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Nitisinone therapy shows beneficial effects on the chondrocytes and extracellular matrix in the osteoarthropathy of alkaptonuria
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Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
Fig 3. Receiver operating curves (ROC) for the various T1r measurements for the femoral (FEM) and acetabular (ACE) cartilage in femoroacetabular (FAI) patients and controls. The local T1r texture analysis within the anterior superior (Ant. Sup.) cluster (black line), with an area under the curve of 0.85, provides a highly reliable detection of acetabular cartilage delamination. Conclusions: The results of this study demonstrate that FAI patients with delamination exhibit altered cartilage texture compared to healthy controls. More specifically, FAI patients exhibit higher T1r radial heterogeneity of the acetabular cartilage in the anterior superior region where delamination occurs, which is consistent with focal cartilage damage. The use of texture based analysis in FAI patients may provide an accurate method (85% accuracy) in detection of acetabular cartilage delamination and thereby, allow clinicians to better counsel FAI patients on treatment options and the prognosis of their condition. In addition, delamination may not be associated with pain in FAI patients, suggesting that pain in these patients may be caused by some other aspect of the pathology. 221 NITISINONE THERAPY SHOWS BENEFICIAL EFFECTS ON THE CHONDROCYTES AND EXTRACELLULAR MATRIX IN THE OSTEOARTHROPATHY OF ALKAPTONURIA J. Mistry y, D. Jackson y, M. Bukhari z, A.M. Taylor y. y Lancaster Univ., Lancaster, United Kingdom; z Royal Lancaster Infirmary, Lancaster, United Kingdom Purpose: Alkaptonuria is a rare autosomal recessive form of osteoarthropathy resulting from deficiency of homogentisate 1,2 dioxygenase (HGD) causing inability to metabolise the tyrosine metabolite; homogentisic acid (HGA). HGA levels in the body become elevated and show high affinity for collagenous tissues, primarily load bearing articular cartilages, where it polymerises and deposits as a dark pigment; this process is ochronosis. Over time ochronosis causes early onset joint arthropathy, mimicking osteoarthritis (OA) in presentation but progressing much more rapidly. Recently a therapy; Nitisinone has been shown to be effective in reducing the causative molecule; HGA, therefore potentially halting the arthropathy. This study aims to examine the effect of the drug Nitisinone on cell number, ochronosis and turnover of the extracellular matrix in an in vitro model of this disease whilst also comparing with the effects of IL-1b. Methods: Cultures of immortalised human chondrocytes C20/A4 were cultured in 24 well plates in DMEM containing HGA, IL-1b, Nitisinone or a combination of these reagents for up to 9 days. The rate of pigmentation was assessed using histology and counted as a number of pigment deposits/mm3, degradation and turnover of the matrix was assessed using DMMB assay and CTX-II ELISA on cell culture supernatants. Statistical analysis was performed using Graphpad Prism by ANOVA and Newman Keuls post-hoc analysis. Results: Cultures showed a significance decrease in the number of cells after 3 days of culture when comparing cultures containing HGA,
compared to any of those which didn’t (p < 0.001). By day 9 of culture similar significance decreases in cell number were seen when comparing cultures containing HGA and those without; Control vs; HGA and vs HGA þ IL-1b (p < 0.001). The rate of pigmentation in these cultures containing HGA showed no significant differences on day 3 or 6, but by day 9 the cultures containing HGA vs those which contained HGA in combination with Nitisinone showed a significantly higher amount of pigmentation. DMMB assay revealed a significant increase in the amount of GAG in supernatants of culture containing HGA alone vs control, IL-1b, Nitisinone or HGAþIL-1bþNTBC cultures. The cultures containing a combination of HGAþIL-1b vs HGA also showed a significant increase in the amount of GAG released. By day 9 the amount of GAG detectable was not significantly different between treatment groups, although the cultures containing HGAþILþNTBC did show a decrease in the amount of GAG compared to HGA and HGAþIL-1b. CTXII showed no significant differences in any treatment groups across 3, 6 or 9 days. Conclusions: This model is the first to test the effect of Nitisinone on the chondrocytes and their extracellular matrix in a model of Alkaptonuria; demonstrating the combined effect on pigmentation rate, cell number and the extracellular matrix. The effect of HGA on cell number shows an early effect on chondrocytes suggesting rapid decrease in chondrocytes at day 3. This is mirrored by loss of GAG from cultures early in the presence of HGA. Nitisinone shows a significant effect in decreasing the pigmentation rate, thus preventing the advancement of disease in these patients. Our results are the first to show the effect of Nitisinone on the release of GAG’s in this in vitro model and provide useful insight into how this drug may protect cartilage in these patients; this also shows some effect of protection of GAG in the presence of IL-1b alone. It also demonstrates