Role of Smooth Muscle Calponin in Vascular Pathobiology

现在有来自复杂的血统追踪研究的无可辩驳的证据表明，血管平滑肌 细胞(VSMC)去分化在许多血管疾病中起重要作用。一份重要的宣言- 这种表型变化的抑制是一系列VSMC限制性基因的表达减弱，包括 神秘的肌钙蛋白(Cnn1)基因。Cnn1的功能已初步在体外或在 一种单基因敲除小鼠，其中几个外显子和内含子被一个新的盒式磁带取代，因为 下面详细描述，混淆了对表型的准确解释。我们支持CRISPR-CAS9 小鼠的基因组编辑系统，以在许多基因的调节元件内设计微妙的替换， 保持所有编码序列完好无损。我们在这里报告了一个显著的表型，其中单个碱基的变化 Cnn1基因上的单个Carg框可取消血管(但不是内脏)SMC中CNN1的表达。饱满 携带人CNN1或CRISPR介导的EN-1激活的BAC可恢复CNN1的表达 内源性Cnn1启动子。Cnn1 Carg突变小鼠，代表了第一个调控元件的动物模型 编辑，也显示VSMC DNA合成增加和收缩能力缺陷。相反，过度- BAC-CNN1的表达抑制VSMC生长，拮抗新生内膜形成，降低VSMC脂质 摄取，并增强收缩基因的表达。有趣的是，CNN1-Ser175似乎是一个关键残基 其中一些作用使其成为CRISPR介导的小鼠编辑的一种有吸引力的氨基酸。其他内容 数据支持CNN1作为ERK信号的支架和分化的VSMC静止状态的效应器。 重要的是，CNN1功能的完整理解是复杂的，因为相关的两个 VSMC基因(Cnn2和Cnn3)。我们假设CNN1与其他CNN亚型协同工作 维持成熟的VSMC分化状态。这一假设将用最先进的技术在三个目标上进行测试 遗传学、疾病动物模型和血管平滑肌细胞功能的分子生理学分析的工具。目标1将 利用Cnn1中的单碱基编辑作为一种新的急性和慢性血管功能丧失的小鼠 疾病模型；互补性研究将在体外和体内进行，以严格归因于 CNN1特异性丢失的典型变化。Aim 2将利用一只特征良好的BAC小鼠来阐明 AIM中使用的血管疾病模型中与Cnn1功能增益相关的功能1.互补性 在Cnn1 KO的背景下，对BAC鼠标的CRISPR编辑将提供对功能的精致洞察 调节CNN1功能的残基。AIM 3将利用令人兴奋的Cnn2和Cnn3小鼠 我们开发的VSMC特定CRE驱动程序首次解决了三个CNN KO鼠标模型的问题，网址为 在基线和急性或慢性血管疾病模型中。这些研究以范式转换为基础 Kos基因的研究方法，为研究与VSMC差异连锁的其他基因家族提供了一个模板。 并将在未来针对VSMC分化受损的疾病中的CNN1的研究提供信息。

There is now incontrovertible evidence from sophisticated lineage tracing studies that vascular smooth muscle cell (VSMC) de-differentiation contributes substantively to a number of vascular diseases. A major manifesta- tion of such phenotypic change is attenuated expression of a battery of VSMC-restricted genes, including the enigmatic smooth muscle calponin (Cnn1) gene. The function of Cnn1 has been studied primarily in vitro or in a single knockout mouse wherein several exons and introns were replaced with a neo cassette which, as detailed below, obfuscates accurate interpretation of phenotypes. We have championed the CRISPR-Cas9 genome editing system in mice to engineer subtle substitutions within regulatory elements of many genes, leaving intact all coding sequences. We report here a remarkable phenotype wherein a single base change of a single CArG box in the Cnn1 locus abolishes expression of CNN1 in vascular (but not visceral) SMC. Full expression of CNN1 is restored with a BAC carrying human CNN1 or CRISPR-mediated activation of the en- dogenous Cnn1 promoter. Cnn1 CArG mutant mice, representing the first animal models of regulatory element edits, also show elevation in VSMC DNA synthesis and defective contractile competence. Conversely, over- expression of BAC-CNN1 suppresses VSMC growth, antagonizes neointimal formation, reduces VSMC lipid uptake, and enhances contractile gene expression. Interestingly, CNN1-Ser175 appears to be a critical residue for some of these effects making it an attractive amino acid for CRISPR-mediated editing in mice. Additional data support CNN1 as a scaffold for ERK signaling and effector of the immotile state of differentiated VSMC. Importantly, a complete understanding of CNN1 function is complicated by robust expression of two related genes (Cnn2 and Cnn3) in VSMC. We hypothesize that CNN1 functions in concert with other CNN isoforms to maintain a mature VSMC differentiated state. This hypothesis will be tested in three aims using state-of-the-art tools in genetics, animal models of disease, and molecular physiology assays of VSMC function. Aim 1 will exploit the single base edit in Cnn1 as a novel loss-of-function mouse in context of acute and chronic vascular disease models; complementation studies will be carried out in vitro and in vivo to rigorously ascribe pheno- typic changes to the specific loss of CNN1. Aim 2 will exploit a well-characterized BAC mouse to elucidate functions associated with Cnn1 gain-of-function in models of vascular disease used in Aim 1. Complementary CRISPR editing of the BAC mouse, in background of Cnn1 KO, will afford exquisite insight into functional residues that mediate CNN1 function. Aim 3 will exploit new floxed Cnn2 and Cnn3 mice with an exciting VSMC-specific Cre driver we have developed to address, for the first time, triple Cnn KO mouse models at baseline and in acute or chronic vascular disease models. These studies, based on paradigm-shifting approaches to gene KOs, serve as a template for the study of other gene families linked to VSMC differen- tiation and will inform future studies targeting CNN1 in diseases where VSMC differentiation is compromised.