Association of circulating gene expression signatures with stiffness following total knee arthroplasty for osteoarthritis: a pilot study.

A subset of patients undergoing total knee arthroplasty (TKA) for knee osteoarthritis develop debilitating knee stiffness (reduced range of motion) for poorly understood reasons. Dysregulated inflammatory and immune responses to surgery correlate with reduced surgical outcomes, but the dysregulated gene signatures in patients with stiffness after TKA are poorly defined. As a consequence, we are limited in our ability to identify patients at risk of developing poor surgical outcomes and develop preventative approaches. In this pilot study we aimed to identify perioperative blood gene signatures in patients undergoing TKA for knee osteoarthritis and its association with early surgical outcomes, specifically knee range of motion. To do this, we integrated clinical outcomes collected at 6 weeks after surgery with transcriptomics analyses in blood samples collected immediately before surgery and at 24 h after surgery. We found that patients with stiffness at 6 weeks after surgery have a more variable and attenuated circulating gene expression response immediately after surgery. Our results suggest that patients with stiffness following TKA may have distinct gene expression signatures detectable in peripheral blood in the immediate postoperative period.

Transcriptomic and epigenomic analyses uncovered Lrrc15 as a contributing factor to cartilage damage in osteoarthritis.

In osteoarthritis (OA), articular chondrocytes display phenotypic and functional changes associated with epigenomic alterations. These changes contribute to the disease progression, which is characterized by dysregulated reparative processes and abnormal extracellular matrix remodeling leading to cartilage degradation. Recent studies using a murine model of posttraumatic OA highlighted the contribution of changes in DNA hydroxymethylation (5hmC) to OA progression. Here, we integrated transcriptomic and epigenomic analyses in cartilage after induction of OA to show that the structural progression of OA is accompanied by early transcriptomic and pronounced DNA methylation (5mC) changes in chondrocytes. These changes accumulate over time and are associated with recapitulation of developmental processes, including cartilage development, chondrocyte hypertrophy, and ossification. Our integrative analyses also uncovered that Lrrc15 is differentially methylated and expressed in OA cartilage, and that it may contribute to the functional and phenotypic alterations of chondrocytes, likely coordinating stress responses and dysregulated extracellular matrix remodeling.

Changes in DNA methylation accompany changes in gene expression during chondrocyte hypertrophic differentiation in vitro.

During osteoarthritis (OA), articular chondrocytes undergo phenotypic changes that resemble developmental patterns characteristic of growth plate chondrocytes. These phenotypic alterations lead to a hypertrophy-like phenotype characterized by altered production of extracellular matrix constituents and increased collagenase activity, which, in turn, results in cartilage destruction in OA disease. Recent studies have shown that the phenotypic instability and dysregulated gene expression in OA are associated with changes in DNA methylation patterns. Subsequent efforts have aimed to identify changes in DNA methylation with functional impact in OA disease, to potentially uncover therapeutic targets. Here, we paired an in vitro 3D/pellet culture system that mimics chondrocyte hypertrophy with RNA sequencing (RNA-Seq) and enhanced reduced representation of bisulfite sequencing (ERRBS) to identify transcriptomic and epigenomic changes in murine primary articular chondrocytes undergoing hypertrophy-like differentiation. We identified hypertrophy-associated changes in DNA methylation patterns in vitro. Integration of RNA-Seq and ERRBS datasets identified associations between changes in methylation and gene expression. Our integrative analyses showed that hypertrophic differentiation of articular chondrocytes is accompanied by transcriptomic and epigenomic changes in vitro. We believe that our integrative approaches have the potential to uncover new targets for therapeutic intervention.

Inducible knockout of CHUK/IKKα in adult chondrocytes reduces progression of cartilage degradation in a surgical model of osteoarthritis.

CHUK/IKKα contributes to collagenase-driven extracellular matrix remodeling and chondrocyte hypertrophic differentiation in vitro, in a kinase-independent manner. These processes contribute to osteoarthritis (OA), where chondrocytes experience a phenotypic shift towards hypertrophy concomitant with abnormal matrix remodeling. Here we investigated the contribution of IKKα to OA in vivo. To this end, we induced specific IKKα knockout in adult chondrocytes in AcanCreERT2/+; IKKαf/f mice treated with tamoxifen (cKO). Vehicle-treated littermates were used as wild type controls (WT). At 12 weeks of age, WT and cKO mice were subjected to the destabilization of medial meniscus (DMM) model of post-traumatic OA. The cKO mice showed reduced cartilage degradation and collagenase activity and fewer hypertrophy-like features at 12 weeks after DMM. Interestingly, in spite of the protection from structural articular cartilage damage, the postnatal growth plates of IKKα cKO mice after DMM displayed abnormal architecture and composition associated with increased chondrocyte apoptosis, which were not as evident in the articular chondrocytes of the same animals. Together, our results provide evidence of a novel in vivo functional role for IKKα in cartilage degradation in post-traumatic OA, and also suggest intrinsic, cell-autonomous effects of IKKα in chondrocytes that control chondrocyte phenotype and impact on cell survival, matrix homeostasis, and remodeling.

Through-Space Shielding Effects of Metal-Complexed Phenyl Rings.

Several naphthalene compounds containing a methyl group in a 1,8-relationship to a metal-complexed phenyl ring bearing various substituents have been synthesized. The through-space shielding effects of the phenyl ring, as a function of substituent and complexing metal species, were monitored by observing the 1H NMR signal of the methyl group located in the shielding zone of the ring. In all cases, the methyl signal was slightly more downfield in the complexes than in the uncomplexed analogues. A comparison of available crystal structures, however, shows that the phenyl ring is slightly closer to the naphthyl methyl group in the complexes than in their metal-free counterparts. X-ray structures and DFT calculations also reveal a slight elongation in the average length of the carbon-carbon bonds of the phenyl ring upon complexation. The effect of substituents on the signal of the naphthyl methyl group is small but discernible in the uncomplexed derivatives, and consistent with our previous report. A similar trend is absent in the corresponding metal complexes, as exemplified by the chromium series, and the effect of the metal appears to be more dominant than that of the substituents. These observations were found to be in line with NMR shift calculations.

Lubricin restoration in a mouse model of congenital deficiency.

OBJECTIVE: Congenital deficiency of the principal boundary lubricant in cartilage (i.e., lubricin, encoded by the gene PRG4) increases joint friction and causes progressive joint failure. This study was undertaken to determine whether restoring lubricin expression in a mouse model would prevent, delay, or reverse the disease process caused by congenital deficiency. METHODS: Using genetically engineered lubricin-deficient mice, we restored gene function before conception or at ages 3 weeks, 2 months, or 6 months after birth. The effect of restoring gene function (i.e., expression of lubricin) on the tibiofemoral patellar joints of mice was evaluated histologically and by ex vivo biomechanical testing. RESULTS: Restoring gene function in mice prior to conception prevented joint disease. In 3-week-old mice, restoring gene function improved, but did not normalize, histologic features of the articular cartilage and whole-joint friction. In addition, cyclic loading of the joints produced fewer activated caspase 3-containing chondrocytes when lubricin expression was restored, as compared to that in littermate mice whose gene function was not restored (nonrestored controls). Restoration of lubricin expression in 2-month-old or 6-month-old mice had no beneficial effect on histopathologic cartilage damage, extent of whole-joint friction, or activation of caspase 3 when compared to nonrestored controls. CONCLUSION: When boundary lubrication is congenitally deficient and cartilage becomes damaged, the window of opportunity for restoring lubrication and slowing disease progression is limited.