Development of a novel therapy with Apolipoprotein E for osteogenesis imperfecta

成骨不全（OI），被称为脆骨病，是一种遗传性疾病，通常由一种 l型胶原蛋白突变。除了骨脆性，生长缺陷是一个关键的肌肉骨骼问题， 喂没有治愈OI。虽然双膦酸盐是强化骨骼的标准治疗方法，但没有 可靠有效的治疗生长障碍，特别是在严重类型的OI患者。此前我们 和其他人已经报道了间充质干细胞/基质细胞（MSC）的系统输注可以刺激骨形成， 在OI患者以及OI模型小鼠中的生长。尽管这种惊人的生长加速， 影响是短暂的。此外，MSC疗法有几个固有的局限性，例如不一致的结果， 不同MSC供体之间的差异，对良好生产规范设施的需求，以及培养的安全性问题- 扩大细胞输注。因此，确定MSC诱导骨生长的机制对于 开发新的无细胞疗法，可以在OI的生长期安全和重复实施 患者我们的研究表明，MSC诱导的骨生长不是MSC的直接作用， 相反，MSC输注诱导血清中刺激软骨细胞的因子的产生， 在生长板中增殖并导致线性骨生长。蛋白质组学和生物信息学分析 来自MSC输注小鼠的血清鉴定载脂蛋白E（ApoE）为该因子的主要候选者。我们 初步研究表明，MSC输注后ApoE血清水平显著升高， 重组ApoE能显著刺激软骨细胞增殖和肥大分化， 软骨细胞培养此外，ApoE在离体器官培养中增强骨生长。这些发现 强有力的证据支持，ApoE基因敲除小鼠的骨骼明显短于 野生型小鼠总的来说，这些发现为我们高度创新的假设提供了严格的前提， ApoE可通过刺激OI中的软骨细胞增殖和分化来改善骨生长缺陷 生长板此外，已经表明ApoE由成熟的成骨细胞产生。在OI中，突变 Col 1表达阻止成骨细胞成熟，结果，成熟成骨细胞的数量减少。 大幅减少。有趣的是，我们的初步研究表明，骨骼中的ApoE水平显着增加， 在OI小鼠中低于野生型对照。这些发现表明，OI导致ApoE的产生减少， 成骨细胞可能在OI生长缺陷的发病机制中起关键作用。因此，我们提出以下建议 具体目的：（1）确定ApoE对OI生长缺陷的治疗作用;（2）检查 成骨细胞产生的ApoE在生长板功能和骨生长中的作用。完成这些 目的是确定ApoE是否可以作为一种新的治疗分子，以促进骨生长在OI 并为确定OI生长缺陷的发病机制提供了新的见解。这个探索性的项目有 彻底改变OI治疗策略的潜力。

Osteogenesis imperfecta (OI), known as a brittle bone disease, is a genetic disorder typically caused by a mutation in type l collagen. In addition to bone fragility, growth deficiency is a critical musculoskeletal issue of OI. There is no cure for OI. While bisphosphonates are the standard treatment to strengthen bones, there is no reliably effective treatment for growth impairment, especially in patients with severe types of OI. Previously, we and others have reported that systemic infusion of mesenchymal stem/stromal cells (MSC) can stimulate bone growth in OI patients as well as in OI model mice. Despite this striking growth acceleration, the therapeutic effects were transient. Moreover, MSC therapy has several inherent limitations such as inconsistent results between different MSC donors, need for Good Manufacturing Practice facilities, and safety concerns of culture- expanded cellular infusions. Thus, identifying the mechanism of MSC-induced bone growth is critical to develop novel cell-free therapies that can be safely and repeatedly implemented during the growth period in OI patients. Our investigation revealed that MSC-induced bone growth was not a direct effect of MSCs themselves; rather, MSC infusion induced production of a factor(s) in the serum which stimulated chondrocyte proliferation in the growth plate and resulted in linear bone growth. Proteomic and bioinformatics analyses of the serum from MSC infused mice identified apolipoprotein E (ApoE) as a primary candidate for the factor. Our preliminary studies demonstrated that ApoE serum levels were significantly elevated after MSC infusion and recombinant ApoE markedly stimulated chondrocyte proliferation and hypertrophic differentiation in chondrocyte culture. Moreover, ApoE enhanced bone growth in ex vivo organ culture. These findings were strongly supported by compelling evidence that ApoE knockout mice display significantly shorter bones than wild type mice. Collectively, these findings provide rigorous premises for our highly innovative hypothesis that ApoE can improve bone growth deficiency by stimulating chondrocyte proliferation and differentiation in the OI growth plate. Additionally, it has been shown that ApoE is produced by mature osteoblasts. In OI, mutated Col1 expression prevents osteoblasts from maturation and as a result, the number of mature osteoblasts is significantly reduced. Interestingly, our preliminary study showed that ApoE levels in bones were significantly lower in OI mice than wild type controls. These findings suggest that reduced production of ApoE by OI osteoblasts may play a critical role in the pathogenesis of OI growth deficiency. Thus, we propose the following specific aims: (1) to determine the therapeutic effects of ApoE for growth deficiency in OI; and (2) to examine the role of ApoE produced by osteoblasts in growth plate function and bone growth. The completion of these aims will determine whether ApoE can serve as a novel therapeutic molecule to enhance bone growth in OI and provide novel insights to identify the pathogenesis of OI growth deficiency. This exploratory project has the potential to completely revolutionize the treatment strategy for OI.