- Email: [email protected]
Suramin protects against osteoarthritis by increasing tissue inhibitor of matrix metalloproteinase-3 and glycosaminoglycans in the articular cartilage
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
Purpose: KIAA1199, also called Cemip for “Cell migration-inducing and hyaluronan-binding protein”, was originally described in the inner ear as a deafness gene. The increase of KIAA1199 expression was also observed in various cancers. In rheumatic diseases, KIAA1199 was overexpressed in the synovial membrane of patients with osteoarthritis (OA) and rheumatoid arthritis compared to non-inflamed membrane. In skin fibroblasts, KIAA1199 was highlighted as a new hyaluronan binding protein that participates to hyaluronic acid depolymerisation independently of CD44 and HYAL enzymes through the clathrin-coated pit pathway. In this work, we investigated the expression of KIAA1199 in human OA cartilage and chondrocytes, as well as signalling pathways modulated by KIAA1199 that could lead to cartilage degradation and OA development. Methods: KIAA1199 expression was assessed ex-vivo by immunohistochemistry on cartilage obtained from human hip fracture, OA hip and OA knee. For in-vitro studies, chondrocytes were isolated from OA knee and seeded in monolayer culture. KIAA1199 and ß-catenin expression level was evaluated by RT-qPCR and western blotting. KIAA1199 expression was silenced using two different specific lentiviral shRNA. An anti-EGFP shRNA was used as control. RNA sequencing analysis was performed on total RNA from silenced and non-silenced human chondrocytes. Differential gene expression was analyzed by a statistical method. Results: First, we assessed the protein expression level of KIAA1199 in human cartilage. Immunohistochemistry showed that KIAA1199 is almost not expressed in cartilage obtained from human hip fracture (i.e. non-OA) (n ¼ 3). Conversely, KIAA1199 protein level is increased in human OA hip (n ¼ 7) and knee (n ¼ 6) cartilage. In-vitro, we observed an accumulation of KIAA1199 at RNA (n ¼ 3) and protein (n ¼ 4) level in chondrocytes after 14 days of monolayer culture compared to freshly isolated chondrocytes. We next investigated the effect of KIAA1199 depletion on the Wnt/ß-catenin pathway. We did not observed any modification in the ß-catenin RNA level in KIAA1199 silenced cells compared to non-silenced cells. However, we detected a significant decrease of the ß-catenin protein expression in KIAA1199 depleted chondrocytes compared to control chondrocytes. In order to bring out signalling pathways modulated by KIAA1199 in human OA chondrocytes, we performed total RNA sequencing. Differential gene expression was analyzed in KIAA1199 depleted cells compared to control cells (n ¼ 5). Among all genes differentially expressed, there were extracellular matrix components lipids, genes implicated in the Wnt/ ß-catenin pathway and in apoptosis. Conclusions: In this work, we demonstrated an increase of KIAA1199 expression in OA cartilage compared to non-OA cartilage as well as in fibrochondrocytes compared to freshly isolated chondrocytes. We found that KIAA1199 could modulate the Wnt/ß-catenin pathway, the expression of extracellular matrix components, lipids and genes implicated in apoptosis. All together, these data suggest that KIAA1199 could play an important role in cartilage degradation during OA development.
