Neutralizing hepatic apolipoprotein E enhances aged bone fracture healing.

Circulating ApoE levels increase with age and deletion of hepatic ApoE expression results in a 95% reduction in circulating ApoE levels in aged mice.

• 24-month-old mice were used as the aged model
• Hepatic ApoE deletion (ΔApoE) mice showed a 95% reduction in circulating ApoE levels
• The liver was identified as the primary source of circulating ApoE relevant to fracture healing
• This establishes a novel liver-to-bone cross-talk axis

Deletion of hepatic ApoE expression significantly improved fracture healing in 24-month-old aged mice.

• 24-month-old ΔApoE mice displayed significantly improved fracture healing compared to aged controls
• The improvement in fracture healing was associated with the 95% reduction in circulating ApoE levels
• Advanced age was confirmed to impair bone fracture healing
• ΔApoE mice served as the primary genetic model to establish ApoE's causal role in impaired aged fracture healing

ApoE treatment of aged bone marrow stromal cells (BMSCs) inhibited osteoblast differentiation in tissue culture models.

• Aged BMSCs were used in in vitro tissue culture models
• Exogenous ApoE treatment inhibited osteoblast differentiation
• RNA-seq, Western blot, immunofluorescence, and RT-PCR analyses were used to characterize the mechanism of inhibition
• These analyses indicated that the Wnt/β-catenin pathway is the target of ApoE-based inhibition

ApoE inhibits osteoblast differentiation through the Wnt/β-catenin signaling pathway, as ApoE had no effect on cultures with stabilized β-catenin levels.

• RNA-seq, Western blot, immunofluorescence, and RT-PCR analyses all pointed to the Wnt/β-catenin pathway as the target of ApoE inhibition
• ApoE had no effect on osteoblast cultures in which β-catenin levels were stabilized, confirming pathway specificity
• This finding demonstrates that ApoE's inhibitory effect on osteoblast differentiation is dependent on β-catenin availability
• The Wnt/β-catenin pathway was validated as the mechanistic target of ApoE in bone cell differentiation

Lrp4 serves as the osteoblast cell surface receptor for ApoE and is required for ApoE-based inhibition of Wnt/β-catenin signaling and osteoblast differentiation.

• Lrp4 expression was determined to be necessary for ApoE-mediated inhibition of osteoblast differentiation
• Loss of Lrp4 expression abolished ApoE-based inhibition of Wnt/β-catenin signaling
• The ApoE-Lrp4-Wnt/β-catenin molecular mechanism was identified as the key signaling axis
• This ApoE-Lrp4-Wnt/β-catenin mechanism was validated in human osteoblast differentiation models

The ApoE-Lrp4-Wnt/β-catenin molecular mechanism was validated in human osteoblast differentiation.

• Human osteoblast differentiation models were used to validate the molecular mechanism identified in mouse models
• The ApoE-Lrp4-Wnt/β-catenin axis was confirmed to operate in human cells
• This cross-species validation supports the translational relevance of the findings
• Human validation was described as an important step toward identifying a translatable therapeutic intervention

Treatment of aged wildtype mice with an ApoE-neutralizing antibody starting 3 days after fracture surgery resulted in fracture calluses with 35% more bone deposition.

• An ApoE-neutralizing antibody (NAb) was identified and used as a therapeutic intervention
• Treatment began 3 days after fracture surgery in aged wildtype mice
• Fracture calluses in NAb-treated mice contained 35% more bone deposition compared to controls
• The treatment was described as 'noninvasive' and 'translatable'
• Aged wildtype (not genetically modified) mice were used, supporting therapeutic applicability