Reversing Age-related Bone Loss by Administration of the Matricellular Protein Cyr61/CCN1

摘要 此前，我们报道了由骨髓合成的无细胞天然细胞外基质(ECM)。 基质细胞，显著促进小鼠和人骨髓间充质干细胞的干细胞性 (BM-MSCs)。更重要的是，老化的骨髓在自我更新、分化和成骨能力方面存在缺陷- MSCs可以通过在年轻的BM-ECM(即，由年轻捐赠者的细胞制成)上培养完全拯救。在……里面 相比之下，老的BM-ECM(即由老年供者的细胞制成)未能同时支持年轻和老年的MSC 更新和分化能力。通过蛋白质组学分析发现，基质细胞蛋白Cyr61是 年轻的细胞外基质丰富，而年老的细胞外基质中缺乏。随后，我们使用基因方法下调或上调 Cyr61在年轻和老年BM-ECM合成过程中的掺入发现Cyr61缺失的年轻ECM 失去了它的能力，而补充了Cyr61的旧ECM获得了保留MSC属性的能力。此外，我们 研究表明，低骨密度(BMD)与骨组织中Cyr61的数量减少有关 年迈的老鼠。更有趣的是，骨骼中Cyr61的存在是间歇性合成代谢作用所必需的。 PTH本身可上调老龄小鼠Cyr61的表达。当前提案的目标是逆转年龄-- 通过补充关键的基质细胞蛋白成分Cyr61/CCN1与老化的骨骼相关的骨丢失 微环境(即干细胞生态位)。我们假设，补充Cyr61到老化的骨基质将 恢复MSC微环境(生态位)支持MSC自我更新、分化和成骨的能力 形成能力，从而减少骨丢失和改善骨合成代谢反应 间歇性甲状旁腺素治疗。我们处于独特的地位来检验这一假设，因为除了我们强大的 初步数据，我们已经建立了一种独特的动物模型，骨特异性Cyr61基因敲除(Cyr61-)小鼠， 这在很小的时候就表现出骨量减少。为了检验这一假设，我们将确定恢复Cyr61的效果 对来自Cyr61或老年野生型(Wt)小鼠的细胞产生的BM-ECM，对其保持/挽救数量的能力 和年轻/老年骨髓间充质干细胞的质量(目标1)。然后我们将测定重组Cyr61(RCyr61)的疗效， 由多壁碳纳米管(MWCNTs)携带，在逆转干细胞数量/质量和骨丢失方面 在Cyr61和老年Wt小鼠中(目标2)。最后，我们将比较rCyr61的有效性，然后是 间歇性甲状旁腺激素与单独甲状旁腺素逆转Cyr61或老年小鼠的骨丢失(目标3)。建议的研究是 高度创新，通过以下途径验证我们的新假设：1)新的动物模型、骨骼特异性Cyr61-和旧重量 小鼠(自然衰老的Cyr61缺乏症)，以确定补充Cyr61是否能逆转BM- MSC功能与骨丢失；2)一种新的载体MWCNTs，它能将rCyr61特异性地运送到骨基质中； 3)检测间歇性甲状旁腺激素前给予Cyr61对骨合成代谢的协同作用 新陈代谢。这项拟议的研究意义重大，因为目前骨质疏松症的治疗主要集中在 平衡破骨细胞/成骨细胞活性，疗效有限，副作用较大。这些研究 具有巨大的潜力，通过提供一种新的治疗方法来造福退伍军人和普通民众 骨质疏松症，专注于恢复老化的干细胞利基，这是以前从未探索过的。

Summary Previously, we reported that cell-free native extracellular matrix (ECM), synthesized by bone marrow (BM) stromal cells, significantly promoted the stemness of mouse and human BM-derived mesenchymal stem cells (BM-MSCs). More importantly, defects in self-renewal, differentiation, and bone formation capacity of aging BM- MSCs can be completely rescued by culture on young BM-ECM (i.e., made by cells from young donors). In contrast, old BM-ECM (i.e., made by cells from old donors) failed to support both young and old MSC self- renewal and differentiation capacity. By proteomic analysis, we found that the matricellular protein Cyr61 was abundant in young ECM but absent in old ECM. Subsequently, we used genetic methods to down- or up-regulate the incorporation of Cyr61 during synthesis of young or old BM-ECM and found that Cyr61-depleted young ECM lost its ability, while Cyr61-replenished old ECM gained its ability, to retain MSC properties. In addition, we showed that low bone mineral density (BMD) was associated with decreased amounts of Cyr61 in bone tissue of old mice. More interestingly, the presence of Cyr61 in bone is required for the anabolic effect of intermittent PTH that by itself up-regulates Cyr61 expression in old mice. The goal of the current proposal is to reverse age- related bone loss by replenishing a critical matricellular protein component, Cyr61/CCN1, to the aging bone microenvironment (i.e., stem cell niche). We hypothesize that replenishing Cyr61 to the aging bone matrix will restore the ability of the MSC microenvironment (niche) to support MSC self-renewal, differentiation, and bone formation capacity, resulting in an attenuation of bone loss and improvement in bone anabolic response to intermittent PTH treatment. We are uniquely positioned to test this hypothesis since, in addition to our strong preliminary data, we have established a unique animal model, bone-specific Cyr61 knockout out (Cyr61‒) mice, which displays osteopenia at an early age. To test the hypothesis, we will determine the effect of restoring Cyr61 to BM-ECM, produced by cells from Cyr61‒ or old wild type (wt) mice, on its ability to retain/rescue the quantity and quality of young-/old-BM-MSCs (Aim 1). Then we will determine the efficacy of recombinant Cyr61 (rCyr61), carried by multi-walled carbon nanotubes (MWCNTs), in reversing both stem cell quantity/quality and bone loss in Cyr61‒ and old wt mice (Aim 2). Finally, we will compare the efficacy of administering rCyr61 followed by intermittent PTH versus PTH alone at reversing bone loss in Cyr61‒ or old mice (Aim 3). The proposed study is highly innovative by testing our novel hypothesis via: 1) novel animal models, bone-specific Cyr61‒ and old wt mice (natural Cyr61 deficiency with aging), to determine if replenishing Cyr61 to the bone ECM can reverse BM- MSC function and bone loss; 2) a novel vehicle, MWCNTs, that specifically delivers rCyr61 to the bone matrix; and 3) testing the synergistic effects of Cyr61 administration prior to intermittent PTH on bone anabolic metabolism. The proposed study is significant because current osteoporosis treatments mainly focus on balancing osteoclast/osteoblast activity and have limited efficacy and considerable side effects. The studies have great potential to benefit both the veteran and general population by providing a new approach for treating osteoporosis that focuses on restoring the aging stem cell niche, which has not been explored previously.