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classified as short term if MRI was performed within 24 hours after a single bout of running, medium-term if between 24 hours and 2 weeks after a single run, and long-term when more than 2 weeks or when studying the effects of multiple running trainings. Outcomes included cartilage thickness, volume, T2-T2* relaxation time, glycosaminoglycans (GAG) content and cartilage lesions. Results: A total of 2420 titles were retrieved and analyzed for appropriateness based on inclusion and exclusion criteria. Twenty-four studies were included in analyses: 23 articles studied the effects of running on knee cartilages, while only 1 investigated hip and ankle cartilages. Thirteen studies were rated as high quality, 8 as moderate quality and 3 as low quality. Short-term effects. Overall, there is strong evidence that a bout of running decreases femoral, tibial and patellar cartilage thickness and volume. Moderate evidence suggests that T2 relaxation time is decreased at the femoral and tibial cartilages immediately after a short bout of running; after a marathon, however, very limited evidence indicates increased T2*. Based on limited evidence, running seems to have no negative effects on pre-existing knee cartilage lesions. Medium-term effects. There is moderate and very limited evidence that running has no detrimental effects on the number or grade of cartilage lesions at the knee and hip, respectively. Long-term effects. Very limited evidence indicates that long-term running has no effect on knee cartilage thickness, but there is conflicting evidence regarding volume. As for T2 relaxation time, limited evidence supports increased values up to 3 months post-marathon. There is very limited evidence that repeated exposure to running through a start-to-run program causes an increase in GAG content at the tibiofemoral cartilage. Moderate evidence indicates that running has no detrimental effects on the number or grade of lesions in the knee in the long term. At the ankle joint, limited evidence suggests that an ultra-endurance running event has no effect on cartilage thickness. Limited evidence also suggests increased T2* relaxation times during the first half of the event, followed by a decrease indicative of partial regeneration. Conclusions: This systematic review highlights the disparity between the reported effects of running on lower limb cartilages depending on study design. According to current evidence, cartilages may present short-term decreases in thickness, volume and relaxation time secondary to temporary loss of fluid following repeated compressions. However, cartilages may well tolerate mechanical loading sustained during running and adapt to repeated exposure. 210 SURAMIN PROTECTS AGAINST OSTEOARTHRITIS BY INCREASING TISSUE INHIBITOR OF MATRIX METALLOPROTEINASE-3 AND GLYCOSAMINOGLYCANS IN THE ARTICULAR CARTILAGE
J.-F. Esculier, L.J. Bouyer, J.-S. Roy. Ctr. for Interdisciplinary Res. in Rehabilitation and Social Integration (CIRRIS), Quebec City, QC, Canada
L.-A. Guns y, M. Kvasnytsia z, G. Kerckhofs z, J. Vandooren x, E. Martens x, G. Opdenakker x, R.J. Lories y, F. Cailotto k, y. y Lab. of Tissue Homeostasis and Disease, Skeletal Biology and Engineering Res. Ctr., Dept. of Dev. and Regeneration, KULeuven, Leuven, Belgium; z Prometheus, Div. of Skeletal Tissue Engineering Leuven, KU Leuven and Tissue Engineering Lab., Skeletal Biology and Engineering Res. Ctr., KU Leuven, Leuven, Belgium; x Lab. of Immunobiology (Rega Inst.), Dept. of Microbiol. and enierie Mol eculaire et Immunology, KULeuven, Leuven, Belgium; k Ing Physiopathologie Articulaire, UMR7561 CNRS-Universit e de Lorraine, Nancy, France
Purpose: While several reviews have examined the long-term association between running and osteoarthritis, no review has explored the effects of running specifically on lower limb cartilages. Therefore, the objective of this systematic review is to summarize the literature with regards to the prospective effects of running on hip, knee and ankle cartilage structure and physiology. Methods: Database searches were performed in Pubmed, Embase, CINAHL, SportDiscus, Web of Science and the Cochrane Central Registry of Controlled Trials up to July 1, 2016. The strategy included a combination of the following keywords: (lower limb, hip, coxofemoral, knee, tibiofemoral, patellofemoral, ankle, foot) AND (running, runner) AND (cartilage, osteoarthritis, magnetic resonance imaging [MRI]). Hand searches of retrieved articles reference lists were also conducted. Fulllength peer-reviewed papers investigating the prospective effects of running on lower limb cartilages in humans using MRI were included. Studies could either evaluate the effects of a single bout of running or repeated exposure. Two raters independently evaluated the methodological quality of each included article using a structured critical appraisal tool. The effects of running on joint imaging variables were
