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.

与衰老相关的骨骼退化与骨微结构的变化、骨密度的丧失有关 骨矿物质密度（BMD）、骨折易感性增加和骨愈合延迟是关键。 这些退行性变化的因素是骨祖细胞（即间充质干细胞）的形成 细胞 [MSC]），随着年龄的增长而减少，并受到局部骨髓中信号的调节 （BM）微环境（生态位）迄今为止，BM生态位中发生的具体变化。 为了解决这一知识空白，陈晓东博士的实验室开发了一种方法。 离体复制 BM-MSC 生态位的培养系统，用于本项目中以比较 骨髓基质细胞产生的 ECM 上培养的 MSC 的生长因子反应 “年轻”（≤25 岁）和“年老”（≥60 岁）供体的 MSC 在“老”ECM 上显示出较少的 BMP-2。 与“年轻”ECM 上的细胞相比，最近的初步数据显示， Cyr61/CCN1 是一种参与调节成骨的基质细胞蛋白，在“老”中缺乏 与“年轻”ECM 相比，年轻 ECM 中 Cyr61 的敲除消除了 BMP-2 反应性，证实了这种蛋白质在成骨中的重要性，此外，还可以治疗老年疾病。 在 ECM 合成之前使用含有 Cyr61 基因的腺病毒对 BM 基质细胞进行恢复 MSC 对 BMP-2 的反应性以及赵等人 (2018) 使用 Cyr61 KO 小鼠（即 Cyr61 KO 由osterix (Osx)驱动的[yKO])表明，相对于野生型(WT)，KO小鼠的BMD降低。 这些发现导致了这样的假设：与衰老相关的骨退化至少部分地发生在 与骨 ECM 中 Cyr61 的耗尽有关，这会对 BM 生态位产生负面影响并减少 在拟议的研究中，目标 1 将评估 Cyr61 耗竭对 MSC 成骨的影响。 3 个月（WT-y）、18 个月（WT-o）和 3 个月的骨表型和 BM 基质蛋白质组。 KO (yKO) 小鼠 WT-o 和 yKO 小鼠的骨基质预计含有较少的 Cyr61，表现出。 与 WT-y 小鼠相比，相似的衰老蛋白质组和降低的 BMD 将评估 由 WT-y、WT-o 和 yKO 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。 预计将显示基质结合的 Cyr61 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讨其机制。 Cyr61 中与衰老相关的变化预计将表明活性 YAP 水平较高[即。 磷酸化]在年轻的MSC中促进Cyr61的表达及其增加的掺入 相比之下，年老的 MSC 预计会表现出较低水平的 YAP 和减少。 ECM 中的 Cyr61 将确定外源性 rhCyr61 是否促进 WT-o 中的腰椎融合。 和 yKO 小鼠，单独或与 rhBMP-2 组合，结果预计表明： rhCyr61 和 rhBMP-2 联合给药将显着改善骨愈合，效果优于 单独使用任一者，特别是在 WT-o 和 yKO 小鼠中，Aim 3 的结果具有较高的翻译效果。 潜力，因为用 Cyr61 补充 BMP-2 将减少实现目标所需的 BMP-2 剂量 总体而言，该项目的结果有潜力。 通过提供创新方法来改善退伍军人医疗保健 在难以实现的条件下（即衰老- 此外，该提案还包含一个完善的计划。 获得骨骼和衰老生物学、蛋白质组学、生物信息学和小动物方面的新技能 手术和有针对性的职业发展，成为一名独立的 VA 研究调查员。