Role of Smooth Muscle Calponin in Vascular Pathobiology

平滑肌钙调蛋白在血管病理学中的作用

• 批准号: 10077575
• 负责人: Joseph M Miano
• 金额: $ 56.04万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-11-07 至 2022-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10077575
• 关键词: Actins; Acute; Address; Alleles; Amino Acids; Aneurysm; Animal Disease Models; Animal Model; Apolipoprotein E; Arterial Injury; Atherosclerosis; Attention; Attenuated; Bacterial Artificial Chromosomes; Binding; Biological Assay; Biology; Blood Vessels; CRISPR/Cas technology; Cell Differentiation process; Cell physiology; Cells; Chronic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Competence; Complement; Cre driver; Cytoskeleton; DNA biosynthesis; Data; Differentiated Gene; Disease; Disease model; Engineering; Exhibits; Exons; Future; Gene Expression; Gene Family; Gene Silencing; Genes; Genetic; Genetic Models; Genetic Transcription; Growth; Hematological Disease; Homeostasis; Human; Impairment; In Vitro; Injury; Introns; Ion Channel; Knock-out; Knockout Mice; Lesion; Link; Lipids; LoxP-flanked allele; MAPK3 gene; Mediating; Modeling; Molecular; Mus; Mutant Strains Mice; Mutation; Nature; Phenotype; Physiology; Point Mutation; Positioning Attribute; Protein Isoforms; Proteins; Regulatory Element; Reporting; Role; Serum Response Factor; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; System; Testing; Thinking; Time; Transcriptional Regulation; Untranslated RNA; Vascular Diseases; Vascular Smooth Muscle; base; calponin; cell growth; design; experimental study; gain of function; gene function; genome editing; human model; in vivo; injured; insight; knockout gene; loss of function; migration; mouse genetics; mouse model; myocardin; novel; novel strategies; overexpression; programs; promoter; response; scaffold; structural genomics; tool; uptake; vascular injury; vascular smooth muscle cell migration

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.

现在，从复杂的谱系追踪研究中有无可争议的证据表明，血管平滑肌 血管平滑肌细胞（VSMC）的去分化实质上有助于许多血管疾病。一个重要的宣言- 这种表型变化的特征是一系列VSMC限制性基因的表达减弱，包括 平滑肌钙调蛋白（Cnn 1）基因。Cnn 1的功能主要在体外或体内进行了研究。 其中几个外显子和内含子被替换为neo盒的单敲除小鼠， 下文详述的模糊了表型的准确解释。我们支持CRISPR-Cas9 基因组编辑系统在小鼠中设计许多基因的调控元件内的细微取代， 留下完整的所有编码序列。我们在这里报告了一个显着的表型，其中一个单一的碱基变化， Cnn 1基因座中的单个CArG盒消除了血管（但不是内脏）SMC中的CNN 1表达。充分 CNN 1的表达用携带人CNN 1的BAC或CRISPR介导的en-CNN 1的活化来恢复。 内源性Cnn 1启动子。Cnn 1 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合成的升高和收缩能力的缺陷。相反，过度- BAC-CNN 1表达抑制VSMC生长，拮抗新生内膜形成，降低VSMC脂质 摄取，并增强收缩基因表达。有趣的是，CNN 1-Ser 175似乎是一个关键的残基， 对于其中一些作用，使其成为小鼠中CRISPR介导的编辑的有吸引力的氨基酸。额外 数据支持CNN 1作为ERK信号传导的支架和分化的VSMC的不动状态的效应物。 重要的是，CNN 1功能的完整理解是复杂的两个相关的稳定表达， 基因（Cnn 2和Cnn 3）。我们假设CNN 1与其他CNN亚型一起发挥作用， 维持成熟的VSMC分化状态。这一假设将在三个目标进行测试，使用最先进的 在遗传学、疾病动物模型和VSMC功能的分子生理学测定中的工具。目标1将 利用Cnn 1中的单碱基编辑作为急性和慢性血管疾病背景下的新型功能丧失小鼠 疾病模型;互补研究将在体外和体内进行，以严格归因于表型， CNN 1特异性丢失的典型变化。目的2将利用一个良好的特点BAC小鼠，以阐明 目的1中使用的血管疾病模型中与Cnn 1功能获得相关的功能。互补 BAC小鼠的CRISPR编辑，在Cnn 1 KO的背景下，将提供对功能的精细洞察。 介导CNN 1功能的残基。Aim 3将开发新的floxed Cnn 2和Cnn 3小鼠， 我们开发的VSMC特定Cre驱动程序首次用于解决三重Cnn KO小鼠模型， 基线和急性或慢性血管疾病模型。这些研究基于范式转换 基因科斯的方法，作为模板的其他基因家族的研究与血管平滑肌细胞增殖， 并将为未来针对CNN 1在VSMC分化受损的疾病中的研究提供信息。