Aging is a main risk factor for osteoarthritis (OA). FoxO transcription factors protect against cellular and organismal aging, and FoxO expression in cartilage is reduced with aging and in OA. To investigate the role of FoxO in cartilage, Col2Cre-FoxO1, 3, and 4 single knockout (KO) and triple KO mice (Col2Cre-TKO) were analyzed. Articular cartilage in Col2Cre-TKO and Col2Cre-FoxO1 KO mice was thicker than in control mice at 1 or 2 months of age. This was associated with increased proliferation of chondrocytes of Col2Cre-TKO mice in vivo and in vitro. OA-like changes developed in cartilage, synovium, and subchondral bone between 4 and 6 months of age in Col2Cre-TKO and Col2Cre-FoxO1 KO mice. Col2Cre-FoxO3 and FoxO4 KO mice showed no cartilage abnormalities until 18 months of age when Col2Cre-FoxO3 KO mice had more severe OA than control mice. Autophagy and antioxidant defense genes were reduced in Col2Cre-TKO mice. Deletion of FoxO1/3/4 in mature mice using Aggrecan(Acan)-CreERT2 (AcanCreERT-TKO) also led to spontaneous cartilage degradation and increased OA severity in a surgical model or treadmill running. The superficial zone of knee articular cartilage of Col2Cre-TKO and AcanCreERT-TKO mice exhibited reduced cell density and markedly decreased In vitro, ectopic FoxO1 expression increased and synergized with transforming growth factor-β stimulation. In OA chondrocytes, overexpression of FoxO1 reduced inflammatory mediators and cartilage-degrading enzymes, increased protective genes, and antagonized interleukin-1β effects. Our observations suggest that FoxO play a key role in postnatal cartilage development, maturation, and homeostasis and protect against OA-associated cartilage damage.

Hemophilic arthropathy is a debilitating condition that can develop as a consequence of frequent joint bleeding despite adequate clotting factor replacement. The mechanisms leading to repeated spontaneous bleeding are unknown. We investigated synovial, vascular, stromal and cartilage changes in response to a single induced hemarthrosis in the FVIII-deficient mouse. We found soft tissue hyperproliferation with marked induction of neoangiogenesis and evolving abnormal vascular architecture. While soft tissue changes were rapidly reversible, abnormal vascularity persisted for months and, surprisingly, was also seen in uninjured joints. Vascular changes in FVIII-deficient mice involved pronounced remodeling with expression of α-Smooth Muscle Actin (SMA), Endoglin (CD105) and vascular endothelial growth factor, as well as alterations of joint perfusion as determined by in vivo imaging. Vascular architecture changes and pronounced expression of α-SMA appeared unique to hemophilia, as these were not found in joint tissue obtained from mouse models of rheumatoid arthritis (RA) and osteoarthritis (OA) and from patients with the same conditions. Evidence that vascular changes in hemophilia were significantly associated with bleeding and joint deterioration was obtained prospectively by dynamic in vivo imaging with musculoskeletal ultrasound and power Doppler of 156 joints (elbows, knees and ankles) in a cohort of 26 patients with hemophilia at baseline and during painful episodes. These observations support the hypothesis that vascular remodeling contributes significantly to bleed propagation and development of hemophilic arthropathy. Based on these findings, the development of molecular targets for angiogenesis inhibition may be considered in this disease.

Objectives Aging is a main risk factor for osteoarthritis (OA), the most prevalent musculoskeletal disorder. Defects in autophagy, an essential cellular homeostasis mechanism, have recently been observed in OA articular cartilage. The objectives of this study were to establish the constitutive level of autophagy activation in normal cartilage and monitor the temporal relationship of changes in autophagy and aging-related cartilage degradation. Methods In GFP-LC3 transgenic mice, GFP-LC3 is ubiquitously expressed, and the accumulation of GFP puncta, representing autophagosomes, was quantified by confocal microscopy as a measure of autophagy activation. Expression of the autophagy proteins Atg5 and LC3 was analyzed by immunohistochemistry. Cartilage cellularity, apoptotic cell death and cartilage structural damage and changes in synovium and bone were examined by histology and immunohistochemistry. Results Basal autophagy activation was detected in young (6 months) mouse liver and knee articular cartilage, with higher levels in cartilage than in liver in the same animals. In aged 28 months old mice, there was a statistically significant reduction in the total number of autophagic vesicles per cell (P < 0.01) and in the total area of vesicles per cell (P < 0.01) compared to young 6 months old mice in articular cartilage. With increasing age, the expression of Atg5 and LC3 decreased, followed by a reduction in cartilage cellularity and an increase in the apoptosis marker PARP p85. Cartilage structural damage progressed in an age-dependent manner, subsequent to autophagy changes. Conclusions Autophagy is constitutively activated in normal cartilage. This is compromised with aging and precedes cartilage cell death and structural damage.

Objective: Osteoarthritis (OA) is a multifactorial disease with etiological heterogeneity. The objective of this study was to classify OA subgroups by generating metabolomic phenotypes from human synovial fluid. Design: Post mortem synovial fluids (n ¼ 75) were analyzed by high performance-liquid chromatography mass spectrometry (LC-MS) to measure changes in the global metabolome. Comparisons of healthy (grade 0), early OA (grades I-II), and late OA (grades III-IV) donor populations were considered to reveal phenotypes throughout disease progression. Results: Global metabolomic profiles in synovial fluid were distinct between healthy, early OA, and late OA donors. Pathways differentially activated among these groups included structural deterioration, glycerophospholipid metabolism, inflammation, central energy metabolism, oxidative stress, and vitamin metabolism. Within disease states (early and late OA), subgroups of donors revealed distinct phenotypes. Synovial fluid metabolomic phenotypes exhibited increased inflammation (early and late OA), oxidative stress (late OA), or structural deterioration (early and late OA) in the synovial fluid. Conclusion: These results revealed distinct metabolic phenotypes in human synovial fluid, provide insight into pathogenesis, represent novel biomarkers, and can move toward developing personalized interventions for subgroups of OA patients.

The WW domain-containing protein 2 ( Wwp2 ) gene, the host gene of miR-140, codes for the Wwp2 protein, which is an HECT-type E3 ubiquitin ligases abundantly expressed in articular cartilage. However, its function remains unclear. Here, we show that mice lacking Wwp2 and mice in which the Wwp2 E3 enzyme is inactivated (Wwp2-C838A) exhibit aggravated spontaneous and surgically induced osteoarthritis (OA). Consistent with this phenotype, WWP2 expression level is downregulated in human OA cartilage. We also identify Runx2 as a Wwp2 substrate and Adamts5 as a target gene, as similar as miR-140. Analysis of Wwp2-C838A mice shows that loss of Wwp2 E3 ligase activity results in upregulation of Runx2-Adamts5 signaling in articular cartilage. Furthermore, in vitro transcribed Wwp2 mRNA injection into mouse joints reduces the severity of experimental OA. We propose that Wwp2 has a role in protecting cartilage from OA by suppressing Runx2-induced Adamts5 via Runx2 poly-ubiquitination and degradation.