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Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes and human articular cartilage explants

Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes... Vascular endothelial growth factor (VEGF) promotes cartilage- PCR. Expression of VEGF and VEGF receptors (VEGFR-1 and degrading pathways, and there is evidence for the involvement VEGFR-2) was quantified by real-time RT-PCR. of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been Synovial fluid from OA patients revealed markedly elevated levels reported. Here, we investigate whether the expression of VEGF of VEGF. Common RT-PCR revealed that the splice variants is modulated by ROS. were present in both immortalized chondrocytes and cartilage discs. In immortalized chondrocytes, stimulation with PMA or SIN-1 caused increases in the levels of VEGF, VEGFR-1 and Aspirates of synovial fluid from patients with osteoarthritis (OA) VEGFR-2 mRNA expression. Cartilage explants produced similar were examined for intra-articular VEGF using ELISA. results, but VEGFR-1 was only detectable after stimulation with Immortalized C28/I2 chondrocytes and human knee cartilage SIN-1. Stimulation with PMA or SIN-1 resulted in a dose- explants were exposed to phorbol myristate acetate (PMA; 0– dependent upregulation of the VEGF protein (as determined 20 μg/ml), which is a ROS inducer, or 3-morpholino- using ELISA) and an increase in the level of NO in the medium. sydnonimine hydrochloride (SIN-1; 0–20 μM), which is a ROS donor. The levels of VEGF protein and nitric oxide (NO) Our findings indicate ROS-mediated induction of VEGF and production were determined in the medium supernatant, using VEGF receptors in chondrocytes and cartilage explants. These ELISA and Griess reagent, respectively. Gene expression of results demonstrate a relationship between ROS and VEGF as VEGF-121 and VEGF-165 was determined by splice variant RT- multiplex mediators in articular cartilage degeneration. ever, the precise signalling pathways in the degradation of Introduction Osteoarthritis (OA) is characterized by a breakdown of the articular cartilage ECM and development of OA are still not extracellular matrix (ECM) of articular cartilage in the affected fully understood. Several studies have demonstrated the joints. The pathogenesis of OA involves multiple aetiologies, involvement of cytokines, such as IL-1 and IL-6, or tumour including mechanical, genetic and biochemical factors. How- necrosis factor (TNF)-α, in addition to proteases, such as AP-1 = activator protein 1; DMEM = Dulbecco's modified Eagle's medium; ECM = extracellular matrix; ELISA = enzyme-linked immunosorbent assay; G3PDH = glyceroaldehyde-3-phosphate dehydrogenase; IL = interleukin; MMP = matrix metalloproteinases; NO = nitric oxide; NTC = no-template control; OA = osteoarthritis; PMA = phorbol myristate acetate; ROS = reactive oxygen species; RT-PCR = reverse transcriptase polymerase chain reaction; SEM = standard error of the mean; SIN-1 = 3-morpholino-sydnonimine hydrochloride; TIMP = tissue inhibitor of matrix metalloproteinase; TNF = tumour necrosis factor; TPA = 12-O-tetradecanoylphorbol-13-acetate; VEGF: vascular endothelial growth factor; VEGFR = VEGF receptor. Page 1 of 8 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 6 Fay et al. matrix metalloproteases (MMPs), in the initiation and progres- imine hydrochloride (SIN-1), which spontaneously decom- .- sion of articular cartilage destruction [1,2]. The imbalance poses to nitric oxide (NO) radicals and O [20]. between activated proteinases and inhibitors ultimately leads to an altered net proteolysis of cartilage components. Once Therefore, the present study was performed to investigate the damaged, articular cartilage has a poor capacity for intrinsic expression of VEGF, VEGF-121 and VEGF-165 splice vari- repair. ants and VEGF receptors (VEGFR-1 and VEGFR-2) after stim- ulation by ROS donors. Knowledge of this expression could Angiogenesis, the development of new blood vessels by lead to a better understanding of the complex role of ROS and sprouting from pre-existing endothelium, is a significant com- VEGF in articular cartilage degeneration. ponent of a wide variety of biological processes [3,4]. How- ever, in rheumatoid arthritis, new capillary blood vessels invade Materials and methods the joints from the emerging synovial pannus and aid in the Reagents destruction of articular cartilage [5], even in the absence of a Chemical reagents were purchased from Sigma (Munich, Ger- causative factor. The most important mediator of angiogenesis many), unless otherwise indicated. is vascular endothelial growth factor (VEGF) [6], which stimu- lates capillary formation in vivo and has direct mitogenic Synovial fluid actions on various cells in vitro [7]. Recent data reveal expres- Samples of synovial fluid from patients with OA (n = 8) were sion of VEGF in OA cartilage and reflect the ability of VEGF to obtained from the Department of Orthopaedic Surgery at the enhance catabolic pathways in chondrocytes by stimulating University Hospital Schleswig-Holstein (Kiel, Germany). Syno- MMP activity and reducing natural MMP inhibitors, that is tis- vial fluid from healthy joints was collected from deceased sue inhibitors of MMPs (TIMPs) [8-11]. These data suggest donors (n = 5) at the Department of Anatomy, Christian-Albre- that, except from the effect of VEGF on proliferation of synovial chts-University (Kiel, Germany). membranes, chondrocyte-derived VEGF promotes catabolic pathways in the cartilage itself, thereby leading to a progres- Chondrocyte monolayer culture sive breakdown of the ECM of articular cartilage. The human C28/I2 chondrocyte cell line was used in monol- ayer culture. These chondrocytes, which were immortalized Recent investigations have revealed the participation of free using SV-40 large T-antigen, continue to express chondro- radicals in the pathogenesis of articular cartilage degradation cyte-specific aggrecan and collagen type II mRNA after multi- [12]. Free radicals are highly reactive in oxidative processes ple subculture [21]. Chondrocytes were seeded (500,000 and are essentially involved in physiological reactions, such as cells/25 cm ) in DMEM supplemented with 10% foetal bovine the cellular respiratory chain. However, uncontrolled release of serum, 10 mM h-(2-hydroxyethyl)-1-piperazinethansulfonacid free radicals can result ultimately in an imbalance, with respect (HEPES) buffer, 1 mM sodium pyruvate, 0.4 mM proline, 20 to their inhibitors or antioxidants. Moreover, free radicals can μg/ml ascorbic acid, 100 U/ml penicillin G, 100 μg/ml strep- stimulate inflammatory pathways or damage lipids, proteins or tomycin and 0.25 μg/ml amphotericin B. After reaching 80% DNA [13]. In the nomenclature of free radicals, the term 'reac- confluence, the cells were rinsed twice with HANK's solution tive oxygen species' (ROS) has prevailed, although ROS can and placed in serum-free medium, containing 0.05% BSA, for be differentiated into reactive nitrogen species and other oxi- subsequent stimulation. dant species. The relationship between ROS and articular car- Human articular cartilage explants tilage degradation is complex and involves multiple pathways [14]. ROS can induce changes in biosynthetic activity [15], in The tissue harvest was approved by the Ethical Commission addition to apoptosis [16]. In addition, ROS can influence of Christian-Albrechts-University of Kiel (Kiel, Germany). Knee transcription factors in chondrocytes and induce the expres- cartilage was obtained from the Institute of Pathology, Chris- sion of catabolic cytokines [17]. However, the evidence for the tian-Albrechts-University, (Kiel, Germany) as post-mortem role of ROS is conflicting, because other investigators have donor tissue. Donors who were 75 years of age or older were demonstrated anti-inflammatory properties of ROS in articular excluded; the average age of the donors was 54 years. Knee cartilage [18]. cartilage was scored, using a modified Collins scale [22], for visual degeneration of grade 2 or less, otherwise the sample The relationship between ROS and VEGF in articular cartilage was rejected. degradation has not been investigated previously. Investiga- tions focusing on the effects of ROS have used ROS donors Cartilage–bone cylinders (11 mm in diameter) from the femo- as stimulants. Phorbol myristate acetate (PMA) activates pro- ral condyles and femoropatellar groove were punched out per- tein kinase C and upregulates nicotinamide adenine dinucle- pendicular to the cartilage surface using Arthrex (Arthrex otide phosphate (NADPH) oxidase, leading to enhanced GmbH, Karlsfeld, Germany) instruments for osteochondral .