Purpose: Drug repositioning is a recent pharmaceutical strategy to discover new uses for market-approved drugs with known safety profiles that can provide quick transition from the laboratory bench to bedside. Here we investigate the influence of suramin, a drug used to treat sleeping sickness and river blindness. Suramin is a polysulfonated compound that is negatively charged and binds to basic side chains of proteins. Suramin has been used in vitro to isolate MMPs and tissue inhibitor of metalloproteinase 3 (TIMP3) from the rat uterus. We therefore hypothesized that suramin may protect the articular cartilage against osteoarthritis by decreasing the catabolic action of tissue destructive enzymes such as matrix metalloproteinases (MMPs) and ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs) through increasing TIMP3 levels in the cartilage matrix. Methods: For in vitro experiments we used the mouse ATDC5 chondroprogenitor cell line and freshly isolated human articular chondrocytes (hAC). ATDC5s were cultured in micromasses (200,000 cells/ 10 ml) in differentiation media (ITS in DMEM/F12 medium for 14 days to induce chondrogenesis; b-glycerophosphate and ascorbic acid in aMEM medium from day 14 till day 21 to induce mineralization) in the
209 THE EFFECTS OF RUNNING ON LOWER LIMB CARTILAGES
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Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
presence or absence of 10 mM suramin. Micromass cultures were analyzed for extracellular matrix components, MMP or ADAMTS induced neo-epitopes (VDIPEN and NTEGE) and key transcription factors involved in differentiation by quantitative PCR, Western blot or immunostaining. MMP activity was determined by gelatin zymography after 1, 7, 14 and 21 days of culture. HACs were cultured in pellets (200,000 cells/ 10 ml) and evaluated by histology and immunohistochemistry. Sulfated glycosaminoglycan (sGAG) content was analyzed with a dimethylmethylene Blue (DMMB) assay. In vivo, the effects of suramin were studied in the papain-induced model of osteoarthritis in C57BL/6 with or without a single intra articular (i.a.) injection of 1 mg suramin. Mice were sacrificed after 7 days and cartilage damage was analyzed by histology (OARSI scoring), and by Hexabrix contrastenhanced nanofocus computed tomography (CE-nanoCT - mice knees: 60 kV, 140 mA, 500 msec exposure time, modus 0, voxel size is 2 mm and for hAC pellets: 60 kV, 240 mA, 750 msec exposure time, modus 1, voxel size is 1 mm) thereby quantifying cartilage volume and GAG content. Results: In the ATDC5 micromasses, TIMP-3 protein levels were 2.3-fold increased. Presence of VDIPEN and NITEGE neoepitopes was decreased, as well as MMP-2 activity (by 23%), thus sGAG content was 50% increased. Noteworthy, gene expression levels of different markers and mineralized content remained unaffected, suggesting a post-translational effect of suramin. In the hAC pellets, TIMP-3 levels increased and neoepitope expression for VDIPEN and NITEGE decreased, again resulting in an increased sGAG content. Papain-triggered osteoarthritis in C57BL/6 mice knees treated with suramin showed increased TIMP-3 and decreased VDIPEN and NITEGE levels. This potential protective mechanism effectively resulted in a 47% decreased damage outcome score at the medial femoral condyle as assessed by histology (n ¼ 13; p ¼ 0.0002; t-test), and a 20% increased effective thickness of non-calcified cartilage as determined by CE nanoCT (n ¼ 5; p ¼ 0.0036; t-test). Conclusions: Our data suggest that suramin has a cartilage protective effect by increasing the amount of TIMP3 in the articular cartilage leading to higher sGAG levels. Therefore, suramin is a new potential osteoarthritic disease modifying agent that appears to act specifically at the level of the cartilage matrix. 211 SYNDECAN-4 DEFICIENCY AFFECTS EXTRACELLULAR ARCHITECTURE OF ARTICULAR CARTILAGE
MATRIX
A. Held y, C. Prein z, A. Aszodi x, D. Kronenberg k, U. Hansen k, H. Clausen-Schaumann z, T. Pap k, J. Bertrand y. y Clinic for Orthopedic Surgery, Magdeburg, Germany; z Dept. of Applied Sci. and Mechatronics, Munich, Germany; x Clinic for Gen., Trauma, Hand and Plastic Surgery, Munich, Germany; k Inst. for Experimental Musculoskeletal Med., Univ. Hosp. Münster, Münster, Germany Purpose: Osteoarthritis (OA) is characterized by a dramatic remodeling of articular cartilage - including the degeneration of the extracellular matrix (ECM) and the loss of chondrocyte phenotypic stability. Altered expression of SOX9 and RUNX2 result in a shift of the major fibrillar matrix component type 2 collagen (COL2) to nonfibrillar COL10. The main proteoglycan aggrecan (ACAN) gets degraded. Gene expression of Syndecan-4 (Sdc4), a transmembrane heparan-sulfate proteoglycan, is upregulated in OA. It can enhance cell sensitivity to ligand binding e.g. cytokines such as IL-1 as well as ECM components. Sdc4 knockout (Sdc4-/-) mice are protected from OA-like cartilage changes and less responsive to IL-1 stimuli. We investigated the role of Sdc4 in the ECM architecture of articular cartilage in vivo and in vitro. Methods: Chondrocytes were isolated from knees of neonatal wild type (WT) or Sdc4-/- mice. Electron microscopy (EM) of primary chondrocytes monolayer cell culture, micromass cell culture, and mouse knees were applied to visualize differences in matrix architecture and fibril formation. Total collagen content was investigated using the QuickZyme Total Collagen Assay. Atomic force microscopy (AFM) was performed to analyze ECM stiffness. Knee joints of 8-week-old mice have been stained for Col2, Lubricin (Prg4) and Lysyl Oxidase (LOX). Expression of chondrocyte phenotype marker genes Sox9, Acan, Col2, Col9 and Col11, hypertrophic marker gene Col10, matrix-associated genes Prg4 and Lox have been analyzed by quantitative Real-time (qRT) PCR.
