Analysis of clusterin expression changes as a biomarker of osteoarthritis

Purpose: The discovery and validation of arthritis-related biomarkers and establishment of methodology for proteomic studies in osteoarthritis (OA) are needed. Proteomics strategies have identified many proteins that may relate to pathological mechanisms of OA, however targeted approaches are required to validate the roles of these proteins. This study aimed to use mass spectrometry and western blotting to identify peptides from several proteins in the secretome of chondrocytes, cartilage explants and osteochondral biopsies treated with inflammatory cytokines over a 2-week period, to evaluate their potential as biomarkers of OA progression. Methods: Healthy cartilage was obtained from fetlock joints of skeletally mature horses, euthanized for unrelated veterinary reasons. Cartilage explants were isolated using a 6 mm biopsy, with discs placed into wells (3 discs per 1 ml DMEM + 1% Pen/Strep) before incubation for 24 hours (37 °C, 5% CO2). After this equilibration period, the media was removed and replaced with either fresh DMEM + 1% Pen/Strep or DMEM supplemented with 1% Pen/Strep containing TNFα and IL-1β both at 10ng/ml. Explants were culture for 7–14 days with the cytokines replaced every 4th day. For cell based assays chondrocytes were isolated from tissue using 70U pronase for 1hr at 37 °C and overnight digestion at 37°C using a 0.2% collagenase II solution. The cell suspension was filtered and washed before being seeded into culture flasks and cultured until confluence was reached (37°C, 5% CO2). Once cultures were established cells were split into two groups: healthy control (DMEM supplemented with 1% Pen/Strep and 10% foetal calf serum) or stimulated cells (DMEM as above plus TNFα and IL-1β both at 10ng/ml). Chondroyctes were cytokine-stimulated for up to one week. Cells were used in experiments up to the 2nd passage. Results: Mass spectrometry data showed that peptides representative of clusterin were found to decrease following 7 days of inflammatory stimulation. Western blotting of secreted proteins in media of cartilage explants or chondrocyte showed that clusterin expression was reduced following 7 days of cytokine treatment. Catabolic matrix metalloproteinase enzymes MMP1, MMP3 and MMP13, as well the matrix component cartilage oligomeric protein (COMP) were all found to have an increased abundance in the media of the cytokine treated samples. This data was supported by qPCR for clusterin gene expression which showed initially mRNA levels increased 3 day after inflammatory stimulation but expression was lost after 7 days. Western blotting of media from the osteochondral biopsies showed an increase in clusterin expression after 7 days of inflammatory stimulation however clusterin protein expression could not be detected after 14 days of treatment, indicating a delayed response compared to cartilage tissue alone. Conclusions: The equine chondrocytes, cartilage explant and osteochondral biopsy models exhibited highest clusterin secretion in untreated cultures. IL-1β and TNFα treatment caused a reduction in clusterin secretion. Clusterin acts as a chaperone to aid protein refolding in situations of stress and is constitutively secreted by mammalian cells. IL-1β and TNFα appear to interrupt clusterin secretion and therefore the protection it may offer healthy functioning cells. Previous studies have reported variable data, with some studies indicating a decrease in clusterin in OA, while others indicate an increase in clusterin expression. Our results suggest the clusterin increases immediately after inflammatory stimulation but is lost after prolonged exposure. Therefore, levels of secreted clusterin may be a candidate biomarker for OA progression

Chondrocyte secretome: a source of novel insights and exploratory biomarkers of osteoarthritis

The extracellular matrix (ECM) of articular cartilage is comprised of complex networks of proteins and glycoproteins, all of which are expressed by its resident cell, the chondrocyte. Cartilage is a unique tissue given its complexity and ability to resist repeated load and deformation. The mechanisms by which articular cartilage maintains its integrity throughout our lifetime is not fully understood, however there are numerous regulatory pathways known to govern ECM turnover in response to mechanical stimuli. To further our understanding of this field, we envision that proteomic analysis of the secretome will provide information on how the chondrocyte remodels the surrounding ECM in response to load, in addition to providing information on the metabolic state of the cell. In this review, we attempt to summarize the recent mass spectrometry-based proteomic discoveries in healthy and diseased cartilage and chondrocytes, to facilitate the discovery of novel biomarkers linked to degenerative pathologies, such as osteoarthritis (OA)

Aggrecanase degradation of type III collagen is associated with clinical knee pain

