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Obesity and high body mass index are associated with a higher incidence of osteoarthritis (OA). The aim of this study is to investigate the involvement of the infrapatellar fat pad (IPFP) in the sub-acute effect of a high fat diet (HFD) on the development of knee-OA. C57BL/6J male mice were fed either a HFD or a normal diet beginning at seven weeks of age. Tissue sections were evaluated with immunohistological analysis. The IPFP was excised, and mRNA expression profiles were compared using real-time RT-PCR analysis. Osteoarthritic changes were initiated in the HFD group after eight weeks of the HFD. Increased synovial cell number and angiogenesis at the anterior edge of the tibial plateau were exhibited prior to osteophyte formation. Quantitative histological analysis indicated that osteophyte volume was significantly increased in the HFD group after eight weeks, along with an increase in the IPFP volume, the size of individual adipocytes and the number of vessels in the IPFP. Histomorphometrical analysis revealed osteophyte area was significantly associated with IPFP area, individual adipocyte area and vascular area. Real-time RT-PCR analysis demonstrated elevated mRNA expression of inflammatory cytokines, growth factor, and adipokines in the IPFP after eight weeks of the HFD. These findings are in parallel with increased expression of the CD68 macrophage marker after eight weeks of the HFD. Expression levels of the adipokines were significantly correlated with expression of TNF-α, VEGF and TGF-β. Immunohistological analysis revealed that the Nampt protein was highly expressed in the IPFP especially around the site of osteophyte formation. Apoptosis and proliferation of chondrocytes were both enhanced at the site of osteophyte formation, indicating higher cell turnover at this region. These observations suggest the IPFP plays a pivotal role in the formation of osteophytes and functions as a secretory organ in response to a HFD.

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Osteoarthritis (OA) is a common disease in the elderly due to an imbalance in cartilage degradation and synthesis. Heterotopic ossification (HO) occurs when ectopic masses of endochondral bone form within the soft tissues around the joints and is triggered by inflammation of the soft tissues. Procyanidin B3 (B3) is a procyanidin dimer that is widely studied due to its high abundance in the human diet and antioxidant activity. Here, we evaluated the role of B3 isolated from grape seeds in the maintenance of chondrocytes in vitro and in vivo. We observed that B3 inhibited H2O2-induced apoptosis in primary chondrocytes, suppressed H2O2- or IL-1ß−induced nitric oxide synthase (iNOS) production, and prevented IL-1ß−induced suppression of chondrocyte differentiation marker gene expression in primary chondrocytes. Moreover, B3 treatment enhanced the early differentiation of ATDC5 cells. To examine whether B3 prevents cartilage destruction in vivo, OA was surgically induced in C57BL/6J mice followed by oral administration of B3 or vehicle control. Daily oral B3 administration protected articular cartilage from OA and prevented chondrocyte apoptosis in surgically-induced OA joints. Furthermore, B3 administration prevented heterotopic cartilage formation near the surgical region. iNOS protein expression was enhanced in the synovial tissues and the pseudocapsule around the surgical region in OA mice fed a control diet, but was reduced in mice that received B3. Together, these data indicated that in the OA model, B3 prevented OA progression and heterotopic cartilage formation, at least in a part through the suppression of iNOS. These results support the potential therapeutic benefits of B3 for treatment of human OA and heterotopic ossification.

Sirtuin 6 (Sirt6) is a mammalian homologue of NAD 1 -dependent histone deacetylase Sir2. Although Sirt62/2 mice exhibit growth retardation, the role of Sirt6 in cartilage metabolism is unclear. The aim of this study was to investigate the Sirt6 signaling pathway in cartilage metabolism. Immunohistological evaluation of the tibial growth plate in Sirt62/2 mice exhibited impaired proliferation and differentiation of chondrocytes, reduced expression of Indian hedgehog (Ihh), and a senescent phenotype. When Sirt6 was knocked down in chondrocytes in vitro, expression of Ihh and its downstream genes were reduced. Impaired differentiation by Sirt6 silencing was completely rescued by administration of a Hh signal agonist. When sirtuins were activated, chondrocyte differentiation was enhanced together with activation of Ihh signal, and these effects were abrogated by Sirt6 silencing. ChIP assay revealed the affinity of ATF4 to the Ihh promoter was markedly decreased by Sirt6 knockdown. These data indicate Sirt6 directly controls proliferation and differentiation of chondrocytes.T he stress-response and chromatin-silencing factor Sir2, a yeast sirtuin, is a NAD 1 -dependent histone deacetylase and is involved in various nuclear actions 1 . Among the seven mammalian sirtuin family members, sirtuin 6 (Sirt6) is localized to the nucleus and is involved in transcriptional silencing, genome stability, and longevity 2,3 . Sirt6 was originally identified as an ADP-ribosyltransferase 4 . Recent studies have demonstrated that the Sirt6 protein is a NAD1-dependent histone 3 deacetylase that targets acetyl-H3K9 and acetyl-H3K56 5,6 . Sirt6 deacetylates histone H3 lysine 9 (H3K9) on telomeres and plays a role in their stability 5 . Human Sirt6 also deacetylates C-terminal binding protein interacting protein (CtIP) and promotes DNA end resection 7 . Recent studies have revealed multiple functions of Sirt6 in the regulation of inflammation and metabolism. Sirt6 inhibits inflammation by suppressing NF-kB target molecules via interaction with the RelA subunit of NF-kB and by deacetylating H3K9 at target promoters 8 . In glycometabolism, Sirt6 functions as a corepressor of the transcription factor Hif1a and inhibits glycolysis through suppression of Hif1a activity 9 . As a result, Sirt6 null mice die before 4 weeks of age due to lethal hypoglycemia 4 .Sirt62/2 mice develop normally for the first two weeks except for reduced body size, which is apparent early after birth 4 . Sirt62/2 mice also exhibit features of premature aging, such as osteopenia and lordokyphosis 4 . Serum insulin-like growth factor 1 (IGF-1) and glucose levels are both markedly reduced in Sirt6 2/2 mice by the age of 24 days 4 . A recent study demonstrated that neural-specific deletion of Sirt6 in mice leads to postnatal growth retardation due to somatotropic attenuation through low growth hormone (GH) and IGF-1 levels 10 . Thus, Sirt6 contributes to skeletal growth in part through the regulation of IGF-1 secretion. However, the local functions of Sirt6 in postnatal gr...