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

项目摘要 细胞外基质（ECM）为心脏结构完整性提供了支架，并在以下方面发挥积极作用： 调节细胞反应。ECM由纤维状蛋白和蛋白聚糖结合细胞外基质的网络组成。 因子、生长因子和ECM修饰酶。衰老的心脏会发生间质纤维化， 几乎没有数据表明，不同的蛋白质在男性和女性心脏的ECM中富集。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，通过由外向内信号传导，其中整合素结合ECM蛋白结构域转化为增殖， 分化或基因表达。另一方面，细胞内整合素尾的活化增加， 它们的ECM结合亲和力，促进粘附、迁移或ECM组装（通过由内向外的信号传导）。在那里- 因此，ECM决定细胞表型，但细胞可以通过改变其组成或结构来改变ECM。 组装件.由于男性和女性ECM组成的深刻差异，我们假设 男性和女性在损伤后组装的修复质量和随后的不良重塑也将不同- 这种差异会随着年龄的增长而加剧。在SA 1中，我们将根据以下因素绘制ECM的差异 年龄（将研究幼龄和老龄动物）、性激素水平（将对动物进行化学卵巢 失败或去势）或炎症水平（将使用CCL 2KO动物;这些小鼠具有降低的白细胞- 细胞浸润并因此减少纤维化）。成纤维细胞、内皮细胞（EC） 从所有这些动物中分离的平滑肌细胞（SMC）将在体外和3D培养中进行研究。的 将使用基质交换方法在3D培养物中测试ECM对细胞表型的影响，其中， 细胞将在来自旧心脏的ECM上培养，反之亦然。并讨论了基体刚度对预拉伸强度的影响。 还将检查ECM蛋白的诱导。同样，人类细胞的生理学也将被研究。在 SA 2，我们将检查小鼠和人细胞中ECM-整联蛋白依赖性表型变化。我们将首先 检查性激素、年龄和炎症对成纤维细胞中整合素表达水平的影响， EC和SMC。然后我们将操纵整合素的表达水平（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阻止- 挖掘这些操作对整合素水平和ECM蛋白合成的影响。在SA 3中，我们将检查 性激素水平和年龄如何影响缺血/再灌注损伤后瘢痕形成和不良重塑 ry。将通过ELISA、质谱法和蛋白质印迹法检查ECM的变化。方法是INNO- 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今后的研究提供一个机制联系。 靶向不足修复性和过度不良纤维化的治疗剂。

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.