Minh Vu Chuong Nguyen

Evaluation of the structure‐modifying effects of diacerein in hip osteoarthritis: ECHODIAH, a three‐year, placebo‐controlled trial

OBJECTIVE To evaluate the ability of diacerein, an interleukin-1beta inhibitor, to slow the progressive decrease in joint space width observed in patients with hip osteoarthritis (OA). METHODS In this randomized, double-blind, placebo-controlled 3-year study, 507 patients with primary OA of the hip (by the American College of Rheumatology criteria) received diacerein (50 mg twice a day) or placebo. The minimal hip joint space width was measured by a central reader on yearly pelvic radiographs, using a 0.1-mm-graduated magnifying glass. RESULTS Baseline characteristics were comparable in the 2 treatment groups (255 patients receiving diacerein, 252 receiving placebo); 238 patients (47%) discontinued the study, mainly because of adverse events in the diacerein group (25% versus 12% with placebo) and because of inefficacy in the placebo group (14% versus 7% with diacerein). The percentage of patients with radiographic progression, defined by a joint space loss of at least 0.5 mm, was significantly lower in patients receiving diacerein than in patients receiving placebo, both in the intent-to-treat analysis and in the completer analysis (50.7% versus 60.4% [P = 0.036] and 47.3% versus 62.3% [P = 0.007], respectively). In those patients who completed 3 years of treatment, the rate of joint space narrowing was significantly lower with diacerein (mean +/- SD 0.18 +/- 0.25 mm/year versus 0.23 +/- 0.23 mm/year with placebo; P = 0.042). Diacerein had no evident effect on the symptoms of OA in this study. However, a post hoc covariate analysis that took into account the use of analgesics and antiinflammatory drugs showed an effect of diacerein on the Lequesne functional index. Diacerein was well tolerated during the 3-year study. The most frequent adverse events were transient changes in bowel habits. CONCLUSION This study confirms previous clinical findings indicating that the demonstration of a structure-modifying effect in hip OA is feasible, and shows, for the first time, that treatment with diacerein for 3 years has a significant structure-modifying effect as compared with placebo, coupled with a good safety profile. The clinical relevance of these findings requires further investigation.

Radiological progression of hip osteoarthritis: definition, risk factors and correlations with clinical status.

OBJECTIVES: To determine a cut off value for changes in radiological joint space width that allowed definition of radiological progression of hip osteoarthritis not related to measurement method errors and, thereafter, to determine factors predictive of radiological progression of hip osteoarthritis and to evaluate the correlations between clinical and radiological parameters. METHODS: A prospective, longitudinal (one year duration), multicentre study was made of patients with osteoarthritis of the hip (American College of Rheumatology criteria). Data on clinical activity (pain, functional impairment), demographic data (age, gender, body mass index), and femoral head migration (superolateral, superomedial, concentric) were collected when the patient entered the study; radiological grade (joint space width in millimetres at the narrowest point using a 0.1 mm graduated magnifying glass, evaluated by a single observer unaware of the chronology of the films) was recorded at the patients entry to the study and after one year. RESULTS: Analysis of the means of the differences between two analyses performed by a single observer of 30 pairs of radiographs (one performed after an interval of one year) (0.06 (SD 0.23)) suggested that a change of more than 0.56 mm (2 SD) after a one year follow up could define progression of osteoarthritis of the hip. Of the 508 patients recruited, 461 (91%) completed the one year follow up and radiological progression was observed in 102 (22%). The factors predictive of radiological progression that were identified in the multivariate analysis were: radiological joint space width at entry < or = 2 mm, superolateral migration of the femoral head, female gender, Lequesnes functional index > 10, age at entry > 65 years (odds ratios 2.11, 4.25, 2.51, 2.66, 1.90, respectively). The level of clinical parameters (pain, functional impairment) and the amount of symptomatic treatment required (non-steroidal anti-inflammatory drugs and analgesic intake) accounted for 20% (p < 0.0001) of the variability of the changes in radiological joint space width over the one year study period. CONCLUSION: These data suggest that radiological progression of hip osteoarthritis could be defined by a change in joint space width of at least 0.6 mm after a one year follow up period, is correlated with the changes in clinical status of the patients, and is related not only to demographic data (age, gender), but also to some specific characteristics of osteoarthritis (localisation, radiological severity, clinical activity).

