Pierre Ranger

Osteoblast-like cells from human subchondral osteoarthritic bone demonstrate an altered phenotype in vitro: Possible role in subchondral bone sclerosis

OBJECTIVE Osteoarthritis (OA) is accompanied by subchondral bone sclerosis. The present study was undertaken to determine whether osteoblast-like cells in patients with OA show an abnormal phenotype that could contribute to this sclerosis. METHODS Explants and primary in vitro osteoblast-like cell cultures were prepared from subchondral bone specimens from OA patients or from bone removed at autopsy from individuals showing no signs of OA or metabolic bone disease. We measured the abundance and activity of urokinase plasminogen activator (uPA), and the levels of PA inhibitor (PAI-1) and insulin-like growth factor 1 (IGF-1) in conditioned media from both explants and osteoblast-like cells. The expression of osteoblast phenotypic biomarkers was also evaluated. RESULTS OA explants showed increased levels and activity of uPA, no changes in PAI-1 abundance, and increases in IGF-1 release, as compared with preparations from normal individuals. In vitro primary osteoblast-like cells showed results similar to the ex vivo findings for uPA, PAI-1, and IGF-1. Primary OA osteoblast-like cells also expressed higher alkaline phosphatase activity and osteocalcin release than normal cells, both under basal conditions and with 1,25(OH)2D3 (1,25-dihydroxyvitamin D) stimulation. Conversely, OA osteoblast-like cells showed blunted cAMP synthesis in response to human parathyroid hormone and prostaglandin E2 in contrast to the finding with normal osteoblast-like cells, a result that could not be attributed to altered adenylate cyclase activity. CONCLUSION Ex vivo and in vitro results indicate similar altered activities of OA osteoblasts as compared with normal cells. This suggests that an altered phenotype of subchondral osteoblasts may be a contributing factor in human OA.

Peroxisome proliferator–activated receptor γ activators inhibit interleukin‐1β–induced nitric oxide and matrix metalloproteinase 13 production in human chondrocytes

OBJECTIVE To determine the effects of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists on interleukin-1 (IL-1) induction of nitric oxide (NO) and matrix metalloproteinase 13 (MMP-13) in human chondrocytes. METHODS PPARgamma expression and synthesis in human chondrocytes were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Chondrocytes were cultured with IL-1beta, tumor necrosis factor alpha (TNFalpha), and IL-17 in the presence or absence of PPARgamma agonists, and NO and MMP-13 synthesis and expression levels were measured. Transient transfection experiments were performed with the 7-kb inducible NO synthase (iNOS) and 1.6-kb MMP-13 human promoters, as well as with the PPARgamma expression vector and the activator protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB) reporter constructs. RESULTS RT-PCR and immunohistochemical analysis revealed that human chondrocytes expressed and produced PPARgamma. Treatment of chondrocytes with PPARgamma ligands BRL 49653 and 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2), but not with PPARalpha ligand Wy 14643, decreased IL-1beta-induced NO and MMP-13 production in a dose-dependent manner. In addition, both iNOS and MMP-13 messenger RNA were inhibited in the presence of 15d-PGJ2. The inhibitory effect of PPARgamma activation was not restricted to IL-1beta, since TNFalpha- and IL-17-induced NO and MMP-13 production were also inhibited by 15d-PGJ2. In transient transfection experiments, we showed that a constitutively active form of mitogen-activated protein kinase kinase kinase 1 (AMEKK-1) induced the MMP-13 and iNOS human promoter activity. This process was reduced by 15d-PGJ2 and further inhibited by cotransfection with a PPARgamma expression vector. Similarly, in a PPARgamma-dependent manner, 15d-PGJ2 inhibited deltaMEKK-1-induced AP-1- and NF-kappaB-luciferase reporter plasmid activation. CONCLUSION The findings of this study demonstrate that PPARgamma agonists inhibit IL-1beta induction of both NO and MMP-13 in human chondrocytes. The inhibition occurs at least at the transcriptional level through a PPARgamma-dependent pathway, probably by interfering with the activation of AP-1 and NF-kappaB.

