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The activity of aggrecanases and release of proteoglycans from human osteoarthritic cartilage as modulated by biochemical characterized collagen hydrolysates
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Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
Figure 2. Changes in medication (Panel A) and nutraceutical (panel B) costs from baseline gender. 577 THE ACTIVITY OF AGGRECANASES AND RELEASE OF PROTEOGLYCANS FROM HUMAN OSTEOARTHRITIC CARTILAGE AS MODULATED BY BIOCHEMICAL CHARACTERIZED COLLAGEN HYDROLYSATES S. Schadow y, V. Simons y, H.-C. Siebert z, G. Lochnit x, J. Steinmeyer y. y Lab. for Experimental Orthopaedics, Dept. of Orthopaedics, JustusLiebig-Univ. of Giessen, Giessen, Germany; z Res. Inst. of Bioinformatics and Nanotechnology, Kiel, Germany; x Dept. of Biochemistry, JustusLiebig-Univ. of Giessen, Giessen, Germany Purpose: Collagen hydrolysates (CH) are mixtures of collagen type I peptides and are popular nutriceuticals used for prophylaxis of osteoarthritis (OA) [Schadow et al. 2013]. Loss of proteoglycans from articular cartilage is a typical hallmark of OA. The aggrecanases ADAMTS4 (a disintegrin and metalloproteinase with a thrombospondin motif) and ADAMTS5 play a central role in the proteolytic degradation of osteoarthritic articular cartilage. Our in silico calculations have revealed that the different collagen type I peptides bind to various docking sites on the aggrecanases so that their activities are affected differently. We also showed that small collagen peptides bind weakly to the a2A-domain of the integrin receptor, and that this may represent another possible € tzel et al. 2012]. mechanism of action [Sto The aim of this study was to test the extent to which two CHs from fish and porcine are able firstly to influence the activity of ADAMTS4 and ADAMTS5 in vitro, and secondly to modulate the release of proteoglycans from human osteoarthritic cartilage explants. Methods: The molecular composition of CHs was determined by mass spectrometry (MALDI-TOF-MS) [Schadow et al. 2013]. The activity of rhADAMTS4 and rhADAMTS5 was determined in the presence of the two CHs (Peptan™ F 5000 HD from Rousselot, and Mobiforte® from Astrid Twardy) using rhAggrecan-IGD as substrate, whereby the proteolytically released aggrecan peptide with the N-terminal sequence ARGSVIL was quantified in a microtiter plate using specific mAb [Steinmeyer et al. 2010]. Cartilage explants from lateral condyles were obtained after knee TEP implantation, and written consent was received from the patients subsequent to approval by the local university ethics committee. After macroscopic evaluation of the arthritic changes using Collins’ procedure, explants were cultivated. The extent of cartilage degradation was determined using explants after a 6 day treatment with 0e10 mg/ml CH with one media change after 3 days. Proteoglycan content was measured using the DMMB method, with TIMP-3 content being determined by ELISA and NO content being measured using the Griess reaction. The vitality of the explants was determined by fluorescence microscopy. The results were compared
with untreated control samples. Each experiment was repeated five times (n ¼ 6). Groups of data were evaluated using one-way analysis of variance (ANOVA) and the Friedman test. Pharmacological data are presented as dotplot with median, and the significance was set to p< 0.05. Results: MALDI-TOF-MS revealed striking differences between the different CH preparations. They differed both in terms of the composition of their mixtures regarding individual peptides, and in terms of their mean molecular weight. For Peptan™ F 5000 HD the mean molecular weight was 3,600 Da, while for Mobiforte® it was slightly smaller at 3,300 Da. Approximately 2% of collagen type I peptide species are found in both CH preparations. Only Mobiforte® significantly and concentration-dependently stimulated the activity of rhADAMTS4 and rhADAMTS5. This probably allosterically-induced stimulation was associated with a significantly increased release of proteoglycans and NO from human cartilage explants, while no effect was measured with respect to TIMP-3 levels. Peptan F™ 5000 HD slightly inhibited the activity of aggrecanases, while no effect was measured for the NO concentrations, proteoglycan release or the levels of TIMP-3. Conclusions: The present study was carried out in order to understand the molecular mechanisms of possible structure-modifying effect of CHs, as has been insinuated [e.g. Oesser et al. 2008]. Our investigation show for the first time that CH preparations from fish (Peptan™ F 5000 HD) markedly differ from those of porcine origin (Mobiforte®) with respect to both their composition of hydrolyzed peptides as well as their biological activities on human chondrocytes. The different biological activities of CH preparations may be due to their different composition of pharmacological active peptide(s), and it remains to be determined whether one or several oligopeptides or even peptidic aggregates are effective. In conclusion, CHs should be thoroughly investigated with respect to their pharmacological effects both in vitro and in vivo before being declared as safe and effective. 