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UMR S_ 1116 Université de Lorraine-INSERM, Vandœuvre-lès-Nancy, FranceFédération de Recherche 3209 Université de Lorraine-CNRS, Vandœuvre-lès-Nancy, France
Centre de Recherche Saint-Antoine INSERM-Université Paris 6, UMR S_938, Paris, FranceInstitut Hospitalo-Universitaire ICAN, Paris, FranceAssistance-Publique des Hôpitaux de Paris, Hôpital Saint-Antoine, Service d'Endocrinologie, Paris, France
UMR 7365 Université de Lorraine-CNRS, Vandœuvre-lès-Nancy, FranceFédération de Recherche 3209 Université de Lorraine-CNRS, Vandœuvre-lès-Nancy, FranceCentre Hospitalo-Universitaire, Nancy, France
UMR 7365 Université de Lorraine-CNRS, Vandœuvre-lès-Nancy, FranceFédération de Recherche 3209 Université de Lorraine-CNRS, Vandœuvre-lès-Nancy, FranceCentre Hospitalo-Universitaire, Nancy, France
UMR S_ 1116 Université de Lorraine-INSERM, Vandœuvre-lès-Nancy, FranceFédération de Recherche 3209 Université de Lorraine-CNRS, Vandœuvre-lès-Nancy, FranceCentre Hospitalo-Universitaire, Nancy, France
UMR 7365 Université de Lorraine-CNRS, Vandœuvre-lès-Nancy, FranceFédération de Recherche 3209 Université de Lorraine-CNRS, Vandœuvre-lès-Nancy, FranceCentre Hospitalo-Universitaire, Nancy, France
Address correspondence and reprint requests to: N. Mercier, Université de Lorraine-Inserm U1116, Faculté de médecine, bat D 1er étage, 9, avenue de la forêt de Haye-BP 184, 54 505 Vandœuvre-lès-Nancy, France. Tel: 33-3-83-68-36-32, 33-3-83-68-36-23; Fax: 33-3-83-68-36-39.
UMR S_ 1116 Université de Lorraine-INSERM, Vandœuvre-lès-Nancy, FranceFédération de Recherche 3209 Université de Lorraine-CNRS, Vandœuvre-lès-Nancy, France
Semicarbazide-sensitive amine oxidase (SSAO) catalyzes the oxidation of primary amines into ammonia and reactive species (hydrogen peroxide, aldehydes). It is highly expressed in mammalian tissues, especially in vascular smooth muscle cells and adipocytes, where it plays a role in cell differentiation and glucose transport. The study aims at characterizing the expression and the activity of SSAO in rat and human articular cartilage of the knee, and to investigate its potential role in chondrocyte terminal differentiation.
Design
SSAO expression was examined by immunohistochemistry and western blot. Enzyme activity was measured using radiolabeled benzylamine as a substrate. Primary cell cultures of rat chondrocytes were treated for 21 days by a specific SSAO inhibitor, LJP 1586. Terminal chondrocyte differentiation markers were quantified by RT-qPCR. The basal and IL1β-stimulated glucose transport was monitored by the entrance of 3[H]2-deoxyglucose in chondrocytes.
Results
SSAO was expressed in chondrocytes of rat and human articular cartilage. SSAO expression was significantly enhanced during the hypertrophic differentiation of chondrocytes characterized by an increase in MMP13 and in alkaline phosphatase (ALP) expressions. SSAO inhibition delayed the late stage of chondrocyte differentiation without cell survival alteration and diminished the basal and IL1β-stimulated glucose transport. Interestingly, SSAO activity was strongly increased in human osteoarthritic cartilage.
Conclusions
SSAO was expressed as an active form in rat and human cartilage. The results suggest the involvement of SSAO in rat chondrocyte terminal differentiation via a modulation of the glucose transport. In man, the increased SSAO activity detected in osteoarthritic patients may trigger hypertrophy and cartilage degeneration.
Semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6), also known as vascular adhesion protein-1 (VAP-1), catalyzes the oxidative deamination of aromatic or aliphatic amines, leading to the formation of the corresponding hydrogen peroxide, aldehyde and ammonia
. Although the physiological role of SSAO is not completely elucidated, the enzyme has been involved in the leukocyte extravasation from the blood to the inflammation site
Chronic benzylamine administration in the drinking water improves glucose tolerance, reduces body weight gain and circulating cholesterol in high-fat diet-fed mice.
