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Role of Cilp1 in Post-Natal Heart Response to Injury

Cilp1 在产后心脏损伤反应中的作用

基本信息

批准号：
10365053
负责人：
Zhi-Ping Liu
金额：
$ 56.07万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10365053
关键词：
Adult Affect Binding Biological Assay Blood Bone Development CD36 gene Cardiac Cartilage Cell Nucleus Cell Proliferation Cell Proliferation Regulation Cicatrix Clinical Collagen Conditioned Culture Media Cues DNA cassette Deposition Development Disease Electric Conductivity Embryonic Development Enterobacteria phage P1 Cre recombinase Extracellular Matrix Fibroblasts Fibrosis Future Gene Expression Gene Expression Regulation Genetic Transcription Growth Factor Growth Factor Receptors Heart Heart Diseases Heart failure Infarction Inflammation Inflammatory Injury Integrins Interleukin-6 Knock-out Knockout Mice Lead Left Ventricular Hypertrophy Liver Liver Fibrosis Measures Molecular Molecular Profiling Mus Mutagenesis Myocardial Infarction Myocardium Myofibroblast N-terminal NFKB Signaling Pathway Necrosis Outcome Pathologic Pathway interactions Pharmaceutical Preparations Phenotype Physiological Process Production Property Proteins Proteomics Public Health Reagent Receptor Cell Recombinants Regenerative capacity Research Risk Factors Role Shapes Signal Pathway Structural Protein Tamoxifen Testing Therapeutic Tissues Transgenic Mice angiogenesis autocrine base cartilage development cell behavior cell type coronary fibrosis cytokine design differential expression effective therapy experimental study genomic locus heart cell heart function heart preservation insight ischemic injury macrophage organ growth overexpression paracrine periostin prevent promoter receptor receptor-mediated signaling response to injury success tendon development therapeutic target transcriptome sequencing

项目摘要

Matricellular proteins are constituents of the extracellular matrix (ECM). They are normally expressed highly during embryonic development but absent/low in adult tissues unless activated by cues for tissue remodeling. Matricellular proteins shape ECM properties through interactions with structural proteins, growth factors, and cell receptors during organ development and differentiation. In an attempt to identify molecular signatures unique to irreversible cardiac fibrosis, we performed proteomics of fibrotic heart and liver and found that cartilage intermediate layer protein 1 (Cilp1) is differentially upregulated in the infarcted heart. Cilp1 is normally associated with bone and cartilage development. Its function and mechanism of action in adult heart diseases are unknown. We generated Cilp1 knockout (KO) mice from commercial Cilp1fl/fl mice and transgenic (Tg) mice with Cilp1 overexpressed in myofibroblasts. While deletion of Cilp1 reduced adverse cardiac remodeling upon myocardial infarction (MI), overexpression of Cilp1 worsened it. Cilp1 is expressed predominantly in cardiac fibroblasts. We hypothesize that fibroblast Cilp1 promotes inflammation and myofibroblast proliferation upon MI injury. We now generated fibroblast conditional fbKO mice (PostnMCM;Cilp1fl/fl and Tcf21MCM;Cilp1fl/fl that contain a tamoxifen inducible Cre-recombinase expression cassette within Periostin (Postn) and Tcf21 genetic locus, respectively). Aim 1. To determine the cell-type specific function of Cilp1 in post-MI cardiac remodeling. We will delete Cilp1 in cardiac fibroblasts before and post-MI day (d) 1 & d4 to investigate its effect on cardiac remodeling, including cardiac function, inflammation, myofibroblast proliferation/differentiation, and collagen remodeling. We will also perform proteomics of myofibroblasts isolated from these mouse hearts. The role of fibroblast Cilp1 in regulation of gene transcription in various heart cell types will be investigated with single-nuclei RNA-seq of infarcted WT and Cilp1 fbKO hearts at post-MI d3. Aim 2. To establish the molecular function of Cilp1 and its mechanism of action. Preliminary studies showed that Cilp1 protein in culture medium promotes myofibroblast proliferation via the mTORC1 pathway and binds scavenge receptor CD36. Cilp1 may interact with cell receptor/growth factor, promoting cell proliferation and inflammatory gene expression via receptor-mediated signaling pathways. To test this hypothesis, we will identify the minimal functional domain(s) of Cilp1 via mutagenesis and potential Cilp1- binding partners using both screen- and candidate-based assays and will establish how Cilp1 may act as a paracrine factor to regulate the cellular phenotypes of various heart cell types via receptor-mediated signaling pathways. We will also measure blood level of Cilp1 in mice before and after an anti-fibrogenic therapy upon MI injury. Matricellular proteins are clinically tractable owing to their accessibility to systemically delivered therapeutic reagents. Our preliminary studies established a pathological role of Cilp1 in post-MI remodeling. This proposal will establish how Cilp1 instructs development and differentiation of heart cells and gene expression to promote adverse remodeling, thus providing mechanistic insight on how to target this protein.

