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Expression of xylosyltransferase-1 is modulated by fibronectin fragment in human articular chondrocytes
S400
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
The walking speed for all groups were 25 m/min. MD: moderate duration walking, HD: high duration walking, EHD extra high duration walking.
cartilage metabolism was poorly characterized. In this study we examined 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f)mediated XT-1 expression mechanism and its signaling pathway, determining the role of 29-kDa FN-f in cartilage matrix synthesis. Methods: Human articular chondrocytes were enzymatically isolated from articular cartilage and cultured in monolayer. In 29-kDa FN-fstimulated chondrocytes, the relative levels of mRNA and protein for XT-1 were analyzed by real-time quantitative reverse transcriptionpolymerase chain reaction and Western blot analysis, respectively. In order to investigate the effects of 29-kDa FN-f on XT-1, human chondrocytes were transfected with small interfering RNAs (siRNAs) targeting TLR-2. Results: The level of aggrecan and XT-1 in human osteoarthritis cartilage was significantly decreased compared to normal cartilage. XT-1 expression in cultured primary articluar chondrocytes showed a periodic oscillation in both mRNA and protein level. 29-kDa FN-f significantly suppressed the mRNA and protein levels of XT-1 at 14 h and 24 h, respectively. Inhibition of mitogen activated protein kinase and nuclear factor-kB signaling pathway restored 29-kDa FN-f-inhibited XT1 expression. Knockdown of toll like receptor-2 (TLR-2) using small interference RNA revealed that the decrease of XT-1 expression by 29kDa FN-f is mediated by TLR-2 signaling pathway. In addition, Sp3, a repressor of XT-1 promoter, was up-regulated by 29-kDa FN-f. Knockdown and overexpression experiments revealed that XT-1 expression was modulated by 29-kDa FN-f-stimulated Sp3 in primary articular chondrocytes. XT- expression was promoted by AP-1 inhibitor and suppressed by AP-1 activator. Both c-jun and c-fos were activated by 29kDa FN-f. Conclusions: These results demonstrated that 29-kDa FN-f plays a detrimental role in the regulation of cartilage extracellular matrix formation including XT-1 expression. 671 THE GROOVE MODEL OF TIBIA-FEMORAL OSTEOARTHRITIS IN THE RAT H.M. de Visser, H. Weinans, K. Coeleveld, M.H. van Rijen, F.P. Lafeber, S.C. Mastbergen. Univ. Med. Ctr. Utrecht, Utrecht, Netherlands
Figure 1. A) Total Modified Mankin Scores: No changes between groups were found (score ranging from zero to 84).B) Partial Modified Mankin Scores: No changes between groups were found (score ranging from zero to 14). C) COX-2 qPCR analysis for synovium: Data were normalized to control group values (dashed grey line). MD: moderate duration walking, HD: high duration walking, EHD extra high duration walking MTP: medial tibial plateau, MFC: medial femoral condyle, LTP: lateral tibial plateau, LFC: lateral femoral condyle, Pat: patella, Grv: femoral grove. * indicated significant difference when compared to control group. 670 EXPRESSION OF XYLOSYLTRANSFERASE-1 IS MODULATED BY FIBRONECTIN FRAGMENT IN HUMAN ARTICULAR CHONDROCYTES M. Lee, M. Choi, H. Hwang, H. Kim. Hallym Univ. Sacred Heart Hosp., Anyang, Republic of Korea Purpose: Xylosyltransferase-1 (XT-1), encoded by xylt1 gene, is an essential anabolic enzyme to catalyze the initial and rate-determining step in glycosaminoglycan chain synthesis. The effect of fibronectin fragments (FN-fs), generated by proteolytic cleavage of FN and known as damage-associated molecular pattern (DAMP) molecules, on
Purpose: Multiple experimental surgical models in small animals like the rat are currently used to study osteoarthritis (OA) and its potential treatment. However, these models have an important limitation as they often induce a permanent trigger during the course of OA, namely instability, counteracting the potential beneficial effects of therapy. Therefore, animal models of intrinsic cartilage damage, without a permanent trigger, have been described to induce joint degeneration and possibly being more sensitive to therapy. An example of such a model is the canine groove model of joint degeneration with features as observed in early OA. In this canine model, damage to the articular cartilage of the weight-bearing areas of the femoral condyles in the knee is the trigger for development of joint changes consistent with early OA. However, longitudinally in-vivo monitoring the disease progression is difficult in canine experiments. Besides, canine experiments can be limited performed due to restricted facilities and costs involved. The groove model can also be applied in the rat, where intrinsic cartilage damage is induced on the femoral trochlea. Here we report a modified groove model of the rat, where intrinsic cartilage damage on the weight bearing surface of the femoral condyles of a rat knee joint was induced, similar to the canine model. The subsequent degenerative joint changes were evaluated. Methods: In Ten male Wistar rats (Charles-River, Sulzfeld, Germany), 16 weeks of age, surgery was performed in one knee joint. During surgery the articular cartilage, of both the trochlear groove and the weight bearing areas of the femoral condyles, is damaged (grooved) without damaging the underlying subchondral bone. The contralateral knee joint served as an internal control. For a period of 12 weeks the subchondral bone parameters (Subchondral plate, trabecular bone and volume fraction) as well as the articular cartilage of the patella are longitudinally monitored by (contrast enhanced-)micro-CT (mCT) (Quatum FX, PerkinElmar, USA). Subsequently, OA severity is assessed, 6 and 12 weeks post-surgery, by the rat OARSI histopathology score by two observers blinded for study group. Directly induced damage by groove surgery was neglected for OA severity assessment. To analyze
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
The walking speed for all groups were 25 m/min. MD: moderate duration walking, HD: high duration walking, EHD extra high duration walking.