Results: Sdc4-/- chondrocytes showed a higher expression of ACAN, SOX9, COL2, COL9 and COL11, but no altered expression of COL10 and LOX in comparison to WT cells. No difference in total amount of collagens has been observed in WT and Sdc4-/- micromass cultures as wells as neonatal knees. Sdc4-/- chondrocytes cultures and articular knee cartilage of 8-week-old mice displayed finer fibers and more interspaces in the ECM compared to WT controls. The superficial layer of articular cartilage of 8-week-old Sdc4-/- mice appeared less frayed in EM, which is supported by Lubricin immunohistological staining showing more positive cells located in deeper layers of Sdc4-/- articular cartilage. In WT cartilage Lubricin staining was observed more in the superficial cartilage zone. The developmental effect of increasing collagen density and ECM stiffness from neonatal to 8-week-old cartilage was less pronounced in Sdc4-/- superficial layer and middle zone. The proteoglycan and collagen distributions were distinguishable in Sdc4-/-, but not in WT samples of 8-week-old knees. LOX was upregulated progressively with OA severity, but was not altered in unchallenged 8week-old WT and Sdc4-/- mouse knees. Conclusions: Sdc4 is an essential proteoglycan in articular cartilage determining the ECM architecture and ultimately mechanical properties of the tissue. Adult Sdc4-/- cartilage resembles stiffness attributes typical for a more juvenile phenotype, thereby presumably preserving chondrocyte phenotype and protecting from OA-like cartilage changes. 212 HIGH OXYGEN LEVELS INCREASE MITOCHONDRIAL LIPIDS IN HUMAN OSTEOARTHRITIC CHONDROCYTES B. Bakker y, G.B. Eijkel z, R.M. Heeren z, M. Karperien y, J.N. Post y, B. Cillero-Pastor z. y Univ. of Twente, Enschede, Netherlands; z Maastricht Univ., Maastricht, Netherlands Purpose: Articular cartilage is generally exposed to a finely regulated gradient of oxygen levels ranging from 8% at the superficial surface to 1% in the deepest layers. While most cartilage research is performed in supraphysiological oxygen levels (19-21%), culturing chondrocytes under hypoxic oxygen levels ( 8%) promotes the chondrogenic phenotype and cartilage-specific matrix formation. Exposure of cells to various oxygen levels has been shown to alter their lipid metabolism. To better understand the chondrocyte’s behavior in response to oxygen, we studied the effect of various oxygen levels on their lipid composition using Matrix Assisted Laser Desorption Ionization mass spectrometry imaging (MALDI-IMS). Methods: Human primary chondrocytes were isolated from cartilage knee biopsies of patients (n ¼ 5) undergoing total knee replacement, expanded in monolayer in normoxia (20% oxygen) or hypoxia (2.5% oxygen) and subsequently cultured in 3D pellets in normoxia or hypoxia for 7 days. To assess which lipids are altered by oxygen and to visualize their spatial distribution, we performed MALDI-IMS on cryosections of chondrocyte pellets in the negative and positive ion mode. Briefly, cell pellets were cryo-sectioned (10 mm section) and sprayed with Norharmane 7 mg/mL in chloroform/methanol (2:1, (v/v)) or a-Cyano-4hydroxycinnamic acid (CHCA) 5 mg/ml in methanol/water/trifluoroacetic acid (70:30:0.01) using a SunCollect (SunChrom). The Synapt HDMS MALDI-Q-TOF (Waters) was used for the MSI experiments with a spatial raster size of 100 mm. Principal component analysis (PCA) and linear discriminant analysis (LDA) were used to search for spectral similarities and differences between the conditions. Biomap software was used to visualize molecular distributions. Tandem MS and the Lipid Maps database were used for molecular identification. Results: MALDI-IMS followed by LDA reveal that normoxic and hypoxic cultures of primary chondrocytes can be easily separated based on their molecular lipid profile. In the negative ion mode spectra phosphatidylglycerols (PG) were the species that had a high contribution in normoxic pellets, whereas in hypoxic pellets phosphatidylinositol (PI) species were the most prominent lipids (Table 1). Spatial mapping of individual molecules revealed that several PI species have the tendency to be more prominent in the center of hypoxic and normoxic pellets where there is generally less oxygen present. PG is a low abundant phospholipid and mostly present in mitochondria where it is a precursor for cardiolipin (CL), a mitochondrial membrane stabilizing lipid.
