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: 10053587
• 负责人: Joseph M Miano
• 金额: $ 57.12万
• 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/10053587
• 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，来自复杂谱系追踪研究的无可争议的证据表明，血管平滑肌 细胞（VSMC）去分化在很大程度上导致了许多血管疾病。一个重要的宣言—— 这种表型变化的原因是一系列 VSMC 限制基因的表达减弱，包括 神秘的平滑肌钙调蛋白（Cnn1）基因。 Cnn1 的功能主要在体外或体内研究 单个基因敲除小鼠，其中几个外显子和内含子被 Neo 盒替换， 下面详细介绍，混淆了表型的准确解释。我们倡导 CRISPR-Cas9 小鼠基因组编辑系统可以在许多基因的调控元件中进行微妙的替换， 保留所有编码序列完整。我们在这里报告了一个显着的表型，其中单个碱基的变化 Cnn1 基因座中的单个 CArG 盒消除了血管（但不是内脏）SMC 中 CNN1 的表达。满的 CNN1 的表达可通过携带人 CNN1 的 BAC 或 CRISPR 介导的 en-介导的激活来恢复。 内源 Cnn1 启动子。 Cnn1 CArG突变小鼠，代表第一个调控元件动物模型 编辑还显示 VSMC DNA 合成升高和收缩能力缺陷。相反，过度 BAC-CNN1 的表达抑制 VSMC 生长、拮抗新内膜形成、降低 VSMC 脂质 吸收并增强收缩基因表达。有趣的是，CNN1-Ser175 似乎是一个关键残基 其中一些效应使其成为小鼠体内 CRISPR 介导编辑的有吸引力的氨基酸。额外的 数据支持 CNN1 作为 ERK 信号传导的支架和分化的 VSMC 不动状态的效应器。 重要的是，由于两个相关的鲁棒表达，对 CNN1 函数的完整理解变得复杂 VSMC 中的基因（Cnn2 和 Cnn3）。我们假设 CNN1 与其他 CNN 亚型协同发挥作用 维持成熟的VSMC分化状态。该假设将使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 VSMC 功能的分子生理学分析工具。目标1将 利用 Cnn1 中的单碱基编辑作为急性和慢性血管疾病中新型功能丧失小鼠 疾病模型；将在体外和体内进行互补研究，以严格归因于现象 CNN1 特定损失的典型变化。目标 2 将利用特征良好的 BAC 小鼠来阐明 目标 1 中使用的血管疾病模型中与 Cnn1 功能获得相关的功能。补充 在 Cnn1 KO 的背景下，对 BAC 小鼠进行 CRISPR 编辑，将提供对功能的敏锐洞察 介导 CNN1 功能的残基。 Aim 3 将利用新的 floxed Cnn2 和 Cnn3 小鼠，带来令人兴奋的结果 我们开发了 VSMC 特定的 Cre 驱动程序，首次解决了三重 Cnn KO 鼠标模型的问题 基线和急性或慢性血管疾病模型。这些研究基于范式转变 基因敲除的方法，作为研究与 VSMC 差异相关的其他基因家族的模板 并将为未来针对 CNN1 治疗 VSMC 分化受损疾病的研究提供信息。