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.

基质细胞蛋白是细胞外基质 (ECM) 的组成部分。它们通常表达量很高 在胚胎发育过程中，但在成人组织中不存在/含量较低，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、生长因子和细胞的相互作用塑造 ECM 特性 器官发育和分化过程中的受体。试图识别独特的分子特征 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 (CILP1) 在梗塞心脏中差异性上调。 Cilp1 通常关联 随着骨骼和软骨的发育。其在成人心脏病中的功能和作用机制尚不清楚。 我们从商业 Cilp1fl/fl 小鼠和带有 Cilp1 的转基因 (Tg) 小鼠中生成了 Cilp1 敲除 (KO) 小鼠 在肌成纤维细胞中过度表达。 Cilp1 的缺失减少了心肌重构的不利影响。 梗塞 (MI) 中，Cilp1 的过度表达会使情况恶化。 Cilp1 主要在心脏成纤维细胞中表达。我们 假设成纤维细胞 Cilp1 在 MI 损伤时促进炎症和肌成纤维细胞增殖。我们现在 生成的成纤维细胞条件 fbKO 小鼠（PostnMCM;Cilp1fl/fl 和 Tcf21MCM;Cilp1fl/fl 含有他莫昔芬 分别在 Periostin (Postn) 和 Tcf21 基因位点内诱导型 Cre 重组酶表达盒。 目标 1. 确定 Cilp1 在 MI 后心脏重塑中的细胞类型特异性功能。我们将删除 Cilp1 在 MI 之前和之后第 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/分化和胶原蛋白重塑。我们还将 对从这些小鼠心脏中分离出的肌成纤维细胞进行蛋白质组学。成纤维细胞 Cilp1 的调节作用 将通过梗死 WT 的单核 RNA-seq 研究各种心脏细胞类型中的基因转录 和 Cilp1 fbKO 心脏在 MI 后 d3。目的2. 建立Cilp1的分子功能及其作用机制 行动。初步研究表明培养基中的Cilp1蛋白通过促进肌成纤维细胞增殖 mTORC1 通路并结合清除受体 CD36。 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。测试 根据这一假设，我们将通过诱变和潜在的 Cilp1- 来识别 Cilp1 的最小功能域 使用基于筛选和基于候选的测定法结合配偶体，并将确定 Cilp1 如何充当 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 抗纤维形成治疗前后小鼠血液中 Cilp1 的水平 受伤。基质细胞蛋白在临床上易于处理，因为它们易于全身递送 治疗试剂。我们的初步研究确定了 Cilp1 在 MI 后重塑中的病理作用。这 该提案将确定 Cilp1 如何指导心脏细胞的发育和分化以及基因表达 促进不良重塑，从而提供有关如何靶向该蛋白质的机制见解。