Nicolas Duval

Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes

BackgroundMMP-13 and IGFBP-5 are important factors involved in osteoarthritis (OA). We investigated whether two highly predicted microRNAs (miRNAs), miR-140 and miR-27a, regulate these two genes in human OA chondrocytes.MethodsGene expression was determined by real-time PCR. The effect of each miRNA on IGFBP-5 and MMP-13 expression/production was evaluated by transiently transfecting their precursors (pre-miRNAs) and inhibitors (anti-miRNAs) into human OA chondrocytes. Modulation of IGFBP-5, miR-140 and miR-27a expression was determined upon treatment of OA chondrocytes with cytokines and growth factors.ResultsIGFBP-5 was expressed in human chondrocytes with its level significantly lower (p < 0.04) in OA. Five computational algorithms identified miR-140 and miR-27a as possible regulators of MMP-13 and IGFBP-5 expression. Data showed that both miRNAs were expressed in chondrocytes. There was a significant reduction (77%, p < 0.01) in miR-140 expression in OA compared to the normal chondrocytes, whereas miR-27a expression was only slightly decreased (23%). Transfection with pre-miR-140 significantly decreased (p = 0.0002) and with anti-miR-140 significantly increased (p = 0.05) IGFBP-5 expression at 24 hours, while pre-miR-27a did not affect either MMP-13 or IGFBP-5. Treatment with anti-miR-27a, but not with anti-miR-140, significantly increased the expression of both MMP-13 (p < 0.05) and IGFBP-5 (p < 0.01) after 72 hours of incubation. MMP-13 and IGFBP-5 protein production followed the same pattern as their expression profile. These data suggest that IGFBP-5 is a direct target of miR-140, whereas miR-27a down-regulates, likely indirectly, both MMP-13 and IGFBP-5.ConclusionThis study is the first to show the regulation of these miRNAs in human OA chondrocytes. Their effect on two genes involved in OA pathophysiology adds another level of complexity to gene regulation, which could open up novel avenues in OA therapeutic strategies.

Histone deacetylase inhibitors suppress interleukin-1β-induced nitric oxide and prostaglandin E2 production in human chondrocytes

OBJECTIVE Overproduction of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) plays an important role in the pathogenesis of osteoarthritis (OA). In the present study, we determined the effect of trichostatin A (TSA) and butyric acid (BA), two histone deacetylase (HDAC) inhibitors, on NO and PGE(2) synthesis, inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 expression, and nuclear factor (NF)-kappaB DNA-binding activity, in interleukin-1beta (IL-1)-stimulated human OA chondrocytes, and on IL-1-induced proteoglycan degradation in cartilage explants. METHODS Chondrocytes were stimulated with IL-1 in the absence or presence of increasing concentrations of TSA or BA. The production of NO and PGE(2) was evaluated using Griess reagent and an enzyme immunoassay, respectively. The expression of iNOS and COX-2 proteins and mRNAs was evaluated using Western blotting and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. Proteoglycan degradation was measured with dimethymethylene blue assay. Electrophoretic mobility shift assay (EMSA) was utilized to analyze the DNA-binding activity of NF-kappaB. RESULTS HDAC inhibition with TSA or BA resulted in a dose-dependent inhibition of IL-1-induced NO and PGE(2) production. IL-17- and tumor necrosis factor-alpha (TNF-alpha)-induced NO and PGE(2) production was also inhibited by TSA and BA. This inhibition correlated with the suppression of iNOS and COX-2 protein and mRNA expression. TSA and BA also prevented IL-1-induced proteoglycan release from cartilage explants. Finally, we demonstrate that the DNA-binding activity of NF-kappaB, was induced by IL-1, but was not affected by treatment with HDAC inhibitors. CONCLUSIONS These data indicate that HDAC inhibitors suppressed IL-1-induced NO and PGE(2) synthesis, iNOS and COX-2 expression, as well as proteoglycan degradation. The suppressive effect of HDAC inhibitors is not due to impaired DNA-binding activity of NF-kappaB. These findings also suggest that HDAC inhibitors may be of potential therapeutic value in the treatment of OA.