The increased synthesis of inducible nitric oxide inhibits IL-1ra synthesis by human articular chondrocytes: possible role in osteoarthritic cartilage degradation*

The degradation of osteoarthritic (OA) cartilage is likely related to the synthesis and the release of catabolic factors by chondrocytes. Nitric oxide (NO) has recently been suggested as playing a role in cartilage degradation. Since NO production is largely dependent on stimulation by IL-1, its effects on factors regulating the IL-1 biological activity, such as IL-1ra, are of the utmost importance. This study examined and compared the level of NO production by normal and OA cartilage and chondrocytes, as well as studied the effect of IL-1-induced NO production on the synthesis and steady-state mRNA of interleukin-1 receptor antagonist (IL-1ra). The NO baseline production by normal cartilage explants was undetectable but inducible by rhIL-1 beta. OA cartilage spontaneously produced NO. About a two-fold increase in NO production was found in OA rhIL-1 beta-stimulated (0.5-100 units/ml) cartilage as compared with the similarly stimulated normal cartilage. on chondrocytes rhIL-1 beta-stimulation (0.5-100 units/ml) produced a dose-dependent enhancement of both NO production and IL-1ra synthesis. Treatment with 200 microM N(g)-monomethyl-L-arginine (L-NMA), a well known NO synthase inhibitor, induced over 70% inhibition of the NO production and a marked increased IL-1ra synthesis (average of 84%) and expression (mRNA level). Inhibition of prostaglandin synthesis by indomethacin had no effect on both the NO production or the IL-1ra level. In the present study, we demonstrated the capacity of OA cartilage to produce a larger amount of NO than the normal controls, both in spontaneous and IL-1-stimulated conditions. These data support the notion that, in vivo, OA chondrocytes are stimulated by factors, possibly IL-1, which in turn may induce the expression of NO synthase, thus the synthesis of NO itself. Importantly, our results showed that the elevation of of NO production may be an important factor in the pathophysiology of OA since it can reduce IL-1ra synthesis by chondrocytes. As such, an increased level of IL-1, associated with a decreased IL-1ra level, may be responsible for the stimulation of OA chondrocytes by this cytokine, leading to an enhancement of cartilage matrix degradation.

4-Hydroxynonenal induces apoptosis in human osteoarthritic chondrocytes: the protective role of glutathione-S-transferase

Introduction4-Hydroxynonenal (HNE) is one of the most abundant and reactive aldehydes of lipid peroxidation products and exerts various effects on intracellular and extracellular signalling cascades. We have previously shown that HNE at low concentrations could be considered as an important mediator of catabolic and inflammatory processes in osteoarthritis (OA). In the present study, we focused on characterizing the signalling cascade induced by high HNE concentration involved in cell death in human OA chondrocytes.MethodsMarkers of apoptosis were quantified with commercial kits. Protein levels were evaluated by Western blotting. Glutathione (GSH) and ATP levels were measured with commercial kits. Glucose uptake was assessed by 2-deoxy-D-[3H]-glucose. The role of GSH-S-transferase A4-4 (GSTA4-4) in controlling HNE-induced chondrocyte apoptosis was investigated by chondrocyte transfection with small interfering RNA (siRNA) or with the expression vector of GSTA4-4.ResultsOur data showed that HNE at concentrations of up to 10 μM did not alter cell viability but was cytotoxic at concentrations of greater than or equal to 20 μM. HNE-induced chondrocyte death exhibited several classical hallmarks of apoptosis, including caspase activation, cytochrome c and apoptosis-induced factor release from mitochondria, poly (ADP-ribose) polymerase cleavage, Bcl-2 downregulation, Bax upregulation, and DNA fragmentation. Our study of signalling pathways revealed that HNE suppressed pro-survival Akt kinase activity but, in contrast, induced Fas/CD95 and p53 expression in chondrocytes. All of these effects were inhibited by an antioxidant, N-acetyl-cysteine. Analysis of cellular energy and redox status showed that HNE induced ATP, NADPH, and GSH depletion and inhibited glucose uptake and citric acid cycle activity. GSTA4-4 ablation by the siRNA method augmented HNE cytotoxicity, but, conversely, its overexpression efficiently protected chondrocytes from HNE-induced cell death.ConclusionOur study provides novel insights into the potential mechanisms of cell death in OA cartilage and suggests the potential role of HNE in OA pathophysiology. GSTA4-4 expression is critically important for cellular defence against oxidative stress-induced cell death in OA cartilage, possibly by HNE elimination.

