Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells That Contribute to the Fibrous Cap

Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including “vascular disease,” “disorder of artery,” and “occlusion of artery,” as well as disease-related cellular functions including “cellular movement” and “cellular growth and proliferation.” In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall.

近年来的全基因组关联研究（genome wide association studies）已鉴定出多个与冠心病（coronary heart disease）发病风险相关的基因。其中TCF21基因编码一种碱性螺旋-环-螺旋转录因子（basic-helix-loop-helix transcription factor），被认为在心外膜祖细胞（epicardial progenitor cells）的发育过程中发挥关键作用——这类祖细胞可分化为冠状动脉平滑肌细胞（SMC）与心脏成纤维细胞（cardiac fibroblasts）。本研究通过对小鼠及人类组织开展报告基因（reporter gene）与免疫定位（immunolocalization）实验，发现成年个体的血管TCF21表达主要局限于冠状动脉相关的外膜细胞（adventitial cells），以及小鼠主动脉近端（proximal aorta）的中膜平滑肌细胞。
针对人类冠状动脉平滑肌细胞（HCASMC, human coronary artery SMC）开展的全基因组RNA测序（RNA-Seq）研究显示，经小干扰RNA（siRNA）敲低TCF21后，可鉴定出多个潜在的下游通路；这些通路的富集术语涉及冠心病（CAD, coronary artery disease），包括"血管疾病""动脉异常""动脉闭塞"，同时还涵盖与疾病相关的细胞功能，如"细胞迁移""细胞生长与增殖"。
在HCASMC中开展的体外实验证实，TCF21的表达可促进细胞增殖与迁移，并抑制平滑肌细胞谱系标志物的表达。借助报告基因与谱系示踪（lineage tracing）技术开展的详细原位表达研究发现，在疾病发生前表达Tcf21的血管壁细胞会迁移至载脂蛋白E敲除小鼠（ApoE-/- mice）与低密度脂蛋白受体敲除小鼠（Ldlr-/- mice）的血管病变区域。尽管Tcf21谱系示踪细胞在早期病变中广泛分布，但在成熟病变中，它们参与形成了包膜下层（subcapsular layer）细胞层，其余细胞则与纤维帽（fibrous cap）相关联。这些谱系示踪的纤维帽细胞会激活平滑肌细胞标志物与生长因子受体基因（growth factor receptor genes）的表达。
综合上述实验数据，本研究提示TCF21可能通过调控迁移至血管病变区域的平滑肌前体细胞的分化状态发挥作用；结合该基因与人类冠心病的关联，上述过程或可作为血管壁相关冠心病发病风险的潜在机制。