Advanced glycation end products induce cell cycle arrest and proinflammatory changes in osteoarthritic fibroblast-like synovial cells
© Franke et al.; licensee BioMed Central Ltd. 2009
Received: 3 March 2009
Accepted: 7 September 2009
Published: 7 September 2009
Advanced glycation end products (AGEs) have been introduced to be involved in the pathogenesis of osteoarthritis (OA). The influence of AGEs on osteoarthritic fibroblast-like synovial cells (FLS) has been incompletely understood as yet. The present study investigates a potential influence of AGE-modified bovine serum albumin (AGE-BSA) on cell growth, and on the expression of proinflammatory and osteoclastogenic markers in cultured FLS.
FLS were established from OA joints and stimulated with AGE-BSA. The mRNA expression of p27Kip1, RAGE (receptor for AGEs), nuclear factor kappa B subunit p65 (NFκB p65), tumor necrosis factor alpha (TNF-α, interleukin-6 (IL-6), receptor activator of NFκB ligand (RANKL) and osteoprotegerin was measured by real-time PCR. The respective protein expression was evaluated by western blot analysis or ELISA. NFκB activation was investigated by luciferase assay and electrophoretic mobility shift assay (EMSA). Cell cycle analysis, cell proliferation and markers of necrosis and early apoptosis were assessed. The specificity of the response was tested in the presence of an anti-RAGE antibody.
AGE-BSA was actively taken up into the cells as determined by immunohistochemistry and western blots. AGE-induced p27Kip1 mRNA and protein expression was associated with cell cycle arrest and an increase in necrotic, but not apoptotic cells. NFκB activation was confirmed by EMSAs including supershift experiments. Anti-RAGE antibodies attenuated all AGE-BSA induced responses. The increased expression of RAGE, IL-6 and TNF-α together with NFκB activation indicates AGE-mediated inflammation. The decreased expression of RANKL and osteoprotegerin may reflect a diminished osteoclastogenic potential.
The present study demonstrates that AGEs modulate growth and expression of genes involved in the pathophysiological process of OA. This may lead to functional and structural impairment of the joints.
Osteoarthritis (OA) is the most common joint disease of middle aged and older people across the world. OA is caused by joint degeneration, a process that includes progressive loss of articular cartilage accompanied by remodelling and sclerosis of subchondral bone, and osteophyte formation. Currently, the pathophysiology of joint degeneration that leads to the clinical syndrome of OA remains poorly understood . Multiple factors for OA initiation and progression have been identified. These factors can be segregated into categories that include hereditary factors, mechanical factors and effects of ageing . Among these, the most important risk factor is age.
In contrast to rheumatoid arthritis (RA), OA is defined as a non-inflammatory arthropathy, due to the absence of neutrophils in the synovial fluid and the lack of systemic manifestations of inflammation. However, morphological changes found in patients with OA include cartilage erosion as well as a variable degree of synovial inflammation. Proinflammatory cytokines have been implicated as important mediators in the disease [2–4]. Fibroblast-like synovial cells (FLS) are involved in osteoarthritic synovial inflammation. FLS activated by proinflammatory cytokines such as TNF-α and IL-1 show marked increases in the release of matrix metalloproteinases that can promote cartilage degradation . On the other hand, FLS itself may be a source of proinflammatory cytokines [6, 7].
Increasing age is accompanied by tissue accumulation of advanced glycation end products (AGEs). AGEs are chemical modifications of proteins by carbohydrates, including metabolic intermediates generated during the Maillard reaction, which are formed during ageing as a physiological process .
Metabolic intermediates accumulate in human articular cartilage and bone through life, and affect biomechanical, biochemical and cellular characteristics of the tissues [9, 10]. AGEs bind to specific proteins. Among these the 'receptor for AGEs', RAGE, a multiligand member of the immunoglobulin superfamily, is the most well known. Today RAGE is considered to be a pattern recognition receptor. RAGE-ligand interaction results in a rapid and sustained cellular activation of nuclear factor kappa B (NFκB), accompanied by subsequent transcription of proinflammatory cytokines and increased expression of the receptor itself [11, 12].
As suggested recently, OA synovitis can be considered to be a common final pathway in a tissue that is easily primed for innate immune responses triggered by cartilage damage [13, 14]. In this context, release of AGE-modified molecules from damaged tissue into the synovium may play a role in the initiation and perpetuation of inflammation and degradation processes. RAGE as well as AGEs are present in the synovial lining, sublining and endothelium of OA synovial tissue [15, 16]. FLS obtained from patients with OA express RAGE and stimulation of these cells with AGEs upregulates metalloproteinases .
