A Labasse

Strontium Ranelate Increases Cartilage Matrix Formation

Based on previous studies showing that strontium ranelate (S12911) modulates bone loss in osteoporosis, it could be hypothesized that this drug also is effective on cartilage degradation in osteoarthritis (OA). This was investigated in vitro on normal and OA human chondrocytes treated or not treated with interleukin‐1β (IL‐1β). This model mimics, in vitro, the imbalance between chondroformation and chondroresorption processes observed in vivo in OA cartilage. Chondrocytes were isolated from cartilage by enzymatic digestion and cultured for 24–72 h with 10−4−10−3 M strontium ranelate, 10−3 M calcium ranelate, or 2 · 10−3 M SrCl2 with or without IL‐1β or insulin‐like growth factor I (IGF‐I). Stromelysin activity and stromelysin quantitation were assayed by spectrofluorometry and enzyme amplified sensitivity immunoassay (EASIA), respectively. Proteoglycans (PG) were quantified using a radioimmunoassay. Newly synthesized glycosaminoglycans (GAGs) were quantified by labeled sulfate (Na235SO4) incorporation. This method allowed the PG size after exclusion chromatography to be determined. Strontium ranelate, calcium ranelate, and SrCl2 did not modify stromelysin synthesis even in the presence of IL‐1β. Calcium ranelate induced stromelysin activation whereas strontium compounds were ineffective. Strontium ranelate and SrCl2 both strongly stimulated PG production suggesting an ionic effect of strontium independent of the organic moiety. Moreover, 10−3 M strontium ranelate increased the stimulatory effect of IGF‐I (10−9 M) on PG synthesis but did not reverse the inhibitory effect of IL‐1β. Strontium ranelate strongly stimulates human cartilage matrix formation in vitro by a direct ionic effect without stimulating the chondroresorption processes. This finding provides a preclinical basis for in vivo testing of strontium ranelate in OA.

Effects of three avocado/soybean unsaponifiable mixtures on metalloproteinases, cytokines and prostaglandin E2 production by human articular chondrocytes.

The in-vitro effects of avocado and soybean unsaponifiable residues on neutral metalloproteinase activity, cytokines and prostaglandin E2 (PGE2) production by human articular chondrocytes were investigated. Avocado and soybean unsaponifiable residues were mixed in three ratios: 1∶2 (A1S2), 2∶1 (A2S1) or 1∶1 (A2S2). Freshly isolated human chondrocytes were cultured for 72 h in the absence or presence of interleukin-1β (IL-1β) (17 ng/ml), with or without unsaponifiable residue mixtures at a concentration of 10 μg/ml. A/S unsaponifiable residues were also tested separately at concentrations of 3.3, 6.6 and 10 μg/ml. All A/S unsaponifiable mixtures reduced the spontaneous production of stromelysin, interleukin-6 (IL-6), interleukin-8 (IL-8) and prostaglandin E2 (PGE2) by chrondrocytes. At concentrations of 3.3 and 6.6 μg/ml, A/S residues, tested separately, were potent inhibitors of the production of IL-8 and PGE2. Nevertheless, only avocado residue inhibited IL-6 production at these concentrations. A/S unsaponifiable mixtures had a more pronounced inhibitory effect on cytokine production than avocado or soybean residues added alone. As anticipated, IL-1β induced a marked release of collagenase, stromelysin, IL-6, IL-8 and PGE2. A/S unsaponifiable mixtures partially reversed the IL-1 effects on chrondrocytes. These findings suggest a potential role for A/S unsaponifiable extracts in mitigating the deleterious effects of IL-1β on cartilage.

