Procollagen Binding Proteins in Age-Dependent LV Remodeling

描述(由申请人提供)： 左心室(LV)结构重构，如左心室质量、体积和几何形状的改变，是预测不良功能和临床结局的重要指标。年龄增大，与并发心血管疾病无关，可能与显著的左室重构有关。在老年动物身上的研究表明，随着年龄的增加，左室向心性重构的发展，细胞外基质(ECM)纤维胶原含量增加，以及显著的舒张期功能异常。然而，高龄导致心脏重构的细胞机制，特别是心肌胶原含量的净增加和舒张期功能障碍的发展，尚未完全确定。心肌成纤维细胞调控心肌胶原ECM的结构和组成，并在前胶原合成、合成后的前胶原加工和胶原降解水平上进行调节。在老年动物中，胶原合成减少，前胶原加工增加，胶原降解介质普遍减少。此外，衰老的成纤维细胞表现出与衰老的成纤维细胞表型一致的增殖率降低。在之前的资助期间，我们发现了一种胶原结合的基质细胞蛋白SPARC的表达增加，这是导致老年心脏胶原含量增加的一个关键因素。我们还发现，SPARC可以减少与心脏成纤维细胞的前胶原相互作用，这可能是通过减少跨膜胶原受体的结合来实现的。根据初步数据，我们已经确定Discoidin结构域受体2(DDR2)是主要的胶原受体，参与与胶原结合并与SPARC相反。由于DDR2和SPARC在前胶原上具有相同的结合部位，我们认为，在老年心肌中，SPARC表达的增加减少了DDR2与前胶原的结合，从而限制了DDR2的结合活性和下游信号通路。前胶原与DDR2结合可诱导成纤维细胞增殖，增加基质金属蛋白酶-2和-13的产生。因此，预计老年心肌中DDR2活性的降低会减少成纤维细胞的增殖和基质金属蛋白酶-2和-13的产生，从而导致衰老的心脏成纤维细胞的衰老表型。在目标1中，我们将确定DDR2活性的降低是否有助于老年成纤维细胞的增殖减少和基质金属蛋白酶-2和13的表达减少，以及是否通过降低老年细胞中SPARC的表达来增强DDR2的活性。Aim 2中的实验将测试，无论是通过DDR2的过度表达还是通过抑制SPARC的表达，增加老年心肌中DDR2的活性，是否通过增加成纤维细胞的增殖和MMP-2和13的产生来逆转体内衰老的成纤维细胞表型。以前的研究主要基于对这些过程的间接测量，例如处理或降解胶原的酶的水平。在目标3中，新开发的体内放射性标记技术将被用于直接量化老年心肌中前胶原加工和降解的变化，并确定改变DDR2或SPARC活性是否影响这些过程。

DESCRIPTION (provided by applicant): Left ventricular (LV) structural remodeling, such as changes in LV mass, volume, and geometry, are important predictors of adverse functional and clinical outcomes. Advancing age, independent of concurrent cardiovascular disease, can be associated with significant LV remodeling. Studies in aged animals have shown that increasing age is associated with development of LV concentric remodeling, increased extracellular matrix (ECM) fibrillar collagen content, and significant abnormalities in diastolic function. However, cellular mechanisms by which advanced age leads to cardiac remodeling, particularly a net increase in myocardial collagen content and the development of diastolic dysfunction, have not been completely defined. Regulation of the structure and composition of the collagenous ECM of the myocardium is controlled by cardiac fibroblasts and is regulated at the levels of procollagen synthesis, post-synthetic procollagen processing, and collagen degradation. In aged animals, collagen synthesis is decreased, procollagen processing is increased, and mediators of collagen degradation are generally decreased. In addition, aged fibroblasts demonstrate reduced rates of proliferation consistent with a senescent fibroblast phenotype. In the previous funding period, we identified that expression of SPARC (Secreted Protein Acidic and Rich in Cysteine/osteonectin/BM40), a collagen-binding matricellular protein, was increased in aged myocardium and is a critical factor contributing to elevated collagen content in aged hearts. We also found that SPARC acts to decrease procollagen interaction with cardiac fibroblasts, presumably through decreased engagement of transmembrane collagen receptors. Based on preliminary data, we have identified Discoidin Domain Receptor 2 (DDR2) as a principle collagen receptor engaged in binding collagen and acting in opposition to SPARC. Because DDR2 and SPARC share the same binding site on procollagen, we propose that, in aged myocardium, increases in SPARC expression reduce DDR2 binding to procollagen thus limiting DDR2 binding activity and down-stream signaling pathways. Procollagen binding to DDR2 has been demonstrated to induce fibroblast proliferation and increase production of matrix metalloproteinases (MMP) -2 and -13. Decreased DDR2 activity in aged myocardium is therefore predicted to decrease fibroblast proliferation and production of MMP-2 and -13 thus contributing to the senescent phenotype indicative of aged cardiac fibroblasts. In Aim 1, we will determine whether reductions in DDR2 activity contribute to decreased proliferation and decreased expression of MMP-2 and 13 in aged fibroblasts and whether DDR2 activity is enhanced by decreasing SPARC expression in aged cells. Experiments in Aim 2 will test whether increasing DDR2 activity in aged myocardium, either through over-expression of DDR2 or inhibition of SPARC expression, reverses the senescent fibroblast phenotype in vivo through increasing fibroblast proliferation and production of MMP-2 and 13. Previous studies that have addressed indices of procollagen processing and degradation in aged myocardium have been based primarily on indirect measurements of these processes such as levels of enzymes that process or degrade collagen. In Aim 3, newly developed in vivo radiolabeling techniques will be used to directly quantify changes in procollagen processing and degradation in aged myocardium and determine whether altering DDR2 or SPARC activity influences these processes.