Role of Cyr61/CCN1 in Mesenchymal Stem Cell Niche and Aging Bone

Cyr61/CCN1 在间充质干细胞生态位和骨老化中的作用

• 批准号: 10369575
• 负责人: Milos Marinkovic
• 金额: --
• 依托单位: SOUTH TEXAS VETERANS HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10369575
• 关键词: Address; Adenoviruses; Age; Aging; Americas; Animals; BMP2 gene; Bioinformatics; Biology of Aging; Bone Density; Bone Marrow; Bone Matrix; Bone structure; Cells; Cues; Data; Disease; Dose; Exhibits; Experimental Designs; Extracellular Matrix; Fifth lumbar vertebra; Fracture; Gene Transfer; Genes; Genetic Engineering; Growth Factor; Harvest; Health; Healthcare; Histology; Immunohistochemistry; Impaired healing; Implant; In Vitro; Injury; Insulin-Like Growth Factor I; Knockout Mice; Knowledge; Laboratories; Lead; Maintenance; Measures; Mentors; Mesenchymal Stem Cells; Methods; Morbidity - disease rate; Mus; Natural regeneration; Operative Surgical Procedures; Osteoblasts; Osteogenesis; Osteoporosis; Phenotype; Population; Predisposition; Production; Proteins; Proteome; Proteomics; Reporting; Research; Research Personnel; Risk; Role; Small Interfering RNA; Spinal Fractures; Stromal Cells; System; Testing; Time; Tissues; Translations; Vertebral column; Veterans; Western Blotting; Woman; age related; aged; base; bone; bone fracture repair; bone healing; bone loss; bone marrow mesenchymal stem cell; bone mass; bone repair; career development; cell behavior; collagen scaffold; comorbidity; debilitating pain; implantation; improved; in vivo Model; innovation; knock-down; men; novel; novel strategies; novel therapeutic intervention; osteoblast differentiation; osteogenic; osteoprogenitor cell; radiological imaging; recombinant human bone morphogenetic protein-2; regenerative; response; side effect; skeletal; skills; stem cell growth; stem cell niche; stem cells; stemness; tomography; wound

Aging-related skeletal degeneration is associated with changes in bone microarchitecture, loss of bone mineral density (BMD), increased susceptibility to fracture, and delayed bone healing. A key factor in these degenerative changes is the formation of osteoprogenitors (i.e. mesenchymal stem cells [MSCs]), which decreases with aging and are regulated by cues in the local bone marrow (BM) microenvironment (niche). To date, the specific changes that occur in the BM niche during aging are unknown. To address this knowledge gap, Dr. Xiao-Dong Chen's lab developed a culture system that reproduces the BM-MSC niche ex vivo, used in this project to compare the growth factor responsiveness of MSCs cultured on ECMs produced by BM stromal cells from “young” (≤25 y/o) and “old” (≥60 y/o) donors. MSCs on “old” ECM displayed less BMP-2 responsiveness compared to cells on “young” ECM. Recent preliminary data showed that Cyr61/CCN1, a matricellular protein involved in regulating osteogenesis, was deficient in “old” compared to “young” ECM and knock-down of Cyr61 in young ECM abrogated BMP-2 responsiveness, confirming the importance of this protein in osteogenesis. Further, treating old BM stromal cells with adenovirus containing the Cyr61 gene prior to ECM synthesis restored MSC responsiveness to BMP-2 and studies by Zhao et al (2018) using Cyr61 KO mice (i.e. Cyr61 KO [yKO]) driven by osterix (Osx) showed that KO mice had reduced BMD relative to wild type (WT). These findings lead to the hypothesis that aging-related bone degeneration is at least partially related to depletion of Cyr61 in bone ECM, which negatively impacts the BM niche and reduces MSC osteogenesis. In the proposed studies, Aim 1 will assess the impact of Cyr61 depletion on bone phenotype and BM matrix proteome in 3 mo. old (WT-y), 18 mo. old (WT-o) and 3 mo. old KO (yKO) mice. The bone matrix of WT-o and yKO mice is expected to contain less Cyr61, exhibit similar aging proteomes, and reduced BMD as compared to WT-y mice. Aim 2 will assess the ability of ECMs, produced by BM stromal cells from WT-y, WT-o and yKO mice, to support MSC growth factor responsiveness to BMP-2 and IGF-1 and osteoblast differentiation. These studies are expected to show that matrix bound Cyr61 determines the ability of an ECM to support MSC responsiveness to growth factors and osteoblast differentiation. Studies to probe the mechanism of aging-related changes in Cyr61 are expected to show that higher levels of active YAP [i.e. de- phosphorylated] in young MSCs promote the expression of Cyr61 and its increased incorporation into young BM-ECM. In contrast, old MSCs are expected to show lower levels of YAP and reduced Cyr61 in the ECM. Aim 3 will determine if exogenous rhCyr61 promotes lumbar fusion in WT-o and yKO mice, either alone or in combination with rhBMP-2. The results are expected to show that co-administration of rhCyr61 and rhBMP-2 will dramatically improve bone healing, better than either one alone, especially in WT-o and yKO mice. The results of Aim 3 have high translation potential as supplementing BMP-2 with Cyr61 will reduce the dose of BMP-2 necessary to achieve fusion and decrease its side-effects. Overall, the results of this project have the potential to significantly improve Veteran health care by providing an innovative approach for regenerating/healing bone under conditions where this is difficult to achieve (i.e. aging- related diseases and co-morbidities). Moreover, the proposal contains a well-developed plan for gaining new skills in bone and aging biology, proteomics, bioinformatics, and small animal surgery and targeted career development to become an independent VA research investigator.