578 INDIAN MEDICINAL PLANT BUTEA MONOSPERMA FLOWER EXTRACT AND ITS BIOACTIVE CONSTITUENT BUTEIN ACTIVATES AUTOPHAGY IN HUMAN OA CHONDROCYTES UNDER PATHOLOGICAL CONDITIONS M.Y. Ansari, S. Haynie, T.M. Haqqi;. Northeast Ohio Med. Univ., Rootstown, OH, USA Purpose: Butea monosperma (Lamk.) plant products have been widely used in Indian subcontinent for medicinal purpose. The flowers are large, 46 cm long and are bright yellowish red to orange red. An extract prepared from Butea monosperma (Lamk.) flower is a rich source of glycosides of Butein, Butrin, Isobutrin which possess strong antioxidant properties. Autophagy is an evolutionary conserved cellular function that plays a central role in clearance of damaged and dysfunctional cellular organelles and aggregates. Autophagy is implicated in several physiological processes including development, aging and disease. Osteoarthritis (OA) is the most common form of whole joint disease characterized by loss of articular cartilage, joint pain and inflammation. Articular cartilage show reduced autophagy with age and an approach to boost autophagy in chondrocytes has been shown to have protective effect in experimental OA. Here we determined whether a standardized extract of Butea monosperma (Lamk.) flowers can activate autophagy in OA chondrocytes and protect OA chondrocytes from IL-1b induced production of ROS and cell death. Methods: Primary human chondrocytes were prepared from undamaged cartilage obtained from OA donors who underwent total knee arthroplasty. Primary chondrocytes were maintained in DMEM/ Ham’sF-12 medium (Lonza, Walkersville, MD) with regular media changes every 3 day. Butea monosperma (Lamk.) flowers extract (BME) was prepared from dried flower powder by MeOH (70%) extraction in Soxhlet extractor overnight. MeOH was removed under reduced pressure using Buchi Rotavapor and lyophilized. Butein was purchased from Alkemist Labs (Costa Mesa, CA). Chondrocytes (1x106 cells/well in 6 well plates) were treated with IL-1b (10ng/ml, R & D Systems) and BME for different time points. Cellular ROS levels were measured by flow cytometry using DCFDA and DHR123 (Life Technologies). Activation of autophagy was assessed by immunoblot analysis of autophagy markers and gene expression profiling using TaqMan assays. Formation of autophagosomes was determined by immunofluorescence staining of LC3 as well as transient expression of LC3-GFP plasmid construct
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
Figure 2. Changes in medication (Panel A) and nutraceutical (panel B) costs from baseline gender. 577 THE ACTIVITY OF AGGRECANASES AND RELEASE OF PROTEOGLYCANS FROM HUMAN OSTEOARTHRITIC CARTILAGE AS MODULATED BY BIOCHEMICAL CHARACTERIZED COLLAGEN HYDROLYSATES S. Schadow y, V. Simons y, H.-C. Siebert z, G. Lochnit x, J. Steinmeyer y. y Lab. for Experimental Orthopaedics, Dept. of Orthopaedics, JustusLiebig-Univ. of Giessen, Giessen, Germany; z Res. Inst. of Bioinformatics and Nanotechnology, Kiel, Germany; x Dept. of Biochemistry, JustusLiebig-Univ. of Giessen, Giessen, Germany Purpose: Collagen hydrolysates (CH) are mixtures of collagen type I peptides and are popular nutriceuticals used for prophylaxis of osteoarthritis (OA) [Schadow et al. 2013]. Loss of proteoglycans from articular cartilage is a typical hallmark of OA. The aggrecanases ADAMTS4 (a disintegrin and metalloproteinase with a thrombospondin motif) and ADAMTS5 play a central role in the proteolytic degradation of osteoarthritic articular cartilage. Our in silico calculations have revealed that the different collagen type I peptides bind to various docking sites on the aggrecanases so that their activities are affected differently. We also showed that small collagen peptides bind weakly to the a2A-domain of the integrin receptor, and that this may represent another possible € tzel et al. 2012]. mechanism of action [Sto The aim of this study was to test the extent to which two CHs from fish and porcine are able firstly to influence the activity of ADAMTS4 and ADAMTS5 in vitro, and secondly to modulate the release of proteoglycans from human osteoarthritic cartilage explants. Methods: The molecular composition of CHs was determined by mass spectrometry (MALDI-TOF-MS) [Schadow et al. 2013]. The activity of rhADAMTS4 and rhADAMTS5 was determined in the presence of the two CHs (Peptan™ F 5000 HD from Rousselot, and Mobiforte® from Astrid Twardy) using rhAggrecan-IGD as substrate, whereby the proteolytically released aggrecan peptide with the N-terminal sequence ARGSVIL was quantified in a microtiter plate using specific mAb [Steinmeyer et al. 2010]. Cartilage explants from lateral condyles were obtained after knee TEP implantation, and written consent was received from the patients subsequent to approval by the local university ethics committee. After macroscopic evaluation of the arthritic changes using Collins’ procedure, explants were cultivated. The extent of cartilage degradation was determined using explants after a 6 day treatment with 0e10 mg/ml CH with one media change after 3 days. Proteoglycan content was measured using the DMMB method, with TIMP-3 content being determined by ELISA and NO content being measured using the Griess reaction. The vitality of the explants was determined by fluorescence microscopy. The results were compared
