Michelle Deberg

Phenotypic characterization of osteoblasts from the sclerotic zones of osteoarthritic subchondral bone.

OBJECTIVE To determine the phenotype of osteoblasts from the sclerotic zones of human osteoarthritic (OA) subchondral bone. METHODS Human osteoblasts were isolated from sclerotic or nonsclerotic areas of subchondral bone and cultured for 14 days in monolayer. The expression of 14 genes was investigated by real-time reverse transcription-polymerase chain reaction. The activities of alkaline phosphatase (AP) and transglutaminases (TGases) were quantified by enzymatic assays. C-terminal type I procollagen propeptide (CPI), interleukin-1beta (IL-1beta), IL-6, IL-8, transforming growth factor beta1 (TGFbeta1), osteocalcin (OC), and osteopontin (OPN) were assayed in the culture medium by immunoassay. RESULTS Gene expression levels of matrix metalloproteinase 13, COL1A1 and COL1A2, OPN, tissue-nonspecific AP, OC, vascular endothelial growth factor, ANKH, TGase 2, factor XIIIA, and dentin matrix protein 1 were significantly up-regulated in sclerotic osteoblasts compared with nonsclerotic osteoblasts. In contrast, parathyroid hormone receptor gene expression was depressed in sclerotic osteoblasts, but bone sialoprotein levels were unchanged. The activities of AP and TGases were increased in sclerotic osteoblasts, while matrix mineralization, revealed by alizarin red staining, was decreased. In parallel, protein synthesis of CPI, OC, OPN, IL-6, IL-8, and TGFbeta1 was significantly higher in sclerotic osteoblasts than in nonsclerotic osteoblasts, while IL-1beta production was similar in both groups. CONCLUSION These findings contribute to a better understanding of the mechanisms involved in subchondral bone sclerosis and identify osteoblasts with an altered phenotype as a potential target for future OA therapies.

Type II collagen markers in osteoarthritis: what do they indicate?

Purpose of reviewWe provide a critical review of recent in-vitro, animal and human clinical studies on type II collagen biomarkers. In describing the human studies, we have applied the BIPED (burden of disease, investigative, prognostic, efficacy of intervention, and diagnostic) classification scheme recently proposed by the Osteoarthritis Biomarkers Network (a consortium of five US National Institutes of Health designated sites). Based on this analysis, we propose an update to the classification of the type II collagen biomarkers. Recent findingsVarious type II collagen epitopes have been described as potential biomarkers for osteoarthritis. Some have demonstrated ability in the following areas: classification of individuals as either diseased or nondiseased; assessment of severity or extent of osteoarthritis; prediction of future onset of osteoarthritis among those without osteoarthritis at baseline or the progression of osteoarthritis among those with existing disease; and monitoring treatment efficacy. SummaryType II collagen biomarkers provide useful information for clinical and research applications. Furthermore, they are promising tools for the monitoring the influence of drug treatment on cartilage metabolism in joint diseases such as osteoarthritis.

Effects of rhein on human articular chondrocytes in alginate beads

This study was designed to investigate the effects of rhein, the active metabolite of diacerhein, on the metabolic functions of human chondrocytes cultured in alginate beads. Enzymatically isolated osteoarthritic (OA) chondrocytes were cultured in alginate beads in a well-defined culture medium for 12 days. Rhein was tested in a range of concentrations comprised between 10(-7) and 4 x 10(-5)M, in the presence or absence of 10(-10)M IL-1beta. Interleukin (IL)-6 and -8, macrophage inflammatory protein (MIP-1beta), stromelysin-1 (MMP-3), aggrecan (AGG), tissue inhibitor of metalloproteinases-1 (TIMP-1), prostaglandin E(2) (PGE(2)) and nitric oxide (NO) productions were assayed. Cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) mRNA steady-state levels were also quantified. In the basal condition, 10(-5)M rhein increased by 46.5% the production of AGG, decreased by 17-30% the production of IL-6, MMP-3, NO and MIP-1beta but enhanced by 50% the production of PGE(2). IL-1beta increased IL-6, IL-8, MIP-1beta, NO, PGE(2) and MMP-3 productions, but inhibited AGG and TIMP-1 synthesis. Rhein partially reversed the effect of IL-1beta on TIMP-1 and NO production, had no effect on AGG, IL-6 and MIP-1beta production, but up-regulated the IL-1beta stimulated PGE(2) production. The COX-2 and iNOS mRNA levels and IL-8 production were not modified by rhein.Overall, these results contribute to explain the clinical efficiency of rhein and give new information on its mechanisms of action.

Fibulin 3 peptides Fib3-1 and Fib3-2 are potential biomarkers of osteoarthritis.