For FLS obtained from patients with RA, it was shown that intraarticular serum amyloid A, which is also a RAGE ligand, could activate NFκB signalling through binding to cell surface RAGE, subsequently associated with increased expression of proinflammatory cytokines . In addition, FLS are substantial sources of the osteoclastogenesis-promoting factor receptor activator of NFκB ligand (RANKL) and its soluble decoy receptor osteoprotegerin .
The influence of AGEs on FLS obtained from patients with OA has been, however, incompletely studied. We used AGE-BSA as a defined model system to study the potential effects on FLS. Our study demonstrates that AGE-BSA induce cell cycle arrest, proinflammatory changes and inhibition of osteoclastogenesis in cultured FLS obtained from OA patients. Thus, the effect of AGEs on FLS may likely contribute to the pathophysiology of OA.
Materials and methods
The following reagents were used for cell isolation and culturing: DMEM (Gibco, Karlsruhe, Germany), RPMI 1640 (Promocell; Heidelberg, Germany), FCS (Lonza, Verviers, Belgium), gentamicin, Hepes (PAA Laboratories, Pasching, Austria), trypsin (Gibco, Karlsruhe, Germany), collagenase P (Roche Diagnostics, Mannheim, Germany) and Dynabeads CD14 (Invitrogen Dynal AS, Oslo, Norway). For the AGE-BSA preparation, fraction V, fatty acid-poor, endotoxin-free type of BSA was used (Calbiochem, La Jolla, CA, USA). For immunohistostaining and western blotting the following were used: primary antibodies anti-CD90 (AS02, Dianova, Hamburg, Germany); anti-CML (Roche Diagnostics, Penzberg, Germany); anti-imidazolone (kindly provided by Toshumitsu Niwa, Japan); anti-p27Kip1 (Cell Signaling Technology, Inc., Danvers, MA, USA); anti-RAGE (SP6366P, Acris Antibodies, Hiddenhausen, Germany); anti-NFκB p65, anti-IκB-αanti-pIκα (Santa Cruz Biotech, Santa Cruz, CA, USA); anti-β-actin and anti-vinculin (Sigma, St. Louis, MO, USA); horseradish peroxidase (HRP)-conjugated secondary antibodies (KPL, Gaithersburg, MD, USA); mouse and rabbit immunoglobulin (DakoCytomation, Glostrup, Denmark); Vectastain® Elite ABC Kits (Vector Laboratories, Burlingame, CA, USA); complete Lysis-M buffer for protein extraction (Roche Diagnostics, Mannheim, Germany); BCA protein assay kit for quantification of total protein (Pierce, Rockford, IL, USA); Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer LAS, Boston, MA, USA).
For cell proliferation and viability the following were used: bromodeoxyuridine (BrdU) and tetrazolium salt 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation kits (Roche Diagnostics, Mannheim, Germany).
For cell cycle and cell death analysis the following were used: Annexin-V-FLUOS Staining Kit (Roche Diagnostics, Mannheim, Germany). For reverse transcriptase and real-time PCR the following were used: RNA lysis buffer, RNeasy Mini Kit, RNase-Free DNase Set (Qiagen, Hilden, Germany) for RNA extraction, Reverse Transcription System (Promega, Madison, WI, USA) for cDNA synthesis, FastStart DNA Masterplus SYBR Green I-Kit (Roche Diagnostics, Mannheim, Germany).
For cytokine measurements in culture supernatants the following were used: human TNF-α and IL-6 ELISA (R&D Systems, Minneapolis, MN, USA), osteoprotegerin and total soluble RANKL (sRANKL) ELISA (Immundiagnostik AG, Bensheim, Germany). For NFκB transactivation assay the following were used: pNFκB-Luc plasmid (Clontech Laboratories Inc., Mountain View, CA, USA), pSV-β-galactosidase plasmid (Promega, Madison, WI, USA), Lipofectamine Plus Reagent (Invitrogen, Carlsbad, CA, USA), Luciferase reporter assay system (Promega, Madison, WI, USA), Luminescent β-gal Reporter System 3 & Detection Kit II (Clontech, Mountain View, CA, USA). For electrophoretic mobility shift assay (EMSA) the following were used: NFκB consensus and mutant oligonucleotides, anti-NFκB p65(A)X (Santa Cruz Biotech, Santa Cruz, CA, USA), T4 Polynucleotide Kinase and Reaction Buffer (New England Biolabs Inc., Ipswich, MA, USA), [γ32P] ATP (Hartmann Analytic GmbH, Braunschweig, Germany), poly d(I-C) (Roche Diagnostics, Mannheim, Germany). For RAGE inhibition the following were used: anti-RAGE antibody (N-16; Santa Cruz Biotech, Santa Cruz, CA, USA).