. Indeed, the activity of the tissue and/or soluble enzyme is altered in several diseases including types I and II diabetes, atherosclerosis, inflammatory liver diseases, or Alzheimer's disease
Hyaline cartilage is the support for bone tissue formation through proliferation, maturation (the early phase of differentiation) and then hypertrophy and mineralization (termed terminal differentiation) of chondrocytes that lead to apoptosis. This terminal differentiation can be monitored by a down-regulation of SOX9 and collagen II, an induction of collagen X and matrix metalloproteinase 13 (MMP-13), and a later increased expression of alkaline phosphatase (ALP) and osteopontin (OPN) involved in cartilage mineralization
. In hyaline articular cartilage, this terminal differentiation process is inhibited to prevent bone formation. Dysfunction of articular chondrocytes leads to cartilage degradation mediated by pro-inflammatory cytokines, and is associated with osteoarthritis. Thus, understanding chondrocyte differentiation is a prerequisite to decipher the mechanistic basis of cartilage degenerative diseases, and to develop new therapeutic opportunities.
have shown that SSAO activity was detectable in rat articular cartilage, but its role was not elucidated. Given the ability of SSAO to generate tissue injury through hydrogen peroxide or aldehyde production
Semicarbazide-sensitive amine oxidase in aortic smooth muscle cells mediates synthesis of a methylglyoxal-AGE: implications for vascular complications in diabetes.
, it is conceivable that this enzyme contributes to cartilage maturation or homeostasis under physiological conditions, or promotes cartilage injury under pathological conditions.
The purpose of this work was to investigate the expression and the activity of SSAO in rat and human chondrocytes. Moreover, we studied the potential implication of SSAO in differentiation process of chondrocytes and glucose transport in vitro.
Materials and methods
Source of human cartilage
Femoral cartilage from condyles were obtained from five patients classified as grades 3 and 4 according to the Kellgren and Lawrence osteoarthritis grading system (four women and one men with a mean age 69 ± 7 years) undergoing total knee joint replacement. The study was approved by our local Research Institution review board (Commission de la Recherche Clinique, UF 9757-CPRC 2004) and conforms to the ethical guidelines of the Declaration of Helsinki. Cartilage biopsies were taken from the injured area (Mankin score 4 to 13) and from the macroscopically unaffected area distant to the damaged zone (Mankin score 2 to 4) measurement of parallel SSAO activity, mRNA expression, and histology was performed. The samples were kept frozen at −80°C until used or placed in paraformaldehyde for histological analysis.
Sources of animal tissues and chondrocyte cells
All experiments were performed in accordance with national animal care guidelines and were pre-approved by a local ethics committee. For the entire study, 35 male Wistar rats (100–150 g) obtained from Charles River (L'Arbresle, France) were housed under controlled temperature and light cycle conditions with food and water ad libitum. Five rats were anesthetized with 3.5% of isoflurane in oxygen and put to death by cervical dislocation. Three were used for histology, enzyme activity and mRNA expression and two for lysyl oxidase activity. The other rats were used for cell cultures.
Isolation of chondrocytes and hypertrophic differentiation protocol
For each cell culture, chondrocytes were isolated from femoral head cartilage of five Wistar rats (100–150 g), as mentioned in
and plated in a growth medium composed of DMEM/F-12 with 1% penicillin/streptomycin, 20 mM l-glutamine, 5.2 nM sodium selenite and 10 μg/ml human transferrin (all provided by Sigma–Aldrich, Saint-Quentin Fallavier, France) containing 10% decomplemented FBS until confluence, in a 37°C, 5% CO2 humidified cell culture incubator. All experiments were performed after the initial passage. At confluence, chondrocytes were switched to growth medium supplemented with 2% FBS, 2 μg/ml insulin and 37.5 μg/ml 2-phospho-l-ascorbic acid for different times up to 21st day, in the absence or in the presence of 1 μM LJP 1586 [Z-3-Fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], a selective SSAO inhibitor
Anti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activity.
. Cells were observed at several time points of the culture by phase contrast microscope (Nikon DIAPHOT 300, Tokyo, Japan) and pictures were recorded (Nis-Elements BR 3.2 software, Champigny sur Marne, France) at magnification ×10. Three to four different cell cultures were used to characterize the terminal differentiation (Fig. 2, Fig. 3), five to six were used to study the LJP effect on chondrocyte terminal differentiation (Fig. 5).
Histological examination of human and rat cartilage and immunohistochemistry
Knee cartilage from three rats and five human condyle samples were fixed for 48 h in 4% (v/v) paraformaldehyde in PBS, decalcified in 0.34 M ethylene diamine tetraacetate in water and embedded in paraffin. Sections (5 μm thick) were stained with Hematoxylin-Erythrosin-Saffron (HES) to evaluate their cellular and morphologic aspect and with Safranin O to evaluate the proteoglycan content. Five human cartilage samples were graded according to the Mankin scale system. A maximal total score of 14 could be assigned by the observer according to structure, cellularity, Safranin-O staining, tidemark integrity (Table I).