that when HGA, the causative molecule in AKU is compared to one of the key mediators of OA; IL-1b, the effect on the cells and their matrix is more severe - giving potential insight into why the arthritis in AKU is more severe and rapidly progressing than that in OA. 222 IMPACT OF NAD-DEPENDENT DEACETYLASE SIRTUIN-1 IN THE OSTEOPHYTE FORMATION AND THE DEGRADATION OF ARTICULAR CARTILAGE IN OSTEOARTHRITIS (OA) K. Yudoh, K. Terauchi, N. Yui, H. Kobayashi, H. Fujiya, H. Niki, H. Musha. St. Marianna Univ. Sch. of Med., Kawasaki City, Japan Purpose: Aging is one of the major pathologic factors associated with osteoarthritis (OA). It has been indicated that the NAD-dependent deacetylase sirtuin-1 (Sirt-1) has an important role in human aging (1). Recent report demonstrated that SIRT-1 insufficiency induced the vascular calcification and artherosclerosis in cardiovascular tissues, suggesting that SIRT-1 may be of benefit in the protection against vascular calcification and maintenance of cardiovascular function with aging (2). In addition, it has been suggested that Sirt-1 promotes osteogenic and chondrogenic differentiation of mesenchymal stem cells (3). However, the impact of Sirt-1 in the osteoarthritic chondrocyte state still remains unclear. We postulated that Sirt-1 regulates a hypertrophic chondrocyte lineage and osteophyte formation through the activation of osteogenic transcription factor Runx2 in osteoarthritic chondrocytes. Since it is well known that Runx2 is a promotor of the activation of cartilage matrix degrading enzyme, matrix metalloproteinase (MMP)-13 (4), Sirt-1 may also influence the expression of MMP-13 in chondrocytes, consequently facilitating the deterioration of articular cartilage in OA. To verify the impact of Sirt-1 in the pathology of OA, we investigated expressions of Sirt-1, Runx2 and MMP-13, and their correlations in human chondrocytes in OA. Methods: i) The expressions of Sirt-1 and Runx2 were histologically analyzed in the articular cartilage tissue of OA model mouse. ii) Human chondrocytes were isolated from articular cartilage tissues from OA patients who underwent the knee joint replacement surgery. Levels of expressions of Runx2, Sirt-1 and MMP-13 were analyzed in the presence or absence of OA related catabolic factor, IL-1beta (10 ng/ml), in human osteoarthritic chondrocytes. The impact of Sirt-1 insufficiency in the chondrocyte activity was also examined in chondrocytes cultures that were treated with Sirt-1 inhibitor. Results: i) The expression of Sirt-1 was ubiquitously observed in chondrocytes, in contrast, Runx2 expressed in the only osteophyte region in the knee joint of OA model mice. ii) IL-1beta up-regulated the expression of Runx2 in osteoarthritic chondrocytes. The IL-1beta-
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
Fig 3. Receiver operating curves (ROC) for the various T1r measurements for the femoral (FEM) and acetabular (ACE) cartilage in femoroacetabular (FAI) patients and controls. The local T1r texture analysis within the anterior superior (Ant. Sup.) cluster (black line), with an area under the curve of 0.85, provides a highly reliable detection of acetabular cartilage delamination. Conclusions: The results of this study demonstrate that FAI patients with delamination exhibit altered cartilage texture compared to healthy controls. More specifically, FAI patients exhibit higher T1r radial heterogeneity of the acetabular cartilage in the anterior superior region where delamination occurs, which is consistent with focal cartilage damage. The use of texture based analysis in FAI patients may provide an accurate method (85% accuracy) in detection of acetabular cartilage delamination and thereby, allow clinicians to better counsel FAI patients on treatment options and the prognosis of their condition. In addition, delamination may not be associated with pain in FAI patients, suggesting that pain in these patients may be caused by some other aspect of the pathology. 221 NITISINONE THERAPY SHOWS BENEFICIAL EFFECTS ON THE CHONDROCYTES AND EXTRACELLULAR MATRIX IN THE OSTEOARTHROPATHY OF ALKAPTONURIA J. Mistry y, D. Jackson y, M. Bukhari z, A.M. Taylor y. y Lancaster Univ., Lancaster, United Kingdom; z Royal Lancaster Infirmary, Lancaster, United Kingdom Purpose: Alkaptonuria is a rare autosomal recessive form of osteoarthropathy resulting from deficiency of homogentisate 1,2 dioxygenase (HGD) causing inability to metabolise the tyrosine metabolite; homogentisic acid (HGA). HGA levels in the body become elevated and show high affinity for collagenous tissues, primarily load bearing articular cartilages, where it polymerises and deposits as a dark pigment; this process is ochronosis. Over time ochronosis causes early onset joint arthropathy, mimicking osteoarthritis (OA) in presentation but progressing much more rapidly. Recently a therapy; Nitisinone has been shown to be effective in reducing the causative molecule; HGA, therefore potentially halting the arthropathy. This study aims to examine the effect of the drug Nitisinone on cell number, ochronosis and turnover of the extracellular matrix in an in vitro model of this disease whilst also comparing with the effects of IL-1b. Methods: Cultures of immortalised human chondrocytes C20/A4 were cultured in 24 well plates in DMEM containing HGA, IL-1b, Nitisinone or a combination of these reagents for up to 9 days. The rate of pigmentation was assessed using histology and counted as a number of pigment deposits/mm3, degradation and turnover of the matrix was assessed using DMMB assay and CTX-II ELISA on cell culture supernatants. Statistical analysis was performed using Graphpad Prism by ANOVA and Newman Keuls post-hoc analysis. Results: Cultures showed a significance decrease in the number of cells after 3 days of culture when comparing cultures containing HGA,