- production of superoxide anions (O ) [19], one of the major transplantation (T-handle bar and punch; AR-1980D-11). The ROS. Another potent ROS donor is 3-morpholino-sydnon- cartilage–bone samples were removed, rinsed in HANK's Page 2 of 8 (page number not for citation purposes) Available online http://arthritis-research.com/content/8/6/R189 solution (supplemented with antibiotics; see below) and Quantitative real-time RT-PCR for VEGF, VEGFR-1 and placed in a microtome holder. After creating a level surface by VEGFR-2 removing superficial tissue, the cartilage tissue was sliced at a Real-time RT-PCR was carried out using a one-step system, thickness of 1 mm. Finally, up to eight explant discs (measur- according to the manufacturer's instructions (QuantiTect ing 3 mm in diameter and 1 mm in thickness) were punched SYBR Green RT-PCR; Qiagen), with 100 ng of total RNA in from each slice. In all subsequent experiments, treatment an i-Cycler (Biorad, Munich, Germany). The temperature pro- groups were location-matched by distributing the explant file included an initial denaturation for 15 minutes at 95°C, fol- discs from a single slice to each of the different groups. lowed by 37 cycles of denaturation at 95°C for 15 seconds, annealing at a temperature of 60°C for 30 seconds, elongation Cartilage explants were equilibrated for 2 days in culture at 72°C (the elongation time depended on the size of the frag- medium (250 μl of high-glucose DMEM with supplements (as ment, that is the number of bp divided by 25 yielded the time above) per explant in a 96-well plate) under free-swelling con- in seconds) and fluorescence monitoring at 72°C. Each cDNA ditions at 37°C in a standard cell-culture environment. Then, sample was analysed for expression of the gene of interest, in cartilage explants were rinsed twice with HANK's solution and addition to G3PDH, with the fluorescent TaqMan 5'-nuclease placed in serum-free medium, containing 0.05% BSA, for sub- assay, using 2 × TaqMan Master Mix (Applied Biosystems, sequent stimulation. For each group, we used eight cartilage Foster City, CA, USA) and 20 × assay-on-demand TaqMan explants in five different experiments. primers and probes in a total volume of 20 μl. Each plate included no-template controls (NTCs). TaqMan human prim- Stimulants ers and probes had the following identification numbers: PMA was used at concentrations of 5, 10 and 20 μg/ml in VEGF, Hs00173626_m1; VEGFR-1, Hs00176573_m1; medium. SIN-1 concentrations were 1, 10 and 20 μM. VEGFR-2, Hs00176676_m1; and G3PDH, Chondrocytes in monolayer culture were exposed to PMA or Hs99999905_m1. The cycle of threshold (C ) for each sam- SIN-1 stimulation for 48 hours and cartilage explants were ple was averaged and normalized to G3PDH. The results were (-ΔΔCT) similarly stimulated for 72 hours. then analysed by comparative ΔΔC method (2 ) for rela- tive quantification of gene expression: Isolation of RNA and cDNA synthesis Total RNA was extracted from immortalized chondrocytes ΔΔC = ΔC (sample) - ΔC (control) T T T using an RNeasy Total RNA Kit (Qiagen, Hilden, Germany). Total RNA from tissue homogenates of cartilage explants was ΔC (sample) = C (sample; target) - C (sample; G3PDH) T T T extracted using the TriZOL Reagent (Invitrogen, Life Technol- ogies, Karlsruhe, Germany). DNA contamination was ΔC (control) = C (control; target) - C (control; G3PDH) T T T destroyed by digestion with RNase-free DNase-I (20 minutes at 25°C; Boehringer, Mannheim, Germany), and cDNA was ELISA generated from 100 ng RNA reacting with 1 μl (20 pmol) of After stimulation with PMA or SIN-1, the conditioned medium oligo(dT) primer (Amersham Biosciences, Amersham, UK) supernatant of each chondrocyte monolayer or cartilage and 0.8 μl of superscript RNase H-reverse transcriptase explant culture was collected. Aliquots were analysed using a (Gibco, Paisley, UK) in 50 μl total volume for 60 minutes at sandwich ELISA (R&D Systems, Minneapolis, MN, USA) to 37°C. For each sample, a control without reverse tran- detect VEGF, and signals were identified by a chemolumines- scriptase was run in parallel to enable assessment of genomic cence reaction (ECL-Plus; Amersham-Pharmacia, Uppsala, DNA contamination. Sweden). Human recombinant VEGF (Repro Tech, Rocky Hill, NJ, USA) served as an internal standard. Aliquots of syn- RT-PCR for VEGF splice variants ovial fluid samples from OA patients were analysed by an iden- For PCR, 4 μl of cDNA was incubated with 30.5 μl water, 4 μl tical procedure. VEGF concentrations were normalized using 25 mM MgCl , 1 μl deoxynucleoside-triphosphate, 5 μl 10 × Bradford reagent (Roti-Quant; Roth, Karlsruhe, Germany). PCR buffer, 0.5 μl (2.5 U) Platinum Taq DNA polymerase (Gibco) and 2.5 μl (10 pmol) of each primer pair. The following Biochemical analysis primers and conditions were applied: VEGF splice variants, 5'- Concentrations of nitrite, the stable end product of NO, were CCA-TGA-ACT-TTC-TGC-TGT-CTT-3' (sense) and 5'-TCG- analysed in the culture medium using Griess reagent, accord- ATC-GTT-CTG-TAT-CAG-TCT-3' (antisense), with 40 cycles ing to the protocol described by Ailland and coworkers [23]. performed at a 55°C annealing temperature. A glyceralde- Results were corrected for the nitrite content of pure medium hyde-3-phosphate-dehydrogenase (G3PDH)-specific primer with or without (blanks) the PMA or SIN-1 stimulants. Data pair (5'-ATC-AAG-AAG-GTG-GTG-AAG-CAGG-3' (sense) were calculated according to the amount of medium and and 5'-TGA-GTG-TCG-CTG-TTG-AAG-TCG-3' (antisense), normalized to the number of cells or cartilage wet weight with 40 cycles at 58°C) served as the internal control (983 (monolayer or tissue explants, respectively) and control group, bp). which was set at 100%. Page 3 of 8 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 6 Fay et al. Figure 1 The VEGF splice variants VEGF-121 and VEGF-165 are detectable by splice-variant RT-PCR To determine whether the splice variants VEGF-121 (526 bp) and VEGF-165 (658 bp) are expressed after stimulation of .