Table IThe cartilage samples were graded according to the Mankin scale system. A maximal total score of 14 could be assigned by the observer according to Structure, Cellularity, Safranin-O staining, Tidemark integrity
For immunohistochemical staining, sections were incubated in 10 mM sodium citrate buffer (pH 6.0) then in 3% H2O2 and eventually in 5% BSA. Slides were incubated with an anti-SSAO antibody (either Santa Cruz Biotechnology, Nanterre, France, for human sections or a polyclonal rabbit antiserum raised against recombinant human VAP-1, that also recognizes mouse and rat VAP-1 for rat sections), or with an anti-Collagen X (Abcam, Paris, France) overnight. The secondary biotinylated rabbit antibody (Dako, Glostrup, Denmark) was incubated with Streptavidin/Horseradish peroxidase and 3,3'-diaminobenzidine (Thermo Scientific, Villebon sur Yvette, France). Positive controls were obtained with adipose tissue whereas the negative controls were performed by replacement the primary antibody by normal rabbit serum. Slides were observed under a DMD 108 Leica microscope with a magnification 10×, 20× and 40×.
RNA isolation from cells in primary culture and tissues
Total RNA from cultured chondrocytes was extracted using the Qiagen RNeasy Mini Kit (Courtaboeuf, France) according to the manufacturer's instructions whereas tissues were crushed in liquid nitrogen and homogenized in TRIzol® reagent (1 ml/100 mg) as indicated by the manufacturer (Ambion, thermo scientific, France). RNA concentrations were determined by Nanodrop (Labtech, Paris, France).
RT- and real time qPCR
cDNAs were synthesized from 1 μg of total RNA (Thermo Scientific). Real-time PCR were carried out with SYBR Green PCR Master Mix (Biorad, Marnes-la-Coquette, France) according to the manufacturer's instructions, using an iCyler iQ thermal cycler (My iQ Single-Color Real-Time PCR Detection System, Bio-Rad). The sequences of primers for the rat and human genes of interest are shown in Table II, Table III, respectively. Each sample was tested in duplicate. The methods of 2−ddCt was used for calculation of the relative expression of the targeting mRNA using RPS29 as house keeping gene
Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes.
Cells homogenates (25 μg of protein) from two different cell cultures were separated in Bio-Rad® TGX™ FastCast 10% Acrylamide gel Kit performed with a Mini-PROTEAN Tetra Cell, Mini Trans-Blot Module and transferred on a Trans-Blot® Turbo™ transfer system (Biorad). The membrane was blocked in 5% (w/v) non-fat dry milk, 0.1% Tween 20 in TBS buffer (10 mM Tris-Base and 15 mM NaCl, pH 7.5) for 1 h at room temperature. The membrane was blotted either with the same polyclonal rabbit antiserum raised against recombinant human VAP-1 or a mouse monoclonal anti-alpha tubulin (Sigma–Aldrich, France) in the blocking solution overnight at 4°C. Then a horseradish peroxidase conjugated secondary antibody (anti-rabbit or anti-mouse, respectively) (Sigma Aldrich) was added in the blocking buffer for 1 h. Immune complexes were visualized using the Clarity™ ECL Western Blotting Substrate, (BioRad) and a Fujifilm Luminescent Image Analyzer LAS4000 System. The intensity of the bands was analysed using Multi Gauge (FUJIFILM, Science Lab 2005). Results were expressed as the SSAO/tubulin ratio.
SSAO activity
SSAO activity was determined in tissue or cell homogenates using 0.1 mM (0.1 μCi) [7-14C]benzylamine hydrochloride (Perkin Elmer, NEC835050UC, Villebon sur Yvette, France) as the substrate, as described in
for 30 min at 37°C in presence of 0.5 mM pargyline hydrochloride (a selective MAO inhibitor, Sigma), and/or 1 mM semicarbazide hydrochloride (SSAO inhibitor, Sigma). [14C]aldehyde was quantified using a TRI-CARB 2100TR Liquid Scintillation Analyzer (Perkin Elmer, Zaventem, Belgium). Duplicates were made for each sample. SSAO enzyme activity, expressed in nmol/h/mg of protein, was calculated from the activity measured in the presence of pargyline minus that measured with pargyline and semicarbazide.
Lysyl oxidase (LOX) activity
The LOX activity was measured as described in the legend of the Supplemental data 1
Glucose transport from at least four different cell cultures was determined by measuring the uptake of 2-deoxy-d-glucose (2-DG) (Sigma Aldrich) as reported in
Impaired glucose transporter-1 degradation and increased glucose transport and oxidative stress in response to high glucose in chondrocytes from osteoarthritic versus normal human cartilage.
