POS0232 IDENTIFICATION OF THE CORE REGULATORY GENE NETWORKS REGULATING SUBCHONDRAL BONE AND MARROW ADIPOSE TISSUE REMODELING IN HUMAN KNEE OA

Abstract

BackgroundSubchondral bone and the marrow adipose tissue (BMAT) contained therein undergo elevated turnover and remodeling during progression of osteoarthritis (OA). BMAT changes, detected as bone marrow lesions (BML) on MRI images, are considered important diagnostic and prognostic imaging biomarkers for OA. However, a comprehensive understanding of the regulatory mechanisms in individual BMAT-resident cell populations that mediate marrow fibrosis and subchondral bone sclerosis is still lacking. Recently generated single-cell transcriptomic atlas of human adipose tissue has opened new avenues for computational bulk tissue cell type deconvolution and dissecting gene regulatory networks.ObjectivesTo infer core gene regulatory networks underlying subchondral bone sclerosis and BMAT remodeling in human knee OA.MethodsWe conducted comparative transcriptomics on bulk transcriptomic profiles of human subchondral bone tissue from three independent studies1,2,3 comprising tibial plateaus from non-OA controls (n=20) and BMLs (n=14), non-sclerotic (n=45) and sclerotic (n=45) regions from patients with medial knee OA. Cell type deconvolution (adipocyte/pre-adipocyte/fibroblast/endothelial/macrophage) of differentially expressed genes (DEGs) was performed using single-cell atlases of human brown and white adipose tissue4. Cell type annotated DEGs were functionally annotated using Gene Ontology enrichment. To infer transcription factors (TFs) regulating gene networks we performed ChIP-Seq enrichment analyses and assessed the presence of TF binding sites in the proximal promoter (-500/+100) sequence of DEGs. Tissue explants were stained en bloc with Oil red O and Hoechst to evaluate distribution of neutral lipids and nuclei in BMAT from non-sclerotic and sclerotic regions.Results534 upregulated and 363 downregulated DEGs were shared between at least two independent datasets. Cell deconvolution revealed the majority of upregulated DEGs were expressed in fibroblasts (38%), pre-adipocytes (27%) and endothelial cells (22%). Dowregulated DEGs were predominantly expressed by endothelial (31%), adipocyte (25%) and fibroblast (16%) cell populations. We inferred major TF regulatory networks driving upregulated DEGs in pre-adipocytes (PRRX1/SNAI2/TWIST1) and fibroblasts (SP7/SMAD4/RUNX2/DLX5) and loss of PPARG/SOX17/SNAI1-driven gene expression in adipocytes and endothelial cells. Pre-adipocyte genes were functionally enriched for collagen fibril organization, ossification and cell migration. Fibroblast genes associated with biomineral tissue development and negative regulation of angiogenesis. Downregulated DEGs were enriched for triglyceride catabolism and sequestration (adipocytes) and regulation of vascular permeability and endothelial cell differentiation (endothelial). Whole mount staining displayed homogenous distribution of neutral lipids in adipocytes and low cell abundance in BMAT from non-sclerotic tissue. In contrast, BMAT from sclerotic tissue showed heterogeneously distributed and lower neutral lipid content as well as high cell abundance (Figure 1).Figure 1.ConclusionThese data provide detailed insight into the cellular and molecular mechanisms underpinning subchondral bone and BMAT remodeling in OA. An expansion of pre-adipocyte populations along altered function of BMAT adipocytes might represent a previously unrecognized mechanism regulating subchondral bone sclerosis. TFs driving core gene regulatory networks might be promising therapeutic targets for knee OA.