Secretome analysis of chondroitin sulfate-treated chondrocytes reveals anti-angiogenic, anti-inflammatory and anti-catabolic properties

Macroscopically normal human knee cartilage from three adult donors (70, 73 and 78 years old) with no history of joint disease was provided by the Tissue Bank and the Autopsy Service at CHU A Coruña for the proteomic analysis. The study was approved by the local ethics committee. Cartilage was processed as previously described [13].

HACs were isolated as described previously [9, 13]. Briefly, cartilage surfaces were rinsed with saline buffer, and scalpels were used to cut parallel vertical sections 5 mm apart from the cartilage surface to the subchondral bone. These cartilage strips were dissected from the bone, and the tissue was incubated with trypsin at 37°C for 10 minutes and then digested with type IV clostridial collagenase. The release of chondrocytes from cartilage was achieved after 16 hours of digestion in an incubator at 37ºC, 5% carbon dioxide.

The isolated chondrocytes were recovered and plated at low density in SILAC DMEM-Flex (Invitrogen, Paisley, UK) deficient in arginine (R) and lysine (K) supplemented with 10% dialyzed fetal bovine serum (Gibco, Invitrogen), 4.5 g/l glucose (Sigma, St. Louis, MO, USA), 2 mM L-glutamine (Sigma), 100 units/ml penicillin and 100 µg/ml streptomycin. In the case of light media, standard L-lysine and L-arginine were used, while in the heavy media, isotope-labeled L-lysine (¹³C₆) and isotope-labeled L-arginine (¹³C₆,¹⁵N₄) were used. For the initial cell expansion, 5×10⁴ chondrocytes from each donor were seeded in two T-25 cell culture flasks (one grown in light medium and one in heavy medium). At confluence cells were recovered from each culture flask by trypsinization and seeded onto two six-multiwell plates (15×10⁴ for well) for cell treatment. Chondrocytes were used at week 3 in primary culture, when 100% of labeling was reached. Verification of complete labeling was performed as previously described [12]. Briefly, a small aliquot of cells cultured in the heavy media were subjected to protein extraction. The extracts were then digested with trypsin and analyzed by nano-scale liquid chromatography (LC)-MS to determine the degree of incorporation by looking for the presence of light peptides. Verification of cell type was carried out by real-time PCR for the analysis of type II collagen mRNA expression under the conditions of study.

The chondroitin sulfate employed in this work is of bovine origin, with a CS content of 99.9% and a molecular weight of 15.12 kDa. Other characteristics (viscosity, sulfation sites, and so forth) have been previously detailed elsewhere [14]. Chondrocyte stimulation for the experiments was carried out following procedures previously described by our group, in which CS and IL-1β concentrations in the chondrocyte cultures were optimized for the proteomic studies [9, 12]. Briefly, cells were washed thoroughly to remove abundant serum proteins and were cultured in serum-free medium with or without chondroitin sulfate (200 μg/ml; Bioibérica, Barcelona, Spain). Two hours later, IL-1β was added to the culture media (5 ng/ml; Sigma). Finally, conditioned media were collected after 48 hours of culture. Cell viability was assessed by Trypan Blue dye exclusion.

Conditioned media obtained from three different donors were analyzed independently. In addition, the off-gel measurements were performed in duplicate to assess the technical reproducibility of the LC-MS set-up.

Conditioned media were collected, centrifuged and filtered using a 0.2 µm filter to ensure removal of any dead cells. Proteins in the individual medium were precipitated with 0.02% sodium deoxycholate for 10 minutes and then with 10% (v/v) trichloroacetic acid overnight at 4ºC. Precipitates were harvested by centrifugation at 13,000 rpm for 15 minutes at 4°C and then washed twice with ice-cold acetone. The protein pellets were dried in air and then resuspended in 6 M urea, 2 M thiourea and 25 mM ammonium bicarbonate. The protein content of the concentrated media was measured using the Bradford reagent from Sigma. Heavy and light samples were then mixed 1:1, and 4 μg of each mixed sample were in-solution reduced, alkylated and digested with trypsin. Digestion was performed overnight with 12.5 ng/l Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA) at 37ºC. The mixtures were acidified with Trifluoroacetic acid (1% final concentration) to stop the enzymatic reaction. The resulted peptides were desalted and filtered through a C18 microcolumn (NuTip; Glygen, Columbia, MD, USA) and finally eluted from the C18 bed using 70% Acetonitrile/0.1% TFA. The organic component was removed by evaporating in a vacuum centrifuge and the peptides were resuspended in 2% Acetonitrile/0.1% TFA. Then 5 µl were injected into a reversed-phase column (Integrafit C18, Proteopep™ II; New Objective, Woburn, MA, USA) for nano-flow LC analysis, using a Tempo nanoLC (Eksigent, Dublin, CA, USA) equipped with a Sun Collect MALDI Spotter/Micro-Fraction Collector (SunChrom GmbH, Friedrichsdorf, Germany).

