OR27-02 The Bone Anabolic Effect Of An Antibody Blocking Oxidized Phospholipids Is Associated With An Increase In Wnt10b In Osteoblasts.

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Abstract Disclosure: M. Palmieri: None. I. Nookaew: None. M.J. Schuller-Almeida: None. H. Kim: None. T.E. Jospeh: None. S.C. Manolagas: None. C.A. O'Brien: None. E. Ambrogini: None. Oxidized phospholipids containing phosphatidylcholine (PC-OxPLs) are toxic molecules present on oxidized low-density lipoproteins (OxLDL) and apoptotic cells; and are pathogenic in many diseases, including osteoporosis. OxPLs decrease Wnt and BMP2 signaling, induce ferroptosis and stimulate pro-inflammatory cytokine production, in atherosclerosis and nonalcoholic steatohepatitis. The natural antibody E06 blocks PC-OxPLs. Transgenic expression of a single chain (scFv) form of the antigen-binding domain of E06 IgM (E06-scFv) increases bone mass in 6-month-old mice and attenuates high fat diet- and age-induced bone loss by increasing osteoblast number and bone formation rate and reducing osteoblast apoptosis. To elucidate the mechanism(s) mediating the bone anabolic effect of E06-scFv we performed bulk-RNA sequencing (bulk-RNA-seq) of vertebral bone and single cell RNA-sequencing (scRNA-seq) of mesenchymal and myeloid cells from long bones of WT and E06-scFv transgenic mice. Earlier bulk-RNA-seq performed in vertebral bone of 22- month-old females had shown that E06-scFv upregulates Wnt10b, but not other Wnt ligands, and the Wnt signaling pathway. We report now that scRNA-seq of mesenchymal cells isolated from 7-month-old female mice revealed that Wnt10b expression is increased by 3-fold (adj-P=3.04E-11) in pre-osteoblasts and osteoblasts from E06-scFv transgenic mice as compared to cells from WT mice. Wnt target genes were also upregulated. An integrated analysis of 9 published scRNA-seq datasets from bone resident mesenchymal and hematopoietic cell types revealed that the majority of Wnt10b in the bone microenvironment originates from cells of the osteoblast lineage. Further, OxLDL decreased Wnt10b and Wnt target genes in calvaria- or bone marrow-derived osteoblastic cell cultures. In contrast, OxLDL did not cause the ferroptosis associated upregulation of Chac1, Ptgs2, Slc7A11, and Acsl4, and the downregulation of Rgs4 in calvaria cells. In addition, no changes in the expression of these genes were detected in the scRNA-seq of mesenchymal cells from E06-scFv compared to WT mice. BMP signaling pathways were also unchanged in this scRNA-seq analysis. Finally, using bulk-RNA-seq of vertebral bone and scRNA-seq of myeloid cells, we could not detect changes in the expression of pro-inflammatory cytokines, such as Tnf-α, Il-6, Il1-α, Il-β, Il-4, Il-12, Lif, Il-23, interferon-α, and interferon-β, between WT and E06-scFv mice. Together, these findings indicate the PC-OxPLs decrease Wnt10b and E06-scFv increases Wnt10b in bone but has no effect on other pathways shown to be affected by OxPLs in other tissues. Thus, Wnt10b may be a critical mediator of the bone anabolic effects of E06-scFv. Presentation: Saturday, June 17, 2023

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