OBJECTIVE This study was undertaken to identify new biomarkers of osteoarthritis (OA) by proteomics analysis and to develop specific immunoassays to detect and quantify them. METHODS Proteomics analysis was performed in urine samples from 10 women (mean±SD age 76.0±5.0 years) undergoing knee replacement surgery due to severe OA and 5 healthy women (mean±SD age 25.6±2.6 years). Protein content was analyzed by 2-dimensional differential gel electrophoresis. Protein spots that exhibited an OA:control abundance ratio of ≥1.5 were identified by mass spectrometry. Specific enzyme-linked immunosorbent assays were developed and validated in serum obtained from 236 healthy subjects ages 20-64 years and from 76 patients with severe radiologic knee OA (mean±SD age 68.8±11.9 years). Immunohistochemical analysis was performed on articular cartilage from tibial plateaus. RESULTS Thirteen proteins within spots that were significantly modified between groups were identified. Two peptides of fibulin 3, named Fib3-1 and Fib3-2, were of particular interest. Two antisera directed against these peptides were used to develop immunoassays. Compared with age-matched healthy subjects, median levels of serum Fib3-1 and Fib3-2 were elevated in OA patients (54.6 pM versus 85.1 pM [P<0.0001] and 144.4 pM versus 191.4 pM [P<0.0001], respectively). Using area under the receiver operating characteristic curve analysis, we demonstrated that Fib3-1 and Fib3-2 levels discriminate between OA and normal populations. Immunostaining revealed the presence of Fib3-1 and Fib3-2 in chondrocytes and in the extracellular matrix of the superficial layer of the fibrillated cartilage. CONCLUSION Our findings indicate that Fib3-1 and Fib3-2 are potential biochemical markers for the diagnosis of OA.

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.

Early decrease of serum biomarkers of type II collagen degradation (Coll2-1) and joint inflammation (Coll2-1 NO2) by hyaluronic acid intra-articular injections in patients with knee osteoarthritis: A research study part of the Biovisco study

To measure the evolution of the serum levels of specific Osteoarthritis (OA) biomarker, Coll2‐1 and Coll2‐1 NO2 in knee osteoarthritic patients after viscosupplementation (VS). Fifty‐one patients with unilateral symptomatic knee were recruited for this prospective open label study. They received three intra‐articular injections of 2 ml of hyaluronic acid (Hylan GF‐20) and were followed for 3 months. Walking pain was evaluated and serum samples were taken at each visit. Coll2‐1 and Coll2‐1 NO2 were measured in the serum using specific immunoassays. Variations over time of each parameter and predictive factor of response were studied. Forty‐five patients were analyzed. The serum concentrations of Coll2‐1 and Coll2‐1 NO2 were significantly higher in KL III/IV patients compared to KL I/II patients at baseline and decreased systematically over time after VS. Its effect was ever more pronounced in patients with KL III/IV. The serum concentration of Coll2‐1 was significantly lower at baseline in responders than in non‐responders. This study suggests a rapid slowdown of type II collagen degradation and joint inflammation after VS with Hylan G‐20 and gives additional information for the validation of accurate biomarkers for OA. The serum level of Coll2‐1 appeared to be a predictive factor for response to treatment.

Collagen Fibril Disruption Occurs Early in Primary Guinea Pig Knee Osteoarthritis

OBJECTIVE A major barrier inhibiting the discovery of structural modifying agents for osteoarthritis (OA) is an incomplete understanding of early disease events. Herein, we investigated the time course of collagen II cleavage and fibril disruption in the well-validated Hartley guinea pig model of spontaneous OA of the knee. METHODS Knee joints of 46 male Hartley guinea pigs were analyzed at 3 weeks, 2, 4, 7, 10, 12, and 18 months of age for histological severity of OA, cartilage collagen fibril disruption by semi-quantitative polarized light microscopy, and expression of type II collagen degradation biomarkers, 9A4 and Coll2-1, by immunohistochemistry. In addition, serum biomarkers specific for collagen II degradation, CTX-II, C2C, and Coll2-1 were quantified. RESULTS Collagen fibril disruption and expression of the collagenase-generated cleavage neoepitope, 9A4, were observed as early as 2 months of age, despite the appearance of histological OA at 4 months of age. Only serum Coll2-1 increased coincident with the early disruption of the collagen fibril between 3 weeks and 7 months, in contrast to serum C2C, which did not change significantly or correlate with histological severity. Inversely, CTX-II declined dramatically from 3 weeks to 4 months and remaining low thereafter, coincident with growth plate turnover. CONCLUSIONS Collagenase cleavage and disruption of the type II collagen network are early OA disease events in this model, preceding histological evidence of proteoglycan loss. The markedly different serum profiles of collagen II-related biomarkers during the early stages of disease development suggest compartmental segregation and temporal regulation of collagen degrading enzymes.