Patient satisfaction needs as related to knee stability and objective findings after ACL reconstruction using the LARS artificial ligament

The purposes of this study are to compare patient satisfaction with the objective measurement of knee stability and assess early complications following ACL reconstruction using a LARS artificial ligament. Forty-seven patients were reviewed 8-45 months after surgery. Assessment was made by the Knee and Osteoarthritis Outcome Score for patient satisfaction, a modified International Knee Documentation Committee form for clinical knee stability, and a Telos stress radiography for PA stability. Complications were assessed at interview and were double-checked with charts. The LARS artificial ligament may be a safe device to reconstruct an ACL tear. Documenting mechanical stability of the knee is inadequate when reporting follow-up studies and a questionnaire assessing patient satisfaction should be added to provide a better picture of the outcome and results.

Abnormal regulation of urokinase plasminogen activator by insulin-like growth factor 1 in human osteoarthritic subchondral osteoblasts.

OBJECTIVE Subchondral bone sclerosis is a common feature of osteoarthritis (OA), but the mechanisms responsible for this condition remain unresolved. We investigated the role of insulin-like growth factor 1 (IGF-1) and urokinase plasminogen activator (uPA) in human osteoblasts from subchondral bone obtained from the tibial plateaus of OA patients and normal individuals. METHODS Primary in vitro osteoblasts were prepared from subchondral bone specimens obtained from OA patients at surgery and from normal individuals at autopsy. Levels of uPA and PA inhibitor 1 (PAI-1) levels were determined under basal conditions and after IGF-1 stimulation in conditioned media from osteoblasts by enzyme-linked immunosorbent assay. The activity of uPA was evaluated by specific substrate hydrolysis and zymography under basal conditions and after plasminogen stimulation, in the presence and absence of added IGF-1. Plasmin activity was also evaluated by specific substrate hydrolysis. RESULTS Levels of uPA released by OA osteoblasts were significantly higher than normal. Addition of IGF-1 to osteoblasts significantly reduced uPA protein levels only in OA patients (P < 0.05). In contrast, the addition of uPA to osteoblasts did not modify IGF-1 levels in either normal or OA osteoblasts. Basal uPA activity was higher in OA than in normal osteoblasts. Interestingly, IGF-1 enhanced basal uPA activity in OA specimens in a dose-dependent manner. Addition of plasminogen promoted uPA activity in both normal and OA osteoblasts via a positive feedback loop due to plasmin generation, since this activity was inhibited by both PAI-1 and alpha2-antiplasmin. Unexpectedly, incubation with IGF-1 inhibited this positive feedback of plasminogen-dependent uPA activity in OA osteoblasts, but not in normal osteoblasts, in a dose-dependent manner. Hence, normal osteoblasts were relatively insensitive to IGF-1, whereas the same treatment reduced both uPA levels and plasminogen-dependent uPA activity in OA osteoblasts while it increased basal uPA activity in OA osteoblasts. This could not be explained by PAI-1 protein levels, which were similar in normal and OA osteoblasts in the presence and absence of IGF-1. IGF-1 also reduced plasmin activity in OA osteoblasts while it did not modify this activity in normal osteoblasts. CONCLUSION These results suggest that in OA osteoblasts, the uPA/plasmin system functions normally, yet IGF-1 inhibits the positive feedback of plasmin on uPA activity. This inhibition may contribute to abnormal IGF-1- and uPA-dependent bone remodeling, ultimately leading to abnormal bone sclerosis in OA.

Comparison of 2 Surgical Techniques of Posterolateral Corner Reconstruction of the Knee

Background Various surgical techniques to treat posterolateral knee instability have been described. To date, the recommended treatment is an anatomical form of reconstruction, in which the 3 key structures of the posterolateral corner are addressed: the lateral collateral ligament, the popliteofibular ligament, and the popliteus tendon. Hypothesis Two methods of surgical reconstruction will restore posterolateral knee instability, in terms of static laxity as well as dynamic 6 degrees of freedom kinematics, to statistically significant levels compared with the intact state. Study Design Controlled laboratory study. Methods Two surgical techniques (A and B) were used to reconstruct the posterolateral structures in 10 cadaveric knees. Static tests were performed on the intact, sectioned, and reconstructed knees at 30° and 90° of flexion for anterior-posterior laxity and external rotational laxity, as well as at 0° and 30° of flexion for varus laxity; dynamic 6 degrees of freedom kinematic testing, through a path of motion from 90° of flexion to full extension, was also performed. Results For the static varus tests, external rotation and varus laxity were significantly increased after the posterolateral structures were cut. Both reconstruction techniques restored external rotation and varus laxity to levels not significantly different from the intact state. For technique B, dynamic testing did not show any significant difference for all degrees of freedom kinematics compared with the intact state. However, for technique A, a significant internal tibial rotation was observed throughout the entire path of motion from 0° to 90° of knee flexion. Conclusions Both surgical techniques for anatomical posterolateral corner reconstruction showed good results in the static laxity tests. The anatomical reconstruction of all structures, including the popliteus tendon, resulted in an abnormal internal tibial rotation during dynamic testing.