There is a lack of biochemical markers for non-invasive and objective assessment of symptomatic osteoarthritis (OA). Aggrecanase activity has been shown to be associated with joint deterioration and symptomatic disease through the degradation of extracellular matrix proteins, such as type III collagen. Our study aimed to identify and develop a novel biomarker by measuring an aggrecanase-mediated type III collagen neoepitope, and correlate levels of this biomarker with OA joint pain. Mass spectrometric analysis of purified type III collagen, degraded by the aggrecanase A Disintigrin and Metalloproteinase with Thrombospondin motif (ADAMTS), revealed a fragment generated by ADAMTS-1, -4 and -8. A monoclonal antibody was raised against the neoepitope of this fragment (COL3-ADAMTS) and a competitive ELISA was developed and tested; using serum samples from a cross-sectional cohort of patients with different degrees of knee OA (n = 261). The COL3/ADAMTS ELISA was technically robust and specific for the ADAMTS-1, -4 and -8 generated neoepitope. COL3/ADAMTS was released form cytokine stimulated synovial cultures, indicating a biologic link between the marker and synovium. In OA patients, serum COL3/ADAMTS was independently associated with pain scores (rho = −0.13–0.17, p < 0.05). This association was associated significantly with the presence of radiographic OA. Together, these data indicate that COL3/ADAMTS could be a marker of early osteoarthritis and the underlining pathology

A new approach to comprehensively evaluate the morphological properties of the human femoral head : example of application to osteoarthritic joint

Osteoarthritis affects the morphological properties of the femoral head. The goal of this study was to develop a method to elucidate whether these changes are localised to discrete regions, or if the reported trends in microstructural changes may be identified throughout the subchondral bone of the human femoral head. Whole femoral heads extracted from osteoarthritic (n = 5) and healthy controls (n = 5) underwent microCT imaging 39 μm voxel size. The subchondral bone plate was virtually isolated to evaluate the plate thickness and plate porosity. The trabecular bone region was divided into 37 volumes of interest spatially distributed in the femoral head, and bone morphometric properties were determined in each region. The study showed how the developed approach can be used to study the heterogeneous properties of the human femoral head affected by a disease such as osteoarthritis. As example, in the superior femoral head osteoarthritic specimens exhibited a more heterogeneous micro-architecture, with trends towards thicker cortical bone plate, higher trabecular connectivity density, higher trabecular bone density and thicker structures, something that could only be observed with the newly developed approach. Bone cysts were mostly confined to the postero-lateral quadrants extending from the subchondral region into the mid trabecular region. Nevertheless, in order to generalise these findings, a larger sample size should be analysed in the future. This novel method allowed a comprehensive evaluation of the heterogeneous micro-architectural properties of the human femoral head, highlighting effects of OA in the superior subchondral cortical and trabecular bone. Further investigations on different stages of OA would be needed to identify early changes in the bone

Subchondral bone and ligament changes precede cartilage degradation in guinea pig osteoarthritis

There is increasing recognition that osteoarthritis (OA) is a complex disease involving the whole synovial joint, rather than the articular cartilage alone, however its aetiology and pathogenesis is not understood. Our initial studies revealed elevated turnover of bone and ligament collagen in human and mouse OA, respectively. To investigate the relative appearance of pathology in cartilage, bone and ligament, we studied the progression of spontaneous OA in the Dunkin–Hartley (DH) guinea pig knee, and compared with age-matched control Bristol Strain 2 (BS2) knees. The classical radiographic OA score of the DH knees compared to BS2 knees was 2-fold higher at 24 weeks of age. The patella perimeter and subchondral bone density was significantly greater in the DHs at 24 and 36 weeks compared to BS2. The femoral intercondylar notch width was found to be significantly lower in the DHs at 24 and 36 weeks, compared to BS2, indicating bone remodelling at the cruciate ligament (CL) insertion site. We found significantly greater laxity of the DH anterior CL at 12, 16 and 20 weeks compared to BS2. This elevated laxity was associated with increased remodelling of the CLs, based on markers of collagen turnover, and occurred prior to bone and cartilage pathology. We propose that the laxity of the CL leads to remodelling of the subchondral bone, and intercondylar notch, due to a change in load through the joint. Remodelling of the CLs and bone occurs prior to and concomitant with histopathological changes in the articular cartilage respectively, demonstrating the fundamental role of the ligament and subchondral bone in the aetiology of knee OA

Factors that influence cartilage implant integration during joint repair

The low spontaneous healing capacity of articular cartilage, the avascular nature, and the acellularity of the interface of damaged cartilage, all these factors limit the integration between the implanted engineered cartilage and native articular cartilage. The disintegration remains an unsolved clinical problem as reported by many implantationstudies, regardless of the type of cells used in creating the implanted engineered cartilage. The purpose of this study was to investigate factors that influence implant integration with native cartilage

Regulation of Smooth Muscle Cell Proliferation by β-Catenin/T-Cell Factor Signaling Involves Modulation of Cyclin D1 and p21 Expression

