Gremlin-1 and BMP-4 Overexpressed in Osteoarthritis Drive an Osteochondral-Remodeling Program in Osteoblasts and Hypertrophic Chondrocytes.

Osteoarthritis (OA) is a whole joint disease characterized by an important remodeling of the osteochondral junction. It includes cartilage mineralization due to chondrocyte hypertrophic differentiation and bone sclerosis. Here, we investigated whether gremlin-1 (Grem-1) and its BMP partners could be involved in the remodeling events of the osteochondral junction in OA. We found that Grem-1, BMP-2, and BMP-4 immunostaining was detected in chondrocytes from the deep layer of cartilage and in subchondral bone of knee OA patients, and was positively correlated with cartilage damage. ELISA assays showed that bone released more Grem-1 and BMP-4 than cartilage, which released more BMP-2. In vitro experiments evidenced that compression stimulated the expression and the release of Grem-1 and BMP-4 by osteoblasts. Grem-1 was also overexpressed during the prehypertrophic to hypertrophic differentiation of murine articular chondrocytes. Recombinant Grem-1 stimulated Mmp-3 and Mmp-13 expression in murine chondrocytes and osteoblasts, whereas recombinant BMP-4 stimulated the expression of genes associated with angiogenesis (Angptl4 and osteoclastogenesis (Rankl and Ccl2). In conclusion, Grem-1 and BMP-4, whose expression at the osteochondral junction increased with OA progression, may favor the pathological remodeling of the osteochondral junction by inducing a catabolic and tissue remodeling program in hypertrophic chondrocytes and osteoblasts.

The nuclear factor-erythroid 2-related factor/heme oxygenase-1 axis is critical for the inflammatory features of type 2 diabetes-associated osteoarthritis.

Epidemiological findings support the hypothesis that type 2 diabetes mellitus (T2DM) is a risk factor for osteoarthritis (OA). Moreover, OA cartilage from patients with T2DM exhibits a greater response to inflammatory stress, but the molecular mechanism is unclear. To investigate whether the antioxidant defense system participates in this response, we examined here the expression of nuclear factor-erythroid 2-related factor (Nrf-2), a master antioxidant transcription factor, and of heme oxygenase-1 (HO-1), one of its main target genes, in OA cartilage from T2DM and non-T2DM patients as well as in murine chondrocytes exposed to high glucose (HG). Ex vivo experiments indicated that Nrf-2 and HO-1 expression is reduced in T2DM versus non-T2DM OA cartilage (0.57-fold Nrf-2 and 0.34-fold HO-1), and prostaglandin E2 (PGE2) release was increased in samples with low HO-1 expression. HG-exposed, IL-1ß-stimulated chondrocytes had lower Nrf-2 levels in vitro, particularly in the nuclear fraction, than chondrocytes exposed to normal glucose (NG). Accordingly, HO-1 levels were also decreased (0.49-fold) in these cells. The HO-1 inducer cobalt protoporphyrin IX more efficiently attenuated PGE2 and IL-6 release in HG+IL-1ß-treated cells than in NG+IL-1ß-treated cells. Greater reductions in HO-1 expression and increase in PGE2/IL-6 production were observed in HG+IL-1ß-stimulated chondrocytes from Nrf-2-/- mice than in chondrocytes from wild-type mice. We conclude that the Nrf-2/HO-1 axis is a critical pathway in the hyperglucidic-mediated dysregulation of chondrocytes. Impairments in this antioxidant system may explain the greater inflammatory responsiveness of OA cartilage from T2DM patients and may inform treatments of such patients.

Knee and hip intra-articular adipose tissues (IAATs) compared with autologous subcutaneous adipose tissue: a specific phenotype for a central player in osteoarthritis.