Purpose: KIAA1199, also called Cemip for “Cell migration-inducing and hyaluronan-binding protein”, was originally described in the inner ear as a deafness gene. The increase of KIAA1199 expression was also observed in various cancers. In rheumatic diseases, KIAA1199 was overexpressed in the synovial membrane of patients with osteoarthritis (OA) and rheumatoid arthritis compared to non-inflamed membrane. In skin fibroblasts, KIAA1199 was highlighted as a new hyaluronan binding protein that participates to hyaluronic acid depolymerisation independently of CD44 and HYAL enzymes through the clathrin-coated pit pathway. In this work, we investigated the expression of KIAA1199 in human OA cartilage and chondrocytes, as well as signalling pathways modulated by KIAA1199 that could lead to cartilage degradation and OA development. Methods: KIAA1199 expression was assessed ex-vivo by immunohistochemistry on cartilage obtained from human hip fracture, OA hip and OA knee. For in-vitro studies, chondrocytes were isolated from OA knee and seeded in monolayer culture. KIAA1199 and ß-catenin expression level was evaluated by RT-qPCR and western blotting. KIAA1199 expression was silenced using two different specific lentiviral shRNA. An anti-EGFP shRNA was used as control. RNA sequencing analysis was performed on total RNA from silenced and non-silenced human chondrocytes. Differential gene expression was analyzed by a statistical method. Results: First, we assessed the protein expression level of KIAA1199 in human cartilage. Immunohistochemistry showed that KIAA1199 is almost not expressed in cartilage obtained from human hip fracture (i.e. non-OA) (n ¼ 3). Conversely, KIAA1199 protein level is increased in human OA hip (n ¼ 7) and knee (n ¼ 6) cartilage. In-vitro, we observed an accumulation of KIAA1199 at RNA (n ¼ 3) and protein (n ¼ 4) level in chondrocytes after 14 days of monolayer culture compared to freshly isolated chondrocytes. We next investigated the effect of KIAA1199 depletion on the Wnt/ß-catenin pathway. We did not observed any modification in the ß-catenin RNA level in KIAA1199 silenced cells compared to non-silenced cells. However, we detected a significant decrease of the ß-catenin protein expression in KIAA1199 depleted chondrocytes compared to control chondrocytes. In order to bring out signalling pathways modulated by KIAA1199 in human OA chondrocytes, we performed total RNA sequencing. Differential gene expression was analyzed in KIAA1199 depleted cells compared to control cells (n ¼ 5). Among all genes differentially expressed, there were extracellular matrix components lipids, genes implicated in the Wnt/ ß-catenin pathway and in apoptosis. Conclusions: In this work, we demonstrated an increase of KIAA1199 expression in OA cartilage compared to non-OA cartilage as well as in fibrochondrocytes compared to freshly isolated chondrocytes. We found that KIAA1199 could modulate the Wnt/ß-catenin pathway, the expression of extracellular matrix components, lipids and genes implicated in apoptosis. All together, these data suggest that KIAA1199 could play an important role in cartilage degradation during OA development.
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classified as short term if MRI was performed within 24 hours after a single bout of running, medium-term if between 24 hours and 2 weeks after a single run, and long-term when more than 2 weeks or when studying the effects of multiple running trainings. Outcomes included cartilage thickness, volume, T2-T2* relaxation time, glycosaminoglycans (GAG) content and cartilage lesions. Results: A total of 2420 titles were retrieved and analyzed for appropriateness based on inclusion and exclusion criteria. Twenty-four studies were included in analyses: 23 articles studied the effects of running on knee cartilages, while only 1 investigated hip and ankle cartilages. Thirteen studies were rated as high quality, 8 as moderate quality and 3 as low quality. Short-term effects. Overall, there is strong evidence that a bout of running decreases femoral, tibial and patellar cartilage thickness and volume. Moderate evidence suggests that T2 relaxation time is decreased at the femoral and tibial cartilages immediately after a short bout of running; after a marathon, however, very limited evidence indicates increased T2*. Based on limited evidence, running seems to have no negative effects on pre-existing knee cartilage lesions. Medium-term effects. There is moderate and very limited evidence that running has no detrimental effects on the number or grade of cartilage lesions at the knee and hip, respectively. Long-term effects. Very limited evidence