Epigenetic dysregulation of articular cartilage during progression of hip femoroacetabular impingement disease.

Femoroacetabular impingement (FAI) is an important trigger of hip osteoarthritis (OA). Epigenetic changes in DNA methyltransferase 3B (DNMT3B) attenuate catabolic gene expression in cartilage hemostasis. This study aimed to examine the articular chondrocyte catabolic state and DNMT3B and 4-aminobutyrate aminotransferase promoter (ABAT) expression during OA progression in FAI. Cartilage samples were collected from the impingement zone of 12 patients with cam FAI (early-FAI) and 12 patients with advanced OA secondary to cam FAI (late-FAI-OA). Five healthy samples were procured from cadavers (ND: nondiseased). Explants were cultured under unstimulated conditions, catabolic stimulus (IL1ß), or anabolic stimulus (TGFß). Histology was performed with safranin-O/fast-green staining. Gene expression was analyzed via qPCR for GAPDH, DNMT3B, ABAT, MMP-13, COL10A1. Methylation specific PCR assessed methylation status at the ABAT promoter. Cartilage samples in early-FAI and late-FAI-OA showed a histological OA phenotype and increased catabolic marker expression (MMP13/COL10A1, ND vs. early-FAI, p = 0.004/p < 0.001, ND vs. late-FAI-OA, p < 0.001/p < 0.001). RT-PCR confirmed DNMT3B underexpression (ND vs. early-FAI, p < 0.001, early-FAI vs. late-FAI-OA, p = 0.016) and ABAT overexpression (ND vs. early-FAI, p < 0.001, early vs. late-FAI-OA, p = 0.035) with advanced disease. End-stage disease showed ABAT promoter hypomethylation. IL1ß stimulus accentuated ABAT promoter hypomethylation and led to further ABAT and catabolic marker overexpression in early-FAI and late-FAI-OA while TGFß normalized these alterations in gene expression. Catabolic and epigenetic molecule expression suggested less catabolism in early-stage disease. Sustained inflammation induced ABAT promoter hypo-methylation causing a catabolic phenotype. Suppression of ABAT by methylation control could be a new target for therapeutic intervention to prevent OA progression in hip FAI.

Whole-genome RNA sequencing identifies distinct transcriptomic profiles in impingement cartilage between patients with femoroacetabular impingement and hip osteoarthritis.

Femoroacetabular impingement (FAI) has a strong clinical association with the development of hip osteoarthritis (OA); however, the pathobiological mechanisms underlying the transition from focal impingement to global joint degeneration remain poorly understood. The purpose of this study is to use whole-genome RNA sequencing to identify and subsequently validate differentially expressed genes (DEGs) in femoral head articular cartilage samples from patients with FAI and hip OA secondary to FAI. Thirty-seven patients were included in the study with whole-genome RNA sequencing performed on 10 gender-matched patients in the FAI and OA cohorts and the remaining specimens were used for validation analyses. We identified a total of 3531 DEGs between the FAI and OA cohorts with multiple targets for genes implicated in canonical OA pathways. Quantitative reverse transcription-polymerase chain reaction validation confirmed increased expression of FGF18 and WNT16 in the FAI samples, while there was increased expression of MMP13 and ADAMTS4 in the OA samples. Expression levels of FGF18 and WNT16 were also higher in FAI samples with mild cartilage damage compared to FAI samples with severe cartilage damage or OA cartilage. Our study further expands the knowledge regarding distinct genetic reprogramming in the cartilage between FAI and hip OA patients. We independently validated the results of the sequencing analysis and found increased expression of anabolic markers in patients with FAI and minimal histologic cartilage damage, suggesting that anabolic signaling may be increased in early FAI with a transition to catabolic and inflammatory gene expression as FAI progresses towards more severe hip OA. Clinical significance:Cam-type FAI has a strong clinical association with hip OA; however, the cellular pathophysiology of disease progression remains poorly understood. Several previous studies have demonstrated increased expression of inflammatory markers in FAI cartilage samples, suggesting the involvement of these inflammatory pathways in the disease progression. Our study further expands the knowledge regarding distinct genetic reprogramming in the cartilage between FAI and hip OA patients. In addition to differences in inflammatory gene expression, we also identified differential expression in multiple pathways involved in hip OA progression.

Otto Aufranc Award: Identification of Key Molecular Players in the Progression of Hip Osteoarthritis Through Transcriptomes and Epigenetics.