. Briefly, mature chondrocytes were cultivated in the absence (Control) or presence of 1 μM LJP 1586 for 21 days and switched to a 4.5 mM glucose-alpha MEM medium 2 days before the experiment. Chondrocytes were pre-incubated in 0.2 % BSA in a Krebs Ringer buffer without glucose, 4 h before the glucose uptake measurement. Some cells were then treated or not with 1 μM LJP 1586, 2h30 before the beginning of the experiment, further started by the addition of [1,23H]-2-DG (0.5 μCi/ml, 28 Ci/mmol, Perkin Elmer). The reaction was stopped after 30 min at 37°C. Radioactivity was determined in solubilized cells plus 2.5 ml of TRI-CARB 2100TR scintillation cocktail (Perkin Elmer). The same experiment was performed with all cells treated with 10 ng/ml IL1β for 24 h which is know to increase glucose transport and to induce inflammation
. Negative controls were obtained by treating cells with 10 μM cytochalasin B (Sigma Aldrich), a specific inhibitor of the facilitated glucose transporters. Results were expressed in nmol of DG/h/μg of protein.
Cell viability
Cell viability was established with 3-(4,5 dimethyl-2-thiazolyl)-2.5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay according to the manufacturer (Sigma Aldrich, Schnendorf, Germany) in cells treated or not for 7, 14 or 21 days with 1 μM of LJP 1586. The results obtained from four independent cell cultures were expressed as the difference between the absorbance at 550 nm (converted dye) and the absorbance at 620 nm (background). These values were directly proportional to the number of living cells in the culture.
Statistical analysis
A detailed description of “n”, the number of independent statistical units is available for each assessment in Supplemental data 2.
All results represent the individual values and the mean ± 95% confidence interval (CI). The 2-tailed significance level was set to P < 0.05. All analyses were performed using NCSS V9 software (NCSS, Kaysville, UT, USA). Considering the exploratory, hypotheses-generating, nature of the study and the small size of samples, Analyses were not adjusted for multiple testing; results were interpreted according to their consistency. In Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, a Kruskal–Wallis test was used for inter-group comparisons. When significant, a Dunn's test was performed in order to identify the significant changes from baseline. Intra-group comparisons were carried out using the Mann–Whitney U test. The brackets appearing under P values indicate the way of comparison LJP-treated vs untreated samples at corresponding time (Fig. 5).
Fig. 1Expression of SSAO in cartilage of normal rat. (A) Histological examination of cross section of Wistar rat knee joint stained with HES (morphology), safranin O (proteoglycan content), and immunostained with the anti-SSAO antibody. Only one representative picture is shown. Arrows indicate anti-SSAO immunostaining. The micrographs were taken at a magnification of 10× (subchondral bone and cartilage zone) and 40× (cartilage zone); the scale bars represent 200 μm. (B) SSAO immunostained of growth plate cartilage with a magnification of 10× and 40×. The scale bars represent 200 μm. (C) SSAO mRNA expression in articular cartilage and in positive controls (adipose tissue and aorta) from three rats. RPS29 mRNA expression was used for normalization. Results are expressed as fold change relative to aorta. (D) SSAO enzyme activity in articular cartilage, adipose tissue and aorta was expressed in nmol/h/mg. All results are presented as the mean value ± 95% CI of three different experiments, each performed in duplicate for real time qPCR and enzyme activity assay. All comparisons are made vs aorta values.
Fig. 2Expression of differentiation-dependent markers in primary cultures of rat chondrocytes. Rat chondrocytes were cultured from confluence (day 0) until day 21 in a differentiation medium. Terminal differentiation of chondrocytes was monitored at day 7, 14 and 21. (A) Representative photographs of chondrocytes in culture during the differentiation protocol. The micrographs were taken at magnification of 10×; the scale bar represents 200 μm. In these conditions, mRNA expression of chondrogenic markers: Sox 9 (B), collagen II (C), aggrecan (D) and mRNA expression of pre-hypertrophic collagen X (E) and hypertrophic markers MMP13 (F) and ALP (G) were quantified. RPS29 mRNA expression was used for standardization, and results are expressed as relative expression to day 0 expression according to delta delta Ct method. In graphs, dots represent the average of duplicate for a single cell culture. The lines show the average values ± 95% CI of three to four different cell cultures. Comparisons are made vs day 0 values.
Fig. 3Differentiation dependent expression and activity of SSAO in primary cultures of rat chondrocytes. (A) SSAO mRNA expression was evaluated by RT-qPCR. (B) SSAO protein expression was analyzed by western blotting in two different cell cultures. Immunoblot intensities for SSAO/Tubulin were quantified by densitometry. Intensities at days 0 were set at 1. A representative result is shown. (C) Enzyme activity was measured in parallel, and expressed in nmol/h/mg. In A and C graphs, dots represent means of duplicates and lines, the average value ± 95% CI of four to five different cell cultures. All comparisons are made vs day 0 values.