Human Adult Chondrocytes Express Hepatocyte Growth Factor (HGF) Isoforms but Not HGF: Potential Implication of Osteoblasts on the Presence of HGF in Cartilage†

HGF is increased in human OA cartilage, possibly from Obs. RT‐PCR shows HGF isoforms are differently regulated between chondrocytes and Ob. A paracrine cross‐talk between subchondral bone and cartilage may occur during OA.

Feature selection using a principal component analysis of the kinematics of the pivot shift phenomenon

The pivot shift test reproduces a complex instability of the knee joint following rupture of the anterior cruciate ligament. The grade of the pivot shift test has been shown to correlate to subjective criteria of knee joint function, return to physical activity and long-term outcome. This severity is represented by a grade that is attributed by a clinician in a subjective manner, rendering the pivot shift test poorly reliable. The purpose of this study was to unveil the kinematic parameters that are evaluated by clinicians when they establish a pivot shift grade. To do so, eight orthopaedic surgeons performed a total of 127 pivot shift examinations on 70 subjects presenting various degrees of knee joint instability. The knee joint kinematics were recorded using electromagnetic sensors and principal component analysis was used to determine which features explain most of the variability between recordings. Four principal components were found to account for most of this variability (69%), with only the first showing a correlation to the pivot shift grade (r = 0.55). Acceleration and velocity of tibial translation were found to be the features that best correlate to the first principal component, meaning they are the most useful for distinguishing different recordings. The magnitudes of the tibial translation and rotation were amongst those that accounted for the least variability. These results indicate that future efforts to quantify the pivot shift should focus more on the velocity and acceleration of tibial translation and less on the traditionally accepted parameters that are the magnitudes of posterior translation and external tibial rotation.

Hepatocyte growth factor induction of collagenase 3 production in human osteoarthritic cartilage: Involvement of the stress‐activated protein kinase/c‐Jun N‐terminal kinase pathway and a sensitive p38 mitogen‐activated protein kinase inhibitor cascade

OBJECTIVE Osteoarthritis (OA) involves both a decreased reparative process and an increased degradative phenomenon. Several cytokines and growth factors are known to facilitate the repair of articular cartilage defects. The hepatocyte growth factor (HGF) present in OA cartilage is suggested to be involved in the cartilage repair process as well as in matrix remodeling and chondrocyte migration, leading to partial reconstruction of articular cartilage. Since cell migration is often correlated with metalloprotease activity, the effect of HGF on collagenase 3 production was studied because of its possible implication in OA cartilage remodeling. METHODS We examined HGF-stimulated collagenase 3 production in human OA chondrocytes by Western and Northern blotting. Furthermore, we explored the intracellular signaling pathways through which HGF induced collagenase 3 production. RESULTS This study showed that HGF stimulated collagenase 3 production in human OA chondrocytes at the transcriptional level, and this induction was mediated by activation of the stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) pathway, but not the p38 mitogen-activated protein kinase (MAPK). The p44/42 MAPKs were also phosphorylated and the use of their specific inhibitor (PD 98059) did not affect HGF-induced collagenase 3 production in OA chondrocytes. Induced collagenase 3 production via the SAPK/JNK pathway was mediated, at least in part, by the TRE site in the promoter, and in the activator protein 1 complex, c-Jun, JunD, and Fra-1 were activated. Surprisingly, further experiments revealed that the specific p38 MAPK inhibitor SB 202190 also inhibited collagenase 3 production early in the HGF-induced process. The 50% inhibitory concentration was as low as 50 nM, which is unlikely to be related to p38 MAPK inhibition (which is usually in the microM range), suggesting the involvement of another kinase sensitive to SB 202190. CONCLUSION This is the first study to show that HGF has the ability to induce both the expression and synthesis of collagenase 3 in OA chondrocytes. The effect is mediated by kinase cascades involving SAPK/JNK and another, unidentified kinase. This study provides novel information implicating a role for HGF in the pathophysiology of OA through its effect on the production of collagenase 3, which is an enzyme that is possibly involved in OA cartilage remodeling.