Synovial tissues were obtained at the time of knee replacements from 15 patients with OA (9 women, 6 men; 64.5 ± 9 years). Informed consent for the study was given by all patients and the study was approved by the local ethics committee.
In an additional experiment, human dermal fibroblasts were used to evaluate the specificity of the RANKL and osteoprotegerin expression data obtained in synovial FLS. Dermal fibroblasts were isolated from small skin pieces obtained from surgical resections performed for a variety of reasons (e.g. removal of subcutaneous lipoms). Histological evaluation showed normal skin structure. The specimens were minced, suspended in DMEM (with 10% FCS and 100 μg/ml gentamicin) and cultured at 37°C in 5% CO2 and 95% air. Outgrowing cells were isolated by trypsination two weeks later and expanded in DMEM with 10% FCS.
Preparation of AGE-BSA
BSA was incubated under sterile conditions at 37°C for 50 days in PBS with and without the addition of glucose (90 mg/ml), then filtrated to remove unbound glucose and glucose degradation products (Millipore Labscale TFF System, Bedford, MA, USA), and lyophilised. After glycation, AGE-BSA was characterised by a 90-fold higher content of Nε-carboxymethyllysine (CML) than control-BSA (12.47 versus 0.14 nmol/mg protein in control-BSA (Co-BSA)) and a 10-fold higher pentosidine concentration (2.3 versus 22.8 pmol/mg protein in Co-BSA). CML was measured by an ELISA (Roche Diagnostics, Mannheim, Germany) and pentosidine by high performance liquid chromatography (Merck-Hitachi, Darmstadt, Germany) as previously described .
After optimising the dose and time course of AGE-BSA treatment all experiments were conducted in RPMI 1640 containing 0.1% FCS supplemented with 5 mg/ml AGE-BSA or 5 mg/ml Co-BSA (corresponding to 75 μmol/l). Cells were incubated for a period of up to seven days at 37°C in a humidified atmosphere of 5% CO2 and 95% air. For histochemical studies, cells were seeded in chamber slides (Nunc, Rochester, NY, USA) and treated as described before. AGE uptake of FLS was confirmed by immunohistochemical staining and western blot analysis for the detection of AGE-modified albumin.
For immunohistochemical staining, cells growing in chamber slides were fixed with 70% ethanol in a glycine buffer (150 mM glycine, 25 mM NaCl, 25 mM HCL) for 20 minutes at -20°C and then incubated with 3% hydrogen peroxide for 10 minutes at room temperature to block endogenous peroxidase. The following primary antibodies were used: anti-CD90, anti-CML and anti-imidazolone. Staining was performed using the Vectastain® Elite ABC Kits and aminoethylcarbazole as a chromogen. Counterstaining was performed with Mayer's haematoxylin. For negative controls, primary antibodies were replaced by rabbit or mouse immunoglobulin in the same concentration as the primary antibody.
Cell proliferation and viability tests
To evaluate the influence of AGE-BSA on the number of cultured cells, FLS were seeded in six-well plates. After 24 hours, the media were changed to RPMI 1640 containing either AGE-BSA or Co-BSA and incubated for a period of up to seven days. On days 1, 2, 3 and 7, cells were detached and counted (CASY Cell Counter, Innovatis, Reutlingen, Germany). To assess the FLS proliferation in response to Co-BSA or AGE-BSA treatment, BrdU incorporation was measured by a colorimetric assay as a parameter for DNA synthesis. For evaluation of cell viability and metabolic activity the MTT assay was used. The assay is based on the cleavage of tetrazolium salt (MTT) to coloured formazan by metabolic active cells, which occurs in viable cells only. FLS were grown in 96-well microtiter plates with 3000 cells per well in RPMI 1640 containing 10% FCS for 24 hours. Then, the media were changed into RPMI containing Co-BSA or AGE-BSA and incubated for another 16 hours. Subsequently, either BrdU or MTT labelling reagent was added for four hours. BrdU incorporation was measured at an absorbance of 450 nm and the solubilised formazan of the MTT assay at 570 nm. Each measurement was performed in six different FLS cell lines with eight per treatment group.
