Development of a novel therapy with Apolipoprotein E for osteogenesis imperfecta

成骨不全(OI)，被称为脆性骨骼疾病，是一种遗传性疾病，通常由 L型胶原基因突变。除了骨骼脆性外，生长缺陷也是一个严重的肌肉骨骼问题 喂。OI是没有治愈方法的。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，特别是在严重的OI类型的患者。此前，我们 另有报道称，全身输注间充质干细胞/基质细胞(MSC)可以刺激骨骼 OI患者和OI模型小鼠的生长。尽管增长速度惊人，但治疗性的 影响是暂时的。此外，MSC治疗有几个固有的局限性，如结果不一致 在不同的MSC捐赠者之间，对良好的制造实践设施的需求，以及对文化的安全担忧- 扩大细胞输注。因此，确定MSC诱导骨生长的机制对于 开发可在OI生长期安全和重复实施的新的无细胞疗法 病人。我们的研究表明，MSC诱导的骨生长不是MSCs的直接作用。 相反，骨髓间充质干细胞输注诱导血清中产生一种刺激软骨细胞的因子(S)。 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 来自骨髓间充质干细胞输注的小鼠的血清确定载脂蛋白E(ApoE)是该因子的主要候选者。我们的 初步研究表明，骨髓间充质干细胞输注后血清载脂蛋白E水平显著升高， 重组载脂蛋白E显著刺激软骨细胞增殖和肥大分化 软骨细胞培养。此外，在体外器官培养中，ApoE促进了骨的生长。这些发现是 令人信服的证据有力地支持了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通过促进OI软骨细胞增殖分化改善骨生长缺陷 生长板。此外，已有研究表明，ApoE是由成熟的成骨细胞产生的。在OI，突变 COL1的表达阻止了成骨细胞的成熟，因此，成熟的成骨细胞的数量 显著减少。有趣的是，我们的初步研究表明，骨骼中的载脂蛋白E水平显著 OI小鼠低于野生型对照组。这些发现表明，OI减少了ApoE的产生 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，我们提出如下建议 具体目的：(1)确定载脂蛋白E对OI生长缺陷的治疗效果；(2)检测 成骨细胞产生的载脂蛋白E在生长板功能和骨生长中的作用这些项目的完成 AIMS将确定ApoE是否可以作为一种新的治疗分子来促进OI的骨生长 为确认OI生长缺陷的发病机制提供了新的见解。这个探索性项目具有 有可能彻底改变OI的治疗策略。

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.