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

与衰老相关的骨骼退行性变与骨微结构的改变、骨密度的丧失 骨密度(BMD)，增加骨折易感性，延迟骨愈合。一把钥匙 这些退行性改变的因素是骨祖细胞(即间充质干细胞)的形成 细胞[MSCs])，随着年龄的增长而减少，并受局部骨髓中的信号调节 (BM)微环境(生态位)。到目前为止，在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，陈晓东博士的实验室开发了一种 体外复制BM-MSC生态位的培养系统，在本项目中用于比较 骨髓基质细胞体外培养对骨髓间充质干细胞生长因子应答的影响 “年轻”(≤25y/o)和“老年”(≥60y/o)捐赠者。“旧”ECM上的MSCs显示较少的BMP-2 与“年轻的”ECM上的细胞相比，反应速度更快。最近的初步数据显示， Cyr61/CCN1是一种参与调控成骨的基质细胞蛋白，在“old”中缺失。 与年轻的细胞外基质相比，年轻的细胞外基质中Cyr61基因的表达缺失了BMP-2 反应性，证实了该蛋白在成骨过程中的重要性。此外，治疗老年人 细胞外基质合成前携带Cyr61基因重组腺病毒的骨髓基质细胞修复间充质干细胞 对BMP-2的反应性以及赵等人(2018)使用Cyr61 KO小鼠(即Cyr61 KO)进行的研究 结果表明，与野生型(WT)相比，KO小鼠的骨密度降低。 这些发现导致了一种假设，即与衰老相关的骨变性至少部分是 与骨骼ECM中Cyr61的耗竭有关，这对BM生态位产生了负面影响，并使 MSC成骨。在拟议的研究中，目标1将评估Cyr61枯竭对 3个月时的骨表型和骨髓基质蛋白质组。旧(WT-Y)，18个月。老(WT-O)和3个月。年长的 KO(YKO)小鼠。WT-o和yKO小鼠的骨基质预计含有较少的Cyr61，展示 与WT-y小鼠相比，相似的衰老蛋白质组和降低的骨密度。目标2将评估 WT-y、WT-o和yKO小鼠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的反应性与成骨细胞分化。这些研究 将显示矩阵界限Cyr61确定ECM支持MSC的能力 对生长因子和成骨细胞分化的反应性。探讨其作用机制的研究 预计Cyr61中与衰老相关的变化将表明更高水平的活跃YAP[即去- 年轻间充质干细胞中的磷酸化]促进Cyr61的表达及其掺入增加 变成年轻的BM-ECM。相比之下，旧的MSCs预计会显示较低的YAP水平和较低的 ECM中的Cyr61。AIM 3将确定外源性rhCyr61是否促进WT-O患者的腰椎融合 和yKO小鼠，单独或与重组人骨形态发生蛋白-2联合。预计结果将表明， 联合应用重组人Cyr61和重组人骨形态发生蛋白-2将显著促进骨愈合，优于 任何一种单独存在，特别是在WT-O和YKO小鼠中。目标3的结果具有很高的平移性 作为补充BMP-2的潜在的Cyr61将减少BMP-2的剂量，以实现 并减少其副作用。总体而言，该项目的成果有可能 通过提供创新的方法，显著改善退伍军人医疗保健 在难以实现的条件下再生/修复骨骼(即老化- 相关疾病和并存)。此外，该提案还包含了一项完善的计划 在骨骼和衰老生物学、蛋白质组学、生物信息学和小动物方面获得新技能 外科手术和有针对性的职业发展，成为一名独立的退伍军人事务部研究调查员。

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.