12538 Blocking Oxidized Phospholipids Improves Bone Mass In Mice By Transcriptional Reprogramming Of Osteoblastic Cells: Analysis At The Single Cell Level

Abstract Disclosure: M. Palmieri: None. I. Nookaew: None. M. Almeida: None. T.E. Joseph: None. C. O'Brien: None. E. Ambrogini: None. Oxidized phospholipids containing phosphatidylcholine (PC-OxPLs), present on oxidized low-density lipoproteins (OxLDL) and apoptotic cells, are pathogenic in many diseases, including osteoporosis. The E06 IgM antibody binds PC-OxPLs and blocks their adverse effects. In our studies, six-month-old female and male mice overexpressing a single chain (scFv) form of the antigen-binding domain of E06 IgM (E06-scFv) have high cancellous and cortical bone mass. In addition, E06-scFv transgenic mice are protected against high fat diet- and age-induced bone loss. E06-scFv increases osteoblast number, bone formation rate and reduces osteoblast apoptosis. To investigate the effect of E06-scFv on osteoblastic cells transcriptional reprogramming, we performed single cell RNA-sequencing (scRNA-seq) of mesenchymal cells from long bones of 6-months old WT and E06-scFv transgenic female and male mice. Based on the total of over 7,500 osteoblastic cells, we focused the analysis on 3 cell clusters, pre-osteoblasts, identified by high levels of osteopontin (Spp1), osteoblasts, identified by high expression of osteocalcin (Bglap) and adipo-CAR expressing the adipogenic marker Adipoq in addition to high expression of the stromal marker Cxcl12. In both males and female transgenic mice, the relative number osteoblasts increased [12.0 % in females and 23.7 % in males; conversely, the relative number of adipo-CAR cells decreased [18.2 % in females and 15.1 % in males] compared to WT. We performed the analysis of functional enrichment-based gene ontology by piano software package and found that, in the three clusters of both male and female transgenic mice, there was an upregulation of gene expression, with a range of 37-125 genes per cluster, related to extracellular matrix organization [p range 0.0003-3.95E-09], bone mineralization [p range 0.04-0.0022] and cell adhesion [p range 0.019-0.00008]. Consistent with these results, bulk RNA sequencing indicated that these ontology terms were downregulated in calvaria-derived osteoblasts treated with OxLDL [extracellular matrix organization p= 1.19E-13; bone mineralization p= 0.0018655; cell adhesion p= 2.73E-12]. Based on these results, we conclude that PC-OxPLs not only affect osteoblast number but also decrease osteoblast activity on a single cell basis. Presentation: 6/1/2024