Neutralizing hepatic apolipoprotein E enhances aged bone fracture healing

Abstract

Advanced age impairs bone fracture healing; the underlying mechanism of this phenomenon remains unknown. We determined that apolipoprotein E (ApoE) increases with age and causes poor fracture healing. After deletion of hepatic ApoE expression (ΔApoE), 24-month-old ΔApoE mice displayed a 95% reduction in circulating ApoE levels and significantly improved fracture healing. ApoE treatment of aged BMSCs inhibited osteoblast differentiation in tissue culture models; RNA-seq, Western blot, immunofluorescence, and RT-PCR analyses indicated that the Wnt/β-catenin pathway is the target of this inhibition. Indeed, we showed that ApoE had no effect on cultures with stabilized β-catenin levels. Next, we determined that Lrp4 serves as the osteoblast cell surface receptor to ApoE, as expression of Lrp4 is required in ApoE-based inhibition of Wnt/β-catenin signaling and osteoblast differentiation. Importantly, we validated this ApoE-Lrp4-Wnt/β-catenin molecular mechanism in human osteoblast differentiation. Finally, we identified an ApoE-neutralizing antibody (NAb) and used it to treat aged, wildtype mice 3 days after fracture surgery resulting in fracture calluses with 35% more bone deposition. Our work here identifies novel liver-to-bone cross-talk and a noninvasive, translatable therapeutic intervention for aged bone regeneration.

References

[1] Meinberg EG, Clark D, Miclau KR, Marcucio R, Miclau T. Fracture repair in the elderly: clinical and experimental considerations. Injury, 2019, 50: S62-S65

[2] Clark D, Nakamura M, Miclau T, Marcucio R. Effects of aging on fracture healing. Curr. Osteoporos. Rep., 2017, 15: 601-608

[3] Xing Z, Lu C, Hu D, Miclau T3rd, Marcucio RS. Rejuvenation of the inflammatory system stimulates fracture repair in aged mice. J. Orthop. Res, 2010, 28: 1000-1006

[4] Wahl EC, et al.. Restoration of regenerative osteoblastogenesis in aged mice: modulation of TNF. J. Bone Min. Res., 2010, 25: 114-123

[5] Mroz TE, Wang JC, Hashimoto R, Norvell DC. Complications related to osteobiologics use in spine surgery: a systematic review. Spine, 2010, 35: S86-104

[6] Lubelski D, et al.. Adverse events with the use of rhBMP-2 in Thoracolumbar and Lumbar Spine Fusions: a 9-year institutional analysis. J. Spinal Disord. Tech., 2015, 28: E277-283

[7] Lubelski D, et al.. Urological complications following use of recombinant human bone morphogenetic protein-2 in anterior lumbar interbody fusion: presented at the 2012 Joint Spine Section Meeting: clinical article. J. Neurosurg. Spine, 2013, 18: 126-131

[8] Baht GS, et al.. Exposure to a youthful circulaton rejuvenates bone repair through modulation of beta-catenin. Nat. Commun., 2015, 6 7131

[9] Vi L, et al.. Macrophage cells secrete factors including LRP1 that orchestrate the rejuvenation of bone repair in mice. Nat. Commun., 2018, 9 5191

[10] Huang, R. et al. Lowering circulating apolipoprotein E levels improves aged bone fracture healing. JCI Insight4, e129144 (2019).

[11] Kraft HG, Menzel HJ, Hoppichler F, Vogel W, Utermann G. Changes of genetic apolipoprotein phenotypes caused by liver transplantation. Implications for apolipoprotein synthesis. J. Clin. Investig., 1989, 83: 137-142

[12] Fu M, et al.. The Hippo signalling pathway and its implications in human health and diseases. Signal. Transduct. Target Ther., 2022, 7: 376

[13] Lane-Donovan C, Herz J. ApoE, ApoE receptors, and the synapse in Alzheimer’s disease. Trends Endocrinol. Metab., 2017, 28: 273-284

[14] Boisvert WA, Spangenberg J, Curtiss LK. Treatment of severe hypercholesterolemia in apolipoprotein E-deficient mice by bone marrow transplantation. J. Clin. Investig., 1995, 96: 1118-1124