- chondrocytes with PMA, a known inducer of O , semiquanti- tative RT-PCR was performed (Figure 2). Both splice variants, VEGF-121 (526 bp) and VEGF-165 (658 bp), are present in immortalized chondrocytes and articular cartilage explants. There were bold signals corresponding to VEGF-121 and fine bands corresponding to VEGF-165. In general, more intense signals were present in the chondrocytes compared with those in cartilage explants. Stimulation with PMA (10 μg/ml) increased the signals of the mRNAs encoding the VEGF splice variants in monolayer chondrocytes and cartilage explants. Co E Co LImpa mpa SA) fro r rison ison m hea of th of th lthy e V e V(Con E EGF GF con con ) or OA t ten ent of syn t of syn patients o ovial flu vial fluiid (a d (as s de determ termin ined by ed by ELISA) from healthy (Con) or OA patients. The level of VEGF is strongly Real-time RT-PCR revealed upregulation of VEGF, increased in the synovial fluid of OA patients. Results are shown as VEGFR-1 and VEGFR-2 mRNA after stimulation with mean ± standard error of the mean; n = 5 (Con) and n = 8 (OA). * p < ROS donors 0.05. Con, control; OA, osteoarthritis; VEGF, vascular endothelial The levels of mRNA encoding VEGF and VEGF receptors growth factor. (VEGFR-1 (flt-1) and VEGFR-2 (KDR, flk-1)) were quantified Statistics by real-time RT-PCR. After challenging with PMA, VEGF All data are shown as mean ± standard error of the mean mRNA was upregulated in monolayer chondrocytes (Figure (SEM), unless indicated otherwise. Differences between ana- 3a). The levels of VEGF were elevated dose-dependently, from lysed data were tested using the Student t test. Significance 4.1-fold at 5 μg of PMA to 15.8-fold at 10 μg of PMA, with a was set to p value of < 0.05. following decrease by 4.4-fold at 20 μg of PMA. Although VEGFR-1 mRNA levels were increased only slightly (1.5-fold, 2.8-fold and 2.4-fold) after PMA stimulation (at 5, 10 and 20 Results Increased levels of VEGF in synovial fluids from patients μg, respectively), the levels of VEGFR-2 mRNA were elevated with OA 2.6-fold, 10.4-fold and 4.6-fold at PMA concentrations of 5, 10 To characterize VEGF in vivo, aspirates of synovial fluid were and 20 μg, respectively. Stimulation of cartilage explants with assessed for VEGF by ELISA. Compared with VEGF concen- 10 and 20 μg of PMA upregulated VEGF mRNA by 2.3-fold trations in healthy joints (36 pg/ml), VEGF concentrations in and 4.9-fold, respectively, compared with the control (Figure the synovial fluid of patients with OA were significantly higher 3b). The level of VEGFR-2 mRNA was unaffected by 10 μg of (2,100 pg/ml, which was nearly 60-fold higher than healthy PMA and increased 2.7-fold by 20 μg of PMA. The level of synovial fluids; control versus OA, p ≤ 0.05 (Figure 1)). VEGFR-1 mRNA was undetectable in cartilage explants after treatment with PMA. Figure 2 Treatment of chondrocytes (monolayer) with SIN-1 resulted in responses similar, in part, to those after PMA stimulation. After treatment with 1, 10 and 20 μM of SIN-1, VEGF mRNA expression was increased 2.2-fold, 19.6-fold and 17.2-fold, respectively (Figure 3c), and VEGFR-2 mRNA expression was elevated 2.0-fold, 15.4-fold and 13.5-fold, respectively. In con- trast to PMA, 1, 10 and 20 μM of SIN-1 enhanced VEGFR-1 mRNA levels 1.2-fold, 9.2-fold and 8.1-fold, respectively, com- pared with the control. In the cartilage explants, 3.1-fold and 9.0-fold increases in VEGF mRNA expression were apparent after treatment with 10 and 20 μM of SIN-1, respectively VE VEG GF F spl spliic ce vari e variants. Expressi ants on of VEGF-121 (526 bp) and VEGF- (Figure 3d). VEGFR-2 mRNA expression was increased by 165 (658 bp) in cartilage explants and immortalized chondrocytes after 1.8-fold and 6.1-fold at 10 and 20 μM of SIN-1, respectively. stimulation with PMA (10 μg/ml) and SIN-1 (10 μM). The splice vari- In contrast to the undetectable level of VEGFR-1 in cartilage ants VEGF-121 and VEGF-165 are detected in cartilage explants and C28/I2 cells. Con, control; PMA, phorbol myristate acetate; SIN-1, 3- explants after PMA treatment, VEGFR-1 mRNA levels slightly morpholino-sydnonimine hydrochloride; VEGF, vascular endothelial increased after treatment with SIN-1 (1.2-fold and 1.8-fold growth factor. increases at 10 and 20 μM of SIN-1, respectively). Page 4 of 8 (page number not for citation purposes) Available online http://arthritis-research.com/content/8/6/R189 Figure 3 Q Qu uan antitati titative ve mRN mRNA A ex expre pressio ssion n o of f VE VEG GF F,, VE VEG GF FR R- -1 an 1 and VE d VEG GF FR-2 R-2 no norm rmaliz alized ed t to o th the c e co ontr ntro oll ( (n n = 1) = 1). Stimulation of immortalized chondrocytes (a) and (c) and articular cartilage explants (b) and (d) with PMA (μg per 1 ml of medium) or SIN-1 (μM). VEGFR-1 is undetectable in (b). mRNA expres- sion of VEGF, VEGFR-1 and VEGFR-2 is upregulated after stimulation with reactive oxygen species donors. Results are shown as mean ± standard error of the mean for five separate experiments. * p < 0.05 versus control. G3PDH, glyceroaldehyde-3-phosphate dehydrogenase; PMA, phorbol myristate acetate; SIN-1, 3-morpholino-sydnonimine hydrochloride; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor. Enhanced VEGF production after stimulation with PMA Increasing nitric oxide content of the medium or SIN-1 (using ELISA) supernatant after stimulation with ROS donors To determine whether the increased levels of VEGF mRNA PMA dose-dependently increased the NO content of culture were reflected in the production of protein by chondrocytes, medium from chondrocytes and, to a lesser extent, cartilage an ELISA was performed to quantify the VEGF content in the explants. In monolayer cultures, NO levels were increased 2.6- medium supernatants (Figure 4). Chondrocyte monolayer con- fold, 3.2-fold and 4.4-fold at 5, 10 and 20 μg/ml of PMA com- trols released 1,100 pg of VEGF per 1 ml of medium and car- pared with the control (control versus PMA, p ≤ 0.05; Figure tilage explant controls released 540 pg of VEGF per 1 ml of 5a). By contrast, the cartilage explants showed no response to medium. Treatment with PMA at concentrations of 5, 10 and low-dose PMA stimulation (5 μg) and only 1.7-fold and 2.4- 20 μg/ml increased VEGF production by 2.4-fold, 3.0-fold and fold increases using 10 and 20 μg of PMA, respectively (con- 3.4-fold, respectively, in monolayer chondrocytes (control ver- trol versus PMA, p ≤ 0.05; Figure 5b). After treatment with sus PMA, p ≤ 0.05) and treatment with SIN-1 at concentra- SIN-1, the NO content showed similar results but the effect tions of 1, 10 and 20 μM increased the level of VEGF by 1.7- was more extended (Figure 5a): in monolayer chondrocytes, fold, 2.5-fold and 2.8-fold, respectively (control versus SIN-1, the NO content was 2.1-fold, 24.8-fold and 41.9-fold higher p ≤ 0.05; Figure 4a). In cartilage explants, no increase in VEGF than control after treatment with 1, 10 and 20 μM of SIN-1 was detected at 5 μg of PMA or 1 μM of SIN-1 (Figure 4b). (control versus SIN-1, p ≤ 0.05). In the cartilage explants, the Using higher concentrations, VEGF production was increased effects were attenuated compared with the monolayer cul- 2.7-fold and 3.8-fold after treatment with PMA at concentra- tures, with 1.7-fold, 2.5-fold and 3.6-fold increases in NO tions of 10 and 20 μg, respectively (control versus PMA, p ≤ content reported after treatment with 1, 10 and 20 μM of SIN- 0.05), and 2.4-fold and 3.7-fold after treatment with SIN-1 at 1, respectively (control versus SIN-1, p ≤ 0.05; Figure 5b). concentrations of 10 and 20 μM, respectively (control versus SIN-1, p ≤ 0.05). Page 5 of 8 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 6 Fay et al. Figure 4 Figure 5 Th The e V VE EG GF F co con nt ten ent o t of f medi medium um s su upe pern rnata atan nt (a t (as det s dete erm rmin ined by ed by EL ELIS ISA) A). Th The N e NO con O conten tent of t of m me ediu dium m supern superna ata tan nt norm t normalize alized d to th to the Con ( e Con (n n = 1). = 1) Stimulation of immortalized chondrocytes (a) and articular cartilage Stimulation of immortalized chondrocytes (a) and articular cartilage explants (b) with PMA (μg per 1 ml of medium) or SIN-1 (μM). The level explants (b) with PMA (μg per 1 ml of medium) or SIN-1 (μM). The level of VEGF protein is increased after stimulation with PMA or SIN-1. of NO is increased after stimulation with reactive oxygen species Results are shown as mean ± standard error of the mean for five sepa- donors. Results are shown as mean ± standard error of the mean for rate experiments. * p < 0.05 versus Con. Con, control; PMA, phorbol five separate experiments. * p < 0.05 versus Con. Con, control; NO, myristate acetate; SIN-1, 3-morpholino-sydnonimine hydrochloride; nitric oxide; PMA, phorbol myristate acetate; SIN-1, 3-morpholino-syd- VEGF, vascular endothelial growth factor. nonimine hydrochloride. Discussion Our findings show that the level of VEGF in synovial joint fluids from patients suffering from OA is 60-fold higher than healthy lage metabolism during rheumatoid arthritis. The initial growth joints. Moreover, our in vitro model revealed that VEGF mRNA and invasion of the synovial pannus tissue contributes to the and protein levels and VEGF receptors are increased by PMA subsequent cartilage destruction. Blockade of VEGFR-I or SIN-1 stimulation in chondrocytes and human articular car- resulted in reduced intensity of clinical manifestations and pre- tilage explants. We conclude that the presence of ROS, or vented joint destruction in a mouse model of rheumatoid arthri- .