A novel target in aged fracture healing

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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.