A novel target in aged fracture healing

老年骨折愈合的新目标

• 批准号: 10798768
• 负责人: Gurpreet Baht
• 金额: $ 41.6万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10798768
• 关键词: Affect; Age; Aging; Antibodies; Apolipoprotein E; Biology; Bone Marrow; Bone Regeneration; Bone Tissue; Bone callus; Cell Culture Techniques; Cell Surface Receptors; Cell surface; Circulation; Clinic; Data; Deposition; Dose; Elderly; Fracture; Health; Healthcare; Hepatocyte; Human; Immunoglobulin G; Impairment; In Vitro; Injury; Intervention; Ligands; Literature; Liver; LoxP-flanked allele; MUSK gene; Malignant neoplasm of liver; Mechanics; Modeling; Morbidity - disease rate; Mus; Neuromuscular Junction; Operative Surgical Procedures; Orthopedics; Osteoblasts; Outcome; Pathway interactions; Patients; Phenotype; Phosphorylation; Population; Proteins; Public Health; Publishing; Receptor Protein-Tyrosine Kinases; Regimen; Reporting; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Stromal Cells; Testing; Therapeutic; Time; Tissues; Transcript; Transgenic Mice; Translating; Treatment Protocols; Work; aged; bone; bone aging; bone fracture repair; bone healing; bone repair; care burden; clinical translation; cohort; cost; improved; inducible Cre; inhibitor; knock-down; male; mineralization; mortality; mouse Cre recombinase; mouse model; neutralizing antibody; novel; optimal treatments; osteoblast differentiation; osteogenic; pre-clinical; prevent; progenitor; protein expression; receptor; single-cell RNA sequencing; therapeutic target; therapy development; tissue culture

ABSTRACT Bone fractures occur in 50% of the population causing significant morbidity and mortality and costing more than $20 billion annually. Advanced age diminishes bone repair capacity and is associated with increased surgical intervention at the time of the injury and subsequently with the need for revisions. The development of therapies aimed at enhancing bone repair would significantly reduce the burden on the geriatric population. We recently identified Apolipoprotein E (ApoE) to be an age-associated inhibitor of fracture repair. ApoE is a circulatory protein and increases in abundance with age in mice and in humans. In our published work, circulating ApoE inhibited osteoblast differentiation and activity decreasing the amount of bone deposited within the fracture callus. The liver produces >90% of the ApoE found in circulation, as such, we delivered siRNA against ApoE using an AAV targeting hepatocytes in aged mice. Circulating levels of ApoE were dramatically decreased and subsequent aged bone healing was greatly improved. This finding serves as a ‘proof of concept’ that ApoE is a viable target to improve aged fracture repair. Within this proposal we will build on these findings identifying interventions that can be translated to clinic to improve aged bone healing. In Aim 1 we will determine whether neutralizing circulating ApoE improves aged fracture healing. We have developed an ApoE-neutralizing antibody. A small cohort of aged male mice underwent fracture injury and were later treated with this antibody IP. Versus IgG-treated mice, the calluses of anti-ApoE treated mice contained higher amounts of bone tissue. In this aim we will identify the optimal regimen and dose of antibody to use and determine how this treatment changes the stages of fracture repair. Furthermore, we have identified ApoE-based inhibition to propagate through osteoblastic muscle-specific kinase (MuSK), a cell-surface tyrosine kinase receptor whose osteogenic expression and function has yet to be reported in the literature. In Aim 2 we will we will use MuSK floxed mice crossed with inducible Cre-recombinase mice to identify the role of osteogenic progenitor and osteoblastic MuSK in bone repair. In Aim 3 we will identify the mechanism by which ApoE decreases osteoblast differentiation. Using in vitro cell culture techniques, we have identified the Yap/Taz pathway to be modulated with ApoE treatment. An osteoblastic cell-surface receptor for this pathway has yet to be identified. We have determined MuSK to potentially serve as such a receptor. Using transgenic mouse models, we will determine functionality of MuSK in osteoblast biology and investigate the role of MuSK in ApoE-based osteoblast inhibition/signal transduction.

摘要 骨折发生在50%的人口中，导致显著的发病率和死亡率， 每年200亿美元高龄会降低骨修复能力，并与手术次数增加有关。 在受伤时进行干预，随后需要进行翻修。治疗方法的发展 旨在增强骨修复的治疗将显著减轻老年人群的负担。我们最近 确定载脂蛋白E（ApoE）是一种与年龄相关的骨折修复抑制剂。ApoE是一种循环系统 在小鼠和人类中，蛋白质的丰度随着年龄的增长而增加。在我们发表的工作中，循环ApoE 抑制成骨细胞分化和活性，减少骨折内沉积的骨量 愈伤组织肝脏产生循环中发现的>90%的ApoE，因此，我们递送了针对ApoE的siRNA。 使用靶向老年小鼠肝细胞的AAV。ApoE的循环水平显著降低， 随后的老化骨愈合大大改善。这一发现可以作为ApoE是一种“概念证明”。 改善老年骨折修复的可行目标。在本提案中，我们将以这些调查结果为基础， 可以转化为临床干预，以改善老年骨愈合。在目标1中，我们将确定 中和循环ApoE改善老化骨折愈合。我们研制出了一种ApoE中和抗体。 一小群老年雄性小鼠经历骨折损伤，随后用该抗体IP治疗。与 IgG处理的小鼠中，抗ApoE处理的小鼠的愈伤组织含有更高量的骨组织。在这一目标中， 我们将确定最佳方案和抗体剂量，并确定这种治疗如何改变 骨折修复的阶段。此外，我们已经确定了基于ApoE的抑制通过 成骨细胞肌肉特异性激酶（MuSK），一种细胞表面酪氨酸激酶受体，其成骨细胞特异性激酶（MSK）是一种细胞表面酪氨酸激酶受体， 表达和功能尚未在文献中报道。在目标2中，我们将使用MuSK floxed小鼠 与诱导型Cre重组酶小鼠杂交，以确定成骨祖细胞和成骨细胞MuSK的作用 在骨修复中。在目标3中，我们将确定ApoE降低成骨细胞分化的机制。 使用体外细胞培养技术，我们已经鉴定了ApoE调节的雅普/Taz通路 治疗该通路的成骨细胞表面受体尚未确定。我们已经确定 MuSK可能作为这样的受体。使用转基因小鼠模型，我们将确定功能 MuSK在成骨细胞生物学中的作用，并研究MuSK在基于ApoE的成骨细胞抑制/信号传导中的作用。 转导