Mitochondrial DNA haplogroups modulate the serum levels of biomarkers in patients with osteoarthritis

Objective To analyse the influence of mitochondrial DNA (mtDNA) haplogroups on serum levels of molecular biomarkers in patients with osteoarthritis (OA). Methods Serum levels of molecular biomarkers of cartilage metabolism (collagen type II markers: C-terminal neoepitope generated by the collagenase-mediated cleavage of collagen type II triple helix (C2C), collagen type II (Coll2-1, and its nitrated form, Coll2-1NO2), procollagen type II (CPII)), synovial metabolism (hyaluronic acid (HA)) and cartilage and synovial turnover (cartilage glycoprotein 39 (YKL-40)) were analysed in 73 patients with OA and 77 healthy controls using ELISAs. All participants had been previously genotyped for the mtDNA haplogroups J, U and H. Non-parametric and multivariate analysis were performed to test the effects of the clinical variables, including gender, age, smoking status, diagnosis, mtDNA haplogroups and radiological Kellgren and Lawrence (K/L) grade on the serum levels of the molecular markers. Results Non-parametric analysis found increased serum levels of HA in patients with OA, while the values for C2C and the C2C/CPII ratio were significantly higher in the healthy controls. A multiple regression analysis showed a relationship between the mtDNA haplogroups and serum levels of the typical collagen type II markers. Carriers of the mtDNA haplogroup H had higher levels while carriers of the mtDNA haplogroup J showed lower levels. Statistically significant interactions between mtDNA haplogroups and diagnosis and between mtDNA haplogroups and radiological K/L grade in the serum levels of molecular markers were also found. Conclusion A new role for mtDNA haplogroups emerges from this work. The results suggest that the mtDNA haplogroups interact significantly with the serum levels of OA-related molecular markers, suggesting the possibility of their use as a complementary assay with these molecular markers.

Coll2-1, Coll2-1NO2 and myeloperoxidase serum levels in erosive and non-erosive osteoarthritis of the hands

OBJECTIVE Erosive osteoarthritis of the hand (EHOA) is thought to be an aggressive variant of hand osteoarthritis (HOA) characterised by prominent local inflammation and radiographic aspects of bone erosions in interphalangeal (IP) joints. However, rare studies have until now investigated the value of biomarkers in these patients. Thus, we determined Coll2-1, a marker of type II collagen denaturation, its nitrated form (Coll2-1NO2) and myeloperoxidase (MPO) levels in serum of patients with EHOA vs non-EHOA and subsequently evaluated their relationships with disease indices of severity and activity. METHODS Coll2-1, Coll2-1NO2 and MPO were measured using specific immunoassays in 82 patients, 57 with EHOA, all females, median age 59 (41-74 yrs) and 20 with non-EHOA, all females, median age 55 (43-73 yrs), fulfilling the American College of Rheumatology (ACR) criteria for hand OA. EHOA was characterized by the presence of at least one central bone erosion on radiograph in the IP joints. Patients were also evaluated for disease duration, number of affected (swollen and painful or tender) joints, radiographic score (RS) by Kallman scale and high sensitivity C-reactive protein (hsCRP). RESULTS Serum levels of MPO were higher in EHOA (230.0 ± 152.1 ng/ml) than in non-EHOA (160.2 ± 111.5 ng/ml, P=0.037). Coll2-1NO2 levels trended towards an elevation in EHOA compared non-EHOA (0.40 ± 0.86 vs 0.22 ± 0.14 nmol/l, P=0.06), while Coll2-1 levels were not different. Correlations were found for disease duration and both MPO (R(2)=0.48, P=0.001) and Coll2-1NO2 (R(2)=0.73, P=0.01) after the splitting of the population in subgroups according to a cut off value above the 50th percentile. A correlation was found between hsCRP and MPO (R(2)=0.57, P=0.01). CONCLUSIONS This study clearly demonstrates an elevation of some serum biomarkers in EHOA, in comparison with non-EHOA. In particular, MPO, hsCRP and the ratio Coll2-1NO2/Coll2-1 discriminated the two subsets of hand osteoarthritis (HOA), and a trend was also observed for Coll2-1NO2. These data suggest that these biomarkers could be helpful for the diagnosis of EHOA.

Mitochondrial DNA haplogroups and serum levels of proteolytic enzymes in patients with osteoarthritis.

Objective To analyse the influence of mitochondrial DNA haplogroups, as well as the radiographic grade, on serum levels of proteolytic enzymes in patients with osteoarthritis (OA). Methods Serum levels of metalloproteinase-1 (MMP-1), MMP-3, MMP-13, myeloperoxidase and cathepsin K were analysed in 73 patients with OA and 77 healthy controls carrying the haplogroups J, U and H, by ELISA. Knee and hip radiographs were classified according to Kellgren and Lawrence (K/L) scoring from grade 0 to grade IV. Non-parametric and multiple regression analyses were performed to test the effects of clinical variables, including gender, age, smoking status, diagnosis, haplogroups and radiological K/L grade on serum levels of these enzymes. Results A significant influence of the haplogroups on the serum levels of MMP-3 and MMP-13 was detected (p=0.027 and p=0.035, respectively). Patients with OA with haplogroup H showed higher serum levels of MMP-3 than healthy controls. Serum levels of MMP-13 were significantly higher in patients with OA (p<0.001), and carriers of the haplogroup J showed lower levels than H carriers. Besides, levels of MMP-13 were proportionally higher in radiological groups B (K/L grade II and III) and C (K/L grade IV) than in group A (K/L grade 0 and I) (p=0.005). Conclusions This study shows that haplogroups have a significant influence on serum levels of MMP-3 and MMP-13. The influence of the haplogroups on serum levels of MMP-3 is clearly dependent on the diagnosis, whereas the influence of the haplogroups on serum levels of MMP-13 is independent of diagnosis.