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

• 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
• 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

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.

项目摘要 细胞外基质（ECM）为心脏结构完整性提供了脚手架，并在 调节细胞反应。 ECM由纤维蛋白和蛋白聚糖结合细胞的网络组成 风光，生长因子和ECM修改酶。衰老的心脏发展出间质纤维化，并 NARY数据表明，在男性和女性心脏中，不同的蛋白质富含ECM。改变 ECM可以影响细胞表型。 ECM细胞通信通过细胞表面受体（例如 整联蛋白通过外部信号传导，在其中整合素结合到ECM蛋白质结构域，转化为Prolifera- 分化，分化或基因表达。另一方面，细胞内整联蛋白尾巴的激活增加 它们的ECM结合亲和力，促进粘附，迁移或ECM组装（通过内而外信号传导）。那里- 首先，ECM决定了细胞表型，但是细胞可以通过更改其组成或 集会。由于男性和女性的ECM组成有很大差异，我们假设 受伤后组装的维修质量以及随后在男性和女性中进行不良改造的质量也将有所不同 - ENT，差异将因衰老而突出。在SA1中，我们将映射ECM的差异 年龄（将研究年轻动物），性激素水平（动物将接受化学卵巢 炎症的衰竭或cast割）或炎症水平（使用CCL2KO动物；这些小鼠降低了白血病 - Cyte浸润，因此减少了纤维化）。成纤维细胞，内皮细胞（ECS）的细胞表型， 从所有这些动物中分离出来的平滑肌细胞（SMC）将在体内和3D培养中进行研究。这 ECM对细胞表型的影响将在3D培养物中使用矩阵交换方法在3D培养物中进行测试，即年轻 细胞将从旧心脏上培养在ECM上，反之亦然。以及矩阵刚度对pro的影响 还将检查ECM蛋白的作用。同样，将研究人类细胞的生理学。在 SA2，我们将检查小鼠和人类细胞中依赖性的ECM-整合素的表型变化。我们将首先 检查性激素，年龄和炎症对成纤维细胞表达水平的影响， ECS和SMC。然后，我们将操纵整联蛋白表达水平（通过下调或过表达）， 并检查细胞表型。最后，我们将操纵雌激素和雄激素受体，并确定 挖掘这些操纵对整合素水平和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/再灌注后的疤痕形成和不良重塑 RY。 ECM的变化将通过ELISA，质谱和蛋白质印迹检查。这种方法是无知的 因为它检查了三个叠加变量（年龄，性激素和炎症）在 ECM和细胞表型。这些研究的成功完成将为未来提供机械链接 靶向不足的修复和过度不良纤维化的治疗剂。