[15] Linton MF, Atkinson JB, Fazio S. Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation. Science, 1995, 267: 1034-1037

[16] Van Eck M, et al.. Bone marrow transplantation in apolipoprotein E-deficient mice. Effect of ApoE gene dosage on serum lipid concentrations, (beta)VLDL catabolism, and atherosclerosis. Arterioscler. Thromb. Vasc. Biol., 1997, 17: 3117-3126

[17] Liao F, et al.. Targeting of nonlipidated, aggregated apoE with antibodies inhibits amyloid accumulation. J. Clin. Investig., 2018, 128: 2144-2155

[18] Liao F, et al.. Anti-ApoE antibody given after plaque onset decreases Abeta accumulation and improves brain function in a mouse model of Abeta amyloidosis. J. Neurosci., 2014, 34: 7281-7292

[19] Kim J, et al.. Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of Abeta amyloidosis. J. Exp. Med., 2012, 209: 2149-2156

[20] Li X, et al.. Sclerostin antibody treatment increases bone formation, bone mass, and bone strength in a rat model of postmenopausal osteoporosis. J. Bone Min. Res., 2009, 24: 578-588

[21] Delgado-Calle J, Sato AY, Bellido T. Role and mechanism of action of sclerostin in bone. Bone, 2017, 96: 29-37

[22] Bartelt A, et al.. Apolipoprotein E-dependent inverse regulation of vertebral bone and adipose tissue mass in C57Bl/6 mice: modulation by diet-induced obesity. Bone, 2010, 47: 736-745

[23] Schilling AF, et al.. Increased bone formation in mice lacking apolipoprotein E. J. Bone Min. Res., 2005, 20: 274-282

[24] Day TF, Guo X, Garrett-Beal L, Yang Y. Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev. Cell, 2005, 8: 739-750

[25] Kim JB, et al.. Bone regeneration is regulated by Wnt signaling. J. Bone Min. Res., 2007, 22: 1913-1923

[26] Duan P, Bonewald LF. The role of the wnt/beta-catenin signaling pathway in formation and maintenance of bone and teeth. Int J. Biochem. Cell Biol., 2016, 77: 23-29

[27] Choi HY, Dieckmann M, Herz J, Niemeier A. Lrp4, a novel receptor for Dickkopf 1 and sclerostin, is expressed by osteoblasts and regulates bone growth and turnover in vivo. PLoS ONE, 2009, 4: e7930

[28] Kim SP, et al.. Lrp4 expression by adipocytes and osteoblasts differentially impacts sclerostin’s endocrine effects on body composition and glucose metabolism. J. Biol. Chem., 2019, 294: 6899-6911

[29] Xiong L, et al.. Lrp4 in osteoblasts suppresses bone formation and promotes osteoclastogenesis and bone resorption. Proc. Natl. Acad. Sci. USA, 2015, 112: 3487-3492

[30] Azzolin L, et al.. YAP/TAZ incorporation in the beta-catenin destruction complex orchestrates the Wnt response. Cell, 2014, 158: 157-170

[31] Harada N, et al.. Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene. EMBO J., 1999, 18: 5931-5942

[32] Baht GS, Nadesan P, Silkstone D, Alman BA. Pharmacologically targeting beta-catenin for NF1 associated deficiencies in fracture repair. Bone, 2017, 98: 31-36

[33] Zhang X, et al.. Rejuvenation of neutrophils and their extracellular vesicles is associated with enhanced aged fracture healing. Aging Cell, 2022, 21 e13651

[34] Huang R, et al.. The role of Meteorin-like in skeletal development and bone fracture healing. J. Orthop. Res., 2022, 40: 2510-2521

[35] Huang R, Vi L, Zong X, Baht GS. Maresin 1 resolves aged-associated macrophage inflammation to improve bone regeneration. FASEB J., 2020, 34: 13521-13532

[36] Huang MJ, et al.. lncRNA ADAMTS9-AS2 controls human mesenchymal stem cell chondrogenic differentiation and functions as a ceRNA. Mol. Ther. Nucleic Acids, 2019, 18: 533-545

[37] Dobin A, et al.. STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 2013, 29: 15-21

[38] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol., 2014, 15 550

RIGHTS & PERMISSIONS

The Author(s)