Fig. 4Chondrocyte viability and glucose transport. Chondrocytes were treated (gray triangles) or not (open circles) from confluence (day 0) with 1 μM LJP 1586, as a selective SSAO inhibitor. (A) Cell viability was determined by MTT reduction at days 0, 7, 14 and 21. Results are expressed as the difference between A560 and A620 nm the mean of four different cell cultures ± 95% CI. (B) Glucose transport was estimated by the entrance rate of 2-DG in chondrocyte, cultured 21 days after confluence as indicated in Methods. Lines indicate the mean ± 95% CI of five distinct cell cultures. Statistics were calculated with respect to control cells. When indicated, the brackets under P values indicate that LJP-treated values were compared vs control values of the corresponding time point.
Fig. 5Effect of SSAO activity inhibition during the course of rat chondrocyte terminal differentiation. Chondrocytes were treated (black bars) or not (gray bars) from confluence (day 0) with 1 μM LJP 1586, as a selective SSAO inhibitor. (A) The SSAO enzyme activity expressed in nmol/h/mg was performed to confirm the efficacy of LJP 1586 in one experiment done in duplicate. The expression of SSAO (B), MMP13 hypertrophic marker (C), ALP (D) and OPN (E) mineralization markers and Collagen X pre-hypertrophic marker (F), were monitored by the determination of mRNA levels expressed as fold change relative to day 0, at days 7, 14 and 21, SSAO mRNA expression at days 7, 14 and 21, relative to day 0. Dots stent for one single cell culture performed in duplicate and lines represent the average value ± 95% CI of five to six different cell cultures. Statistics were calculated with respect to the day 0. The brackets under P values indicate that values from LJP-treated cells were compared vs control ones of the corresponding time point.
SSAO is expressed in chondrocytes of rat cartilage
Figure 1(A) shows a typical cross section of the cartilage of the knee joint. The cartilage matrix exhibited a good cellularity and high glycosaminoglycan content evaluated with HES and Safranin-O staining, respectively.
Immunochemical staining using an anti-SSAO antibody, revealed the presence of the enzyme in chondrocytes and in the marrow of the sub-chondral bone [Fig. 1(A)]. SSAO was also detected in growth plate in a more intensive manner in hypertrophic chondrocytes [Fig. 1(B)]. In femoral head and knee cartilage, the expression of mRNA encoding SSAO and SSAO activity were low, when compared to that of other SSAO-rich tissues (thoracic aorta, perigonadal adipose tissue)[Fig. 1(C–D)]. In terms of specific activity, the value was up to 3 to 7- fold less than in aorta taken as a reference tissue. Altogether, these experiments show that SSAO is expressed in joint and growth plate cartilages, and its activity is moderate.
Expression of SSAO as a function of cell differentiation in primary cultures of chondrocytes
The chondrogenic terminal differentiation of a primary culture model has been enhanced by insulin
In vitro chondrocyte differentiation using costochondral chondrocytes as a source of primary rat chondrocyte cultures: an improved isolation and cryopreservation method.
and was followed as a function of time along with that of protein markers (SOX9, collagen II, collagen X, MMP13, ALP, OPN), described in the literature
In Fig. 2(A), confluence stage (day 0) could be observed using a phase-contrast microscope. Cells had a round shape as normal chondrocytes. At days 7, 14 and 21, chondrocytes became progressively larger compared to day 0. At day 21, chondrocytes exhibited a hypertrophic-like shape.
Expression of SOX9 mRNA was decreasing by 41 % after one and 2 weeks of culture [Fig. 2(B)] whereas a dramatic and significant decrease in collagen II [Fig. 2(C)] was noticed at days 14 and 21. The same tendency was followed for aggrecan [Fig. 2(D)] without reaching a statistical significance… As expected, collagen X gene expression [Fig. 2(E)] increased significantly until day 14, then decreased at day 21, in agreement with the transition from the pre-hypertrophic toward the hypertrophic phenotype of the chondrocyte. By contrast, MMP13 [Fig. 2(F)] which is a strong terminal differentiation marker was significantly higher at day 14 when compared to day 0, and continued to progressively increase at day 21. This fitted with their functional involvement in the hypertrophic and mineralizing phenotype of chondrocytes. The kinetics of ALP [Fig. 2(G)], and OPN (data not shown) expression followed the same profile but did not reach the statistical significance. Interestingly, SSAO mRNA [Fig. 3(A)], SSAO protein expression [Fig. 3(B)] and enzyme activity [Fig. 3(C)] were progressively enhanced in parallel, leading to a 3-fold increase in transcript levels and a 4-fold change in enzyme activity after 21 days in culture. These results indicate that SSAO expression and activity appears to be closely associated with the differentiation of chondrocytes that emerge concomitantly with the pre-hypertrophic phenotype, and continue to increase during the late stage of this process.