We previously observed that stimulation of vascular smooth muscle cell (VSMC) proliferation with growth factors is associated with dismantling of cadherin junctions and nuclear translocation of β-catenin. In this study we demonstrate directly that growth factors stimulate β-catenin/T-cell factor (TCF) signaling in primary VSMCs. To determine whether β-catenin/TCF signaling regulates VSMC proliferation via modulation of the β-catenin/TCF responsive cell cycle genes, cyclin D1 and p21, we inhibited β-catenin/TCF signaling by adenoviral-mediated over-expression of N-Cadherin, ICAT (an endogenous inhibitor of β-catenin/TCF signaling), or a dominant negative (dn) mutant of TCF-4. N-cadherin, ICAT or dnTCF-4 over-expression significantly reduced proliferation of isolated human VSMCs by approximately 55%, 80%, and 45% respectively. Similar effects were observed in human saphenous vein medial segments where proliferation was reduced by approximately 55%. Transfection of dnTCF-4 in the ISS10 human VSMC line significantly lowered TCF and cyclin D1 reporter activity but significantly elevated p21 reporter activity, indicating regulation of these genes by β-catenin/TCF signaling. In support of this, over-expression of N-cadherin, ICAT or dnTCF-4 in isolated human VSMCs significantly lowered levels of cyclin D1 mRNA and protein levels. In contrast, over-expression of N-Cadherin, ICAT or dnTCF4 significantly elevated p21 mRNA and protein levels. In summary, we have demonstrated that increasing N-cadherin and inhibiting β-catenin/TCF signaling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We therefore suggest that the N-cadherin and β-catenin/TCF signaling pathway is a key modulator of VSMC proliferation via regulation of these 2 β-catenin/TCF responsive genes

SAT0062 Metabolomics and metabolic function analysis of the secretome of articular cartilage and chondrocytes in response to pro-inflammatory cytokines

Background Chondrocytes rely primarily on glycolysis to meet their energy requirements, but can support cell survival and matrix synthesis during periods of nutrient stress by enhancing glycolysis with mitochondrial respiration. Accessing this ‘spare respiratory capacity’ requires optimal mitochondrial function. Impaired mitochondrial function is implicated in osteoarthritis (OA). Metabolic adaptation is evident in early-stage OA, however cartilage from late-stage disease does not seem to have this flexibility. A deeper understanding of these complex metabolic pathways may identify new markers of disease stage, and support therapeutic strategies for treating OA. Objectives Metabolomics has the potential to reveal pathological pathways and identify novel biomarkers. The aim was to identify metabolic processes involved in early stage disease by analysis of metabolites and metabolic function in pro-inflammatory models of cartilage degradation. Methods Macroscopically normal articular cartilage was obtained from equine and bovine metacarpophalangeal joints. Equine cartilage explants (n=6), and primary chondrocytes seeded at 105,000/cm2 (n=4), were cultured for 7 days in serum-free DMEM (Gibco) with or without 10 ng/ml equine interleukin-1β (IL-1β) and 10 ng/ml tumour necrosis factor-α (TNF-α). Secretome metabolite levels were measured using AbsoluteIDQ p180 targeted metabolomics kit (Biocrates), with Waters Xevo TQ-S mass spectrometer coupled to an Acquity UPLC system. PCA and OPLS-DA were performed using SIMCA-P v12.0 software. Metabolic function of primary equine (n=9) and bovine chondrocytes (n=3) was determined using Seahorse XFp and XFe24 analyzers. Cells were treated with species-specific 10 ng/ml IL-1β and/or 10 ng/ml TNF-α for 18 hour, and metabolically challenged with the Mito Stress Test. Metabolite levels, and oxygen consumption rates, were normalised to total cell protein, and values analysed by ANOVA with Tukey’s multiple comparison post-tests. Results Cytokine treatment decreased proline, ornithine and alpha-aminoadipic acid (p<0.0001) in explant secretome. Citrulline increased with cytokine treatment (p<0.0001) and glutamate, present in DMEM, was also elevated (p<0.0001). Metabolomic analysis of chondrocyte secretome showed that glutamine decreased (p<0.02) with cytokine treatment whereas citrulline was elevated (p<0.003). Metabolic analysis showed that cytokine treatment reduced basal respiration and negated spare respiratory capacity in chondrocytes (p<0.01), and the effect was due to IL-1β alone. Conclusions Explant metabolites which decreased with cytokine treatment are all downstream of glutamate. With elevated glutamate, this suggests that cytokines inhibit glutamate uptake and metabolism. Elevated citrulline in cell and explant models may be attributed to disruption of the urea cycle via induction of nitric oxide synthase. IL-1β alone negated spare respiratory capacity, and chondrocytes remained glycolytic. In conclusion, cytokines disrupt glutamate and citrulline metabolism, normally tightly regulated mitochondrial pathways, and IL-1β alone is responsible for the metabolic switch. These metabolic pathways could provide markers of early-stage inflammatory disease