Toward Preventing Arthritis: A Comparison of Synovial RNA in Osteoarthritic Ankles and Knees

Abstract

Category: Ankle; Ankle Arthritis; Basic Sciences/Biologics; Trauma Introduction/Purpose: Osteoarthritis (OA) of the lower limb is a debilitating, incurable, expensive and prevalent condition in people over 45. Although ankle posttraumatic OA (PTOA) has the highest incidence, primary OA is increasingly recognized. The difference in OA incidence between lower limb joints is partly driven by differences in cartilage architecture and chondrocyte function at each site, with ankle chondrocytes showing reduced responsiveness to inflammatory mediators and more metabolic activity than those in the knee. Synovium also contributes to the pathogenesis of OA, however its potentially differential role in knee versus ankle OA remains an unanswered question. We aim to understand the molecular contribution of synovial dysregulation in the progression of ankle and knee OA to uncover candidate pathways for the development of targeted therapeutic paradigms. Methods: After obtaining informed consent from patients undergoing total knee or ankle replacement, basic surgical, past medical history, and imaging data was collected. Synovium samples were harvested during surgery and processed for RNA. Bulk 150 bp, paired-end RNA seq was conducted using standard protocols using the Illumina NovaSeq6000 platform. After quality control trimming, reads were aligned to human reference genome (GRCh38) using STAR (v 2.5) using the Maximum Mappable Prefix method and HTSeq (V0.6.1) was used to index mappable reads per gene. Counts were corrected for gene length using the FPKM method. Differential expression was determined using the edgeR (v 3.16.5) package for R, after adjustment using a scaling normalization factor and P-values were corrected using the Benjamini and Hochberg method. Similarities between samples were determined using Spearman Correlation and degree of similarity was visualized considering the first three Principal Components (PC). Results: RNA was obtained from 6 end-stage ankle OA, 1 end-stage knee OA patients. Of the ankle samples, two were PTOA and the remaining four were primary OA. The PTOA and the primary OA samples showed high sample correlation regardless of class (correlation coefficients ranged from 0.844 to 0.941). In contrast, the total knee sample was less correlated with the ankle samples (correlation coefficients ranged from 0.719 to 0.878). By using the first three PC, we observed that there was no obvious differentiation among the ankle OA samples, but there was clear separation between ankle and total knee samples. We observed an enrichment of genes annotated to the Gene Ontology clusters 'Extracellular Structure Organization' and 'Inflammatory Response' when comparing the two types of OA. Conclusion: These initial pilot results of an ongoing study support our hypothesis that synovial transcriptome differs between the knee and ankle in end-stage osteoarthritis and that anatomic site is a larger driver of synovial expression differences than OA type. This is supported by previous differences reported between ankle and knee chondrocytes in end-stage OA. These findings emphasize the importance of investigating synovial tissue to better understand the microenvironment of the ankle joint and pathogenesis of all etiologies of OA. The ultimate goal is the development of clinical tests and therapeutics aimed at slowing or discontinuing the degenerative process in ankle arthritis.