A new ex vivo human model of osteoarthritis cartilage calcification.

OBJECTIVE: Cartilage pathologic calcification is a hallmark of osteoarthritis (OA). Here, we aimed to describe a new ex vivo human model to study the progression of cartilage calcification. METHOD: Cartilage explants (n = 11), as well as primary chondrocytes (n = 3), were obtained from OA patients undergoing knee replacement. Explants and chondrocytes were cultured in control (NT) or calcification (CM) medium (supplemented with ascorbic acid and ß-glycerophosphate). Calcification was evaluated by micro-CT scan at day 0 and 21 in explants, and by Alizarin red staining in chondrocyte monolayers. Raman spectrometry allowed characterization of the crystal type. Interleukin-6 (IL-6) secretion in explant and cell supernatants was measured by ELISA. Finally, matrix degradation was evaluated by Safranin-O staining of explant sections and by glycosaminoglycans (GAG) release in supernatants. RESULTS: Micro-CT scan showed calcifications in all explants at baseline (day 0), which in the CM group increased significantly in number and size after 21 days compared with the NT group. Raman spectrometry revealed that crystals were exclusively basic calcium phosphate crystals (carbonated hydroxyapatite) both in NT and CM. IL-6 secretion was significantly increased in calcifying conditions. Finally, CM significantly increased cartilage catabolism as assessed by decreased Safranin-O staining of tissue explants and increased GAG release in supernatants. CM effects (enhanced calcification, IL-6 secretion and proteoglycans turn-over) were recapitulated in vitro in OA chondrocytes. CONCLUSIONS: We have described a new ex vivo human model of cartilage calcification that can summurize the triad of events seen during osteoarthritis progression, i.e. calcification, inflammation, and cartilage degradation. This model will allow the identification of new anti-calcification compounds.

Relationships between Circulating Sclerostin, Bone Marrow Adiposity, Other Adipose Deposits and Lean Mass in Post-Menopausal Women.

Sclerostin is a Wnt signaling pathway inhibitor that negatively regulates bone formation. Bone-marrow-derived stromal cell (BMSC) differentiation is influenced by the Wnt pathway, leading to the hypothesis that higher levels of sclerostin might be associated with an increase in bone marrow adiposity (BMA). The main purpose of this study was to determine whether a relationship exists between circulating sclerostin and BMA in post-menopausal women with and without fragility fractures. The relationships between circulating sclerostin and body composition parameters were then examined. The outcomes measures included vertebral and hip proton density fat fraction (PDFF) using the water fat imaging (WFI) MRI method; DXA scans; and laboratory measurements, including serum sclerostin. In 199 participants, no significant correlations were found between serum sclerostin and PDFF. In both groups, serum sclerostin was correlated positively with bone mineral density (R = 0.27 to 0.56) and negatively with renal function (R = -0.22 to -0.29). Serum sclerostin correlated negatively with visceral adiposity in both groups (R = -0.24 to -0.32). Serum sclerostin correlated negatively with total body fat (R = -0.47) and appendicular lean mass (R = -0.26) in the fracture group, but not in the controls. No evidence of a relationship between serum sclerostin and BMA was found. However, serum sclerostin was negatively correlated with body composition components, such as visceral adiposity, total body fat and appendicular lean mass.

Raman microspectroscopy reveals unsaturation heterogeneity at the lipid droplet level and validates an in vitro model of bone marrow adipocyte subtypes.

Bone marrow adipocytes (BMAds) constitute the most abundant stromal component of adult human bone marrow. Two subtypes of BMAds have been described, the more labile regulated adipocytes (rBMAds) and the more stable constitutive adipocytes (cBMAds), which develop earlier in life and are more resilient to environmental and metabolic disruptions. In vivo, rBMAds are enriched in saturated fatty acids, contain smaller lipid droplets (LDs) and more readily provide hematopoietic support than their cBMAd counterparts. Mouse models have been used for BMAds research, but isolation of primary BMAds presents many challenges, and thus in vitro models remain the current standard to study nuances of adipocyte differentiation. No in vitro model has yet been described for the study of rBMAds/cBMAds. Here, we present an in vitro model of BM adipogenesis with differential rBMAd and cBMAd-like characteristics. We used OP9 BM stromal cells derived from a (C57BL/6xC3H)F2-op/op mouse, which have been extensively characterized as feeder layer for hematopoiesis research. We observed similar canonical adipogenesis transcriptional signatures for spontaneously-differentiated (sOP9) and induced (iOP9) cultures, while fatty acid composition and desaturase expression of Scd1 and Fads2 differed at the population level. To resolve differences at the single adipocyte level we tested Raman microspectroscopy and show it constitutes a high-resolution method for studying adipogenesis in vitro in a label-free manner, with resolution to individual LDs. We found sOP9 adipocytes have lower unsaturation ratios, smaller LDs and higher hematopoietic support than iOP9 adipocytes, thus functionally resembling rBMAds, while iOP9 more closely resembled cBMAds. Validation in human primary samples confirmed a higher unsaturation ratio for lipids extracted from stable cBMAd-rich sites (femoral head upon hip-replacement surgery) versus labile rBMAds (iliac crest after chemotherapy). As a result, the 16:1/16:0 fatty acid unsaturation ratio, which was already shown to discriminate BMAd subtypes in rabbit and rat marrow, was validated to discriminate cBMAds from rBMAd in both the OP9 model in vitro system and in human samples. We expect our model will be useful for cBMAd and rBMAd studies, particularly where isolation of primary BMAds is a limiting step.

Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur.

Osteoporosis is characterized by a bone loss associated to an increased bone marrow adiposity; however, it is still unclear what kind of lipids are involved. Therefore, the main purpose of this study was to see if there is any local bone lipid changes related to osteoporosis, by using the ovariectomy-induced osteoporosis (OVX) rat model. Female SD rats (operated at 6 months of age for skeletal maturity) were divided in control SHAM and OVX groups (n = 6/group) and maintained for 9 month post-surgery. Lipids were analyzed in two compartments of femoral diaphyses: bone marrow (BM) and mineralized tissue (MT), by chromatographic methods. As expected, osteoporotic femurs had a larger BM mass associated with a two-fold increase of lipid content. The MT had a similar lipid enrichment, indicating that adiposity affected the mineral part as well. The main lipids concerned were triglycerides, sphingomyelin, phosphatidylcholine and phosphatidylserine in BM, and triglycerides and cholesterol esters in MT. The increase of both energy-storage and membrane-associated lipids in BM suggested that cell number and/or size was enhanced to allow more triglyceride storage. Interestingly, in MT of osteoporotic femurs, sphingomyelin was decreased, suggesting that its catabolism could be linked to osteoporosis. In both femoral compartments, fatty acid profiles were enriched in 14:0 and 16:1, lowered in 18:0 and 20:4 n-6, and two-fold higher stearoyl-CoA desaturase indexes (16:1/16:0 and 18:1/18:0 ratios), suggesting an increased de novo lipogenesis in osteoporotic femurs. Thus, the present study is first to report local changes of individual lipids in rat osteoporotic femurs and suggests that osteoporosis is a pathologic condition associated with an enhanced de novo lipogenesis. Further studies will be needed to better understand the consequences of these lipid changes in osteoporotic bones.

Analysis of the interactome of Schistosoma mansoni histone deacetylase 8.

BACKGROUND: Histone deacetylase 8 from Schistosoma mansoni (SmHDAC8) is essential to parasite growth and development within the mammalian host and is under investigation as a target for the development of selective inhibitors as novel schistosomicidal drugs. Although some protein substrates and protein partners of human HDAC8 have been characterized, notably indicating a role in the function of the cohesin complex, nothing is known of the partners and biological function of SmHDAC8. METHODOLOGY/PRINCIPAL FINDINGS: We therefore employed two strategies to characterize the SmHDAC8 interactome. We first used SmHDAC8 as a bait protein in yeast two-hybrid (Y2H) screening of an S. mansoni cDNA library. This allowed the identification of 49 different sequences encoding proteins. We next performed co-immunoprecipitation (Co-IP) experiments on parasite extracts with an anti-SmHDAC8 antibody. Mass spectrometry (MS) analysis allowed the identification of 160 different proteins. CONCLUSIONS/SIGNIFICANCE: SmHDAC8 partners are involved in about 40 different processes, included expected functions such as the cohesin complex, cytoskeleton organization, transcriptional and translational regulation, metabolism, DNA repair, the cell cycle, protein dephosphorylation, proteolysis, protein transport, but also some proteasome and ribosome components were detected. Our results show that SmHDAC8 is a versatile deacetylase, potentially involved in both cytosolic and nuclear processes.

Chromatin regulation in schistosomes and histone modifying enzymes as drug targets.

Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.

Chromatin regulation in schistosomes and histone modifying enzymes as drug targets

Only one drug is currently available for the treatment and control of schistosomiasis and the increasing risk of selecting strains of schistosome that are resistant to praziquantel means that the development of new drugs is urgent. With this objective we have chosen to target the enzymes modifying histones and in particular the histone acetyltransferases and histone deacetylases (HDAC). Inhibitors of HDACs (HDACi) are under intense study as potential anti-cancer drugs and act via the induction of cell cycle arrest and/or apoptosis. Schistosomes like other parasites can be considered as similar to tumours in that they maintain an intense metabolic activity and rate of cell division that is outside the control of the host. We have shown that HDACi can induce apoptosis and death of schistosomes maintained in culture and have set up a consortium (Schistosome Epigenetics: Targets, Regulation, New Drugs) funded by the European Commission with the aim of developing inhibitors specific for schistosome histone modifying enzymes as novel lead compounds for drug development.