OBJECTIVES: Compared with subcutaneous adipose tissue (SCAT), infrapatellar fat pad (IFP), the main knee intra-articular adipose tissue (IAAT), has an inflammatory phenotype in patients with osteoarthritis (OA). We phenotyped suprapatellar fat pad (SPFP) and hip acetabular fat pad (AFP), two other IAATs, to determinate the unique signature of IAATs compared with SCAT. METHODS: IFP, SPFP, AFP and autologous SCAT were obtained from patients with OA during total knee (n=38) or hip replacement (n=5). Fibrosis and adipocyte area were analysed by histology and vascularisation, leucocyte and mast cell infiltration were analysed by immunohistochemistry for von Willebrand factor, leucocytes and tryptase, respectively. Secretion of interleukin (IL)-6, IL-8 and prostaglandin E2 (PGE2) was assessed by ELISA. The mRNA expression of adipocyte-associated genes (ATGL, LPL, PPAR-γ, FABP4 and CD36) and developmental genes (SFRP2, HoxC9 and EN1) was determined. The inflammatory response of isolated fibroblast-like synoviocytes (FLS) to autologous IFP and SPFP conditioned media was examined. RESULTS: Fibrosis, vascularisation and leucocyte and mast cell infiltration were greater in IAATs than SCAT, and levels of IL-6, IL-8 and PGE2 were greater in all IAATs than SCAT. IFP and SPFP induced a similar inflammatory response to FLS. Adipocyte area was smaller in IAATs than SCAT. Adipocyte-associated and developmental genes showed a similar gene expression pattern in all IAATs, different from SCAT. CONCLUSIONS: IFP but also SPFP and AFP (gathered under the term 'IAAT') may play a deleterious role in OA by affecting joint homeostasis because of their inflammatory phenotype and their close interaction with synovium in the same functional unit.

Inhibition of matrix metalloproteinase-3 and -13 synthesis induced by IL-1beta in chondrocytes from mice lacking microsomal prostaglandin E synthase-1.

Joint destruction in arthritis is in part due to the induction of matrix metalloproteinase (MMP) expression and their inhibitors, especially MMP-13 and -3, which directly degrade the cartilage matrix. Although IL-1ß is considered as the main catabolic factor involved in MMP-13 and -3 expression, the role of PGE(2) remains controversial. The goal of this study was to determine the role of PGE(2) on MMP synthesis in articular chondrocytes using mice lacking microsomal PGE synthase-1 (mPGES-1), which catalyses the rate-limiting step of PGE(2) synthesis. MMP-3 and MMP-13 mRNA and protein expressions were assessed by real-time RT-PCR, immunoblotting, and ELISA in primary cultures of articular chondrocytes from mice with genetic deletion of mPGES-1. IL-1ß-induced PGE(2) synthesis was dramatically reduced in mPGES-1(-/-) and mPGES-1(+/-) compared with mPGES-1(+/+) chondrocytes. A total of 10 ng/ml IL-1ß increased MMP-3 and MMP-13 mRNA, protein expression, and release in mPGES-1(+/+) chondrocytes in a time-dependent manner. IL-1ß-induced MMP-3 and MMP-13 mRNA expression, protein expression, and release decreased in mPGES-1(-/-) and mPGES-1(+/-) chondrocytes compared with mPGES-1(+/+) chondrocytes from 8 up to 24 h. Otherwise, MMP inhibition was partially reversed by addition of 10 ng/ml PGE(2) in mPGES-1(-/-) chondrocytes. Finally, in mPGES-1(-/-) chondrocytes treated by forskolin, MMP-3 protein expression was significantly decreased compared with wild-type, suggesting that PGE(2) regulates MMP-3 expression via a signaling pathway dependent on cAMP. These results demonstrate that PGE(2) plays a key role in the induction of MMP-3 and MMP-13 in an inflammatory context. Therefore, mPGES-1 could be considered as a critical target to counteract cartilage degradation in arthritis.

Alpha-7 Nicotinic Receptor Dampens Murine Osteoblastic Response to Inflammation and Age-Related Osteoarthritis.