基质细胞蛋白是细胞外基质（ECM）的组成部分。它们通常表达得很高，在胚胎发育期间，但在成体组织中不存在/低，除非被组织重塑的线索激活。基质细胞蛋白通过与结构蛋白、生长因子和细胞生长因子的相互作用形成ECM特性。器官发育和分化过程中的受体。为了确定一种独特的分子特征，不可逆的心脏纤维化，我们对纤维化的心脏和肝脏进行了蛋白质组学研究，发现软骨中间层蛋白1（Cilp 1）在梗塞心脏中差异性上调。Cilp 1通常与骨骼和软骨发育。它在成人心脏病中的作用和机制尚不清楚。我们从商业化的Cilp 1fl/fl小鼠和Cilp 1转基因（Tg）小鼠中产生Cilp 1敲除（KO）小鼠在肌成纤维细胞中过表达。而Cilp 1的缺失减少了心肌梗死后的不良心脏重构， Cilp 1主要在心肌成纤维细胞中表达。我们假设成纤维细胞Cilp 1在MI损伤时促进炎症和肌成纤维细胞增殖。我们现在产生成纤维细胞条件fbKO小鼠（PostnMCM; Cilp 1fl/fl和Tcf 21 MCM; Cilp 1fl/fl，其含有他莫昔芬分别在骨膜蛋白（Postn）和Tcf 21基因座内的可诱导Cre重组酶表达盒）。目标1.探讨Cilp 1在心肌梗死后心脏重构中的细胞类型特异性功能。我们将删除Cilp 1 在心肌梗死前和后第1天和第4天的心脏成纤维细胞中，研究其对心脏重塑的影响，包括心脏功能、炎症、肌成纤维细胞增殖/分化和胶原重塑。我们还将对从这些小鼠心脏中分离的肌成纤维细胞进行蛋白质组学研究。成纤维细胞Cilp 1在调节细胞凋亡中的作用将用梗塞WT的单核RNA-seq研究各种心脏细胞类型中的基因转录和MI后第3天的Cilp 1 fbKO心脏。目标二。目的探讨Cilp 1的分子功能及其作用机制。行动上初步研究表明，培养基中的Cilp 1蛋白通过以下途径促进肌成纤维细胞增殖： mTORC 1通路并结合CD 36受体。Cilp 1可能与细胞受体/生长因子相互作用，通过受体介导的信号通路促进细胞增殖和炎症基因表达。测试基于这一假设，我们将通过诱变鉴定Cilp 1的最小功能结构域和潜在的Cilp 1- 结合伙伴使用筛选和候选人为基础的测定，并将建立如何Cilp 1可以作为一个旁分泌因子通过受体介导的信号传导调节各种心脏细胞类型的细胞表型途径。我们还将测量MI后抗纤维化治疗前后小鼠中Cilp 1的血液水平损伤基质细胞蛋白由于其可获得性而在临床上易于处理，治疗试剂我们的初步研究确立了Cilp 1在心肌梗死后重构中的病理作用。这该提案将确定Cilp 1如何指导心脏细胞的发育和分化以及基因表达，促进不良重塑，从而提供关于如何靶向这种蛋白质的机制见解。