Modulation of OPG, RANK and RANKL by human chondrocytes and their implication during osteoarthritis

OBJECTIVES Earlier studies suggest the involvement of osteoprotegerin (OPG), RANK and RANK ligand (RANKL) in OA subchondral bone metabolism; however, few studies have looked at their functional consequences on chondrocytes. We compared the expression/production of OPG, RANK and RANKL on human normal and OA chondrocytes, and evaluated, on OA chondrocytes, their modulation by some catabolic factors. Furthermore, the role of OPG and RANKL on the production of catabolic/anabolic factors was assessed. METHODS Expression was determined using real-time PCR, production of RANK and RANKL by flow cytometry and that of OPG by ELISA. Modulation of these factors was determined upon treatment with IL-1beta, TNF-alpha and PGE(2). The functional consequences were examined following treatment with soluble RANKL or OPG-Fc (OPG without the heparin-binding domain). RESULTS OPG, RANK and RANKL were expressed and produced by human chondrocytes. Membranous RANK was produced only by an OA chondrocyte subpopulation (29%) localized throughout the cartilage. The OPG/RANKL ratio was significantly (P = 0.05) reduced on the OA chondrocytes, whereas the RANK/RANKL ratio was significantly (P < 0.03) increased. OPG and membranous RANKL levels were significantly enhanced by IL-1beta, TNF-alpha and PGE(2), whereas membranous RANK was significantly increased only with IL-1beta. Administration of soluble RANKL had no effect on the OA chondrocytes. However, addition of OPG-Fc significantly stimulated MMP-13 (P = 0.05) and protease-activated receptor-2 (PAR-2) (P < 0.04) production. CONCLUSIONS Our findings showed that human chondrocytes express and produce OPG, RANK and RANKL. OA chondrocyte treatment with catabolic factors pointed towards an increased biological effect of OPG. Interestingly, OPG appears to be involved in OA progression by increasing two catabolic factors involved in cartilage pathophysiology.

Differential modulation of RANKL isoforms by human osteoarthritic subchondral bone osteoblasts: Influence of osteotropic factors

BACKGROUND Osteoarthritis (OA) is the most common human joint disease. Recent studies suggest that an abnormal subchondral bone metabolism is intimately involved in the genesis of this disease. Bone remodelling is tightly regulated by a molecular triad composed of OPG/RANK/RANKL. RANKL exists as 3 isoforms: RANKL1, 2, and 3. RANKL1 and 2 enhance osteoclastogenesis whereas RANKL3 inhibits this phenomenon. We previously reported that human OA subchondral bone osteoblasts can be discriminated into two subgroups according to their level of PGE2 [low (L) or high (H)]. Moreover, we also showed that L-OA osteoblasts express higher levels of total RANKL compared to H-OA osteoblasts. In this study, we investigated the level of membranous RANKL, comparing L- and H-OA subchondral bone osteoblasts, as well as its modulation by osteotropic factors. The impact of the modulation of RANKL1 and 3 on the membranous RANKL level was also studied. METHODS Gene expression was determined using real-time PCR for RANKL1 and semi-quantitative PCR for RANKL3. Membranous RANKL was measured by flow cytometry. The modulation of membranous RANKL and RANKL isoforms was monitored on the L- and H-OA osteoblasts and also following treatment with osteotropic factors, including vitamin D3 (50 nM), IL-1beta (100 pg/ml), TNF-alpha (5 ng/ml), PGE2 (500 nM), PTH (100 nM), IL-6 (10 ng/ml) and IL-17 (10 ng/ml). RESULTS Membranous RANKL levels were significantly increased in L-OA osteoblasts compared to normal (p<0.01) and H-OA (p<0.05). The gene expression level of the RANKL1 profile was reminiscent of the membranous RANKL level. Although RANKL3 gene expression was lower on the H-OA osteoblasts than on normal and L-OA osteoblasts (p<0.03), the overall outcome favoured RANKL1. Treatment with the tested factors showed a significant increase in membranous RANKL on the L-OA osteoblasts, with the exception of PTH and IL-17. Interestingly in this subpopulation, the RANKL3 gene expression level was significantly increased upon PTH and IL-17 treatment. No effect of the tested osteotropic factors was found on the H-OA. CONCLUSION Our findings showed that the normal, L- and H-OA subchondral bone osteoblasts differentially express membranous RANKL and RANKL isoforms, and that treatment with osteotropic factors generally favours increased membranous localization of RANKL on L-OA compared to H-OA osteoblasts. This phenomenon appears to take place through differential modulation of each RANKL isoform.