indicates that long-term running has no effect on knee cartilage thickness, but there is conflicting evidence regarding volume. As for T2 relaxation time, limited evidence supports increased values up to 3 months post-marathon. There is very limited evidence that repeated exposure to running through a start-to-run program causes an increase in GAG content at the tibiofemoral cartilage. Moderate evidence indicates that running has no detrimental effects on the number or grade of lesions in the knee in the long term. At the ankle joint, limited evidence suggests that an ultra-endurance running event has no effect on cartilage thickness. Limited evidence also suggests increased T2* relaxation times during the first half of the event, followed by a decrease indicative of partial regeneration. Conclusions: This systematic review highlights the disparity between the reported effects of running on lower limb cartilages depending on study design. According to current evidence, cartilages may present short-term decreases in thickness, volume and relaxation time secondary to temporary loss of fluid following repeated compressions. However, cartilages may well tolerate mechanical loading sustained during running and adapt to repeated exposure. 210 SURAMIN PROTECTS AGAINST OSTEOARTHRITIS BY INCREASING TISSUE INHIBITOR OF MATRIX METALLOPROTEINASE-3 AND GLYCOSAMINOGLYCANS IN THE ARTICULAR CARTILAGE
J.-F. Esculier, L.J. Bouyer, J.-S. Roy. Ctr. for Interdisciplinary Res. in Rehabilitation and Social Integration (CIRRIS), Quebec City, QC, Canada
L.-A. Guns y, M. Kvasnytsia z, G. Kerckhofs z, J. Vandooren x, E. Martens x, G. Opdenakker x, R.J. Lories y, F. Cailotto k, y. y Lab. of Tissue Homeostasis and Disease, Skeletal Biology and Engineering Res. Ctr., Dept. of Dev. and Regeneration, KULeuven, Leuven, Belgium; z Prometheus, Div. of Skeletal Tissue Engineering Leuven, KU Leuven and Tissue Engineering Lab., Skeletal Biology and Engineering Res. Ctr., KU Leuven, Leuven, Belgium; x Lab. of Immunobiology (Rega Inst.), Dept. of Microbiol. and enierie Mol eculaire et Immunology, KULeuven, Leuven, Belgium; k Ing Physiopathologie Articulaire, UMR7561 CNRS-Universit e de Lorraine, Nancy, France
Purpose: While several reviews have examined the long-term association between running and osteoarthritis, no review has explored the effects of running specifically on lower limb cartilages. Therefore, the objective of this systematic review is to summarize the literature with regards to the prospective effects of running on hip, knee and ankle cartilage structure and physiology. Methods: Database searches were performed in Pubmed, Embase, CINAHL, SportDiscus, Web of Science and the Cochrane Central Registry of Controlled Trials up to July 1, 2016. The strategy included a combination of the following keywords: (lower limb, hip, coxofemoral, knee, tibiofemoral, patellofemoral, ankle, foot) AND (running, runner) AND (cartilage, osteoarthritis, magnetic resonance imaging [MRI]). Hand searches of retrieved articles reference lists were also conducted. Fulllength peer-reviewed papers investigating the prospective effects of running on lower limb cartilages in humans using MRI were included. Studies could either evaluate the effects of a single bout of running or repeated exposure. Two raters independently evaluated the methodological quality of each included article using a structured critical appraisal tool. The effects of running on joint imaging variables were
Purpose: Drug repositioning is a recent pharmaceutical strategy to discover new uses for market-approved drugs with known safety profiles that can provide quick transition from the laboratory bench to bedside. Here we investigate the influence of suramin, a drug used to treat sleeping sickness and river blindness. Suramin is a polysulfonated compound that is negatively charged and binds to basic side chains of proteins. Suramin has been used in vitro to isolate MMPs and tissue inhibitor of metalloproteinase 3 (TIMP3) from the rat uterus. We therefore hypothesized that suramin may protect the articular cartilage against osteoarthritis by decreasing the catabolic action of tissue destructive enzymes such as matrix metalloproteinases (MMPs) and ADAMTS (A disintegrin and metalloproteinase with thrombospondin motifs) through increasing TIMP3 levels in the cartilage matrix. Methods: For in vitro experiments we used the mouse ATDC5 chondroprogenitor cell line and freshly isolated human articular chondrocytes (hAC). ATDC5s were cultured in micromasses (200,000 cells/ 10 ml) in differentiation media (ITS in DMEM/F12 medium for 14 days to induce chondrogenesis; b-glycerophosphate and ascorbic acid in aMEM medium from day 14 till day 21 to induce mineralization) in the