LC eluate was deposited onto an Opti-TOF LC MALDI target plate (1,534-spot format; ABSciex, Framingham, MA, USA) with a speed of one spot per 15 seconds. Before spotting, the LC microfractions were mixed with MALDI matrix (3 mg/ml α-cyano-4-hydroxycinnamic acid in 70% Acetonitrile and 0.1% TFA containing 10 fmol/μl angiotensin as internal standard). Peptide-containing LC spots were analyzed in a 4800 MALDI-TOF/TOF instrument (ABSciex) with a 200 Hz repetition rate (Nd:YAG laser). MS full-scan spectra were acquired from 800 to 4,000 m/z. A total of 1,500 laser shots were accumulated for each time-of-flight MS spectrum at an optimized fixed laser setting. Tandem MS mode was operated with 1 kV collision energy with CID gas (air) over a range of 60 to -20 m/z of the precursor mass value. The precursor mass window was 300 ppm (full width at half-maximum) in relative mode. A minimum of 800 and a maximum of 1,500 laser shots were accumulated with laser stop conditions set at 10 product ion peaks of signal-to-noise ratio >100 at an optimized, fixed laser setting with metastable suppressor option on. Data-dependent tandem MS settings included acquisition of up to 20 of the most intense ion signals per spot. If two or more consecutive spots in an LC run with precursor m/z were within 200 ppm tolerance, the spot with the maximum signal-to-noise ratio was subjected to tandem MS analysis.

Peptide and protein identification and comparative quantification were performed using the Protein Pilot software vs 3.0 (ABSciex) with Paragon Algorithm. MS/MS data was searched against the UniProt/Swiss-Prot database of protein sequences (August 2010; Swiss-Prot, Geneva, Switzerland), using the following parameters: sample type set as SILAC (Lis+6, Arg+10), cysteine alkylation with Iodoacetamide, urea denaturation, one missed cleavage allowed in trypsin digestion and focus in biological modifications. Only proteins with a threshold >95% confidence (>1.3 unused score) were considered for protein identification. Data were normalized for mixing error by bias corrections.

Total RNA was isolated from chondrocytes (5×10⁵ per well) using Trizol Reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's instructions. cDNA was synthesized from 1 μg total RNA, using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany) in accordance with the manufacturer's instructions, and was analyzed by quantitative real-time PCR. The quantitative real-time PCR assay was performed in the LightCycler 480 instrument (Roche Applied Science) using 96-well plates. Primers for thrombospondin-1 (TSP1), TNFα-induced protein (TSG6) and the housekeeping genes, HPRT1 and RPLP0, were designed using the Universal Probe Library tool from the Roche website [15]. Primer sequences were as follows: TSP1 forward, 5'-GCTGCACTGAGTGTCACTGTC-3'; TSP1 reverse, 5'-TCAGGAACTGTGGCATTGG-3'; TSG6 forward, 5'-GCTAGAGGCAGCCAGAAAAA-3'; TSG6 reverse, 5'-ATCCAACTCTGCCCTTAGCC-3'; HPRT1 forward, 5'-TGACCTTGATTTATTTTGCATACC-3'; HPRT1 reverse, 5'-CGAGCAAGACGTTCAGTCCT-3'; RPLP0 forward, 5'-TCTACAACCCTGAAGTGCTTGAT-3', PRPL0 reverse 5'-CAATCTGCAGACAGACACTGG-3'. The results were analyzed using the LightCycler 480 software release 1.5.0 (Roche Applied Science), which automatically recorded the threshold cycle (Ct). An untreated cell sample (basal) was used as the calibrator; the fold-change for this sample was 1.0. Target gene Ct values were normalized against HPRT1 and RPLP0. Data were analyzed using the 2^-ΔΔCt method and expressed as the fold-change of the test sample compared with the basal condition [16].

