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

基本信息

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
项目类别：
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

项目摘要

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.

现在有无可争议的证据来自复杂的谱系追踪研究，即血管平滑肌细胞（VSMC）去分化会导致多种血管疾病。主要宣言这种表型变化的影响会减弱一系列VSMC限制基因的表达，包括神秘的平滑肌钙蛋白（CNN1）基因。 CNN1的功能已被研究主要是在体外或一只单一的淘汰鼠标，其中几个外显子和内含子被一个新盒式盒子代替，该盒子下面详细说明，对表型的准确解释混淆。我们拥护CRISPR-CAS9 小鼠的基因组编辑系统，以在许多基因的调节元素中设计微妙的取代，保留完整的所有编码序列。我们在这里报告了一个非凡的表型 CNN1基因座中的一个carg盒消除了血管（但不是内脏）SMC中CNN1的表达。满的 CNN1的表达通过携带人CNN1的BAC或CRISPR介导的En-的激活来恢复 Dogenous CNN1启动子。 CNN1 CARG突变小鼠，代表调节元件的第一个动物模型编辑，还显示VSMC DNA合成和有缺陷的收缩能力的升高。相反，过度 BAC-CNN1的表达抑制VSMC的生长，拮抗新内膜的形成，减少VSMC脂质吸收并增强收缩基因表达。有趣的是，CNN1-SER175似乎是关键残留物对于其中一些效果，使其成为小鼠CRISPR介导的编辑的有吸引力的氨基酸。额外的数据支持CNN1作为ERK信号传导和分化VSMC的Immotile状态效应子的支架。重要的是，通过两种相关的鲁棒表达使对CNN1功能的完全理解变得复杂 VSMC中的基因（CNN2和CNN3）。我们假设CNN1与其他CNN同工型一起起作用保持成熟的VSMC差异化状态。该假设将在三个目标中使用最先进的目的进行检验 VSMC功能的遗传学，疾病动物模型和分子生理测定的工具。目标1意志在急性和慢性血管的情况下，将CNN1中的单个碱基编辑作为一种新颖的功能丧失鼠标疾病模型；互补研究将在体外和体内进行严格归纳 CNN1的特定损失的典型变化。 AIM 2将利用良好的BAC小鼠来阐明在AIM 1中使用的血管疾病模型中与CNN1功能获得的功能。互补在CNN1 KO的背景下，BAC鼠标的CRISPR编辑将为功能提供精致的见解介导CNN1功能的残基。 AIM 3将用令人兴奋的我们开发的VSMC特异性CRE驱动程序是第一次解决三重CNN KO鼠标模型基线以及急性或慢性血管疾病模型。这些研究，基于范式移动基因KO的方法，作为研究与VSMC不同的其他基因家族的模板 tiation并将告知针对CNN1在VSMC分化受到损害的疾病中的未来研究。