Proteomics based mapping of cardiac extracellular matrix to define sex and age-dependent changes

基于蛋白质组学的心脏细胞外基质图谱来定义性别和年龄依赖性变化

基本信息

批准号：
10751473
负责人：
KATARZYNA A. CIESLIK
金额：
$ 60.13万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10751473
关键词：
Adhesions Affect Affinity Age Aging Androgen Receptor Animals Area Basement membrane Binding Cardiac Cardiac Myocytes Castration Cell Communication Cell Separation Cell Surface Receptors Cell physiology Cells Chemicals Cicatrix Data Defect Deposition Down-Regulation EFRAC Endothelial Cells Enzyme-Linked Immunosorbent Assay Enzymes Estrogen Receptors Extracellular Matrix Extracellular Matrix Proteins Failure Female Fibrillar Collagen Fibroblasts Fibrosis Functional disorder Future Gene Expression Genetic Transcription Geometry Goals Gonadal Steroid Hormones Grant Growth Factor Heart Heart failure Hormone Receptor Hormones Human Individual Infarction Inflammation Injury Integrin Binding Integrins Ischemia Leucocytic infiltrate Link Maps Mass Spectrum Analysis Modeling Molecular Mus Myocardial Infarction Ovary Patients Perivascular Fibrosis Phenotype Physiology Play Process Production Proliferating Protein Biosynthesis Proteins Proteoglycan Proteomics Reagent Reperfusion Injury Reperfusion Therapy Research Role Severities Sex Differences Signal Transduction Smooth Muscle Myocytes Sorting Tail Tertiary Protein Structure Testing Tissues Translating Ventricular Western Blotting age difference age related aged cell type cytokine druggable target experimental study injured injury and repair innovation interstitial male migration older women overexpression preservation repaired response scaffold sex therapeutic target three dimensional cell culture

项目摘要

PROJECT SUMMARY The extracellular matrix (ECM) provides a scaffold for cardiac structural integrity and plays an active role in modulating cellular responses. ECM consists of a network of fibrillar proteins and proteoglycans binding cyto- kines, growth factors, and ECM-modifying enzymes. The aging heart develops interstitial fibrosis, and prelimi- nary data demonstrate that different proteins are enriched in ECM in the male and female heart. An altered ECM can affect cellular phenotypes. The ECM-cell communication occurs via cell surface receptors such as integrins, through outside-in signaling, where integrin-binding to ECM protein domains translates into prolifera- tion, differentiation, or gene expression. On the other hand, activation of the intracellular integrin tail increases their ECM-binding affinity, promoting adhesion, migration, or ECM assembly (by inside-out signaling). There- fore, ECM dictates a cellular phenotype, but cells can modify ECM in response by changing its composition or assembly. Because of profound differences in ECM composition in males and females, we hypothesize that the quality of repair assembled after injury and subsequent adverse remodeling in males and females will also be differ- ent, and the differences will be accentuated by aging. In SA1, we will map the differences in ECM dependent on age (young and old animals will be studied), sex hormone level (animals will be subjected to chemical ovary failure or castration) or level of inflammation (CCL2KO animals will be used; these mice have reduced leuko- cyte infiltration and therefore reduced fibrosis). The cellular phenotype of fibroblasts, endothelial cells (ECs), and smooth muscle cells (SMCs) isolated from all these animals will be studied ex vivo and in 3D culture. The effect of ECM on cell phenotype will be tested in 3D cultures using the matrix swap approach, where i.e. young cells will be cultured on ECM from the old hearts and vice versa. And the effect of matrix stiffness on the pro- duction of ECM protein will be examined as well. Similarly, the physiology of human cells will be studied. In SA2, we will examine ECM-integrin dependent phenotypic changes in mouse and human cells. We will first examine the effect of sex hormones, age, and inflammation on the levels of integrin expression in fibroblasts, ECs, and SMCs. Then we will manipulate the integrin expression level (via downregulation or overexpression), and examine the cellular phenotype. Finally, we will manipulate estrogen and androgen receptors and deter- mine the effect of these manipulations on integrin levels and ECM protein synthesis. In SA3, we will examine how sex hormone levels and age affect scar formation and adverse remodeling after ischemia/reperfusion inju- ry. Changes in ECM will be examined via ELISA, mass spectrometry, and western blot. The approach is inno- vative because it examines the role of three superimposed variables (age, sex hormone, and inflammation) on ECM and cellular phenotypes. Successful completion of these studies will provide a mechanistic link for future therapeutics targeting insufficient reparative and excessive adverse fibrosis.

项目概要细胞外基质（ECM）为心脏结构完整性提供了支架，并在调节细胞反应。 ECM 由纤维蛋白和蛋白聚糖网络组成，结合细胞驱动因子、生长因子和 ECM 修饰酶。老化的心脏会出现间质纤维化，并且预先大量数据表明，男性和女性心脏的 ECM 中富含不同的蛋白质。一个改变的 ECM 可以影响细胞表型。 ECM-细胞通讯通过细胞表面受体发生，例如整合素，通过由外向内的信号传导，整合素与 ECM 蛋白结构域的结合转化为增殖- 化、分化或基因表达。另一方面，细胞内整合素尾部的激活增加它们的 ECM 结合亲和力，促进粘附、迁移或 ECM 组装（通过由内而外的信号传导）。那里- 因此，ECM 决定了细胞表型，但细胞可以通过改变其组成或结构来修改 ECM 作为响应。集会。由于男性和女性 ECM 成分存在显着差异，我们假设男性和女性受伤后组装的修复质量以及随后的不良重塑也会有所不同。随着年龄的增长，这种差异会更加明显。在 SA1 中，我们将映射 ECM 的差异，具体取决于年龄（年轻和年老的动物将被研究），性激素水平（动物将接受化学卵巢失败或去势）或炎症水平（将使用 CCL2KO 动物；这些小鼠的白细胞减少）细胞浸润，从而减少纤维化）。成纤维细胞、内皮细胞 (EC) 的细胞表型从所有这些动物中分离出的平滑肌细胞 (SMC) 将在离体和 3D 培养中进行研究。这 ECM 对细胞表型的影响将使用矩阵交换方法在 3D 培养物中进行测试，即年轻细胞细胞将在旧心脏的 ECM 上培养，反之亦然。以及基体刚度对亲的影响 ECM 蛋白的产生也将被检查。同样，还将研究人体细胞的生理学。在 SA2，我们将检查小鼠和人类细胞中 ECM 整合素依赖性表型变化。我们首先会检查性激素、年龄和炎症对成纤维细胞整合素表达水平的影响， EC 和 SMC。然后我们将操纵整合素表达水平（通过下调或过度表达），并检查细胞表型。最后，我们将操纵雌激素和雄激素受体并阻止探究这些操作对整合素水平和 ECM 蛋白质合成的影响。在 SA3 中，我们将检查性激素水平和年龄如何影响缺血/再灌注损伤后的疤痕形成和不良重塑里。 ECM 的变化将通过 ELISA、质谱法和蛋白质印迹法进行检查。该方法是创新的因为它检查了三个叠加变量（年龄、性激素和炎症）对 ECM 和细胞表型。这些研究的成功完成将为未来提供机制联系针对修复不足和过度不良纤维化的治疗。