Poster 112: Epigenetic Dysregulation during the OA Progression in Hip FAI
Authors: Kamenaga Tomoyuki , Shen Jie , Wu May , Brophy Robert , O’Keefe Regis , Clohisy John , Pascual-Garrido Cecilia 
Published: August 2023

Objectives: Femoroacetabular impingement (FAI) is considered a leading cause of early hip OA, but initial molecular mechanisms that lead FAI patients to OA progression remains to be elucidated. Recent articles revealed that epigenetic changes are associated with OA progression, specifically, DNA methyltransferase 3B (DNMT3B) plays a critical role on cartilage homeostasis by attenuating catabolic gene expression. This study aimed to examine the catabolic state of articular chondrocyte (ACs) during OA progression in hip FAI disease, particularly the expressions of DNMT3B and 4-aminobutyrate aminotransferase (ABAT, downstream of DNMT3B), to assess how epigenetic dysregulation contributes to OA progression. Methods: Full-thickness cartilage samples were collected from the impingement zone. In total, twelve patients underwent hip preservation surgery for cam FAI (early-FAI) and twelve patients underwent total hip replacement for advanced OA secondary to cam FAI (late FAI-OA) were included in this study. As a non-disease (ND) group, five healthy samples were procured from hip joint cadavers. The explants were cultured in unstimulated conditions or catabolic stimulus (IL1β). Histological analyses was performed with safranin-O/fast-green. Gene expression was analyzed via qPCR for GAPDH, DNMT3B, ABAT, MMP-13, and COL10A1. Protein expression was analyzed by western blotting analysis for DNMT3B and ABAT. Methylation specific PCR was performed to assess the methylation status at ABAT promoter (epigenetic change). Results: The early FAI group included younger subjects with lower Tönnis grade than the late FAI OA group; however, there were no significant differences in gender, BMI, and α-angle between groups. Cartilage samples in early-FAI and late FAI-OA showed a histological OA phenotype (Figure 1A). RT-PCR confirmed increased expression of MMP-13 and COL10A1 in early and late FAI-OA (Figure 1B), confirming cartilage degeneration, catabolism, and chondrocyte hypertrophy when compared to ND group. Interestingly, RT-PCR revealed that expression of DNMT3B gradually declined as the disease progressed (ND vs early p<0.001, early vs late p=0.016), while ABAT increased as the disease progressed (ND vs early p<0.001, early vs late p=0.035, Figure 1B). IF staining confirmed that DNMT3B was abundantly expressed in chondrocytes of normal cartilage but the expression gradually decreased with disease progression, contrary ABAT expression gradually increased as the disease progressed (Figure 1C). These results were supported by protein expression in western blotting analysis (Figure 1D). In MSP analysis, the human ABAT promoter is characterized by a typical CpG island (Figure 2A, blue area). Additionally, there was hypomethylation at the ABAT promoter during end stage of disease (ND vs early p=0.022, early vs late p=0.042) (Figure 2B). Catabolic stimulus with IL1β for early FAI and late FAI-OA, accentuated the ABAT promoter hypomethylation (early FAI: nontreatment vs IL1β p=0.056, late FAI-OA: nontreatment vs IL1β p=0.015, Figure 2C) and resulted in further overexpression of ABAT and catabolic markers and decreased expression of DNMT3B (Figure 3A-C). These findings are supported by protein expression in western-blotting analysis (Figure 3D). Conclusions: In the present study, histological findings were similar in early and late FAI, but DNMT3B expression levels gradually decreased with OA progression, while ABAT and catabolic marker expression levels increased, suggesting a less catabolic phenotype in the early stage of disease. MSP analysis confirmed ABAT promoter hypo-methylation (epigenetically dysregulation) as hip OA progresses. Notably, in the current study, the inflammatory stimulus with IL1β further reduced DNA methylation at the ABAT promoter and resulted in a concomitant excessive increase of ABAT and catabolic markers in human hip OA ACs. This study suggests, for the first time, that cartilage degeneration in the impingement zone in hip FAI is progressive at the molecular level and may be controlled in part by an epigenetic dysregulation. Understanding the mechanism of OA disease in hip FAI will potentially provide concomitant interventional treatment to hip pre-OA disease.