Cell cycle analysis and evaluation of cell death
For cell cycle analysis FLS were harvested after 4, 8, 16, 24 and 48 hours after Co-BSA or AGE-BSA treatment, then stained with propidiumiodide and analysed by a flow cytometer (FACSCalibur, Becton Dickinson, Franklin Lake, NJ, USA). To investigate whether AGE-BSA induces early apoptosis and necrosis, FLS were stained with annexin-V-fluorescein and propidiumiodide simultaneously after one, two, three and seven days of Co-BSA or AGE-BSA incubation. Cell pellets were resuspended in Annexin FLUOS labelling solution (20 μl annexin-V-FLUOS® labelling reagent and 20 μl propidiumiodide in 1 ml incubation buffer®) and incubated for 15 minutes at room temperature. Then, 0.5 ml incubation buffer® was added per 106 cells. Analysis was performed using 488 nm excitation and a 515 nm band pass filter for fluorescein detection and a filter of more than 600 nm for propidiumiodide detection.
Reverse transcriptase and real-time PCR
Total cellular RNA was extracted from treated FLS after direct lyses in the culture flasks using an RNA isolation kit according to the manufacturer's instructions. The standard protocol was supplemented by DNase digestion by using the corresponding RNase-Free DNase Set. RNA yield and purity was determined by measuring the absorbance at 260 and 280 nm. Complementary DNA (cDNA) was synthesised from 3 μg of total RNA with the Reverse Transcription System.
DNA sequences of the sense and antisense primers for real-time PCR analysis and cycler conditions
Annealing temperature (°C)
Number of cycles
Product size (bp)
Western blot analysis
For western blot analysis, FLS stimulated with Co-BSA or AGE-BSA for 48 hours were lysed in complete Lysis-M buffer and the protein concentrations were determined using the BCA protein assay kit. In selected experiments, RAGE activation was blocked by addition of an anti-RAGE antibody to the cells 24 hours prior to AGE-BSA addition (N-16, 20 ng/ml). After incubating the protein extracts in sodium dodecyl sulphate (SDS) sample buffer at 100°C for five minutes, aliquots of 20 μg protein/lane were electrophoresed in a 12% acrylamide SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidene fluoride membrane using a semidry transfer cell (Bio-Rad Laboratories, Hercules, CA, USA). Nonspecific binding sites were blocked for one hour with 5% BSA in Tris-buffered saline (Tris, pH 7.4) and 0.1% Tween-20 followed by overnight incubation at 4°C in primary antibodies to CML (polyclonal rabbit), p27Kip1 (polyclonal rabbit), RAGE (polyclonal rabbit), NFκB p65 (monoclonal mouse), IκB-α, pIkB-α or to β-actin/vinculin (monoclonal mouse). The membrane was then washed four times for five minutes in Tris buffer containing 0.1% Tween-20, and incubated with the corresponding HRP-linked secondary antibody (KPL). Detection of peroxidase was performed with an enhanced chemiluminescent reagent (Western Lightning Chemiluminescence Reagent Plus). For imaging and digitisation the LAS-3000 imaging system (Fujifilm Life Science, Düsseldorf, Germany) was used. For quantification, the band densities were measured using the TotalLab TL120 Software (Nonlinear Dynamics, Newcastle, UK) and normalised for the respective densities of β-actin bands as loading controls.
Measurement of TNF-α, IL-6, sRANKL and osteoprotegerin release
To assess the release of the proinflammatory cytokines IL-6 and TNF-α in FLS culture supernatants, concentrations were determined using cytokine-specific ELISA kits (R&D Systems, Minneapolis, MN, USA). For measurement the respective levels of the osteoclastogenesis-promoting factor sRANKL and its soluble decoy receptor osteoprotegerin, total sRANKL and osteoprotegerin ELISA kits (Immundiagnostik AG, Bensheim, Germany) were used. FLS were stimulated in six-well plates with Co-BSA or AGE-BSA for 48 hours. The conditioned media were harvested and stored at -80°C until the measurements were performed. Then, cells were detached and counted. The results were corrected by the numbers of FLS in the wells.