- activation of production of O , is responsible for the observed tis [26]. It is obvious that tissues other than cartilage are results. Thus, VEGF accumulation in the synovial fluid is, at participating in these processes, especially the surrounding least in part, cartilage-derived. synovial tissue. This reflects the findings of Felson and coworkers [27], who declared OA to be a disease involving Inflammation in OA is known to be associated with activation the whole joint. In summary, these data support the role of of host angiogenesis [24]. VEGF is one of the most potent VEGF in mediating destructive processes in articular joints proangiogenic stimuli of neovascularization. Furthermore, the and encouraged us to investigate the relationship between capacity of VEGF to mediate chemotaxis, raise vascular per- VEGF expression and ROS in articular cartilage. Focusing on meability for neutrophil influx and activate MMPs in chondro- other cell types, such as glomerular podocytes, endothelial cytes suggests a central function in catabolic pathways of OA cells and skeletal muscle fibres, a correlation between ROS joints [9,25]. In addition to the involvement of VEGF in the and VEGF is described, but the results were contradictory development of OA, VEGF has biological importance in carti- [28,29]. Page 6 of 8 (page number not for citation purposes) Available online http://arthritis-research.com/content/8/6/R189 Here, we demonstrate increased VEGF mRNA expression and line, designed part of the study and contributed to the draft production in cultured human chondrocytes and articular car- manuscript. The manuscript has been read and approved by tilage explants after challenge by ROS donors. We conclude all authors. that the observed increase in VEGF content in synovial fluid of OA joints is produced partly by articular chondrocytes and Acknowledgements The authors would like to thank Inka Kronenbitter, Ursula Mundt, Frank consistent with previous findings from this and other laborato- Lichte and Sonja Seiter for their excellent technical assistance. The T- ries, which showed an increase in VEGF content in OA carti- handle bar to perform tissue harvest from the donors' knees was a gen- lage [8,10,11]. The observation that the concentration of erous gift from Arthrex , Karlsfeld, Germany. This work was funded, in VEGF is positively correlated to joint destruction and vascular- part, by the Research Promotion of Faculty of Medicine, Kiel University, ization of synovial membrane in rheumatoid arthritis [30] sug- Kiel, Germany, and Deutsche Forschungsgemeinschaft (DFG; Pu 214/ gests the potential impact of VEGF in the pathophysiology of 4-2, Pu 214/3-2 and Pu 214/5-2). MG's research was funded by a grant OA. from the National Institutes of Health (R01-AG22021). Our demonstration of ROS-mediated induction of the ang- References 1. Fernandes JC, Martel-Pelletier J, Pelletier JP: The role of iogenic factor VEGF in human chondrocytes and articular car- cytokines in osteoarthritis pathophysiology. Biorheology 2002, tilage explants is consistent with prior reports showing that 39:237-246. NO stimulates VEGF production in chondrocytes [31]. How- 2. Burrage PS, Mix KS, Brinckerhoff CE: Matrix metalloproteinases: role in arthritis. Front Biosci 2006, 11:529-543. ever, we observed different responses to the two ROS donors. 3. Ferrara N: Role of vascular endothelial growth factor in the reg- The NO content after stimulation with SIN-1 was up to tenfold ulation of angiogenesis. Kidney Int 1999, 56:794-814. higher than after stimulation with PMA and only SIN-1 induced 4. 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Ayache N, Boumediene K, Mathy-Hartert M, Reginster JY, Henrotin Y, Pujol JP: Expression of TGF-betas and their receptors is dif- ferentially modulated by reactive oxygen species and nitric Authors' contributions oxide in human articular chondrocytes. Osteoarthritis Cartilage JV, DV, CW, BK and TP performed the experiments and con- 2002, 10:344-352. tributed to the draft manuscript; DV and TP contributed 18. Mathy-Hartert M, Martin G, Devel P, Deby-Dupont G, Pujol JP, Reginster JY, Henrotin Y: Reactive oxygen species downregu- equally to the present work. MG established the C28/I2 cell Page 7 of 8 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 6 Fay et al. late the expression of pro-inflammatory genes by human chondrocytes. Inflamm Res 2003, 52:111-118. 19. Li JM, Mullen AM, Yun S, Wientjes F, Brouns GY, Thrasher AJ, Shah AM: Essential role of the NADPH oxidase subunit p47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor-alpha. Circ Res 2002, 90:143-150. 20. de Groot H, Hegi U, Sies H: Loss of alpha-tocopherol upon exposure to nitric oxide or the sydnonimine SIN-1. FEBS Lett 1993, 315:139-142. 21. Goldring MB, Birkhead JR, Suen LF, Yamin R, Mizuno S, Glowacki J, Arbiser JL, Apperley JF: Interleukin-1 beta-modulated gene expression in immortalized human chondrocytes. J Clin Invest 1994, 94:2307-2316. 22. Muehleman C, Bareither D, Huch K, Cole AA, Kuettner KE: Prev- alence of degenerative morphological changes in the joints of the lower extremity. Osteoarthritis Cartilage 1997, 5:23-37. 23. Ailland J, Kampen WU, Schunke M, Trentmann J, Kurz B: Beta irra- diation decreases collagen type II synthesis and increases nitric oxide production and cell death in articular chondrocytes. Ann Rheum Dis 2003, 62:1054-1060. 24. Cerimele F, Brown LF, Bravo F, Ihler GM, Kouadio P, Arbiser JL: Infectious angiogenesis: Bartonella bacilliformis infection results in endothelial production of angiopoetin-2 and epider- mal production of vascular endothelial growth factor. Am J Pathol 2003, 163:1321-1327. 25. Gruber BL, Marchese MJ, Kew R: Angiogenic factors stimulate mast-cell migration. Blood 1995, 86:2488-2493. 26. De Bandt M, Ben Mahdi MH, Ollivier V, Grossin M, Dupuis M, Gaudry M, Bohlen P, Lipson KE, Rice A, Wu Y, et al.: Blockade of vascular endothelial growth factor receptor I (VEGF-RI), but not VEGF-RII, suppresses joint destruction in the K/BxN model of rheumatoid arthritis. J Immunol 2003, 171:4853-4859. 27. Felson DT, Lawrence RC, Dieppe PA, Hirsch R, Helmick CG, Jor- dan JM, Kington RS, Lane NE, Nevitt MC, Zhang Y, et al.: Oste- oarthritis: new insights. Part 1: the disease and its risk factors. Ann Intern Med 2000, 133:635-646. 28. Lee EY, Chung CH, Kim JH, Joung HJ, Hong SY: Antioxidants ameliorate the expression of vascular endothelial growth fac- tor mediated by protein kinase C in diabetic podocytes. Neph- rol Dial Transplant 2006, 21:1496-1503. 29. Kosmidou I, Xagorari A, Roussos C, Papapetropoulos A: Reactive oxygen species stimulate VEGF production from C(2)C(12) skeletal myotubes through a PI3K/Akt pathway. Am J Physiol Lung Cell Mol Physiol 2001, 280:L585-L592. 30. Nagashima M, Yoshino S, Ishiwata T, Asano G: Role of vascular endothelial growth factor in angiogenesis of rheumatoid arthritis. J Rheumatol 1995, 22:1624-1630. 31. Turpaev K, Litvinov D, Dubovaya V, Panasyuk A, Ivanov D, Prassolov V: Induction of vascular endothelial growth factor by nitric oxide in cultured human articular chondrocytes. Bio- chimie 2001, 83:515-522. Page 8 of 8 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Arthritis Research & Therapy Springer Journals

Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes and human articular cartilage explants

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Springer Journals
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2006 Fay et al.; licensee BioMed Central Ltd.