with untreated control samples. Each experiment was repeated five times (n ¼ 6). Groups of data were evaluated using one-way analysis of variance (ANOVA) and the Friedman test. Pharmacological data are presented as dotplot with median, and the significance was set to p< 0.05. Results: MALDI-TOF-MS revealed striking differences between the different CH preparations. They differed both in terms of the composition of their mixtures regarding individual peptides, and in terms of their mean molecular weight. For Peptan™ F 5000 HD the mean molecular weight was 3,600 Da, while for Mobiforte® it was slightly smaller at 3,300 Da. Approximately 2% of collagen type I peptide species are found in both CH preparations. Only Mobiforte® significantly and concentration-dependently stimulated the activity of rhADAMTS4 and rhADAMTS5. This probably allosterically-induced stimulation was associated with a significantly increased release of proteoglycans and NO from human cartilage explants, while no effect was measured with respect to TIMP-3 levels. Peptan F™ 5000 HD slightly inhibited the activity of aggrecanases, while no effect was measured for the NO concentrations, proteoglycan release or the levels of TIMP-3. Conclusions: The present study was carried out in order to understand the molecular mechanisms of possible structure-modifying effect of CHs, as has been insinuated [e.g. Oesser et al. 2008]. Our investigation show for the first time that CH preparations from fish (Peptan™ F 5000 HD) markedly differ from those of porcine origin (Mobiforte®) with respect to both their composition of hydrolyzed peptides as well as their biological activities on human chondrocytes. The different biological activities of CH preparations may be due to their different composition of pharmacological active peptide(s), and it remains to be determined whether one or several oligopeptides or even peptidic aggregates are effective. In conclusion, CHs should be thoroughly investigated with respect to their pharmacological effects both in vitro and in vivo before being declared as safe and effective. 578 INDIAN MEDICINAL PLANT BUTEA MONOSPERMA FLOWER EXTRACT AND ITS BIOACTIVE CONSTITUENT BUTEIN ACTIVATES AUTOPHAGY IN HUMAN OA CHONDROCYTES UNDER PATHOLOGICAL CONDITIONS M.Y. Ansari, S. Haynie, T.M. Haqqi;. Northeast Ohio Med. Univ., Rootstown, OH, USA Purpose: Butea monosperma (Lamk.) plant products have been widely used in Indian subcontinent for medicinal purpose. The flowers are large, 46 cm long and are bright yellowish red to orange red. An extract prepared from Butea monosperma (Lamk.) flower is a rich source of glycosides of Butein, Butrin, Isobutrin which possess strong antioxidant properties. Autophagy is an evolutionary conserved cellular function that plays a central role in clearance of damaged and dysfunctional cellular organelles and aggregates. Autophagy is implicated in several physiological processes including development, aging and disease. Osteoarthritis (OA) is the most common form of whole joint disease characterized by loss of articular cartilage, joint pain and inflammation. Articular cartilage show reduced autophagy with age and an approach to boost autophagy in chondrocytes has been shown to have protective effect in experimental OA. Here we determined whether a standardized extract of Butea monosperma (Lamk.) flowers can activate autophagy in OA chondrocytes and protect OA chondrocytes from IL-1b induced production of ROS and cell death. Methods: Primary human chondrocytes were prepared from undamaged cartilage obtained from OA donors who underwent total knee arthroplasty. Primary chondrocytes were maintained in DMEM/ Ham’sF-12 medium (Lonza, Walkersville, MD) with regular media changes every 3 day. Butea monosperma (Lamk.) flowers extract (BME) was prepared from dried flower powder by MeOH (70%) extraction in Soxhlet extractor overnight. MeOH was removed under reduced pressure using Buchi Rotavapor and lyophilized. Butein was purchased from Alkemist Labs (Costa Mesa, CA). Chondrocytes (1x106 cells/well in 6 well plates) were treated with IL-1b (10ng/ml, R & D Systems) and BME for different time points. Cellular ROS levels were measured by flow cytometry using DCFDA and DHR123 (Life Technologies). Activation of autophagy was assessed by immunoblot analysis of autophagy markers and gene expression profiling using TaqMan assays. Formation of autophagosomes was determined by immunofluorescence staining of LC3 as well as transient expression of LC3-GFP plasmid construct