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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：
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
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/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

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血管平滑肌