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1478-6354
DOI
10.1186/ar2102
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Abstract

Vascular endothelial growth factor (VEGF) promotes cartilage- PCR. Expression of VEGF and VEGF receptors (VEGFR-1 and degrading pathways, and there is evidence for the involvement VEGFR-2) was quantified by real-time RT-PCR. of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been Synovial fluid from OA patients revealed markedly elevated levels reported. Here, we investigate whether the expression of VEGF of VEGF. Common RT-PCR revealed that the splice variants is modulated by ROS. were present in both immortalized chondrocytes and cartilage discs. In immortalized chondrocytes, stimulation with PMA or SIN-1 caused increases in the levels of VEGF, VEGFR-1 and Aspirates of synovial fluid from patients with osteoarthritis (OA) VEGFR-2 mRNA expression. Cartilage explants produced similar were examined for intra-articular VEGF using ELISA. results, but VEGFR-1 was only detectable after stimulation with Immortalized C28/I2 chondrocytes and human knee cartilage SIN-1. Stimulation with PMA or SIN-1 resulted in a dose- explants were exposed to phorbol myristate acetate (PMA; 0– dependent upregulation of the VEGF protein (as determined 20 μg/ml), which is a ROS inducer, or 3-morpholino- using ELISA) and an increase in the level of NO in the medium. sydnonimine hydrochloride (SIN-1; 0–20 μM), which is a ROS donor. The levels of VEGF protein and nitric oxide (NO) Our findings indicate ROS-mediated induction of VEGF and production were determined in the medium supernatant, using VEGF receptors in chondrocytes and cartilage explants. These ELISA and Griess reagent, respectively. Gene expression of results demonstrate a relationship between ROS and VEGF as VEGF-121 and VEGF-165 was determined by splice variant RT- multiplex mediators in articular cartilage degeneration. ever, the precise signalling pathways in the degradation of Introduction Osteoarthritis (OA) is characterized by a breakdown of the articular cartilage ECM and development of OA are still not extracellular matrix (ECM) of articular cartilage in the affected fully understood. Several studies have demonstrated the joints. The pathogenesis of OA involves multiple aetiologies, involvement of cytokines, such as IL-1 and IL-6, or tumour including mechanical, genetic and biochemical factors. How- necrosis factor (TNF)-α, in addition to proteases, such as AP-1 = activator protein 1; DMEM = Dulbecco's modified Eagle's medium; ECM = extracellular matrix; ELISA = enzyme-linked immunosorbent assay; G3PDH = glyceroaldehyde-3-phosphate dehydrogenase; IL = interleukin; MMP = matrix metalloproteinases; NO = nitric oxide; NTC = no-template control; OA = osteoarthritis; PMA = phorbol myristate acetate; ROS = reactive oxygen species; RT-PCR = reverse transcriptase polymerase chain reaction; SEM = standard error of the mean; SIN-1 = 3-morpholino-sydnonimine hydrochloride; TIMP = tissue inhibitor of matrix metalloproteinase; TNF = tumour necrosis factor; TPA = 12-O-tetradecanoylphorbol-13-acetate; VEGF: vascular endothelial growth factor; VEGFR = VEGF receptor. Page 1 of 8 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 6 Fay et al. matrix metalloproteases (MMPs), in the initiation and progres- imine hydrochloride (SIN-1), which spontaneously decom- .- sion of articular cartilage destruction [1,2]. The imbalance poses to nitric oxide (NO) radicals and O [20]. between activated proteinases and inhibitors ultimately leads to an altered net proteolysis of cartilage components. Once Therefore, the present study was performed to investigate the damaged, articular cartilage has a poor capacity for intrinsic expression of VEGF, VEGF-121 and VEGF-165 splice vari- repair. ants and VEGF receptors (VEGFR-1 and VEGFR-2) after stim- ulation by ROS donors. Knowledge of this expression could Angiogenesis, the development of new blood vessels by lead to a better understanding of the complex role of ROS and sprouting from pre-existing endothelium, is a significant com- VEGF in articular cartilage degeneration. ponent of a wide variety of biological processes [3,4]. How- ever, in rheumatoid arthritis, new capillary blood vessels invade Materials and methods the joints from the emerging synovial pannus and aid in the Reagents destruction of articular cartilage [5], even in the absence of a Chemical reagents were purchased from Sigma (Munich, Ger- causative factor. The most important mediator of angiogenesis many), unless otherwise indicated. is vascular endothelial growth factor (VEGF) [6], which stimu- lates capillary formation in vivo and has direct mitogenic Synovial fluid actions on various cells in vitro [7]. Recent data reveal expres- Samples of synovial fluid from patients with OA (n = 8) were sion of VEGF in OA cartilage and reflect the ability of VEGF to obtained from the Department of Orthopaedic Surgery at the enhance catabolic pathways in chondrocytes by stimulating University Hospital Schleswig-Holstein (Kiel, Germany). Syno- MMP activity and reducing natural MMP inhibitors, that is tis- vial fluid from healthy joints was collected from deceased sue inhibitors of MMPs (TIMPs) [8-11]. These data suggest donors (n = 5) at the Department of Anatomy, Christian-Albre- that, except from the effect of VEGF on proliferation of synovial chts-University (Kiel, Germany). membranes, chondrocyte-derived VEGF promotes catabolic pathways in the cartilage itself, thereby leading to a progres- Chondrocyte monolayer culture sive breakdown of the ECM of articular cartilage. The human C28/I2 chondrocyte cell line was used in monol- ayer culture. These chondrocytes, which were immortalized Recent investigations have revealed the participation of free using SV-40 large T-antigen, continue to express chondro- radicals in the pathogenesis of articular cartilage degradation cyte-specific aggrecan and collagen type II mRNA after multi- [12]. Free radicals are highly reactive in oxidative processes ple subculture [21]. Chondrocytes were seeded (500,000 and are essentially involved in physiological reactions, such as cells/25 cm ) in DMEM supplemented with 10% foetal bovine the cellular respiratory chain. However, uncontrolled release of serum, 10 mM h-(2-hydroxyethyl)-1-piperazinethansulfonacid free radicals can result ultimately in an imbalance, with respect (HEPES) buffer, 1 mM sodium pyruvate, 0.4 mM proline, 20 to their inhibitors or antioxidants. Moreover, free radicals can μg/ml ascorbic acid, 100 U/ml penicillin G, 100 μg/ml strep- stimulate inflammatory pathways or damage lipids, proteins or tomycin and 0.25 μg/ml amphotericin B. After reaching 80% DNA [13]. In the nomenclature of free radicals, the term 'reac- confluence, the cells were rinsed twice with HANK's solution tive oxygen species' (ROS) has prevailed, although ROS can and placed in serum-free medium, containing 0.05% BSA, for be differentiated into reactive nitrogen species and other oxi- subsequent stimulation. dant