Activation of proteinase-activated receptor 2 in human osteoarthritic cartilage upregulates catabolic and proinflammatory pathways capable of inducing cartilage degradation: a basic science study

Proteinase-activated receptors (PARs) belong to a family of G protein-coupled receptors. PARs are activated by a serine-dependent cleavage generating a tethered activating ligand. PAR-2 was shown to be involved in inflammatory pathways. We investigated the in situ levels and modulation of PAR-2 in human normal and osteoarthritis (OA) cartilage/chondrocytes. Furthermore, we evaluated the role of PAR-2 on the synthesis of the major catabolic factors in OA cartilage, including metalloproteinase (MMP)-1 and MMP-13 and the inflammatory mediator cyclooxygenase 2 (COX-2), as well as the PAR-2-activated signalling pathways in OA chondrocytes. PAR-2 expression was determined using real-time reverse transcription-polymerase chain reaction and protein levels by immunohistochemistry in normal and OA cartilage. Protein modulation was investigated in OA cartilage explants treated with a specific PAR-2-activating peptide (PAR-2-AP), SLIGKV-NH2 (1 to 400 μM), interleukin 1 beta (IL-1β) (100 pg/mL), tumor necrosis factor-alpha (TNF-α) (5 ng/mL), transforming growth factor-beta-1 (TGF-β1) (10 ng/mL), or the signalling pathway inhibitors of p38 (SB202190), MEK1/2 (mitogen-activated protein kinase kinase) (PD98059), and nuclear factor-kappa B (NF-κB) (SN50), and PAR-2 levels were determined by immunohistochemistry. Signalling pathways were analyzed on OA chondrocytes by Western blot using specific phospho-antibodies against extracellular signal-regulated kinase 1/2 (Erk1/2), p38, JNK (c-jun N-terminal kinase), and NF-κB in the presence or absence of the PAR-2-AP and/or IL-1β. PAR-2-induced MMP and COX-2 levels in cartilage were determined by immunohistochemistry. PAR-2 is produced by human chondrocytes and is significantly upregulated in OA compared with normal chondrocytes (p < 0.04 and p < 0.03, respectively). The receptor levels were significantly upregulated by IL-1β (p < 0.006) and TNF-α (p < 0.002) as well as by the PAR-2-AP at 10, 100, and 400 μM (p < 0.02) and were downregulated by the inhibition of p38. After 48 hours of incubation, PAR-2 activation significantly induced MMP-1 and COX-2 starting at 10 μM (both p < 0.005) and MMP-13 at 100 μM (p < 0.02) as well as the phosphorylation of Erk1/2 and p38 within 5 minutes of incubation (p < 0.03). Though not statistically significant, IL-1β produced an additional effect on the activation of Erk1/2 and p38. This study documents, for the first time, functional consequences of PAR-2 activation in human OA cartilage, identifies p38 as the major signalling pathway regulating its synthesis, and demonstrates that specific PAR-2 activation induces Erk1/2 and p38 in OA chondrocytes. These results suggest PAR-2 as a potential new therapeutic target for the treatment of OA.