MeCP2 is critical for maintaining mature neuronal networks and global brain anatomy during late stages of postnatal brain development and in the mature adult brain

Mutations in the X-linked gene, methyl-CpG binding protein 2 (Mecp2), underlie a wide range of neuropsychiatric disorders, most commonly, Rett Syndrome (RTT), a severe autism spectrum disorder that affects approximately one in 10,000 female live births. Because mutations in the Mecp2 gene occur in the germ cells with onset of neurological symptoms occurring in early childhood, the role of MeCP2 has been ascribed to brain maturation at a specific developmental window. Here, we show similar kinetics of onset and progression of RTT-like symptoms in mice, including lethality, if MeCP2 is removed postnatally during the developmental stage that coincides with RTT onset, or adult stage. For the first time, we show that brains that lose MeCP2 at these two different stages are actively shrinking, resulting in higher than normal neuronal cell density. Furthermore, we show that mature dendritic arbors of pyramidal neurons are severely retracted and dendritic spine density is dramatically reduced. In addition, hippocampal astrocytes have significantly less complex ramified processes. These changes accompany a striking reduction in the levels of several synaptic proteins, including CaMKII α/β, AMPA, and NMDA receptors, and the synaptic vesicle proteins Vglut and Synapsin, which represent critical modifiers of synaptic function and dendritic arbor structure. Importantly, the mRNA levels of these synaptic proteins remains unchanged, suggesting that MeCP2 likely regulates these synaptic proteins post-transcriptionally, directly or indirectly. Our data suggest a crucial role for MeCP2 in post-transcriptional regulation of critical synaptic proteins involved in maintaining mature neuronal networks during late stages of postnatal brain development.

Molecular markers of cartilage breakdown and synovitis at baseline as predictors of structural progression of hip osteoarthritis. The ECHODIAH* Cohort

Objective: To determine whether systemic markers of bone, cartilage, and synovium can predict structural progression of osteoarthritis (OA). Methods: Patients with painful hip OA were treated with diacerein or placebo in a multicentre, prospective, double blind, 3 year follow up trial. The following information was collected at entry: demographics, characteristics of hip OA, and 10 markers: N-propeptides of collagen types I and III, cartilage oligomeric matrix protein, YKL-40, hyaluronan (sHA), matrix metalloproteinases-1 and -3, C reactive protein, C-terminal crosslinking telopeptides of collagen types I and II (uCTX-II). Radiographs were obtained at entry and every year. Structural progression was defined as a joint space decrease ⩾0.5 mm or requirement for total hip replacement. Grouped survival analysis was performed with time to structural progression as dependent variable, and clinical data, radiographic findings, treatment groups (diacerein versus placebo), and markers as explanatory measures. Results: In the 333 patients in whom all markers were measured, high functional impairment, a joint space width <2 mm, and lateral migration of the femoral head at baseline increased the risk of progression, but diacerein had a protective effect (relative risk = 0.75; 95% confidence interval (CI) 0.54 to 0.96). In addition, patients in whom uCTX-II and sHA were in the upper tertile had a relative risk of progression of 3.73 (95% CI 2.48 to 5.61) compared with patients with markers in the two lower tertiles. Conclusion: In this large cohort, combined measurements of uCTX-II and sHA were a new predictor of the structural progression of hip OA.