species. The relationship between ROS and articular car- Human articular cartilage explants tilage degradation is complex and involves multiple pathways [14]. ROS can induce changes in biosynthetic activity [15], in The tissue harvest was approved by the Ethical Commission addition to apoptosis [16]. In addition, ROS can influence of Christian-Albrechts-University of Kiel (Kiel, Germany). Knee transcription factors in chondrocytes and induce the expres- cartilage was obtained from the Institute of Pathology, Chris- sion of catabolic cytokines [17]. However, the evidence for the tian-Albrechts-University, (Kiel, Germany) as post-mortem role of ROS is conflicting, because other investigators have donor tissue. Donors who were 75 years of age or older were demonstrated anti-inflammatory properties of ROS in articular excluded; the average age of the donors was 54 years. Knee cartilage [18]. cartilage was scored, using a modified Collins scale [22], for visual degeneration of grade 2 or less, otherwise the sample The relationship between ROS and VEGF in articular cartilage was rejected. degradation has not been investigated previously. Investiga- tions focusing on the effects of ROS have used ROS donors Cartilage–bone cylinders (11 mm in diameter) from the femo- as stimulants. Phorbol myristate acetate (PMA) activates pro- ral condyles and femoropatellar groove were punched out per- tein kinase C and upregulates nicotinamide adenine dinucle- pendicular to the cartilage surface using Arthrex (Arthrex otide phosphate (NADPH) oxidase, leading to enhanced GmbH, Karlsfeld, Germany) instruments for osteochondral .- production of superoxide anions (O ) [19], one of the major transplantation (T-handle bar and punch; AR-1980D-11). The ROS. Another potent ROS donor is 3-morpholino-sydnon- cartilage–bone samples were removed, rinsed in HANK's Page 2 of 8 (page number not for citation purposes) Available online http://arthritis-research.com/content/8/6/R189 solution (supplemented with antibiotics; see below) and Quantitative real-time RT-PCR for VEGF, VEGFR-1 and placed in a microtome holder. After creating a level surface by VEGFR-2 removing superficial tissue, the cartilage tissue was sliced at a Real-time RT-PCR was carried out using a one-step system, thickness of 1 mm. Finally, up to eight explant discs (measur- according to the manufacturer's instructions (QuantiTect ing 3 mm in diameter and 1 mm in thickness) were punched SYBR Green RT-PCR; Qiagen), with 100 ng of total RNA in from each slice. In all subsequent experiments, treatment an i-Cycler (Biorad, Munich, Germany). The temperature pro- groups were location-matched by distributing the explant file included an initial denaturation for 15 minutes at 95°C, fol- discs from a single slice to each of the different groups. lowed by 37 cycles of denaturation at 95°C for 15 seconds, annealing at a temperature of 60°C for 30 seconds, elongation Cartilage explants were equilibrated for 2 days in culture at 72°C (the elongation time depended on the size of the frag- medium (250 μl of high-glucose DMEM with supplements (as ment, that is the number of bp divided by 25 yielded the time above) per explant in a 96-well plate) under free-swelling con- in seconds) and fluorescence monitoring at 72°C. Each cDNA ditions at 37°C in a standard cell-culture environment. Then, sample was analysed for expression of the gene of interest, in cartilage explants were rinsed twice with HANK's solution and addition to G3PDH, with the fluorescent TaqMan 5'-nuclease placed in serum-free medium, containing 0.05% BSA, for sub- assay, using 2 × TaqMan Master Mix (Applied Biosystems, sequent stimulation. For each group, we used eight cartilage Foster City, CA, USA) and 20 × assay-on-demand TaqMan explants in five different experiments. primers and probes in a total volume of 20 μl. Each plate included no-template controls (NTCs). TaqMan human prim- Stimulants ers and probes had the following identification numbers: PMA was used at concentrations of 5, 10 and 20 μg/ml in VEGF, Hs00173626_m1; VEGFR-1, Hs00176573_m1; medium. SIN-1 concentrations were 1, 10 and 20 μM. VEGFR-2, Hs00176676_m1; and G3PDH, Chondrocytes in monolayer culture were exposed to PMA or Hs99999905_m1. The cycle of threshold (C ) for each sam- SIN-1 stimulation for 48 hours and cartilage explants were ple was averaged and normalized to G3PDH. The results were (-ΔΔCT) similarly stimulated for 72 hours. then analysed by comparative ΔΔC method (2 ) for rela- tive quantification of gene expression: Isolation of RNA and cDNA synthesis Total RNA was extracted from immortalized chondrocytes ΔΔC = ΔC (sample) - ΔC (control) T T T using an RNeasy Total RNA Kit (Qiagen, Hilden, Germany). Total RNA from tissue homogenates of cartilage explants was ΔC (sample) = C (sample; target) - C (sample; G3PDH) T T T extracted using the TriZOL Reagent (Invitrogen, Life Technol- ogies, Karlsruhe, Germany). DNA contamination was ΔC (control) = C (control; target) - C (control; G3PDH) T T T destroyed by digestion with RNase-free DNase-I (20 minutes at 25°C; Boehringer, Mannheim, Germany), and cDNA was ELISA generated from 100 ng RNA reacting with 1 μl (20 pmol) of After stimulation with PMA or SIN-1, the conditioned medium oligo(dT) primer (Amersham Biosciences, Amersham, UK) supernatant of each chondrocyte monolayer or cartilage and 0.8 μl of superscript RNase H-reverse transcriptase explant culture was collected. Aliquots were analysed using a (Gibco, Paisley, UK) in 50 μl total volume for 60 minutes at sandwich ELISA (R&D Systems, Minneapolis, MN, USA) to 37°C. For each sample, a control without reverse tran- detect VEGF, and signals were identified by a chemolumines- scriptase was run in parallel to enable assessment of genomic cence reaction (ECL-Plus; Amersham-Pharmacia, Uppsala, DNA contamination. Sweden). Human recombinant VEGF (Repro Tech, Rocky Hill, NJ, USA) served as an internal standard. Aliquots of syn- RT-PCR for VEGF splice variants ovial fluid samples from OA patients were analysed by an iden- For PCR, 4 μl of cDNA was incubated with 30.5 μl water, 4 μl tical procedure. VEGF concentrations were normalized using 25 mM MgCl , 1 μl deoxynucleoside-triphosphate, 5 μl 10 × Bradford reagent (Roti-Quant; Roth, Karlsruhe, Germany). PCR buffer, 0.5 μl (2.5 U) Platinum Taq DNA polymerase (Gibco) and 2.5 μl (10 pmol) of each primer pair. The following Biochemical analysis primers and conditions were applied: VEGF splice variants, 5'- Concentrations of nitrite, the stable end product of NO, were CCA-TGA-ACT-TTC-TGC-TGT-CTT-3' (sense) and 5'-TCG- analysed in the culture medium using Griess reagent, accord- ATC-GTT-CTG-TAT-CAG-TCT-3' (antisense), with 40 cycles ing to the protocol described by Ailland and coworkers [23]. performed at a 55°C annealing temperature. A glyceralde- Results were corrected for the nitrite content of pure medium hyde-3-phosphate-dehydrogenase (G3PDH)-specific primer with or without (blanks) the PMA or SIN-1 stimulants. Data pair (5'-ATC-AAG-AAG-GTG-GTG-AAG-CAGG-3' (sense) were calculated according to the amount of medium and and 5'-TGA-GTG-TCG-CTG-TTG-AAG-TCG-3' (antisense), normalized to the number of cells or cartilage wet weight with 40 cycles at 58°C) served as the internal control (983 (monolayer or tissue explants, respectively) and control group, bp). which was set at 100%. Page 3 of 8 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 6 Fay et al. Figure 1 The VEGF splice variants VEGF-121 and VEGF-165 are detectable by splice-variant RT-PCR To determine whether the splice variants VEGF-121 (526 bp) and VEGF-165 (658 bp) are expressed after stimulation of .