BACKGROUND: This study aimed: (1) to compare the transcriptome profile of articular cartilage in cam-FAI (early stage) to advanced OA secondary to cam-FAI (late stage) and (2) to investigate epigenetic changes through the expression of DNA methylation enzymes DNMT3B, DNMT1, and DNMT3A and peroxisome proliferator-activated receptor gamma (PPARγ) in human cartilage samples during the progression of hip OA. METHODS: Full-thickness cartilage samples were collected from the anterolateral head-neck junction (impingement zone) of 22 patients (9 early-FAI and 13 late-FAI). RNA sequencing and in vitro cartilage cultures with histological analysis and immunohistochemistry staining for PPARγ and DNMT3B were performed. Target gene validation was confirmed with RT-PCR. RESULTS: Fifty genes and 42 pathways were identified differentially between early and late-FAI (fold change <-1.5 or >1.5, P < .01). PPARγ and DNMT3B were gradually suppressed with disease progression. Contrarily, disease progression induced expression of DNMT1/3A. CONCLUSION: By comparing comprehensive gene expression in early and late stage hip degeneration at the whole-genome level, distinct transcriptome profiles for early and late stage disease were identified along with key molecular contributors to the progression of hip OA. Preservation of endogenous PPARγ may have therapeutic potential to delay or prevent hip OA.

Genes associated with inflammation and bone remodeling are highly expressed in the bone of patients with the early-stage cam-type femoroacetabular impingement.

BACKGROUND: Recent studies have shown high expression levels of certain inflammatory, anabolic, and catabolic genes in the articular cartilage from the impingement zone of the hips with femoroacetabular impingement (FAI), representing an increased metabolic state. Nevertheless, little is known about the molecular properties of bone tissue from the impingement zone of hips with FAI. METHODS: Bone tissue samples from patients with early-stage cam-type FAI were collected during hip arthroscopy for treatment of cam-type FAI. Control bone tissue samples were collected from six patients who underwent total hip replacement because of a femoral neck fracture. Quantitative real-time polymerase chain reaction (PCR) was performed to determine the gene expression associated with inflammation and bone remodeling. The differences in the gene expression in bone tissues from the patients with early-stage cam-type FAI were also evaluated based on clinical parameters. RESULTS: In all, 12 patients with early-stage cam-type FAI and six patients in the control group were included in this study. Compared to the control samples, the bone tissue samples from patients with FAI showed higher expression levels of interleukin-6 (IL-6), alkaline phosphatase (ALP), receptor activator of nuclear factor-kB ligand (RANKL), and osteoprotegerin (OPG) (P < 0.05). IL-1 expression was detected only in the control group. On the other hand, there was no significant difference in IL-8 expression between the patients with FAI and the control group. The patients with FAI having a body mass index (BMI) of >24 kg/m2 showed higher ALP expression (P < 0.05). Further, the expression of IL-6 and ALP was higher in the patients with FAI in whom the lateral center-edge angle was >30° (P < 0.05). CONCLUSIONS: Our results indicated the metabolic condition of bone tissues in patients with early-stage cam-type FAI differed from that of normal bone in the femoral head-neck junction. The expression levels of the genes associated with inflammation and bone remodeling were higher in the bone tissue of patients with early-stage cam-type FAI than in the patients with normal bone tissue.

Autophagy protects chondrocytes from glucocorticoids-induced apoptosis via ROS/Akt/FOXO3 signaling.

OBJECTIVE: Glucocorticoids (GCs) have been widely used in the management of osteoarthritis (OA) and rheumatoid arthritis (RA). Nevertheless, there has been some concern about their ability of increasing reactive oxygen species (ROS) in the cartilage. Forkhead-box class O (FOXO) transcription factors have been proved to have a protective role in chondrocytes through regulation of autophagy and defending oxidative stress. The objective of this study was to investigate the role of FOXO3 in Dex-induce up-regulation of ROS. DESIGN: Healthy cartilages debris from six patients were used for chondrocytes culture. After the treatment of dexamethasone (Dex), the ROS levels, autophagic flux, the expression of FOXO3 in chondrocytes were measured. RNA interference technique was also used to determine the role of FOXO3 in Dex-induced autophagy. The metabolism of the extra-cellular matrix was also investigated. THE RESULTS: Dex increased intracellular ROS level, the expression of Akt, FOXO3 as well as autophagy flux in human chondrocytes. The expression of aggrecanases also increased after the treatment of Dex. Catalase, the ROS scavenger, suppressed Dex-induced up-regulation of autophagy flux and expression of aggrecanases and Akt. MK-2206 and LY294002, the PI3K/Akt inhibitors, repressed Dex-induced up-regulation of FOXO3. Silencing FOXO3 resulted in down-regulation of Dex-induced autophagy. Moreover, knockdown of FOXO3 increased Dex-induced apoptosis as well as ROS levels in chondrocytes. In addition, up-regulation of autophagy by Rapamycin resulted in decreasing ROS level in chondrocytes. CONCLUSION: Dex could advance the degenerative process in cartilage. Autophagy was induced in response to Dex-induced up-regulation of ROS via ROS/Akt/FOXO3 signal pathway.