The inhibition of SSAO activity delays the rat chondrocyte differentiation
In order to determine whether SSAO activity affects chondrocyte phenotype, the cells were exposed or not for 7, 14, or 21 days to LJP 1586 (1 μM). We verified that the LJP concentration used (1 μM) did not significantly change the viability of chondrocytes after 21 days in culture [Fig. 4(A)]. Indeed, at this concentration, LJP 1586 did not inhibit the activity of LOX which belongs to the same enzyme family, in the aorta or in cartilage [Supplemental data 2]. Under these conditions, SSAO activity was completely abolished [Fig. 5(A)], while its expression remained preserved [Fig. 5(B)]. Interestingly, LJP 1586 decreased significantly the expression of MMP13 [Fig. 5(C)] at day 21 following confluence. ALP [Fig. 5(D)] and OPN [Fig. 5(E)] followed the same tendency. By contrast, expression of collagen X [Fig. 5(F)] was still 3-fold increased after 21 days of culture in the presence of the inhibitor. This result suggests that LJP 1586 delayed the terminal maturation in vitro.
Glucose transport
Figure 4(B) shows that an incubation with LJP 1586 for 21 days in control cells decreased by 3.5 times the entrance of 2-deoxygucose in the cells. In control mature chondrocytes (21 days after confluence), an acute LJP 1586-treatment (2h30) tends to diminish the glucose transport but failed to rich a significant level. Hypertrophic chondrocytes were treated with 10 ng/ml IL1β, known to activate glucose transport
and to generate an inflammatory response in chondrocyte, and glucose transport was measured in the absence or with LJP 1586 for 2h30 or from confluence (Day 0). The glucose transport was dramatically and significantly decreased after both LJP treatments. Thus, a modulation of glucose transport by SSAO could represent a mechanism by which SSAO could regulate chondrocyte hypertrophy in basal or inflammatory conditions.
SSAO expression in human cartilage from osteoarthritic patients
The expression and activity of SSAO were investigated in cartilage samples from five patients undergoing knee prosthesis for osteoarthritis. The OA Mankin score is presented in Table IV. Large inter-individual variations in SSAO activity were observed between the five patients included in the study [Fig. 6(C)–(F)]. However, compared to cartilage present in healthy area, cartilage of the damaged zone was characterized by a markedly higher SSAO protein expression [Fig. 6(B)] and activity [Fig. 6(C)] associated with injured cartilage characterized by a lower cellularity and glycosaminoglycan content [Fig. 6(A)–(B)]. A regression curve (r = 0.722) has been found between SSAO activity and Mankin scoring without reaching statistical correlation (data not shown). Cartilage of the damage zone presented a significant increase terminal differentiation markers such as collagen X [Fig. 6(A)–(B),(D)], MMP13 [Fig. 6(E)] and OPN [Fig. 6(F)], as expected in osteoarthritis lesions
Increased type II collagen degradation and very early focal cartilage degeneration is associated with upregulation of chondrocyte differentiation related genes in early human articular cartilage lesions.
. These observations corroborate the results obtained in rat chondrocytes in vitro in which hypertrophy was associated with the increased SSAO expression and activity.
Table IVOA Mankin scores of less injured and more injured zones of condyles from five patients
Fig. 6Expression of SSAO in human OA cartilage. The OA cartilage samples were obtained from knee joints of five patients. For each patient, a zone of preserved (Mankin score 2–4) or damaged cartilage (Mankin score 4 to 13) were taken for histology, SSAO activity and mRNA expression. (A) less affected or (B) damaged Human cartilage examination with HES (morphology), safranin O (proteoglycan content) staining and immunohistochemical analysis using anti-type X collagen and anti-SSAO antibodies. Representative micrographs of one patient were shown at a magnification of 10×, 20× and 40×. The scale bars represent 200 μm. SSAO enzyme activity in nmol/h/mg (C) and expression of collagen X (D), MMP13 (E), and OPN (F) mRNA in 2–4 Mankin score zone (open circles) and 4–13 Mankin score zone (gray triangles) cartilage were measured in five patients which means ± 95% CI are symbolized by lines. Results (D–F) are expressed as the relative expression of mRNA to the 2–4 score zone of each patient.
This work shows that both human and rat articular cartilage express an active form of SSAO. SSAO was associated with the differentiation process of chondrocytes in vitro. Strikingly, addition of a potent and selective SSAO inhibitor appears to delay chondrocyte maturation. SSAO also modulates glucose transport in rat chondrocytes. Finally, a higher expression of SSAO was found in rat hypertrophic chondrocytes and its activity is increased in some human osteoarthritic cartilage.