- chondrocytes with PMA, a known inducer of O , semiquanti- tative RT-PCR was performed (Figure 2). Both splice variants, VEGF-121 (526 bp) and VEGF-165 (658 bp), are present in immortalized chondrocytes and articular cartilage explants. There were bold signals corresponding to VEGF-121 and fine bands corresponding to VEGF-165. In general, more intense signals were present in the chondrocytes compared with those in cartilage explants. Stimulation with PMA (10 μg/ml) increased the signals of the mRNAs encoding the VEGF splice variants in monolayer chondrocytes and cartilage explants. Co E Co LImpa mpa SA) fro r rison ison m hea of th of th lthy e V e V(Con E EGF GF con con ) or OA t ten ent of syn t of syn patients o ovial flu vial fluiid (a d (as s de determ termin ined by ed by ELISA) from healthy (Con) or OA patients. The level of VEGF is strongly Real-time RT-PCR revealed upregulation of VEGF, increased in the synovial fluid of OA patients. Results are shown as VEGFR-1 and VEGFR-2 mRNA after stimulation with mean ± standard error of the mean; n = 5 (Con) and n = 8 (OA). * p < ROS donors 0.05. Con, control; OA, osteoarthritis; VEGF, vascular endothelial The levels of mRNA encoding VEGF and VEGF receptors growth factor. (VEGFR-1 (flt-1) and VEGFR-2 (KDR, flk-1)) were quantified Statistics by real-time RT-PCR. After challenging with PMA, VEGF All data are shown as mean ± standard error of the mean mRNA was upregulated in monolayer chondrocytes (Figure (SEM), unless indicated otherwise. Differences between ana- 3a). The levels of VEGF were elevated dose-dependently, from lysed data were tested using the Student t test. Significance 4.1-fold at 5 μg of PMA to 15.8-fold at 10 μg of PMA, with a was set to p value of < 0.05. following decrease by 4.4-fold at 20 μg of PMA. Although VEGFR-1 mRNA levels were increased only slightly (1.5-fold, 2.8-fold and 2.4-fold) after PMA stimulation (at 5, 10 and 20 Results Increased levels of VEGF in synovial fluids from patients μg, respectively), the levels of VEGFR-2 mRNA were elevated with OA 2.6-fold, 10.4-fold and 4.6-fold at PMA concentrations of 5, 10 To characterize VEGF in vivo, aspirates of synovial fluid were and 20 μg, respectively. Stimulation of cartilage explants with assessed for VEGF by ELISA. Compared with VEGF concen- 10 and 20 μg of PMA upregulated VEGF mRNA by 2.3-fold trations in healthy joints (36 pg/ml), VEGF concentrations in and 4.9-fold, respectively, compared with the control (Figure the synovial fluid of patients with OA were significantly higher 3b). The level of VEGFR-2 mRNA was unaffected by 10 μg of (2,100 pg/ml, which was nearly 60-fold higher than healthy PMA and increased 2.7-fold by 20 μg of PMA. The level of synovial fluids; control versus OA, p ≤ 0.05 (Figure 1)). VEGFR-1 mRNA was undetectable in cartilage explants after treatment with PMA. Figure 2 Treatment of chondrocytes (monolayer) with SIN-1 resulted in responses similar, in part, to those after PMA stimulation. After treatment with 1, 10 and 20 μM of SIN-1, VEGF mRNA expression was increased 2.2-fold, 19.6-fold and 17.2-fold, respectively (Figure 3c), and VEGFR-2 mRNA expression was elevated 2.0-fold, 15.4-fold and 13.5-fold, respectively. In con- trast to PMA, 1, 10 and 20 μM of SIN-1 enhanced VEGFR-1 mRNA levels 1.2-fold, 9.2-fold and 8.1-fold, respectively, com- pared with the control. In the cartilage explants, 3.1-fold and 9.0-fold increases in VEGF mRNA expression were apparent after treatment with 10 and 20 μM of SIN-1, respectively VE VEG GF F spl spliic ce vari e variants. Expressi ants on of VEGF-121 (526 bp) and VEGF- (Figure 3d). VEGFR-2 mRNA expression was increased by 165 (658 bp) in cartilage explants and immortalized chondrocytes after 1.8-fold and 6.1-fold at 10 and 20 μM of SIN-1, respectively. stimulation with PMA (10 μg/ml) and SIN-1 (10 μM). The splice vari- In contrast to the undetectable level of VEGFR-1 in cartilage ants VEGF-121 and VEGF-165 are detected in cartilage explants and C28/I2 cells. Con, control; PMA, phorbol myristate acetate; SIN-1, 3- explants after PMA treatment, VEGFR-1 mRNA levels slightly morpholino-sydnonimine hydrochloride; VEGF, vascular endothelial increased after treatment with SIN-1 (1.2-fold and 1.8-fold growth factor. increases at 10 and 20 μM of SIN-1, respectively). Page 4 of 8 (page number not for citation purposes) Available online http://arthritis-research.com/content/8/6/R189 Figure 3 Q Qu uan antitati titative ve mRN mRNA A ex expre pressio ssion n o of f VE VEG GF F,, VE VEG GF FR R- -1 an 1 and VE d VEG GF FR-2 R-2 no norm rmaliz alized ed t to o th the c e co ontr ntro oll ( (n n = 1) = 1). Stimulation of immortalized chondrocytes (a) and (c) and articular cartilage explants (b) and (d) with PMA (μg per 1 ml of medium) or SIN-1 (μM). VEGFR-1 is undetectable in (b). mRNA expres- sion of VEGF, VEGFR-1 and VEGFR-2 is upregulated after stimulation with reactive oxygen species donors. Results are shown as mean ± standard error of the mean for five separate experiments. * p < 0.05 versus control. G3PDH, glyceroaldehyde-3-phosphate dehydrogenase; PMA, phorbol myristate acetate; SIN-1, 3-morpholino-sydnonimine hydrochloride; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor. Enhanced VEGF production after stimulation with PMA Increasing nitric oxide content of the medium or SIN-1 (using ELISA) supernatant after stimulation with ROS donors To determine whether the increased levels of VEGF mRNA PMA dose-dependently increased the NO content of culture were reflected in the production of protein by chondrocytes, medium from chondrocytes and, to a lesser extent, cartilage an ELISA was performed to quantify the VEGF content in the explants. In monolayer cultures, NO levels were increased 2.6- medium supernatants (Figure 4). Chondrocyte monolayer con- fold, 3.2-fold and 4.4-fold at 5, 10 and 20 μg/ml of PMA com- trols released 1,100 pg of VEGF per 1 ml of medium and car- pared with the control (control versus PMA, p ≤ 0.05; Figure tilage explant controls released 540 pg of VEGF per 1 ml of 5a). By contrast, the cartilage explants showed no response to medium. Treatment with PMA at concentrations of 5, 10 and low-dose PMA stimulation (5 μg) and only 1.7-fold and 2.4- 20 μg/ml increased VEGF production by 2.4-fold, 3.0-fold and fold increases using 10 and 20 μg of PMA, respectively (con- 3.4-fold, respectively, in monolayer chondrocytes (control