Effects of exogenous IL-1β, TNFα, IL-6, IL-8 and LIF on cytokine production by human articular chondrocytes

Summary Cytokines are potent regulators of the chondrocyte functions. Some of them are produced by chondrocytes and interact to regulate cartilage metabolism. In this study, we investigated the production of interleukin-1 β (IL-1 β ), IL-6, IL-8 and leukemia inhibitory factor (LIF) by human chondrocytes and examined the modulation of their secretion by exogenous cytokines. Human articular chondrocytes were isolated from their extracellular matrix by a triple successive enzymatic digestion of the cartilage. Subsequently, chondrocytes were stimulated by increased amounts of human recombinant cytokines [IL-1 β , tumour necrosis factor α (TNF α ), IL-8, LIF, IL-6]. IL-1 β , IL-6, IL-8 and LIF were assayed into culture media and inside cell extracts by specific enzyme amplified sensitivity immunoassays (EASIAs). Under these experimental conditions, we have identified various interactions between cytokines. IL- β and TNF α highly stimulated IL-6, LIF and IL-8 productions. IL-6 decreased IL-8 synthesis and increased LIF production. IL-8 slightly enhanced IL-6 production. Finally, LIF stimulated IL-1 β , IL-6 and IL-8 productions. Using neutralizing antibodies against IL-1, we demonstrated that the effects of LIF were secondary to the stimulation by LIF of IL-1 β production by the chondrocytes. In conclusion, chondrocytes secrete a variety of immunocompetent cytokines including IL-1 β , IL-6, IL-8 and LIF that can interact to regulate chondrocytes metabolism. These results also define new biological activities of LIF and IL-6, and raise questions concerning their role in the pathogenesis of joint diseases.

One-year follow-up of Coll2-1, Coll2-1NO2 and myeloperoxydase serum levels in osteoarthritis patients after hip or knee replacement

Objectives: To determine Coll2-1, Coll2-1NO2 and myeloperoxydase (MPO) levels in serum of patients with knee or hip osteoarthritis (OA) before the surgery, 3 months and 1 year after knee or hip replacement. Methods: Coll2-1, Coll2-1NO2 and MPO were measured in 103 patients with isolated symptomatic knee or hip OA candidates for joint replacement. Sera were taken the day before surgery, 3 months and 1 year after hip or knee replacement. Coll2-1 and Coll2-1NO2 immunohistochemistry was performed on biopsies removed from cartilage lesions. Results: Immunostainings revealed the extensive presence of Coll2-1 and Coll2-1NO2 in the superficial layer of fibrillated cartilage and around some chondrocytes clusters. Three months after joint replacement, Coll2-1 and MPO serum levels were decreased and even reached the reference value for Coll2-1. By contrast, Coll2-1NO2 levels remained elevated. At 1-year follow-up, Coll2-1 levels remained at the reference value, MPO levels were similar to those measured at 3 months, and Coll2-1NO2 levels were unchanged and comparable to the pre-surgery values. However, in patients with pre-surgery values above the median (more than 0.42 nM), Coll2-1NO2 levels significantly and progressively decreased post-operatively, but tended towards an increase in patients with pre-surgery Coll2-1NO2 values below the median. Conclusions: The normalisation of Coll2-1 levels 3 months after surgery indicates that Coll2-1 is a disease-specific marker that is sensitive to the structural changes occurring in a single joint. Furthermore, the immunohistochemical findings are consistent with the concept that the major source of serum Coll2-1 is the damaged articular cartilage. Finally, serum MPO levels decreased after joint replacement indicating that neutrophil activation occurs in OA joints, even in the late stage of the disease.

Collagen catabolism through Coll2-1 and Coll2-1NO2 and myeloperoxidase activity in marathon runners

To determine the influence of marathon on the serum levels of two markers of cartilage degradation, Coll2-1 and its nitrated form, Coll2-1NO2, and of a marker of neutrophils activation, the myeloperoxidase (MPO).Coll2-1, Coll2-1NO2, total and active MPO were measured in 98 marathon runners without joint pain and with an average age of 47 years. Sera were taken at rest right before the departure and within 30 min after the marathon. The subjects were submitted to a questionnaire concerning their physical activity and their life style.The levels of Coll2-1, Coll2-1NO2 and active MPO were not affected by age, body mass index, sex or performance. The levels of total MPO were higher in female than in male (p < 0.05), but were not affected by the other parameters. After the marathon, Coll2-1 and Coll2-1NO2 concentrations were slightly but systematically decreased. The total and active MPO concentrations were increased by 2 to 3-fold in comparison to the pre-marathon values (p < 0.001 for total and active MPO). The active MPO/total MPO ratio was significantly enhanced after the marathon (p < 0.001). The variation of total MPO during the marathon was negatively correlated with the training time per week (r = −0.34; p = 0.009).The serum levels of Coll2-1 and Coll2-1NO2 were slightly decreased by marathon, indicating that intensive running could reduce cartilage catabolism. Furthermore, Coll2-1NO2 was not correlated with the total and active MPO indicating that Coll2-1 nitration did not result of a systemic oxidative phenomenon but reflects local changes.