与年龄相关的骨骼退化与骨微结构的变化、骨密度的丧失、骨密度的降低和骨密度的降低有关。 骨矿物质密度（BMD）、增加骨折的易感性和延迟骨愈合。一个关键 这些退行性变化中的一个因素是骨祖细胞（即间充质干细胞）的形成 细胞[MSC]），随着年龄的增长而减少，并受局部骨髓中的线索调节 (BM)微环境（niche）到目前为止，在BM生态位中发生的具体变化， 年龄不详。为了解决这一知识缺口，陈晓东博士的实验室开发了一种 培养系统，再现BM-MSC生态位离体，在这个项目中使用，以比较 骨髓基质细胞产生的ECM上培养的MSC的生长因子反应性 “年轻”（≤25岁）和“老年”（≥60岁）供体。“旧”ECM上的MSC显示较少的BMP-2 与“年轻”ECM上的细胞相比，最近的初步数据显示， Cyr 61/CCN 1是一种参与调控骨生成的基质细胞蛋白， 与“年轻”ECM相比，年轻ECM中Cyr 61的敲低废除了BMP-2 反应性，证实了这种蛋白质在成骨中的重要性。此外，治疗老年人 在ECM合成之前用含有Cyr 61基因的腺病毒处理BM基质细胞恢复MSC 对BMP-2的反应性以及Zhao等人（2018）使用Cyr 61 KO小鼠（即Cyr 61 KO）进行的研究 [yKO]）显示KO小鼠相对于野生型（WT）具有降低的BMD。 这些发现导致了这样一种假设，即与年龄相关的骨退化至少部分是 与骨ECM中Cyr 61的消耗有关，其对BM生态位产生负面影响， MSC成骨。在拟议的研究中，目标1将评估Cyr 61耗竭对 骨表型和BM基质蛋白质组。年龄（WT-y），18个月。年龄（WT-0）和3个月。老 KO（yKO）小鼠。预期WT-0和yKO小鼠的骨基质含有较少的Cyr 61，表现出更高的细胞增殖能力。 相似的老化蛋白质组，并且与WT-γ小鼠相比BMD降低。目标2将评估 由来自WT-γ、WT-0和γ KO小鼠的BM基质细胞产生的ECM支持MSC的能力 生长因子对BMP-2和IGF-1的反应以及成骨细胞分化。这些研究 预期显示基质结合Cyr 61决定ECM支持MSC的能力， 对生长因子和成骨细胞分化的反应。探索机制的研究 Cyr 61中与衰老相关的变化的研究表明，较高水平的活性雅普[即脱- 磷酸化]促进Cyr 61的表达及其增加的掺入 植入年轻的BM-ECM相比之下，预期老MSC显示较低水平的雅普和降低的细胞增殖。 ECM中的Cyr 61。目的3将确定外源性rhCyr 61是否促进WT-0中的腰椎融合 和yKO小鼠，单独或与rhBMP-2组合。预计结果将显示， rhCyr 61和rhBMP-2的联合给药将显著改善骨愈合，优于 单独使用，尤其是在WT-0和yKO小鼠中。目标3的结果具有高翻译性 潜在的补充BMP-2与Cyr 61将减少剂量的BMP-2所必需的实现 减少其副作用。总的来说，该项目的成果有可能 通过提供创新方法， 在难以实现的条件下再生/愈合骨（即老化- 相关疾病和共病）。此外，该提案包含一个精心制定的计划， 获得骨骼和衰老生物学，蛋白质组学，生物信息学和小动物的新技能 手术和有针对性的职业发展，成为一个独立的VA研究调查员。