ver- trol versus PMA, p ≤ 0.05; Figure 5b). After treatment with sus PMA, p ≤ 0.05) and treatment with SIN-1 at concentra- SIN-1, the NO content showed similar results but the effect tions of 1, 10 and 20 μM increased the level of VEGF by 1.7- was more extended (Figure 5a): in monolayer chondrocytes, fold, 2.5-fold and 2.8-fold, respectively (control versus SIN-1, the NO content was 2.1-fold, 24.8-fold and 41.9-fold higher p ≤ 0.05; Figure 4a). In cartilage explants, no increase in VEGF than control after treatment with 1, 10 and 20 μM of SIN-1 was detected at 5 μg of PMA or 1 μM of SIN-1 (Figure 4b). (control versus SIN-1, p ≤ 0.05). In the cartilage explants, the Using higher concentrations, VEGF production was increased effects were attenuated compared with the monolayer cul- 2.7-fold and 3.8-fold after treatment with PMA at concentra- tures, with 1.7-fold, 2.5-fold and 3.6-fold increases in NO tions of 10 and 20 μg, respectively (control versus PMA, p ≤ content reported after treatment with 1, 10 and 20 μM of SIN- 0.05), and 2.4-fold and 3.7-fold after treatment with SIN-1 at 1, respectively (control versus SIN-1, p ≤ 0.05; Figure 5b). concentrations of 10 and 20 μM, respectively (control versus SIN-1, p ≤ 0.05). Page 5 of 8 (page number not for citation purposes) Arthritis Research & Therapy Vol 8 No 6 Fay et al. Figure 4 Figure 5 Th The e V VE EG GF F co con nt ten ent o t of f medi medium um s su upe pern rnata atan nt (a t (as det s dete erm rmin ined by ed by EL ELIS ISA) A). Th The N e NO con O conten tent of t of m me ediu dium m supern superna ata tan nt norm t normalize alized d to th to the Con ( e Con (n n = 1). = 1) Stimulation of immortalized chondrocytes (a) and articular cartilage Stimulation of immortalized chondrocytes (a) and articular cartilage explants (b) with PMA (μg per 1 ml of medium) or SIN-1 (μM). The level explants (b) with PMA (μg per 1 ml of medium) or SIN-1 (μM). The level of VEGF protein is increased after stimulation with PMA or SIN-1. of NO is increased after stimulation with reactive oxygen species Results are shown as mean ± standard error of the mean for five sepa- donors. Results are shown as mean ± standard error of the mean for rate experiments. * p < 0.05 versus Con. Con, control; PMA, phorbol five separate experiments. * p < 0.05 versus Con. Con, control; NO, myristate acetate; SIN-1, 3-morpholino-sydnonimine hydrochloride; nitric oxide; PMA, phorbol myristate acetate; SIN-1, 3-morpholino-syd- VEGF, vascular endothelial growth factor. nonimine hydrochloride. Discussion Our findings show that the level of VEGF in synovial joint fluids from patients suffering from OA is 60-fold higher than healthy lage metabolism during rheumatoid arthritis. The initial growth joints. Moreover, our in vitro model revealed that VEGF mRNA and invasion of the synovial pannus tissue contributes to the and protein levels and VEGF receptors are increased by PMA subsequent cartilage destruction. Blockade of VEGFR-I or SIN-1 stimulation in chondrocytes and human articular car- resulted in reduced intensity of clinical manifestations and pre- tilage explants. We conclude that the presence of ROS, or vented joint destruction in a mouse model of rheumatoid arthri- .- activation of production of O , is responsible for the observed tis [26]. It is obvious that tissues other than cartilage are results. Thus, VEGF accumulation in the synovial fluid is, at participating in these processes, especially the surrounding least in part, cartilage-derived. synovial tissue. This reflects the findings of Felson and coworkers [27], who declared OA to be a disease involving Inflammation in OA is known to be associated with activation the whole joint. In summary, these data support the role of of host angiogenesis [24]. VEGF is one of the most potent VEGF in mediating destructive processes in articular joints proangiogenic stimuli of neovascularization. Furthermore, the and encouraged us to investigate the relationship between capacity of VEGF to mediate chemotaxis, raise vascular per- VEGF expression and ROS in articular cartilage. Focusing on meability for neutrophil influx and activate MMPs in chondro- other cell types, such as glomerular podocytes, endothelial cytes suggests a central function in catabolic pathways of OA cells and skeletal muscle fibres, a correlation between ROS joints [9,25]. In addition to the involvement of VEGF in the and VEGF is described, but the results were contradictory development of OA, VEGF has biological importance in carti- [28,29]. Page 6 of 8 (page number not for citation purposes) Available online http://arthritis-research.com/content/8/6/R189 Here, we demonstrate increased VEGF mRNA expression and line, designed part of the study and contributed to the draft production in cultured human chondrocytes and articular car- manuscript. The manuscript has been read and approved by tilage explants after challenge by ROS donors. We conclude all authors. that the observed increase in VEGF content in synovial fluid of OA joints is produced partly by articular chondrocytes and Acknowledgements The authors would like to thank Inka Kronenbitter, Ursula Mundt, Frank consistent with previous findings from this and other laborato- Lichte and Sonja Seiter for their excellent technical assistance. The T- ries, which showed an increase in VEGF content in OA carti- handle bar to perform tissue harvest from the donors' knees was a gen- lage [8,10,11]. The observation that the concentration of erous gift from Arthrex , Karlsfeld, Germany. This work was funded, in VEGF is positively correlated to joint destruction and vascular- part, by the Research Promotion of Faculty of Medicine, Kiel University, ization of synovial membrane in rheumatoid arthritis [30] sug- Kiel, Germany, and Deutsche Forschungsgemeinschaft (DFG; Pu 214/ gests the potential impact of VEGF in the pathophysiology of 4-2, Pu 214/3-2 and Pu 214/5-2). MG's research was funded by a grant OA. from the National Institutes of Health (R01-AG22021). Our demonstration of ROS-mediated induction of the ang- References 1. Fernandes JC, Martel-Pelletier J, Pelletier JP: The role of iogenic factor VEGF in human chondrocytes and articular car- cytokines in osteoarthritis pathophysiology. Biorheology 2002, tilage explants is consistent with prior reports showing that 39:237-246. NO stimulates VEGF production in chondrocytes [31]. How- 2. Burrage PS, Mix KS, Brinckerhoff CE: Matrix metalloproteinases: role in arthritis. Front Biosci 2006, 11:529-543. ever, we observed different responses to the two ROS donors. 3. Ferrara N: Role of vascular endothelial growth factor in the reg- The NO content after stimulation with SIN-1 was up to tenfold ulation of angiogenesis. Kidney Int 1999, 56:794-814. higher than after stimulation with PMA and only SIN-1 induced 4. 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Journal

Arthritis Research & TherapySpringer Journals

Published: Dec 1, 2006

Keywords: Rheumatology; Orthopedics

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