NFκB transactivation assay
To test whether AGE-mediated NFκB activation leads to target gene binding and activation in vivo, FLS were transfected with the pNFκB-Luc reporter plasmid together with the pSV-β-galactosidase plasmid. The pNFκB plasmid contains four copies of the κ enhancer fused to the herpes simplex virus thymidine kinase promoter. Activation results in transcription of the luciferase gene. For transfection, FLS were seeded 24 hours before in six-well plates in RPMI/10%FCS. Then, cells were transfected with 4 μg pNFκB-Luc and the same amount pSV-β-galactosidase under serum-free conditions using Lipofectamine and Plus Reagent. After adding the transfection mix gently and drop wise, FLS were incubated over night. Subsequently, cells were stimulated with Co-BSA or AGE-BSA for 24 hours as appropriate. Luciferase activities were measured using a luciferase reporter assay system according to the manufacturer's protocol with a luminometer (LUMIstar OPTIMA, BMG Labtech GmbH, Offenburg, Germany). Luciferase activities were normalised to β-galactosidase activities determined by the corresponding Luminescent β-gal Reporter System 3 & Detection Kit II according to the manufacturer's instructions.
Electrophoretic mobility shift assay for NFκB
FLS isolated from three different patients were grown on 100 mm dishes in RPMI with 10% FCS. To block RAGE activation, an anti-RAGE antibody was added to the cells 24 hours prior to AGE-BSA addition (20 ng/ml). Then cells were treated for one day with Co-BSA, AGE-BSA or AGE-BSA together with the RAGE-blocking antibody. In addition, TNF-α-stimulated FLS (10 ng/ml TNF-α for two hours) were used as a positive control for NFκB activation. EMSA of nuclear extracts was performed as previously described . In detail, cells were washed with ice-cold PBS and lysed in 500 μl buffer (15 mM Tris-HCl, pH 7.9, 10 mM KCl, 2 mM MgCl2, 0.1 mM EGTA, 0.1 mM EDTA, 0.5 mM PMSF, 0.15% NP-40). Lysates were incubated on ice for 15 minutes, passed through a 26-gauge syringe and centrifuged at 5000 rpm for five minutes. The supernatant, containing the cytoplasmic proteins, was removed and 25 μl of nuclear extraction buffer (20 mM Tris-HCl pH 7.9, 0.4 M NaCl, 1 mM MgCl2, 5 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 0.1% NP-40, 10% glycerol and an appropriate amount of protease cocktail inhibitors) were added to the pellet. Nuclei were incubated for 30 minutes on ice followed by centrifugation at 13,000 rpm for 30 minutes. The protein concentration was measured and the samples were aliquoted and stored at -80°C.
The double stranded NFκB consensus oligonucleotide was end-labelled using T4 polynucleotide kinase and [γ-32P] ATP (5000 Ci/mmol) followed by purification over a G-25 Sephadex column (GE Healthcare, Piscataway, NJ, USA). Binding reaction was carried out for 30 minutes at an ambient temperature and consisted of 3 μg of nuclear proteins, binding buffer (15 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM DTT), 2 μg of poly(dI-dC), 3 μg BSA and 40 fmol of labelled probe (450,000 cpm) in a total volume of 20 μl. In competition assays, the 100-fold molar excess of unlabelled oligonucleotides (NFκB consensus and mutant oligonucleotides, AP1 consensus oligonucleotide) were added 30 minutes prior to the addition of labelled probe.
The following sequences were used:
NFκB consensus 5'-AGTTGAGGGGACTTTCCCAGGC-3',
NFκB mutant 5'-AGTTGAGGCGACTTTCCCAGGC-3',
AP1 consensus 5'-CGCTTGATGACTCAGCCGGAA-3'
The supershift antibody (400 ng) against NFκB p65 (Santa Cruz Biotech, Santa Cruz, CA, USA) was added to the reaction 30 minutes before the administration of the labelled probe.
The protein-DNA complexes were resolved on 6% polyacrylamide gel in Tris/Borate/EDTA (TBE)-buffer.
All data are reported as means ± standard error of the mean. Statistical analysis was performed using SPSS 15 for Windows (SPSS, Chicago, IL, USA). Results were analysed with the Kruskal-Wallis test followed by the Mann-Whitney U-test. P values less than 0.05 were considered significant.