Oligodendrocyte Lineage Cells Contribute Unique Features to Rett Syndrome Neuropathology

Mutations in the methyl-CpG binding protein 2 gene, Mecp2, affect primarily the brain and lead to a wide range of neuropsychiatric disorders, most commonly Rett syndrome (RTT). Although the neuropathology of RTT is well understood, the cellular and molecular mechanism(s), which lead to the disease initiation and progression, has yet to be elucidated. RTT was initially attributed only to neuronal dysfunction, but our recent studies and those of others show that RTT is not exclusively neuronal but rather also involves interactions between neurons and glia. Importantly, studies have shown that MeCP2-restored astrocytes and microglia are able to attenuate the disease progression in otherwise MeCP2-null mice. Here we show that another type of glia, oligodendrocytes, and their progenitors are also involved in manifestation of specific RTT symptoms. Mice that lost MeCP2 specifically in the oligodendrocyte lineage cells, although overall normal, were more active and developed severe hindlimb clasping phenotypes. Inversely, restoration of MeCP2 in oligodendrocyte lineage cells, in otherwise MeCP2-null mice, although only mildly prolonging their lifespan, significantly improved the locomotor deficits and hindlimb clasping phenotype, both in male and female mice, and fully restored the body weight in male mice. Finally, we found that the level of some myelin-related proteins was impaired in the MeCP2-null mice. Expression of MeCP2 in oligodendrocytes of these mice only partially restored their expression, suggesting that there is a non–cell-autonomous effect by other cell types in the brains on the expression of myelin-related proteins in oligodendrocytes.

Sex differences in hip osteoarthritis: results of a longitudinal study in 508 patients

Objective: To evaluate sex differences in the clinical and structural presentation, and natural history of hip OA. Methods: A multicentre, prospective, longitudinal, five year follow up study of 508 patients (302 women, 206 men, mean age 63 (7) years) with painful hip OA. Data collected were baseline demographics, symptomatic, therapeutic, and structural variables; symptomatic variables and changes in joint space width (JSW) during the first year’s follow up; requirement for total hip arthroplasty (THA) between the end of the first and fifth years. Statistical analysis: evaluation of sex differences (a) at baseline, in the main characteristics of hip OA using multivariate logistic regression; (b) during the first year of follow up, in the radiological progression of the disease; (c) during the five years of follow up, in the requirement for THA using Kaplan-Meier curves and the log rank test, and of the parameters related to THA, using a multivariate Cox analysis. Results: At entry, women presented more frequently than men with polyarticular OA (mean (SD) articular score 306 (162) v 235 (127)), and superomedial migration of the femoral head (40% v 19%), and had more severe symptomatic disease (patient’s overall assessment 46 (23) v 40 (26)). The change in JSW did not differ between women and men after one year, but a greater proportion of women had rapid structural progression (OR=2.34, 95% CI 1.1 to 5.2). THA was performed more often in women. Multivariate analysis suggested that the decision to perform surgery was related more closely to the symptomatic and structural severity of the disease than to the sex of the patient. Conclusion: Hip OA in women is more frequently part of a polyarticular OA, and displays greater symptomatic and structural severity.

Heme Oxygenase-1 Regulates Matrix Metalloproteinase MMP-1 Secretion and Chondrocyte Cell Death via Nox4 NADPH Oxidase Activity in Chondrocytes

Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22phox heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis.

Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b 558; and (iii) to determine the S100A8 consensus site involved in cytochrome b 558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b 558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase activation.

Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4

NADPH oxidase Nox4 is expressed in a wide range of tissues and plays a role in cellular signaling by providing reactive oxygen species (ROS) as intracellular messengers. Nox4 oxidase activity is thought to be constitutive and regulated at the transcriptional level; however, we challenge this point of view and suggest that specific quinone derivatives could modulate this activity. In fact, we demonstrated a significant stimulation of Nox4 activity by 4 quinone derivatives (AA-861, tBuBHQ, tBuBQ, and duroquinone) observed in 3 different cellular models, HEK293E, T-REx™, and chondrocyte cell lines. Our results indicate that the effect is specific toward Nox4 versus Nox2. Furthermore, we showed that NAD(P)H:quinone oxidoreductase (NQO1) may participate in this stimulation. Interestingly, Nox4 activity is also stimulated by reducing agents that possibly act by reducing the disulfide bridge (Cys226, Cys270) located in the extracellular E-loop of Nox4. Such model of Nox4 activity regulation could provide new insight into the understanding of the molecular mechanism of the electron transfer through the enzyme, i.e., its potential redox regulation, and could also define new therapeutic targets in diseases in which quinones and Nox4 are implicated.

Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 ± 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 ± 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 ± 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 ± 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the importance of this domain.