Interestingly, this enzyme is strongly expressed in highly vascularized tissues such as arteries
Molecular cloning of a major mRNA species in murine 3T3 adipocyte lineage. differentiation-dependent expression, regulation, and identification as semicarbazide-sensitive amine oxidase.
, as also in the bone marrow from the sub-chondral plate. However in cartilage, a conjunctive tissue devoid of vascular network, we confirmed that SSAO was present at a lower level however, as previously suggested
. We also showed that SSAO was also strongly expressed in hypertrophic chondrocytes from the rat growth plate in vivo, which corresponds to their terminal differentiation stage. Recently, an early expression of SSAO/VAP-1 protein was detected in cartilage sites, mainly in the vertebrae and the ribs, during mouse
embryonic development, at a higher intensity in chondrocytes that exhibited an active differentiating process compared to resting chondrocytes. This suggests an implication of SSAO in the building of the cartilage network and chondrocyte terminal differentiation.
To study this hypothesis, a chondrocyte primary cell culture model, permissive for terminal differentiation
In vitro chondrocyte differentiation using costochondral chondrocytes as a source of primary rat chondrocyte cultures: an improved isolation and cryopreservation method.
, was used and characterized by a typical profile expression of various markers: a decrease in SOX9 and type II collagen, an increase in pre- and hypertrophic markers (type X collagen, MMP-13 respectively) and mineralized cartilage (ALP)
. Under these conditions, SSAO expression and activity were increased as a function of time, with a maximal increase at day 21, and was paralleled by the expression profile of the MMP13 hypertrophic chondrocyte marker. These data were corroborated by the morphological cellular changes observed by microscopy.
To establish whether SSAO could be involved in terminal differentiation as already shown in other cell types
Anti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activity.
without any toxicity toward the chondrocyte viability. In the presence of LJP 1586 for 21 days, the MMP-13 expression was decreased whereas that of collagen X was still 3-fold increased. It was associated with enhanced chondrocyte viability. These data show that LJP 1586 exposure delays the chondrocyte terminal maturation which maintain a pre-hypertrophy characterized by a high level of collagen X. These results suggest that SSAO would not only be a marker of hypertrophic differentiation, but would also participate in this process, at least in vitro.
Several mechanisms could be evocated. SSAO belongs to the same enzymatic family as LOX, and was suggested to cross-link extracellular matrix proteins
Interaction of L-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions.
. Thus, SSAO might have a role in the maturation and maintenance of some extracellular matrix proteins. Interestingly LOX like-2, a member of the LOX family, regulates chondrocyte terminal differentiation and is re-expressed in a model of fracture healing
. However, we verified that LOX activity was not inhibited by LJP 1586. Thus, this result strongly suggests that SSAO inhibition and not LOX inhibition was responsible for the delay of the late chondrocyte differentiation.
We and others have previously shown that SSAO activation promotes adipocyte maturation
Facilitative glucose transporters in articular chondrocytes. Expression, distribution and functional regulation of GLUT isoforms by hypoxia, hypoxia mimetics, growth factors and pro-inflammatory cytokines.
Abnormal ambient glucose levels inhibit proteoglycan core protein gene expression and reduce proteoglycan accumulation during chondrogenesis: possible mechanism for teratogenic effects of maternal diabetes.
Immunocytochemical demonstration of glucose transporters in epiphyseal growth plate chondrocytes of young rats in correlation with autoradiographic distribution of 2-deoxyglucose in chondrocytes of mice.
. We showed in basal condition that SSAO inactivation during chondrocyte terminal maturation inhibited more than 70% of the glucose entrance in the cells. The acute SSAO inhibition (2h30) tends to decrease the glucose transport. These results suggest that SSAO could increase terminal differentiation by activation of glucose transport.
Under physiological conditions, articular cartilage homeostasis is maintained to prevent progressive cartilage degeneration through an inhibition of hypertrophic differentiation. Activation of chondrocyte hypertrophy, plays a role in the initiation and progression of cartilage degeneration
and leads to osteoarthritis. We showed that articular cartilage from osteoarthritic patients exhibited a marked increase in SSAO activity in the degenerative tissue associated with an increase in hypertrophic markers, when compared to cartilage removed outside of the lesion. This observation let us hypothesize that the enhanced SSAO activity could be part of the osteoarthritis pathophysiology in accelerating hypertrophic differentiation through several potential mechanisms: the production of reactive products (aldehydes and H2O2), exacerbation of inflammation, and the activation of glucose transport in chondrocyte. On the other hand, reactive oxygen species induce chondrocyte hypertrophy
Evidence linking chondrocyte lipid peroxidation to cartilage matrix protein degradation. Possible role in cartilage aging and the pathogenesis of osteoarthritis.
through the diminution of leukocyte exit, demonstrating the contribution of SSAO to the pathogenesis of arthritis in vivo. The pro-inflammatory cytokine IL1β regulates glucose transport
Impaired glucose transporter-1 degradation and increased glucose transport and oxidative stress in response to high glucose in chondrocytes from osteoarthritic versus normal human cartilage.
