这些作用的分子途径。总之，这些研究将显著扩大我们对SMC在CHD病理生物学中作用的理解。 在目标1中，我们将使用条件性Tcf 21敲除和TCF 21转基因过表达Apo E-/-小鼠来确定Tcf 21表达的变化如何影响动脉粥样硬化斑块内前体SMC的募集和表型，以及这些变化如何影响斑块结构。我们将使用经典的组织学和免疫组织化学来检查斑块的组成和结构，特别关注保护性纤维帽。我们将通过诱导型荧光报告基因跟踪Tcf 21表达细胞。 在目标2中，我们将使用从Tcf 21报告小鼠中分离的细胞来表征Tcf 21谱系示踪细胞的体内分子表型。Tcf 21特异性荧光报告基因将永久标记Tcf 21表达细胞，并允许在不同时间点对其进行鉴定 在疾病进展期间。这些Tcf 21谱系示踪细胞将使用激光捕获显微切割从主动脉根部病变中分离出来，并将进行RNA测序，这将在斑块发展期间的进行性阶段产生细胞基因表达的读数。这将使这些细胞的分子表型，并将揭示重要的途径，形成发展脆弱的动脉粥样硬化斑块。

 DESCRIPTION (provided by applicant): Although approximately 40% of the risk for coronary heart disease (CHD) can be attributed to genetic variation, we currently understand only a fraction of genetic variants that contribute to this risk. Recent genome wide association studies (GWAS) have pointed to CHD-associated loci that could be contributing to CHD pathobiology via completely unexpected mechanisms, raising the possibility of new therapeutic strategies. Our group has recently shown that one of these CHD-associated polymorphisms directly affects expression of the TCF21 gene, implicating it in CHD pathobiology. TCF21 is a basic helix-loop-helix transcription factor that is found in the developing coronary vasculature, where it plays a critical role in determining cell fate decisions in precursor smooth muscle cells (SMCs). Tcf21-expressing cells are present in the adventitia of human and murine coronary arteries and the aortic media. In response to atherosclerotic disease in the ApoE-/- mouse model, these Tcf21-expressing cells migrate into the developing lesion and align at the fibrous cap, where they show evidence of SMC differentiation. This is especially intriguing given that SMCs are thought to strengthen the fibrous cap structure, which protects against plaque rupture and myocardial infarction. However, the role of Tcf21 in these precursor SMCs is unknown. The series of experiments described herein aim to determine how Tcf21 modulates the phenotype of precursor SMCs and lesion composition in vivo, and to elucidate the molecular pathways by which these effects are mediated. Together, these studies will significantly expand our understanding of the role of SMCs in CHD pathobiology. In Aim 1, we will use conditional Tcf21 knockout and TCF21 transgenic over-expressing Apo E-/- mice to determine how changes in Tcf21 expression affect precursor SMC recruitment and phenotype within the atherosclerotic plaque, and how these changes affect plaque architecture. We will use classical histology and immunohistochemistry to examine plaque composition and structure, with a special focus on the protective fibrous cap. We will track Tcf21-expressing cells via an inducible fluorescent reporter gene. In Aim 2, we will use cells isolated from Tcf21 reporter mice to characterize the in vivo molecular phenotype of Tcf21 lineage-traced cells. A Tcf21-specific fluorescent reporter gene will permanently label Tcf21-expressing cells and allow their identification at various time points during disease progression. These Tcf21 lineage-traced cells will be isolated from aortic root lesions using laser capture microdissection and will undergo RNA sequencing, which will produce a readout of cellular gene expression at progressive stages during plaque development. This will allow molecular phenotyping of these cells and will shed light on important pathways that shape the developing vulnerable atherosclerotic plaque.