A3 ONE YEAR FOLLOW-UP OF COLL 2-1, COLL 2-1 NO2AND MYELOPEROXYDASE SERUM LEVELS IN OSTEOARTHRITIC PATIENTS AFTER HIP OR KNEE REPLACEMENT

OBJECTIVES To determine Coll2-1, Coll2-1NO(2) and myeloperoxydase (MPO) levels in serum of patients with knee or hip osteoarthritis (OA) before the surgery, 3 months and 1 year after knee or hip replacement. METHODS Coll2-1, Coll2-1NO(2) and MPO were measured in 103 patients with isolated symptomatic knee or hip OA candidates for joint replacement. Sera were taken the day before surgery, 3 months and 1 year after hip or knee replacement. Coll2-1 and Coll2-1NO(2) immunohistochemistry was performed on biopsies removed from cartilage lesions. RESULTS Immunostainings revealed the extensive presence of Coll2-1 and Coll2-1NO(2) in the superficial layer of fibrillated cartilage and around some chondrocytes clusters. Three months after joint replacement, Coll2-1 and MPO serum levels were decreased and even reached the reference value for Coll2-1. By contrast, Coll2-1NO(2) levels remained elevated. At 1-year follow-up, Coll2-1 levels remained at the reference value, MPO levels were similar to those measured at 3 months, and Coll2-1NO(2) levels were unchanged and comparable to the pre-surgery values. However, in patients with pre-surgery values above the median (more than 0.42 nM), Coll2-1NO(2) levels significantly and progressively decreased post-operatively, but tended towards an increase in patients with pre-surgery Coll2-1NO(2) values below the median. CONCLUSIONS The normalisation of Coll2-1 levels 3 months after surgery indicates that Coll2-1 is a disease-specific marker that is sensitive to the structural changes occurring in a single joint. Furthermore, the immunohistochemical findings are consistent with the concept that the major source of serum Coll2-1 is the damaged articular cartilage. Finally, serum MPO levels decreased after joint replacement indicating that neutrophil activation occurs in OA joints, even in the late stage of the disease.

Comparison of the Effects of Nimesulide and Nimesulide‐L‐lysine on PGE2 Production by COX‐1 and COX‐2 and on Chondrocyte Metabolism In‐vitro

Nimesulide, a non-steroidal anti-inflammatory drug and one of a promising class of selective COX-2 inhibitors, has a very interesting therapeutic profile. Unfortunately, it is poorly soluble in water, which leads to important difficulties in the formulation of injectable solutions. This problem can also affect the bioavailability of nimesulide. To increase the aqueous solubility of the drug a nimesulide-L-lysine salt was synthesized in our laboratory; its aqueous solubility was greater than that of nimesulide (solubility in purified water 7.5 mg mL−1, and 0.01 mg mL-1, respectively). The aim of this study was to compare the anti-inflammatory profiles of nimesulide and nimesulide-L-lysine salt in a two-step in-vitro investigation. First, we evaluated the COX-2 selectivity of the drugs by a method using purified COX-1 and COX-2 enzymes. In a second step we evaluated the effects of the drugs on the production of prostaglandin E2 (PGE2) and proteoglycan by chondrocytes from man. The results obtained confirmed the COX-2 selectivity of the two compounds. Nimesulide-L-lysine had the same anti-inflammatory profile as nimesulide on chondrocyte cultures and better water solubility. Nimesulide-L-lysine should, therefore, be used to prepare injectable preparations and should ameliorate bioavailability after oral treatments.