Role of Cilp1 in Post-Natal Heart Response to Injury

Cilp1 在产后心脏损伤反应中的作用

• 批准号: 10580830
• 负责人: Zhi-Ping Liu
• 金额: $ 54.47万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 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/10580830
• 关键词: Adult; Affect; Binding; Biological Assay; Blood; Blood Vessels; Bone Development; CD36 gene; Cardiac; Cartilage; Cell Communication; 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; Heart; Heart Diseases; Heart failure; Infarction; Inflammation; Inflammatory; Injury; Integrins; Interleukin-6; Knock-out; Knockout Mice; Left Ventricular Hypertrophy; Liver; Liver Fibrosis; Macrophage; 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; Proliferating; Property; Protein Secretion; 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; cartilage development; cell behavior; cell type; coronary fibrosis; cytokine; design; differential expression; effective therapy; experimental study; genomic locus; heart cell; heart function; heart preservation; inducible Cre; insight; ischemic injury; organ growth; overexpression; paracrine; periostin; postnatal; prevent; promoter; receptor; receptor-mediated signaling; response to injury; single nucleus RNA-sequencing; success; tendon development; therapeutic target

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.

基质蛋白是细胞外基质(ECM)的组成部分。它们通常高度表达 在胚胎发育期间，但在成人组织中缺失/低水平，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、生长因子和细胞相互作用形成ECM特性 器官发育和分化过程中的受体。为了尝试识别独有的分子特征 不可逆转的心肌纤维化，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(Cilp1)在心肌梗死后表达上调。Cilp1通常与 伴随着骨骼和软骨的发育。其在成人心脏病中的作用和作用机制尚不清楚。 我们从商品Cilp1fl/fl小鼠和Cilp1转基因(TG)小鼠中获得了Cilp1基因敲除(KO)小鼠 在肌成纤维细胞中过表达。而Cilp1的缺失则减少了对心肌的不良心脏重构 心肌梗死(MI)，Cilp1过表达加重心肌梗死。Cilp1主要在心脏成纤维细胞中表达。我们 假设心肌梗死损伤时成纤维细胞Cilp1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性fbKO小鼠(PostnMCM；Cilp1fl/fl和Tcf21MCM；Cilp1fl/fl含有他莫昔芬 可诱导的Cre-重组酶表达盒分别位于Periostin(Postn)和TCF21基因座内)。 目的1.探讨Cilp1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Cilp1 在心肌梗死前和心肌梗死后第1天和第4天的心脏成纤维细胞中，研究其对心脏重构的影响，包括 心功能、炎症、肌成纤维细胞增殖/分化和胶原重构。我们还将 对从这些小鼠心脏分离的肌成纤维细胞进行蛋白质组学研究。成纤维细胞Cilp1在调控中的作用 在不同类型的心脏细胞中的基因转录的变化将用梗死灶的单核rna-seq来研究。 心肌梗死后d3的Cilp1fbKO心。目的2.建立Cilp1的分子功能及其作用机制。 行动。初步研究表明，培养液中的Cilp1蛋白通过促进肌成纤维细胞增殖 MTORC1途径与清道夫受体CD36结合。Cilp1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。为了测试 在这一假设下，我们将通过突变确定Cilp1的最小功能结构域(S)和潜在的Cilp1- 使用基于筛选和基于候选人的分析来绑定合作伙伴，并将确定Cilp1如何作为 旁分泌因子通过受体介导的信号转导调节不同类型心肌细胞表型 小路。我们还将检测心肌梗塞抗纤维化治疗前后小鼠血中Cilp1的水平。 受伤。基质细胞蛋白在临床上是易于处理的，因为它们可通过系统输送 治疗试剂。我们的初步研究证实了Cilp1在心肌梗死后重塑中的病理作用。这 提案将确定Cilp1如何指导心脏细胞的发育和分化以及基因表达 促进不利的重塑，从而为如何靶向这种蛋白质提供机制上的洞察。