Introduction: Osteoarthritis (OA) is a whole-joint disease characterized by a low-grade inflammation that is involved in both cartilage degradation and subchondral bone remodeling. Since subchondral bone has a cholinergic innervation and that acetylcholine (Ach) might have an anti-inflammatory effect through the α7 nicotinic Ach receptor (α7nAchR), we aimed (i) to determine the expression of non-neuronal cholinergic system and nicotinic receptor subunits by murine and human osteoblasts, (ii) to address the role of α7nAchR in osteoblastic response to inflammation, and (iii) to study the role of α7nAchR in a spontaneous aging OA model. Methods: Primary cultures of WT and α7nAchR knock-out mice (Chrna7-/-) murine osteoblasts and of subchondral bone human OA osteoblasts were performed. The expressions of the non-neuronal cholinergic system and of the nAchR subunits were assessed by PCR. In vitro, IL1ß-stimulated WT, Chrna7-/-, and human osteoblasts were pretreated with nicotine. At 24 h, expressions of interleukin-6 (IL6) and metalloproteinase-3 and -13 (MMP), RANK-ligand (RANKL), and osteoprotegerin (OPG) were quantified by qPCR and ELISA. Spontaneous aging OA was evaluated and compared between male WT and Chrna7-/- mice of 9 and 12 months. Results: Murine WT osteoblasts express the main components of the cholinergic system and α7 subunit composing α7nAchR. Nicotine partially prevented the IL1ß-induced expression and production of IL6, MMP3, and RANKL in WT osteoblasts. The effect for IL6 and MMP was mediated by α7nAchR since nicotine had no effect on Chrna7-/- osteoblasts while the RANKL decrease persisted. Chrna7-/- mice displayed significantly higher cartilage lesions than their WT counterparts at 9 and 12 months, without difference in subchondral bone remodeling. Human OA osteoblasts also expressed the non-neuronal cholinergic system and α7 subunit as well as CHRFAM7A, the dominant negative duplicate of Chrna7. Nicotine pretreatment did not significantly reduce IL6 and MMP3 production in IL-1ß-stimulated human osteoarthritic osteoblasts (n = 4), possibly due to CHRFAM7A. Conclusion: Cholinergic system counteracts murine osteoblastic response to IL-1ß through α7nAchR. Since α7nAchR deletion may limit cartilage degradation during murine age-related OA, enhancing cholinergic system could be a new therapeutic target in OA but may depend on CHRFAM7A expression.

The Role of the Non-neuronal Cholinergic System in Inflammation and Degradation Processes in Osteoarthritis.

OBJECTIVE: The non-neuronal cholinergic system represents non-neuronal cells that have the biochemical machinery to synthetize de novo and/or respond to acetylcholine (ACh). We undertook this study to investigate this biochemical machinery in chondrocytes and its involvement in osteoarthritis (OA). METHODS: Expression of the biochemical machinery for ACh metabolism and nicotinic ACh receptors (nAChR), particularly α7-nAChR, in human OA and murine chondrocytes was determined by polymerase chain reaction and ligand-binding. We investigated the messenger RNA expression of the human duplicate α7-nACh subunit, called CHRFAM7A, which is responsible for truncated α7-nAChR. We assessed the effect of nAChR on chondrocytes activated by interleukin-1ß (IL-1ß) and the involvement of α7-nAChR using chondrocytes from wild-type (WT) and α7-deficient Chrna7-/- mice. The role of α7-nAChR in OA was explored after medial meniscectomy in WT and Chrna7-/- mice. RESULTS: Human and murine chondrocytes express the biochemical partners of the non-neuronal cholinergic system and a functional α7-nAChR at their cell surface (n = 5 experiments with 5 samples each). The expression of CHRFAM7A in human OA chondrocytes (n = 23 samples) correlated positively with matrix metalloproteinase 3 (MMP-3) (r = 0.38, P < 0.05) and MMP-13 (r = 0.48, P < 0.05) expression. Nicotine decreased the IL-1ß-induced IL-6 and MMP expression, in a dose-dependent manner, in WT chondrocytes but not in Chrna7-/- chondrocytes. Chrna7-/- mice that underwent meniscectomy (n = 7) displayed more severe OA cartilage damage (mean ± SD Osteoarthritis Research Society International [OARSI] score 4.46 ± 1.09) compared to WT mice that underwent meniscectomy (n = 9) (mean ± SD OARSI score 3.05 ± 0.9; P < 0.05). CONCLUSION: The non-neuronal cholinergic system is functionally expressed in chondrocytes. Stimulation of nAChR induces antiinflammatory and anticatabolic activity through α7-nAChR, but the anticatabolic activity may be mitigated by truncated α7-nAChR in human chondrocytes. In vivo experiments strongly suggest that α7-nAChR has a protective role in OA.