Gait adaptation in chronic anterior cruciate ligament-deficient patients: Pivot-shift avoidance gait

BACKGROUND A variety of biomechanical adaptations of the knee during gait have been reported in ACL-deficient patients to cope with anteroposterior knee instability. However, strategies to prevent rotatory knee instability are less recognized. We hypothesized that ACL-deficient patients would make distinctive gait changes to prevent anterolateral rotatory knee instability. Specifically, we hypothesized that during the terminal stance phase of the gait cycle, ACL-deficient patients would reduce the internal rotation knee joint moment and exhibit a higher knee flexion angle. We call this altered gait a pivot-shift avoidance gait. We also hypothesized that patients would not be able to adapt their knee biomechanics as efficiently at a fast gait speed. METHODS Twenty-nine patients with chronic ACL deficiency and 15 healthy volunteers took part in a treadmill gait analysis. The terminal stance phase was analyzed under both comfortable and fast gait speed conditions. FINDINGS At both gait speeds, ACL-deficient patients significantly reduced the internal rotation knee joint moment and showed larger knee flexion angles during the terminal stance phase of the gait cycle than did the control group. However, the difference in the minimum knee flexion angle between groups under the fast gait speed condition was not statistically significant. INTERPRETATION ACL-deficient patients adopted the proposed pivot-shift avoidance gait, possibly to prevent anterolateral rotatory knee instability. The patients were not able to adapt their knee biomechanics as effectively during fast-paced walking. This study reinforces the pertinence of gait analysis in ACL-deficient knees to acquire more information about the function of the knee joint.

Accounting for velocity of the pivot shift test manoeuvre decreases kinematic variability

The pivot shift test is the only clinical test which correlates with knee function following rupture of the ACL. A grade is given to the pivot shift in a subjective manner, leading to efforts to quantify the bone movements and correlate them to the grade. However, the dynamic and unconstrained nature of the manoeuvre introduces important kinematic variability. Our main objective was to develop a method to lessen the variability attributable to clinician technique, therefore increasing inter-grade differences. Three different orthopaedic surgeons each performed the pivot shift test on 12 subjects. Knee joint kinematics were recorded using electromagnetic motion capture devices. Inter-clinician variability was quantified and a method was developed to diminish it, using the angular velocity of flexion. This method was then applied to a larger population composed of 127 knees with various degrees of instability, evaluated by one of eight different orthopaedic surgeons. The clinical grades given by the clinicians were in almost perfect agreement (kappa=0.83). Normalization of kinematic parameters using the angular velocity of knee joint flexion produced by the clinicians reduced the intra-clinician variability by 20%, resulting in an intra-class correlation coefficient (ICC) of 0.52, up from 0.41 before normalization. This allowed for more significant differences between the grades of pivot shift. Simple normalisation of pivot shift kinematics using the angular velocity of flexion reduces clinician-related variability and allows for significant differences between the different grades. These results are an important step towards developing an objective measurement tool for the pivot shift phenomenon.

Objective grading of the pivot shift phenomenon using a support vector machine approach

The pivot shift test is the only clinical test that has been shown to correlate with subjective criteria of knee joint function following rupture of the anterior cruciate ligament. The grade of the pivot shift is important in predicting short- and long-term outcome. However, because this grade is established by a clinician in a subjective manner, the pivot shifts value as a clinical tool is reduced. The purpose of this study was to develop a system that will objectively grade the pivot shift test based on recorded knee joint kinematics. Fifty-six subjects with different degrees of knee joint stability had the pivot shift test performed by one of eight different orthopaedic surgeons while their knee joint kinematics were recorded. A support vector machine based algorithm was used to objectively classify these recordings according to a clinical grade. The grades established by the surgeons were used as the gold standard for the development of the classifier. There was substantial agreement between our classifier and the surgeons in establishing the grade (weighted kappa=0.68). Seventy-one of 107 recordings (66%) were given the same grade and 96% of the time our classifier was within one grade of that given by the surgeons. Moreover, grades 0 and 1 were distinguished from grade 2 to 3 with 86% sensitivity and 90% specificity. Our results show the feasibility of automatically grading the pivot shift in a manner similar to that of an experienced clinician, based on knee joint kinematics.