Inhibition of interleukin‐1β–induced matrix metalloproteinases 1 and 13 production in human osteoarthritic chondrocytes by prostaglandin D2

OBJECTIVE To investigate the effects of prostaglandin D2 (PGD2) on interleukin-1beta (IL-1beta)-induced matrix metalloproteinase 1 (MMP-1) and MMP-13 expression in human chondrocytes and the signaling pathways involved in these effects. METHODS Chondrocytes were stimulated with IL-1 in the presence or absence of PGD2, and expression of MMP-1 and MMP-13 proteins was evaluated by enzyme-linked immunosorbent assay. Messenger RNA (mRNA) expression and promoter activity were analyzed by real-time reverse transcription-polymerase chain reaction and transient transfections, respectively. The role of the PGD2 receptors D prostanoid receptor 1 (DP1) and chemoattractant receptor-like molecule expressed on Th2 cells (CRTH2) was evaluated using specific agonists and antibody-blocking experiments. The contribution of the cAMP/protein kinase A (PKA) pathway was determined using cAMP-elevating agents and PKA inhibitors. RESULTS PGD2 decreased in a dose-dependent manner IL-1-induced MMP-1 and MMP-13 protein and mRNA expression as well as their promoter activation. DP1 and CRTH2 were expressed and functional in chondrocytes. The effect of PGD2 was mimicked by BW245C, a selective agonist of DP1, but not by 13,14-dihydro-15-keto-PGD2, a selective agonist of CRTH2. Furthermore, treatment with an anti-DP1 antibody reversed the effect of PGD2, indicating that the inhibitory effect of PGD2 is mediated by DP1. The cAMP-elevating agents 8-Br-cAMP and forskolin suppressed IL-1-induced MMP-1 and MMP-13 expression, and the PKA inhibitors KT5720 and H89 reversed the inhibitory effect of PGD2, suggesting that the effect of PGD2 is mediated by the cAMP/PKA pathway. CONCLUSION PGD2 inhibits IL-1-induced production of MMP-1 and MMP-13 by chondrocytes through the DP1/cAMP/PKA signaling pathway. These data also suggest that modulation of PGD2 levels in the joint may have therapeutic potential in the prevention of cartilage degradation.

Activation of the receptor EphB4 by its specific ligand ephrin B2 in human osteoarthritic subchondral bone osteoblasts.

OBJECTIVE Abnormal subchondral bone metabolism is involved in osteoarthritis (OA). It has been suggested that ephrin B2 and its specific receptor EphB4 participate in bone homeostasis. We previously reported that human OA subchondral bone osteoblasts could be classified into 2 subpopulations: low (L), having proresorption properties, and high (H), having proformation properties. The purpose of this study was to investigate the importance of the ephrin system in OA subchondral bone osteoblasts. METHODS The presence of the EphB4 receptor was determined by immunohistochemistry, and its expression level, modulation upon treatment, and consequences of activation by ephrin B2 were determined by quantitative polymerase chain reaction. The effects of ephrin B2 activation of the EphB4 receptor on bone resorption activity were also determined. EphB4 receptor activation signaling pathways were investigated by specific enzyme-linked immunosorbent assay. RESULTS EphB4 receptors were present in subchondral bone osteoblasts and osteocytes. Compared with normal and H-OA osteoblasts, EphB4 receptor expression levels were significantly increased in L-OA osteoblasts, with no difference between normal and H-OA osteoblasts. EphB4 receptor levels in L-OA osteoblasts were significantly up-regulated by prostaglandin E2 (PGE2) and interleukin-17 (IL-17). Ephrin B2, PGE2, and IL-17 significantly inhibited bone resorption activity in these cells. EphB4 activation by ephrin B2 significantly inhibited the expression of IL-1beta, IL-6, matrix metalloproteinase 1 (MMP-1), MMP-9, MMP-13, and RANKL, but not MMP-2 and osteoprotegerin. EphB4 receptor activation significantly inhibited the phosphatidylinositol 3-kinase/Akt pathway. CONCLUSION This study is the first to provide evidence that EphB4 receptor activation by ephrin B2 in OA subchondral bone could affect abnormal metabolism in this tissue by inhibiting resorption factors and their activities. Ephrin B2 could be targeted as a specific therapeutic approach in the development of a disease-modifying OA drug.