compared to any of those which didn’t (p < 0.001). By day 9 of culture similar significance decreases in cell number were seen when comparing cultures containing HGA and those without; Control vs; HGA and vs HGA þ IL-1b (p < 0.001). The rate of pigmentation in these cultures containing HGA showed no significant differences on day 3 or 6, but by day 9 the cultures containing HGA vs those which contained HGA in combination with Nitisinone showed a significantly higher amount of pigmentation. DMMB assay revealed a significant increase in the amount of GAG in supernatants of culture containing HGA alone vs control, IL-1b, Nitisinone or HGAþIL-1bþNTBC cultures. The cultures containing a combination of HGAþIL-1b vs HGA also showed a significant increase in the amount of GAG released. By day 9 the amount of GAG detectable was not significantly different between treatment groups, although the cultures containing HGAþILþNTBC did show a decrease in the amount of GAG compared to HGA and HGAþIL-1b. CTXII showed no significant differences in any treatment groups across 3, 6 or 9 days. Conclusions: This model is the first to test the effect of Nitisinone on the chondrocytes and their extracellular matrix in a model of Alkaptonuria; demonstrating the combined effect on pigmentation rate, cell number and the extracellular matrix. The effect of HGA on cell number shows an early effect on chondrocytes suggesting rapid decrease in chondrocytes at day 3. This is mirrored by loss of GAG from cultures early in the presence of HGA. Nitisinone shows a significant effect in decreasing the pigmentation rate, thus preventing the advancement of disease in these patients. Our results are the first to show the effect of Nitisinone on the release of GAG’s in this in vitro model and provide useful insight into how this drug may protect cartilage in these patients; this also shows some effect of protection of GAG in the presence of IL-1b alone. It also demonstrates that when HGA, the causative molecule in AKU is compared to one of the key mediators of OA; IL-1b, the effect on the cells and their matrix is more severe - giving potential insight into why the arthritis in AKU is more severe and rapidly progressing than that in OA. 222 IMPACT OF NAD-DEPENDENT DEACETYLASE SIRTUIN-1 IN THE OSTEOPHYTE FORMATION AND THE DEGRADATION OF ARTICULAR CARTILAGE IN OSTEOARTHRITIS (OA) K. Yudoh, K. Terauchi, N. Yui, H. Kobayashi, H. Fujiya, H. Niki, H. Musha. St. Marianna Univ. Sch. of Med., Kawasaki City, Japan Purpose: Aging is one of the major pathologic factors associated with osteoarthritis (OA). It has been indicated that the NAD-dependent deacetylase sirtuin-1 (Sirt-1) has an important role in human aging (1). Recent report demonstrated that SIRT-1 insufficiency induced the vascular calcification and artherosclerosis in cardiovascular tissues, suggesting that SIRT-1 may be of benefit in the protection against vascular calcification and maintenance of cardiovascular function with aging (2). In addition, it has been suggested that Sirt-1 promotes osteogenic and chondrogenic differentiation of mesenchymal stem cells (3). However, the impact of Sirt-1 in the osteoarthritic chondrocyte state still remains unclear. We postulated that Sirt-1 regulates a hypertrophic chondrocyte lineage and osteophyte formation through the activation of osteogenic transcription factor Runx2 in osteoarthritic chondrocytes. Since it is well known that Runx2 is a promotor of the activation of cartilage matrix degrading enzyme, matrix metalloproteinase (MMP)-13 (4), Sirt-1 may also influence the expression of MMP-13 in chondrocytes, consequently facilitating the deterioration of articular cartilage in OA. To verify the impact of Sirt-1 in the pathology of OA, we investigated expressions of Sirt-1, Runx2 and MMP-13, and their correlations in human chondrocytes in OA. Methods: i) The expressions of Sirt-1 and Runx2 were histologically analyzed in the articular cartilage tissue of OA model mouse. ii) Human chondrocytes were isolated from articular cartilage tissues from OA patients who underwent the knee joint replacement surgery. Levels of expressions of Runx2, Sirt-1 and MMP-13 were analyzed in the presence or absence of OA related catabolic factor, IL-1beta (10 ng/ml), in human osteoarthritic chondrocytes. The impact of Sirt-1 insufficiency in the chondrocyte activity was also examined in chondrocytes cultures that were treated with Sirt-1 inhibitor. Results: i) The expression of Sirt-1 was ubiquitously observed in chondrocytes, in contrast, Runx2 expressed in the only osteophyte region in the knee joint of OA model mice. ii) IL-1beta up-regulated the expression of Runx2 in osteoarthritic chondrocytes. The IL-1beta-