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.

与衰老有关的骨骼变性与骨微结构的变化有关，丧失 骨矿物质密度（BMD），增加骨折的敏感性以及延迟的骨骼愈合。钥匙 这些退化性变化的因素是骨基因生成剂的形成（即间充质茎 细胞[MSC]），随着衰老的降低，受局部骨髓中的线索调节 （BM）微环境（利基）。迄今为止，BM利基市场中发生的具体变化 衰老是未知的。为了解决这一知识差距，小钟博士实验室开发了 复制BM-MSC利基离体的培养系统，该项目用于比较该项目 BM基质细胞产生的ECMS培养的MSC的生长因子反应能力 “年轻”（≤25y/o）和“旧”（≥60y/o）供体。 “旧” ECM上的MSC显示出较少的BMP-2 与“ Young” ECM上的细胞相比，反应性。最近的初步数据表明 CYR61/CCN1是一种参与调节成骨的基质蛋白，在“旧”中缺乏 与年轻ECM中的“ Young” ECM和CYR61的击倒相比，BMP-2废除了 反应性，证实了该蛋白在成骨中的重要性。此外，对待老了 在ECM合成之前，含有CYR61基因的腺病毒的BM基质细胞恢复了MSC Zhao等人（2018）对BMP-2和研究的响应能力，使用CYR61 KO小鼠（即CYR61 KO [yko]）由Osterix（OSX）驱动，表明KO小鼠相对于野生型（WT）降低了BMD。 这些发现导致了以下假设：与衰老相关的骨变性至少部分是部分 与骨ECM中CYR61的耗竭有关，骨ECM对BM的利基市场产生负面影响并减少 MSC成骨。在拟议的研究中，AIM 1将评估CYR61部署对 3个月中的骨表型和BM基质蛋白质组。旧（WT-Y），18 mo。旧（WT-O）和3个月。老的 KO（YKO）小鼠。 WT-O和YKO小鼠的骨基质预计将含有较少的CYR61，展出 与WT-Y小鼠相比，相似的衰老蛋白质组和BMD降低。 AIM 2将评估 来自WT-Y，WT-O和YKO小鼠的BM基质细胞产生的ECM的能力，支持MSC 生长因子对BMP-2和IGF-1和成骨细胞分化的响应能力。这些研究 预计将表明矩阵结合的CYR61决定了ECM支持MSC的能力 对生长因子和成骨细胞分化的反应。研究探测机制的研究 CYR61中与衰老相关的变化的变化预计将表明较高水平的活性YAP [即de 磷酸化]在年轻的MSC中促进CYR61的表达及其工业化的增加 进入年轻的BM-ECM。相比之下，旧的MSC预计将显示较低的YAP和减少 ECM中的CYR61。 AIM 3将确定外源性Rhcyr61是否促进WT-O中的腰融合 和Yko小鼠，无论是单独还是与RHBMP-2结合使用。结果预计将表明 RHCYR61和RHBMP-2的共同给药将显着改善骨骼愈合，比 要么一个人，尤其是在WT-O和YKO小鼠中。 AIM 3的结果具有较高的翻译 用CYR61补充BMP-2的潜力将减少实现BMP-2的剂量 融合并减少其副作用。总体而言，该项目的结果有可能 通过提供一种创新的方法来显着改善资深医疗保健 在难以实现的条件下，再生/愈合骨（即衰老 - 相关疾病和合并症）。此外，该提案包含一个完善的计划 为了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 手术并针对职业发展成为独立的VA研究研究者。