Western blot analyses were performed utilizing standard procedures. Briefly, 20 μg secreted proteins and 50 μg intracellular proteins were loaded and resolved using 10% SDS-PAGE. The separated proteins were then transferred to polyvinylidene fluoride membranes (Immobilon P; Millipore Co., Bedford, MA, USA) by electroblotting and probed with specific antibodies against TSP1 (Santa Cruz Biotechnology, Santa Cruz, CA), TSG6 (Abnova, Taipei, Taiwan), MMP1 and MMP3 (Abcam, Cambridge, UK). Immunoreactive bands were detected and housekeeping control GAPDH (Sigma). Immunoreactive bands were detected by chemiluminescence using corresponding horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection reagents (GE Healthcare, Uppsala, Sweden), and then digitized using the LAS 3000 image analyzer (Fujifilm, Tokyo, Japan). For secretome samples, equivalent loadings were verified by Ponceau Red (Sigma) staining after transference (data not shown). Quantitative changes in band intensities were evaluated using ImageQuant 5.2 software (GE Healthcare).

Cartilage shavings from three healthy donors different from those selected for the proteomics strategy were cut into 6 mm discs using a sterile biopsy punch, and one disc/well was placed into 96-well plates containing 200 µl serum-free DMEM. Plates were incubated for 48 hours with 200 µg/ml CS in presence of IL-1β (5 ng/ml). Frozen samples were then cut at 4 μm with a cryostat (Leica Microsystems, Barcelona, Spain) for immunohistochemical analysis. Sections were incubated with primary antibody to detect the presence of TSP1 (1:50; Santa Cruz Biotechnology, Heidelberg, Germany). The peroxidase/DAB ChemMate™ DAKO EnVision™ detection kit (Dako, Barcelona, Spain) was used to determine antigen-antibody interactions. Negative staining controls were achieved by omitting the primary mAb. Samples were visualized using an optical microscope.

Each experiment was repeated at least three times. The statistical significance of the differences between mean values was determined using a two-tailed t-test, considering P ≤ 0.05 significant. In the proteomic analysis, normalization tools and the statistical package from Protein Pilot software were employed (ABSciex). We considered statistically significant only those changes with P ≤ 0.05 and a ratio ≥1.2 (or ≤0.83). Where appropriate, results are expressed as the mean ± standard error.

Given the key role of chondrocytes in ECM synthesis and turnover, and also the importance of these mechanisms for tissue maintenance (which are disturbed in OA and other joint diseases), we examined the effect of CS in the subset of proteins secreted by chondrocytes (secretome) in an inflammatory environment (IL-1β). Inflammatory molecules, such as proinflammatory cytokines, are critical mediators of the disturbed metabolism and enhance the catabolism of joint tissue involved in OA pathophysiology [21]. For this purpose, supernatants from IL-1β-stimulated chondrocytes, with or without CS treatment, were collected after 48 hours of incubation and were analyzed. Owing to the low complexity of the secretome samples, we carried out a monodimensional approach: we combined equal amounts of proteins from the experimental conditions to be compared (treated or untreated with CS, both in the presence of the cytokine), and then these samples were digested in solution with trypsin. The correspondent tryptic peptides were separated by LC and the peptides were subsequently eluted and subjected to mass spectrometry analysis (MALDI-MS/MS).

This procedure resulted in the identification of 75 proteins present in the culture media of IL-1β-treated cells with statistical confidence (73 with Protein Pilot score ≥2). Some of them had not been previously reported to be secreted by chondrocytes [12], but they were found in serum [22] and/or synovial fluid [23] of OA patients and thus possess putative biomarker value. A complete list of these proteins is shown in Table 1. The majority of the identified secreted proteins were cartilage ECM proteins, or proteins with well-established matrix functions. Furthermore, several mediators of the inflammatory response were detected. The molecular function of the identified proteins was categorized by GeneOntology and is shown in Figure 1. The most abundant proteins identified in the samples (in terms of Protein Pilot hits, see Table 1) included well-known cartilage-related proteins, namely fibronectin (FN1) and chitinase-3-like protein 1 (CHI3L1), as well as ECM degradative enzymes, such as stromelysin-1 (MMP3) and interstitial collagenase (MMP1).

By these means we were able to relatively quantify all the identified proteins with statistical significance. To confirm our findings and exclude the possibility of any quantification differences arising from SILAC labeling [24], the whole experiment was replicated with treatment conditions crossed over (swapping the labeled state of the perturbed cells). Finally, among the identified proteins, 18 presented a significant alteration of their levels due to the pharmacological treatment (six increased and 12 decreased), which are listed in Table 2. We detected the modulation of proteins involved in several processes, such as cartilage ECM structural organization (three proteins, including noncollagenous proteins and proteoglycans), ECM remodeling (four proteins, including proteases and their inhibitors), immune response (six proteins) and angiogenesis (two proteins).