cartilage metabolism was poorly characterized. In this study we examined 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f)mediated XT-1 expression mechanism and its signaling pathway, determining the role of 29-kDa FN-f in cartilage matrix synthesis. Methods: Human articular chondrocytes were enzymatically isolated from articular cartilage and cultured in monolayer. In 29-kDa FN-fstimulated chondrocytes, the relative levels of mRNA and protein for XT-1 were analyzed by real-time quantitative reverse transcriptionpolymerase chain reaction and Western blot analysis, respectively. In order to investigate the effects of 29-kDa FN-f on XT-1, human chondrocytes were transfected with small interfering RNAs (siRNAs) targeting TLR-2. Results: The level of aggrecan and XT-1 in human osteoarthritis cartilage was significantly decreased compared to normal cartilage. XT-1 expression in cultured primary articluar chondrocytes showed a periodic oscillation in both mRNA and protein level. 29-kDa FN-f significantly suppressed the mRNA and protein levels of XT-1 at 14 h and 24 h, respectively. Inhibition of mitogen activated protein kinase and nuclear factor-kB signaling pathway restored 29-kDa FN-f-inhibited XT1 expression. Knockdown of toll like receptor-2 (TLR-2) using small interference RNA revealed that the decrease of XT-1 expression by 29kDa FN-f is mediated by TLR-2 signaling pathway. In addition, Sp3, a repressor of XT-1 promoter, was up-regulated by 29-kDa FN-f. Knockdown and overexpression experiments revealed that XT-1 expression was modulated by 29-kDa FN-f-stimulated Sp3 in primary articular chondrocytes. XT- expression was promoted by AP-1 inhibitor and suppressed by AP-1 activator. Both c-jun and c-fos were activated by 29kDa FN-f. Conclusions: These results demonstrated that 29-kDa FN-f plays a detrimental role in the regulation of cartilage extracellular matrix formation including XT-1 expression. 671 THE GROOVE MODEL OF TIBIA-FEMORAL OSTEOARTHRITIS IN THE RAT H.M. de Visser, H. Weinans, K. Coeleveld, M.H. van Rijen, F.P. Lafeber, S.C. Mastbergen. Univ. Med. Ctr. Utrecht, Utrecht, Netherlands
Figure 1. A) Total Modified Mankin Scores: No changes between groups were found (score ranging from zero to 84).B) Partial Modified Mankin Scores: No changes between groups were found (score ranging from zero to 14). C) COX-2 qPCR analysis for synovium: Data were normalized to control group values (dashed grey line). MD: moderate duration walking, HD: high duration walking, EHD extra high duration walking MTP: medial tibial plateau, MFC: medial femoral condyle, LTP: lateral tibial plateau, LFC: lateral femoral condyle, Pat: patella, Grv: femoral grove. * indicated significant difference when compared to control group. 670 EXPRESSION OF XYLOSYLTRANSFERASE-1 IS MODULATED BY FIBRONECTIN FRAGMENT IN HUMAN ARTICULAR CHONDROCYTES M. Lee, M. Choi, H. Hwang, H. Kim. Hallym Univ. Sacred Heart Hosp., Anyang, Republic of Korea Purpose: Xylosyltransferase-1 (XT-1), encoded by xylt1 gene, is an essential anabolic enzyme to catalyze the initial and rate-determining step in glycosaminoglycan chain synthesis. The effect of fibronectin fragments (FN-fs), generated by proteolytic cleavage of FN and known as damage-associated molecular pattern (DAMP) molecules, on
Purpose: Multiple experimental surgical models in small animals like the rat are currently used to study osteoarthritis (OA) and its potential treatment. However, these models have an important limitation as they often induce a permanent trigger during the course of OA, namely instability, counteracting the potential beneficial effects of therapy. Therefore, animal models of intrinsic cartilage damage, without a permanent trigger, have been described to induce joint degeneration and possibly being more sensitive to therapy. An example of such a model is the canine groove model of joint degeneration with features as observed in early OA. In this canine model, damage to the articular cartilage of the weight-bearing areas of the femoral condyles in the knee is the trigger for development of joint changes consistent with early OA. However, longitudinally in-vivo monitoring the disease progression is difficult in canine experiments. Besides, canine experiments can be limited performed due to restricted facilities and costs involved. The groove model can also be applied in the rat, where intrinsic cartilage damage is induced on the femoral trochlea. Here we report a modified groove model of the rat, where intrinsic cartilage damage on the weight bearing surface of the femoral condyles of a rat knee joint was induced, similar to the canine model. The subsequent degenerative joint changes were evaluated. Methods: In Ten male Wistar rats (Charles-River, Sulzfeld, Germany), 16 weeks of age, surgery was performed in one knee joint. During surgery the articular cartilage, of both the trochlear groove and the weight bearing areas of the femoral condyles, is damaged (grooved) without damaging the underlying subchondral bone. The contralateral knee joint served as an internal control. For a period of 12 weeks the subchondral bone parameters (Subchondral plate, trabecular bone and volume fraction) as well as the articular cartilage of the patella are longitudinally monitored by (contrast enhanced-)micro-CT (mCT) (Quatum FX, PerkinElmar, USA). Subsequently, OA severity is assessed, 6 and 12 weeks post-surgery, by the rat OARSI histopathology score by two observers blinded for study group. Directly induced damage by groove surgery was neglected for OA severity assessment. To analyze