. As we noticed either a chonic or an acute LJP 1586 inhibition of IL1β-stimulated glucose transport in rat hypertrophic chondrocytes, we suggest that a SSAO inhibition during osteoarthritis could be a potentially valuable strategy to limit over chondrocyte terminal differentiation an inflammation observed in osteoarthritis. An exacerbated activity of SSAO during inflammation could be implicated in the regulation of glucose transport during OA. It is also tempting to speculate that SSAO could represent a biomarker of inflamed joints. The follow-up of SSAO levels in serum and synovial liquid of patients from an early to a late stage of osteoarthritis could represents an important issue. Development of inhibitors targeting SSAO for treating chronic inflammatory disorders is in expansion, showing that SSAO/VAP-1 is a promising anti-inflammatory target.
Limitations of the study: for cell cultures, although the same response has always been obtained, the intensity varied and the kinetic slightly shifted from one cell culture to another generating some variability. Some results failed to reach the statistical significance with non parametric tests, considering the small independent statistical units (“n”). Nevertheless, the main conclusions of this study were not affected.
Conclusions
In physiological conditions, SSAO that is expressed in rat and human cartilage may participate in chondrocyte terminal differentiation via glucose transport. The observation of a higher SSAO activity in damaged cartilage from some OA patient suggests a possible role in the development of the disease.
Contributions
AF and NM participated in study design, data acquisition, analysis, interpretation and manuscript preparation; AP performed the histological study on human cartilage and participated in interpretation and manuscript preparation; AB participated to the study design, data analysis, interpretation and performed the PCR in human samples; PG and DM provided the human samples and performed the Kellgren–Lawrence grade of OA, SJ provided the LJP 1586 inhibitor. BF, PL and JM participated in study design, interpretation and manuscript preparation. All authors were involved in revising the manuscript; each of them read and approved the final version of the manuscript.
Competing interests
The authors declare that they have no competing interests.
Role of the funding source
This work was supported by grants from the Région Lorraine, the Communauté Urbaine du Grand Nancy (CUGN) and the Fédération de Recherche 3209.
Acknowledgments
Thanks to Carlos Labat, (UMR S_ 1116 Université de Lorraine-INSERM, Vandœuvre-lès-Nancy, France) and to Dr Renaud FAY, Institut Louis Mathieu, CHU (Vandoeuvre-les-Nancy, France) to provide assistance with statistical analysis of the data.
Appendix A. Supplementary data
The following are the supplementary data related to this article:
Chronic benzylamine administration in the drinking water improves glucose tolerance, reduces body weight gain and circulating cholesterol in high-fat diet-fed mice.
Semicarbazide-sensitive amine oxidase in aortic smooth muscle cells mediates synthesis of a methylglyoxal-AGE: implications for vascular complications in diabetes.
Anti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activity.
Inorganic pyrophosphate generation by transforming growth factor-beta-1 is mainly dependent on ANK induction by Ras/Raf-1/extracellular signal-regulated kinase pathways in chondrocytes.
Impaired glucose transporter-1 degradation and increased glucose transport and oxidative stress in response to high glucose in chondrocytes from osteoarthritic versus normal human cartilage.
In vitro chondrocyte differentiation using costochondral chondrocytes as a source of primary rat chondrocyte cultures: an improved isolation and cryopreservation method.
Increased type II collagen degradation and very early focal cartilage degeneration is associated with upregulation of chondrocyte differentiation related genes in early human articular cartilage lesions.
Molecular cloning of a major mRNA species in murine 3T3 adipocyte lineage. differentiation-dependent expression, regulation, and identification as semicarbazide-sensitive amine oxidase.
Interaction of L-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions.
Facilitative glucose transporters in articular chondrocytes. Expression, distribution and functional regulation of GLUT isoforms by hypoxia, hypoxia mimetics, growth factors and pro-inflammatory cytokines.
Abnormal ambient glucose levels inhibit proteoglycan core protein gene expression and reduce proteoglycan accumulation during chondrogenesis: possible mechanism for teratogenic effects of maternal diabetes.
Immunocytochemical demonstration of glucose transporters in epiphyseal growth plate chondrocytes of young rats in correlation with autoradiographic distribution of 2-deoxyglucose in chondrocytes of mice.
Evidence linking chondrocyte lipid peroxidation to cartilage matrix protein degradation. Possible role in cartilage aging and the pathogenesis of osteoarthritis.
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