Contribution of adipocyte precursors in the phenotypic specificity of intra-articular adipose tissues in knee osteoarthritis patients.

BACKGROUND: Intra-articular adipose tissues (IAATs) are involved in osteoarthritis (OA) pathophysiology. We hypothesize that mesenchymal cells residing in IAATs may account for the specific inflammatory and metabolic patterns in OA patients. METHODS: Adipocyte precursors (preadipocytes and dedifferentiated fat cells (DFATc)) from IAATs (infrapatellar and suprapatellar fat pads) and autologous subcutaneous adipose tissues (SCATs) were isolated from knee OA patients. The ability of these precursors to differentiate into adipocytes was assessed by oil red O staining after 14 days of culture in adipogenic medium. The gene expression of adipocyte-related transcription factors (C/EBP-α and PPAR-γ) and development-related factors (EN1 and SFRP2) were analyzed. The inflammatory pattern was assessed by RT-qPCR and ELISA (interleukin 6 (IL-6), IL-8, Cox2, and prostaglandin E2 (PGE2)) after a 24-h stimulation by IL-1ß (1 ng/mL) and by conditioned medium from OA synovium. RESULTS: IAAT preadipocytes displayed a significantly higher ability to differentiate into adipocytes and expressed significantly more C/EBP-α mRNA than SCAT preadipocytes. IAAT preadipocytes expressed significantly less EN-1 and SFRP2 mRNA than SCAT preadipocytes. Unstimulated IAAT preadipocytes displayed a less inflammatory pattern (IL-6, IL-8, and Cox2/PGE2) than SCAT preadipocytes. In contrast, the response of IAAT preadipocytes to an inflammatory stimulus (IL-1ß and conditioned media of OA synovium) was exacerbated compared to that of SCAT preadipocytes. Similar results were obtained with DFATc. CONCLUSION: IAAT adipocyte precursors from OA patients have a specific phenotype, which may account for the unique phenotype of OA IAATs. The exacerbated response of IAAT preadipocytes to inflammatory stimulation may contribute to OA pathophysiology.

Mechanical stress and prostaglandin E2 synthesis in cartilage.

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE2) is one of the main catabolic factors involved in OA in which metalloproteinase (MMP) is crucial for cartilage degradation. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy-prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE2. Among the isoforms described, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. We investigated the regulation of the COX, PGES and 15-PGDH and MMP-2, MMP-9 and MMP-13 genes by mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) from 2 to 24 h. After determination of the PGE2 release in the media, mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blot respectively. Mechanical compression of cartilage explants significantly increased PGE2 production in a time dependent manner. This was not due to the synthesis of IL-1, since pretreatment with IL1-Ra did not alter the PGE2 synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE2 synthesis after 18 hours suggesting that PGE2 synthesis could be altered by the induction of 15-PGDH expression. MAPK are involved in signaling, since specific inhibitors partially inhibited COX-2 and mPGES-1 expressions. Lastly, compression induced MMP-2, -9, -13 mRNA expressions in cartilage. We conclude that dynamic compression induces pro-inflammatroy mediators release and matrix degradating enzymes synthesis. Notably, compression increases mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.

Induction of nerve growth factor expression and release by mechanical and inflammatory stimuli in chondrocytes: possible involvement in osteoarthritis pain.