Characterisation of FLS and AGE uptake
For characterisation of FLS cultured from synovial tissues, the presence of the fibroblastic marker protein CD90 (Thy-1) was demonstrated in Co-BSA as well as in AGE-BSA treated cells (Figure 1a). Cells grown in AGE-BSA and Co-BSA show the typical spindle-shaped form of fibroblasts (Figure 1a). Immunohistochemical staining for CML and imidazolone, representative members of the AGE family, demonstrates AGE-BSA uptake into the cytoplasm of the FLS (Figure 1a). This observation was confirmed by western blotting. AGE-BSA-treated FLS showed a strong accumulation of intracellular CML compared with cells receiving Co-BSA (Figure 1b).
Cell proliferation, cell viability, cell cycle and evaluation of cell death
NFκB p65 expression and activation
As shown in Figure 10b, AGE-BSA, but not Co-BSA treatment, results in NFκB activation and the formation of NFκB-DNA complexes. When RAGE activation was blocked by the anti-RAGE antibody, AGE-BSA-treated FLS showed only marginally NFκB binding as reflected by the lower intense band in comparison to AGE-BSA stimulation alone. This result clearly demonstrates that the AGE-induced NFκB activation in FLS was caused by AGE-RAGE interactions. The specificity of NFκB binding in these experiments was confirmed by supershifts using the NFκB p65 antibody and also the supershifted band was reduced in the presence of the anti-RAGE antibody.
Expression of the proinflammatory cytokines TNF-α and IL-6
To investigate the protein release of both cytokines into the culture supernatants, ELISA assays were used. The measured levels were normalised to the number of cells in the wells. Supernatants from six individual cell lines were examined. Significantly increased TNF-α and IL-6 levels were found in the media of AGE-BSA-stimulated FLS compared with Co-BSA treatment (AGE-BSA versus Co-BSA stimulation: TNF-α 12.7 ± 2.0 versus 6.2 ± 0.5 pg/105 cells; IL-6 173 ± 38 versus 81 ± 14 pg/105 cells). The relative increase of TNF-α and IL-6 release in the media of AGE-BSA treated cells in comparison to Co-BSA is shown in Figure 11c.
Expression of RANKL and osteoprotegerin
The molecular and cellular mechanisms of OA are still not completely understood. However, the importance of synovitis in the pathophysiology of OA is increasingly recognised. Chronic inflammatory changes with the production of proinflammatory cytokines are described as a feature of synovial membranes from patients with early OA . Compared with late OA, increased mononuclear cell infiltration and overexpression of proinflammatory mediators (such as TNF-α and NFκB) were seen in synovium during the early phase of the disease, which may reflect increased activation of interrelated pathophysiological pathways that contribute to progressive joint damage . FLS are involved in osteoarthritic synovial inflammation. FLS obtained from OA patients revealed a steady-state expression of TNF-α and IL-6 in culture . FLS express RANKL and osteoprotegerin and therefore have the potential to participate in osteoclastogenesis.
AGEs extensively accumulate in cartilage collagen with age . In a canine model of OA it has been shown that higher cartilage AGE levels increase the severity of OA and thus provide evidence for a molecular mechanism by which ageing predisposes to the development of OA . Cartilage degradation induces the release of AGE-modified molecules. Access of these molecules to the synovium may affect synovial cells by direct AGE-cell contact. Studying molecular mechanisms of AGEs in vivo is difficult and therefore we relied on cell cultures of human FLS as an accepted in vitro model. We investigated the capacity of AGE-BSA to modulate cell cycle, proinflammatory changes and osteoclastogenesis in cultured FLS obtained from patients with OA.
We used AGE-BSA as a model system with standardised concentrations of specific AGEs such as CML and pentosidine. AGE-BSA is actively incorporated into cultured FLS and induced to cell cycle arrest, decreased cell proliferation and viability, but also increased expression of RAGE, NFκB p65 and of the proinflammatory cytokines TNF-α and IL-6. In contrast, AGE-BSA decreased expression of RANKL and osteoprotegerin in FLS. Although it is possible that FLS in vivo may be exposed to AGE-modified albumin under pathophysiological conditions, it is more likely that AGE-modified collagens may be potential culprits in OA. However, it has been difficult to reliable glycate very large proteins such as collagens and great variations from batch to batch of CML and pentosidine content were noted. Therefore, we relied on AGE-BSA for these in vitro studies to define basic molecular mechanisms of how AGEs may influence FLS.