209 THE EFFECTS OF RUNNING ON LOWER LIMB CARTILAGES
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Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
presence or absence of 10 mM suramin. Micromass cultures were analyzed for extracellular matrix components, MMP or ADAMTS induced neo-epitopes (VDIPEN and NTEGE) and key transcription factors involved in differentiation by quantitative PCR, Western blot or immunostaining. MMP activity was determined by gelatin zymography after 1, 7, 14 and 21 days of culture. HACs were cultured in pellets (200,000 cells/ 10 ml) and evaluated by histology and immunohistochemistry. Sulfated glycosaminoglycan (sGAG) content was analyzed with a dimethylmethylene Blue (DMMB) assay. In vivo, the effects of suramin were studied in the papain-induced model of osteoarthritis in C57BL/6 with or without a single intra articular (i.a.) injection of 1 mg suramin. Mice were sacrificed after 7 days and cartilage damage was analyzed by histology (OARSI scoring), and by Hexabrix contrastenhanced nanofocus computed tomography (CE-nanoCT - mice knees: 60 kV, 140 mA, 500 msec exposure time, modus 0, voxel size is 2 mm and for hAC pellets: 60 kV, 240 mA, 750 msec exposure time, modus 1, voxel size is 1 mm) thereby quantifying cartilage volume and GAG content. Results: In the ATDC5 micromasses, TIMP-3 protein levels were 2.3-fold increased. Presence of VDIPEN and NITEGE neoepitopes was decreased, as well as MMP-2 activity (by 23%), thus sGAG content was 50% increased. Noteworthy, gene expression levels of different markers and mineralized content remained unaffected, suggesting a post-translational effect of suramin. In the hAC pellets, TIMP-3 levels increased and neoepitope expression for VDIPEN and NITEGE decreased, again resulting in an increased sGAG content. Papain-triggered osteoarthritis in C57BL/6 mice knees treated with suramin showed increased TIMP-3 and decreased VDIPEN and NITEGE levels. This potential protective mechanism effectively resulted in a 47% decreased damage outcome score at the medial femoral condyle as assessed by histology (n ¼ 13; p ¼ 0.0002; t-test), and a 20% increased effective thickness of non-calcified cartilage as determined by CE nanoCT (n ¼ 5; p ¼ 0.0036; t-test). Conclusions: Our data suggest that suramin has a cartilage protective effect by increasing the amount of TIMP3 in the articular cartilage leading to higher sGAG levels. Therefore, suramin is a new potential osteoarthritic disease modifying agent that appears to act specifically at the level of the cartilage matrix. 211 SYNDECAN-4 DEFICIENCY AFFECTS EXTRACELLULAR ARCHITECTURE OF ARTICULAR CARTILAGE
MATRIX
A. Held y, C. Prein z, A. Aszodi x, D. Kronenberg k, U. Hansen k, H. Clausen-Schaumann z, T. Pap k, J. Bertrand y. y Clinic for Orthopedic Surgery, Magdeburg, Germany; z Dept. of Applied Sci. and Mechatronics, Munich, Germany; x Clinic for Gen., Trauma, Hand and Plastic Surgery, Munich, Germany; k Inst. for Experimental Musculoskeletal Med., Univ. Hosp. Münster, Münster, Germany Purpose: Osteoarthritis (OA) is characterized by a dramatic remodeling of articular cartilage - including the degeneration of the extracellular matrix (ECM) and the loss of chondrocyte phenotypic stability. Altered expression of SOX9 and RUNX2 result in a shift of the major fibrillar matrix component type 2 collagen (COL2) to nonfibrillar COL10. The main proteoglycan aggrecan (ACAN) gets degraded. Gene expression of Syndecan-4 (Sdc4), a transmembrane heparan-sulfate proteoglycan, is upregulated in OA. It can enhance cell sensitivity to ligand binding e.g. cytokines such as IL-1 as well as ECM components. Sdc4 knockout (Sdc4-/-) mice are protected from OA-like cartilage changes and less responsive to IL-1 stimuli. We investigated the role of Sdc4 in the ECM architecture of articular cartilage in vivo and in vitro. Methods: Chondrocytes were isolated from knees of neonatal wild type (WT) or Sdc4-/- mice. Electron microscopy (EM) of primary chondrocytes monolayer cell culture, micromass cell culture, and mouse knees were applied to visualize differences in matrix architecture and fibril formation. Total collagen content was investigated using the QuickZyme Total Collagen Assay. Atomic force microscopy (AFM) was performed to analyze ECM stiffness. Knee joints of 8-week-old mice have been stained for Col2, Lubricin (Prg4) and Lysyl Oxidase (LOX). Expression of chondrocyte phenotype marker genes Sox9, Acan, Col2, Col9 and Col11, hypertrophic marker gene Col10, matrix-associated genes Prg4 and Lox have been analyzed by quantitative Real-time (qRT) PCR.