INTRODUCTION: Nerve growth factor (NGF) level is increased in osteoarthritis (OA) joints and is involved in pain associated with OA. Stimuli responsible for NGF stimulation in chondrocytes are unknown. We investigated whether mechanical stress and proinflammatory cytokines may influence NGF synthesis by chondrocytes. METHODS: Primary cultures of human OA chondrocytes, newborn mouse articular chondrocytes or cartilage explants were stimulated by increasing amounts of IL-1ß, prostaglandin E2 (PGE2), visfatin/nicotinamide phosphoribosyltransferase (NAMPT) or by cyclic mechanical compression (0.5 Hz, 1 MPa). Before stimulation, chondrocytes were pretreated with indomethacin, Apo866, a specific inhibitor of NAMPT enzymatic activity, or transfected by siRNA targeting visfatin/NAMPT. mRNA NGF levels were assessed by real-time quantitative PCR and NGF released into media was determined by ELISA. RESULTS: Unstimulated human and mouse articular chondrocytes expressed low levels of NGF (19.2 ± 8.7 pg/mL, 13.5 ± 1.0 pg/mL and 4.4 ± 0.8 pg/mL/mg tissue for human and mouse articular chondrocytes and costal explants, respectively). Mechanical stress induced NGF release in conditioned media. When stimulated by IL-1ß or visfatin/NAMPT, a proinflammatory adipokine produced by chondocytes in response to IL-1ß, a dose-dependent increase in NGF mRNA expression and NGF release in both human and mouse chondrocyte conditioned media was observed. Visfatin/NAMPT is also an intracellular enzyme acting as the rate-limiting enzyme of the generation of NAD. The expression of NGF induced by visfatin/NAMPT was inhibited by Apo866, whereas IL-1ß-mediated NGF expression was not modified by siRNA targeting visfatin/NAMPT. Interestingly, PGE2, which is produced by chondrocytes in response to IL-1ß and visfatin/NAMPT, did not stimulate NGF production. Consistently, indomethacin, a cyclooxygenase inhibitor, did not counteract IL-1ß-induced NGF production. CONCLUSIONS: These results show that mechanical stress, IL-1ß and extracellular visfatin/NAMPT, all stimulated the expression and release of NGF by chondrocytes and thus suggest that the overexpression of visfatin/NAMPT and IL-1ß in the OA joint and the increased mechanical loading of cartilage may mediate OA pain via the stimulation of NGF expression and release by chondrocytes.

Induction of an inflammatory and prodegradative phenotype in autologous fibroblast-like synoviocytes by the infrapatellar fat pad from patients with knee osteoarthritis.

OBJECTIVE: The infrapatellar fat pad (IFP) of the knee joint has an inflammatory phenotype in osteoarthritis (OA). Its close proximity to the synovial membrane suggests that the IFP could be involved in the induction of OA synovitis. This study was undertaken to investigate the response of fibroblast-like synoviocytes (FLS) to autologous IFP and subcutaneous adipose tissue (SCAT) from patients with severe knee OA. METHODS: Samples of IFP, SCAT, and autologous synovial membrane tissue close to the IFP were harvested during surgery from 28 patients with end-stage knee OA. FLS from 14 patients were stimulated with autologous IFP- or SCAT-conditioned medium, and levels of messenger RNA (mRNA) expression and protein release of interleukin-6 (IL-6), IL-8, secretory phospholipase A2 (sPLA2 ), cytosolic PLA2 , cyclooxygenase 2 (COX-2), microsomal prostaglandin E synthase, prostaglandin E2 (PGE2 ), and matrix metalloproteinases (MMPs) 1, 3, 9, and 13 were evaluated. Both IFP- and SCAT-conditioned medium were evaluated by enzyme-linked immunosorbent assay for secretion of IL-6, soluble IL-6 receptor (sIL-6R), IL-8, tumor necrosis factor α (TNFα), PGE2 , IL-1ß, and interferon-γ. In addition, OA FLS were treated with PGE2 receptor antagonists to evaluate the contribution of IFP-derived PGE2 to the inflammatory response of FLS to the IFP. RESULTS: Stimulation of OA FLS with IFP-conditioned medium induced the mRNA expression and protein release of IL-6, IL-8, sPLA2 , COX-2, PGE2 , and MMPs 1, 3, 9, and 13. The extent of stimulation was consistently stronger with IFP-conditioned medium than with SCAT-conditioned medium. Moreover, secretion of IL-6, sIL-6R, IL-8, TNFα, and PGE2 was greater in IFP-conditioned medium than in SCAT-conditioned medium, especially PGE2 , whose secretion was 75-fold stronger in IFP-conditioned medium (P < 0.0001). PGE2 receptor antagonists dose-dependently inhibited the release of IL-6, IL-8, and PGE2 by IFP-stimulated FLS. CONCLUSION: This study showed that the IFP has a potential role in the induction of synovial inflammation in patients with severe knee OA. Furthermore, secretion of PGE2 by the IFP may be involved in the OA inflammatory process.