Interestingly, we found distinctively in CS-treated cells a global decrease of immunity-related proteins, degradative enzymes (such as MMP1, MMP2, and MMP3), and some ECM structural proteins (such as fibronectin (FN1) and chitinase-3-like protein 1 (CHI3L1)). Among those proteins described in our previous work as increased by IL-1β [12], which were now decreased by CS, we found FN1 and CHI3L1, two components of normal cartilage matrix (Figure 2). Synthesis and release of both proteins and fragments is often increased in cartilage that is undergoing repair or remodeling, and they have been investigated as markers of cartilage damage in OA [25‐27]. Interestingly, the release of FN1 and CHI3L2 from chondrocytes was also detected in a previous proteomic analysis from our group, which aimed to evaluate the differential effect of three distinct CS molecules in chondrocytes [28]. In that work, the presence of these proteins in the chondrocyte secretomes was caused by treatment with a CS of porcine origin, which appeared to trigger catabolic effects in chondrocytes by increasing also the abundance of matrix metalloproteinases (MMP1 and MMP3). On the contrary, treatment with bovine CS (the one employed in the present study) did not have any effect on the release of these four proteins.

We also performed a database search, using STRING software, to visualize protein interactions on the set of CS-modulated proteins and further elucidate its effect on chondrocytes (Figure 3). The role of CS in counteracting the IL-1β-mediated increase of some proteins was also detected for three degradative enzymes and three members of the complement pathway (Figure 2 and Table 2). Recently, a central role for the inflammatory complement system in the pathogenesis of OA has been identified [29]. Expression and activation of complement is abnormally high in human osteoarthritic joints. We show in this study how CS could reduce inflammation directly by decreasing the presence of several complement components (CFAB, CLUS, CO3, C1S and C1R), and also indirectly by increasing proteins such as TSG6. This protein plays a crucial role in ECM formation, inflammatory cell migration and cell proliferation. TSG6 is also a key component of a negative feedback loop operating through the protease network that reduces matrix degradation during the OA process [30]. The mechanism driven by TSG6 leads to a decrease in pro-matrix metalloproteinase activation, which might protect cartilage from extensive degradation even in the presence of acute inflammation (represented in our case by a high level of IL-1β). Western blot analyses were performed to confirm the detected increase of TSG6 caused by CS treatment. As shown in Figure 4, CS increased the amount of TSG6 secreted by chondrocytes, and this increase correlates with a decline in MMP1 and MMP3 levels. These results point to the increase of TSG6 as a putative mediator of the reduction in pro-matrix metalloproteinase activation, suggesting an important role of this mechanism for the anti-catabolic effect of CS.

A remarkable increase of TSP1, an angiogenesis inhibitor, was detected as a consequence of the CS treatment and counteracting the effect of IL-1β. This result is consistent with our previously observed increase of TSP1 protein driven by CS in the absence of IL-1β stimulation, although in osteoarthritic chondrocytes [28]. TSP1 overexpression reduces inflammation and neovascularization in the OA joint [31]. In our previous study on IL-1β-stimulated chondrocytes, TSP1 presented a ratio of zero [12], indicating a cytokine-dependent dramatic decrease of its release from these cells. IL-1β is a well-recognized angiogenic factor, so the possibility that an increased concentration of IL-1β in OA synovial fluid may reduce the TSP1 expression in severe stages of OA cannot be excluded. The selective inhibition of angiogenesis - also confirmed by the decrease of lactadherin, a protein that promotes vascular endothelial growth factor-dependent neovascularization [32] - demonstrates a novel mechanism of action of CS according to recent results obtained in synoviocytes [33].

The data obtained in the SILAC analysis need to be validated for differences in protein expression profiles before the biological roles of the modulated proteins are extensively studied. We therefore performed additional studies in order to verify the altered abundance of TSP1 in CS-treated chondrocytes. Interestingly, TSP1 is a multifunctional adhesive glycoprotein present in articular cartilage and synthesized by articular chondrocytes [34], whose gene transfer suppresses the disease progression of experimental OA [31]. The inhibitory effect of TSP-1 on angiogenesis has been largely described [35]. Owing to the pivotal role of angiogenesis in OA physiopathology [36], we decided to verify TSP1 gene expression level in CS-treated chondrocytes stimulated with IL-1β by real time-PCR analysis, and also in cells without cytokine stimulation. As shown in Figure 5A, CS upregulates TSP1 already in the absence of IL-1β. When the cytokine is present, CS is capable of counteracting its suppressive effect on TSP1 in chondrocytes. Furthermore, TSP1 protein levels were also evaluated in chondrocyte conditioned media (secretome) and cellular extracts (proteome) by western blot analyses and in cartilage explant culture by immunohistochemistry (Figure 5). The increase of TSP1 protein observed both in cell and tissue cultures following CS treatment suggests the possible mechanism through which this drug could exert an anti-angiogenic action.