Decreased proliferation and viability induced by AGEs is reported for a variety of cells including cardiac and skin fibroblasts, tubular epithelial cells and podocytes [29–32]. For podocytes, it was shown that the cell cycle inhibitor protein p27Kip1 is involved in AGE-induced cell cycle arrest. Thus, we measured the p27Kip1 mRNA and protein expression in AGE-stimulated FLS and found significantly increased levels indicating the important role of p27Kip1 expression in AGE-BSA-mediated cell cycle arrest in FLS. The observation that AGE-BSA induces necrosis and late apoptosis but not early apoptosis in FLS agrees with these findings because cells arrested in the cell cycle become sensitive for necrosis. The release of potential proinflammatory material from necrotic FLS may participate in inflammatory responses. In contrast, AGE-mediated apoptosis has been recently described for fibroblasts and osteoblasts [33, 34]. In these studies, CML-modified collagen was used and not AGE-BSA, which is modified with a variety of AGE structures including CML. This may explain the different AGE-induced mechanisms.
Our data clearly demonstrate that AGEs increase the inflammatory potential of FLS by activating RAGE and NFκB, which leads to increased expression of proinflammatory cytokines TNF-α and IL-6. Our experiments using the anti-RAGE antibody clear showed that this receptor is necessary for AGE-BSA mediated p27Kip1 induction as well as NFκB activation.
These data together with the AGE-induced upregulation of metalloproteinases reported by Steenvoorden and colleagues may be evidence for the capacity of AGEs to modulate FLS towards inflammation and cartilage degradation and amplify OA . As the RAGE gene promoter region contains NFκB binding sites NFκB activation could increase the RAGE mRNA expression . In addition to the fact that binding of AGE to RAGE activates NFκB, TNF-α activates RAGE expression through NFκB activation . These may suggest a self-perpetuating cycle among AGE, RAGE and NFκB signalling, and cytokines. Moreover, TNF-α and IL-1 have the ability to promote the synthesis and release of matrix metalloproteinases .
The enhanced release of AGE-modified molecules during cartilage degradation into the synovium and the activation of FLS through AGE-RAGE interaction may initiate inflammatory responses. In RA, activated NFκB in FLS is involved in the regulation of inflammatory cytokines, adhesion molecules and matrix-grading enzymes, but it also mediates resistance of FLS against apoptosis . Thus, the observation that AGE-BSA failed to induce apoptosis in FLS from OA patients and rather mediates necrosis may also be caused by AGE-induced NFκB activation.
FLS are an integral part of the RANK/RANKL interaction system, which primarily initiates the maturation, activation and stimulation of osteoclasts. Osteophyte formation and subchondral bone sclerosis are evident for disturbed subchondral bone remodelling towards enhanced tissue formation in OA. AGE-BSA reduces the RANKL and osteoprotegerin expression in our study. These findings were specific for FLS from OA patients and was not observed in human dermal fibroblasts. In a recent contribution Wittrant and colleagues report that high glucose levels (25 mM) inhibit RANKL expression and osteoclast differentiation and formation . In addition, AGE-BSA totally inhibited osteoclastogenesis in rabbit and mouse osteoclasts . These findings support our data that AGE-BSA plays an important role in OA.
In summary, the present study demonstrates that AGEs modulate FLS growth and expression of genes involved in the pathophysiological process of OA. This may reflect a molecular mechanism by which inflammation and tissue degradation in OA continues and leads to functional and structural impairment of the joints.
advanced glycation end products
AGE-modified bovine serum albumin
bovine serum albumin
complementary deoxyribonucleic acid
Dulbecco's modified Eagle medium
electrophoretic mobility shift assay
enzyme-linked immunosorbent assay
fetal calf serum
fibroblast-like synovial cells
glyceraldehyde 3-phosphate dehydrogenase
3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide
nuclear factor kappa B
polymerase chain reaction
receptor for AGEs
receptor activator of NFκB ligand
reactive oxygen species
sodium dodecyl sulphate
tumour necrosis factor alpha.
The authors are thankful to Dr Raimund Kinne (Experimental Rheumatology Unit, Department of Orthopedics, Friedrich-Schiller-University Jena) for giving us the opportunity to learn the methods of synovial cell isolation and culturing in his group.
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