Results: Sdc4-/- chondrocytes showed a higher expression of ACAN, SOX9, COL2, COL9 and COL11, but no altered expression of COL10 and LOX in comparison to WT cells. No difference in total amount of collagens has been observed in WT and Sdc4-/- micromass cultures as wells as neonatal knees. Sdc4-/- chondrocytes cultures and articular knee cartilage of 8-week-old mice displayed finer fibers and more interspaces in the ECM compared to WT controls. The superficial layer of articular cartilage of 8-week-old Sdc4-/- mice appeared less frayed in EM, which is supported by Lubricin immunohistological staining showing more positive cells located in deeper layers of Sdc4-/- articular cartilage. In WT cartilage Lubricin staining was observed more in the superficial cartilage zone. The developmental effect of increasing collagen density and ECM stiffness from neonatal to 8-week-old cartilage was less pronounced in Sdc4-/- superficial layer and middle zone. The proteoglycan and collagen distributions were distinguishable in Sdc4-/-, but not in WT samples of 8-week-old knees. LOX was upregulated progressively with OA severity, but was not altered in unchallenged 8week-old WT and Sdc4-/- mouse knees. Conclusions: Sdc4 is an essential proteoglycan in articular cartilage determining the ECM architecture and ultimately mechanical properties of the tissue. Adult Sdc4-/- cartilage resembles stiffness attributes typical for a more juvenile phenotype, thereby presumably preserving chondrocyte phenotype and protecting from OA-like cartilage changes. 212 HIGH OXYGEN LEVELS INCREASE MITOCHONDRIAL LIPIDS IN HUMAN OSTEOARTHRITIC CHONDROCYTES B. Bakker y, G.B. Eijkel z, R.M. Heeren z, M. Karperien y, J.N. Post y, B. Cillero-Pastor z. y Univ. of Twente, Enschede, Netherlands; z Maastricht Univ., Maastricht, Netherlands Purpose: Articular cartilage is generally exposed to a finely regulated gradient of oxygen levels ranging from 8% at the superficial surface to 1% in the deepest layers. While most cartilage research is performed in supraphysiological oxygen levels (19-21%), culturing chondrocytes under hypoxic oxygen levels ( 8%) promotes the chondrogenic phenotype and cartilage-specific matrix formation. Exposure of cells to various oxygen levels has been shown to alter their lipid metabolism. To better understand the chondrocyte’s behavior in response to oxygen, we studied the effect of various oxygen levels on their lipid composition using Matrix Assisted Laser Desorption Ionization mass spectrometry imaging (MALDI-IMS). Methods: Human primary chondrocytes were isolated from cartilage knee biopsies of patients (n ¼ 5) undergoing total knee replacement, expanded in monolayer in normoxia (20% oxygen) or hypoxia (2.5% oxygen) and subsequently cultured in 3D pellets in normoxia or hypoxia for 7 days. To assess which lipids are altered by oxygen and to visualize their spatial distribution, we performed MALDI-IMS on cryosections of chondrocyte pellets in the negative and positive ion mode. Briefly, cell pellets were cryo-sectioned (10 mm section) and sprayed with Norharmane 7 mg/mL in chloroform/methanol (2:1, (v/v)) or a-Cyano-4hydroxycinnamic acid (CHCA) 5 mg/ml in methanol/water/trifluoroacetic acid (70:30:0.01) using a SunCollect (SunChrom). The Synapt HDMS MALDI-Q-TOF (Waters) was used for the MSI experiments with a spatial raster size of 100 mm. Principal component analysis (PCA) and linear discriminant analysis (LDA) were used to search for spectral similarities and differences between the conditions. Biomap software was used to visualize molecular distributions. Tandem MS and the Lipid Maps database were used for molecular identification. Results: MALDI-IMS followed by LDA reveal that normoxic and hypoxic cultures of primary chondrocytes can be easily separated based on their molecular lipid profile. In the negative ion mode spectra phosphatidylglycerols (PG) were the species that had a high contribution in normoxic pellets, whereas in hypoxic pellets phosphatidylinositol (PI) species were the most prominent lipids (Table 1). Spatial mapping of individual molecules revealed that several PI species have the tendency to be more prominent in the center of hypoxic and normoxic pellets where there is generally less oxygen present. PG is a low abundant phospholipid and mostly present in mitochondria where it is a precursor for cardiolipin (CL), a mitochondrial membrane stabilizing lipid.