Molecular characterization of articular cartilage from young adults with femoroacetabular impingement.

BACKGROUND: Femoroacetabular impingement is a frequent cause of hip pain and may lead to secondary osteoarthritis, yet little is known about the molecular events linking mechanical hip impingement and articular cartilage degeneration. The first goal of this study was to quantify the expression of inflammatory cytokine and chemokine, matrix-degrading, and extracellular matrix genes in articular cartilage harvested from control hips and hips with femoroacetabular impingement and end-stage osteoarthritis. The second goal was to analyze the relative expression of these genes in articular cartilage harvested at various stages of osteoarthritis. METHODS: Cartilage samples were obtained from thirty-two hips undergoing hip preservation surgery for femoroacetabular impingement or hip arthroplasty. Three control cartilage samples were also analyzed. Specimens were graded intraoperatively with regard to the severity of cartilage damage, the radiographic osteoarthritis grade was recorded, and quantitative RT-PCR (real-time polymerase chain reaction) was performed to determine relative gene expression. RESULTS: Except for interleukin-1ß (IL-1ß) and CXCL2, the mRNA (messenger RNA) expression of all other chemokine (IL-8, CXCL1, CXCL3, CXCL6, CCL3, and CCL3L1), matrix-degrading (matrix metalloproteinase [MMP]-13 and ADAMTS-4), and structural matrix (COL2A1 [collagen, type II, alpha] and ACAN [aggregan]) genes was higher overall in cartilage from hips with femoroacetabular impingement compared with hips with osteoarthritis and normal controls. The differences reached significance (p ≤ 0.05) for seven of these ten quantified genes, with CXCL3, CXCL6, and COL2A1 being elevated in the femoroacetabular impingement group compared with only the control group and IL-8, CCL3L1, ADAMTS-4, and ACAN being elevated compared with both the osteoarthritis and control groups. When samples were grouped according to the stage of the degenerative cascade, mRNA expression was relatively higher in one of the two middle stages of femoroacetabular impingement (chondromalacia or cleavage/thinning), with the difference reaching significance for IL-8, CXCL2, CXCL3, CCL3L1, and ACAN. ACAN expression was diminished in hips with osteoarthritis compared with femoroacetabular impingement but elevated compared with the control articular cartilage. CONCLUSIONS: Articular cartilage from the impingement zone of hips with femoroacetabular impingement (and particularly those hips in the cleavage/thinning stage) expressed higher levels of certain inflammatory, anabolic, and catabolic genes, representing a heightened metabolic state. CLINICAL RELEVANCE: The articular cartilage from the impingement zone of hips with femoroacetabular impingement was metabolically hyperactive, supporting the concept that such impingement is a structural precursor to hip osteoarthritis.

Human hip impingement morphology: an evolutionary explanation.

We examined the morphology of mammalian hips asking whether evolution can explain the morphology of impingement in human hips. We describe two stereotypical mammalian hips, coxa recta and coxa rotunda. Coxa recta is characterised by a straight or aspherical section on the femoral head or head-neck junction. It is a sturdy hip seen mostly in runners and jumpers. Coxa rotunda has a round femoral head with ample head-neck offset, and is seen mostly in climbers and swimmers. Hominid evolution offers an explanation for the variants in hip morphology associated with impingement. The evolutionary conflict between upright gait and the birth of a large-brained fetus is expressed in the female pelvis and hip, and can explain pincer impingement in a coxa profunda. In the male hip, evolution can explain cam impingement in coxa recta as an adaptation for running.