Abstract
Objectives:
Hip Femoroacetabular impingement (FAI) has a high prevalence of chondral lesions, and the severity of chondral damage is associated with an increased risk of postoperative OA progression, but the mechanisms of cartilage degeneration in OA progression remains unclear. Peroxisome proliferator- activated receptor-gamma (PPARγ) is widely expressed in healthy articular chondrocytes (ACs) and plays an essential role in cartilage homeostasis. Recent investigations into OA pathogenesis have revealed that hyper or hypomethylation (by DNMTs) of OA susceptibility genes are involved in OA progression, especially downregulation of PPARγ has been linked to OA progression. This study aimed to investigate the expression of PPARγ and DNA methyltransferases enzymes (DNMT1 and DNMT3A) during progression of hip disease and 2) methylation changes in the PPARγ promoter area during progression of hip disease.
Methods:
Full-thickness cartilage samples were collected from the impingement zone. 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. As a non-disease (ND) group, six healthy samples were procured from hip joint cadavers. Cartilage explants were cultured in unstimulated conditions or catabolic stimulus (IL1β) with or without DNA demethylating agent 5-Aza-2’- deoxycytidine (5Aza). Histological analysis was performed with safranin O/fast green. Gene expression was analyzed via qPCR for GAPDH, DNMT1, DNMT3A, PPARγ, MMP13, and COL10A1. Methylation specific PCR (MSP) was performed to assess the methylation status at PPARγ promoter site. Chromatin immunoprecipitation (ChIP) assay was performed to confirm an interaction between DNMT3A and the PPAR promoter region.
Results:
The early FAI group included younger subjects than the late FAI OA group. Cartilage samples in early and late FAI-OA displayed a histological OA phenotype (Figure 1A). ACs in early and late FAI-OA displayed increased expression of catabolism and chondrocyte hypertrophy markers (MMP13: ND vs early p<0.001, ND vs late p<0.001, COL10A1: ND vs early p<0.001, ND vs late p<0.001, Figure. 1B).
Contrary, expression of PPARγ gradually declined as the disease progressed (ND vs early p=0.001, early vs late p=0.003, Figure. 1B). IF analysis also displayed a gradual decrease of the PPARγ expression during disease progression (ND vs early p=0.001, early vs late p=0.041, Figure 1C). In the assessment of the epigenetic mechanisms of aberrant DNA methylation in OA progression, overexpression of DNMT1 and DNMT3A levels was observed with disease progression in RT-PCR (DNMT1: ND vs early p<0.001, early vs late p=0.041, DNMT3A: ND vs early p=0.015, early vs late p=0.053,Figure 2A) and IF analysis (DNMT1: ND vs early p<0.001, early vs late p<0.001, DNMT3A: ND vs early <0.001, early vs late p=0.008, Figure 2B). Next, in MSP analysis, human PPARγ promoter was featured by typical CpG islands (Figure 3A). As the OA progresses, hypermethylation at PPARγ promoter (ND: 9%, Early-FAI: 34%, Late FAI-OA: 48%) was observed (Figure 3B), and ChIP assay confirmed DNMT3A interaction with the CpG PPARγ promoter area (Figure 3C). IL1β stimulus increased PPARγ promoter methylation for early (34% to 57%, p=0.016, Figure 3D) and late stages (48% to 79%, p=0.001, Figure 3D) resulting in suppression of PPARγ expression (P=0.073, Figure 3F). Contrary, treatment with 5Aza (a DNA demethylator) brought the level of methylation back to 37% for early FAI (p=0.033, Figure 3D) and 46% for late FAI-OA (p<0.001, Figure 3D), reversed the PPARγ suppression induced by IL1β (early- FAI p=0.009, and late FAI-OA ACs p<0.001 Figure 3F) and decreased expression of MMP-13 and COL10A1 induced by IL1β (early-FAI p=0.009, Figure 4C and late FAI-OA ACs p<0.001 Figure 3F). PPARγ inhibitor rescued cells from the catabolic phenotype. (Fig.3E, F), suggesting that restoration of PPARγ by 5Aza contributes significantly to the cartilage protective effect.
Conclusions:
This current study demonstrates that PPARγ expression was gradually suppressed via promoter hypermethylation during OA progression in hip FAI. Moreover, the inflammatory stimulus with IL1β further increased catabolic markers and enhanced PPARγ suppression via promoter excessive hypermethylation in human hip ACs. Notably, DNA demethylating agent (5 aza) prevents the IL1β induced PPARγ promoter hypermethylation rescuing PPARγ and alleviating cartilage degenerative damage. Certainly, for patients with symptomatic hip FAI, surgical correction should be prioritized to suppress inflammation, but our observations suggests that critical epigenetic dysregulation occurs during the progression of hip OA. Future investigations will be needed to confirm if regulation of these epigenetic changes could potentially complement surgical treatment to minimize OA progression in hip FAI.
