- Email: [email protected]
Characterization of synovial mast cells in knee osteoarthritis: Association with synovitis and clinical parameters
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Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
which allows determination of trabecular and cortical subchondral bone morphometric parameters as well as hyaline and calcified cartilage thickness This technic correlates very well with histological staining assessment (respectively R2 ¼ .94 and .82 for hyaline and calcified cartilage thickness). This technic also allows to assess a proteoglycan (PG) content estimation (inverse relationship between attenuation and PG content). The material level properties (indentation depth, Young’s modulus and working energy(area under the force displacement curve)) of the distal femur was evaluated using bioindentation with Bioindenter equipment (CSM Instrument, Switzerland). Indentation tests corresponding to around 75% of the whole cartilage thickness were performed with an accuracy lower than 9% for all investigated parameters. Results: Systemic IGF-I was decreased in LP group (18%, p<.001). As expected, in LP group, SB compartment was altered: trabecular SB mass was decreased (10%, p<.01) as well as SB cortical plate thickness of the medial condyle (12%, p<.05). Despite no morphologic changes of the cartilage of the medial condyle (thickness tended to decrease by 8% but not significantly), hyaline cartilage biomechanical properties (force, elastic modulus and working energy) were respectively decreased by 47, 58 and 41% (p<.01). We did not observed alterations of PG. Conclusions: We identified degradation of hyaline cartilage material level properties and a SB cortical thickening as well as trabecular SB deterioration with only mild alteration of hyaline cartilage in rats fed a low protein diet. Since these parameters have been documented to be altered in a similar way in early OA, we suggest that alteration of the somatotropic axis induced by protein malnutrition could predispose to OA. Deterioration of cartilage material level properties could be the result of cartilage collagen network disorganization as it has been already described in bone as well as alteration of pattern of joint loads distribution. The alteration of SB mass also reflects alteration of bone cellular activities which could also alter cartilage metabolism as already described in OA and SB interplay. Since protein malnutrition is frequent in elderly this mechanism could be relevant in human. 97 EFFECT OF THE OTAGO EXERCISES ON POSTURAL BALANCE AND FEAR OF FALLING AMONG OLDER FALLERS WITH KNEE OSTEOARTHRITIS S. Mat y, C.T. Ng z, M.P. Tan y. y Dept. of Med., Faculty of Med., Univ. of Malaya, Kuala Lumpur, Malaysia; z Dept. of Rheumatology and Immunology, Singapore, Singapore Purpose: This study evaluated the effectiveness of an individualized home-based exercise program to improve postural balance, falls risk and fear of falling in older fallers with knee osteoarthritis (OA). Methods: This was a substudy of the Malaysian Falls Assessment and Intervention Trial (MyFAIT), a randomized controlled study. A subanalysis was conducted for participants recruited to the study who had radiological OA, with evidence of gait and balance disorder determined by a timed-up and go score of over 13.5s. Participants randomized to the intervention arm received home-based, individualized Otago exercises, the control received conventional treatment. Posturagraphy test, Knee Injury and Osteoarthritis outcome score (KOOS) and short FES-I were completed at baseline and 6 months. Results: Results from 41 participants (exercise ¼ 17; control ¼ 24) were included. The exercise group had better significantly better directional control compared to control group (mean score: 67.26 vs 58.72, P ¼ 0.044) at 6 months. Within-group analysis indicated that postural control parameters results (mean change: rising index: 8.94; 95% confidence interval, 17.38e 0.49; P ¼ 0.039; reaction time : 0.15; 95% confidence interval, 0.29e 0.001; P ¼ 0.048, maximal excursion: 6.88; 95% confidence interval, 0.69e13.06; P ¼ 0.032, directional control: 10.13; 95% confidence interval, 2.55e17.70; P ¼ 0.012) and short FES-I score (mean change: short FES-I: 3.12 ; 95% confidence interval, 5.93e 0.30; P ¼ 0.032) improved significantly in intervention group. No significant difference in pre and post measurements were found among the control group. Conclusions: Home-based strengthening exercise benefited older fallers with OA and gait and balance disorders in improving their directional control. It also helped in reducing fear of falling and improving postural balance. Our study was not powered for falls outcomes. Future research should therefore consider evaluating targeted exercise interventions in fallers with OA and gait and balance disorders in a larger randomized-controlled study.
Angiogenesis & Synovial Tissue Biology 98 CHARACTERIZATION OF SYNOVIAL MAST CELLS IN KNEE OSTEOARTHRITIS: ASSOCIATION WITH SYNOVITIS AND CLINICAL PARAMETERS B. de Lange-Brokaar y, M. Kloppenburg y, S. Andersen y, A. Dorjee y, E. Yusuf y, L. Herb-van Toorn y, H. Kroon y, A.-M. Zuurmond z, V. Stojanovic-Susulic x, J. Bloem y, R. Nelissen y, R. Toes y, A. Ioan-Facsinay y. y Leiden Univ. Med. Ctr., Leiden, Netherlands; z TNO, Leiden, Netherlands; x Janssen Res. & Dev., Spring House, PA, USA Purpose: Mast cells have been previously suggested to be more abundant in OA synovium compared to RA synovium, in contrast to all other synovial inflammatory cells investigated. Moreover, mast cell degranulation products have been identified in synovial fluid of OA patients, indicating a possible activation of these cells in OA. In this study, we sought evidence for a possible role of these cells in OA by quantifying and characterizing mast cells in the osteoarthritic (OA) synovium using immunofluorescence and investigating their association with clinical parameters in comparison with rheumatoid arthritis (RA) samples. Methods: Patients with knee OA, fulfilling the ACR criteria, and RA patients, fulfilling the ACR criteria, were included. Synovial tissues of 56 symptomatic OA and 49 RA patients were obtained. Of these, 22 OA and 23 RA patients underwent arthroscopy and had no indication for joint replacement and are therefore referred to as “established” patients. The rest of the patients underwent arthroplasty and are referred to as “endstage” patients. Two to three paraffin slides were used to quantify inflammation using haematoxylin and eosin staining (synovitis score 0e9), and numbers of (degranulated) mast cells (per 10 high-power fields) using double immunofluorescence for CD117 and tryptase. Average scores per patient were used for analysis. Degranulated mast cells were defined as CD117þ or CD117-tryptaseþ cells for which at least 2 tryptase granules next to the cells were readily detectable. All other tryptaseþ cells were defined as non-degranulated mast cells. Knee radiographs of OA patients were scored according to the Kellgren and Lawrence (KL) system and pain was determined in OA patients at baseline by visual analogue scale (VAS). Results: Mast cells were readily detectable in both OA and RA synovial tissues and were present mostly in the sublining layer. Median (range) of mast cells was significantly higher in OA samples (45 (1e168)) compared to RA samples (4 (1e47)) (p-value < 0.001), while no differences were observed between established and end-stage OA, or established and end-stage RA samples. OA synovium contained more mast cells than RA synovium both in established and end-stage disease samples. All samples contained approximately 17% degranulated mast cells, irrespective of disease and disease stage. As expected, a lower median (range) synovitis score was present in OA (2.5 (0e6.0)) compared to RA (4.6 (0e8.0)) and this held true when established OA and RA, or end-stage OA and RA were compared. The synovitis score was significantly correlated with the number of mast cells (in OA Spearman’s rho (p-value) 0.3 (0.023) and RA 0.5 (p-value < 0.001)) in both diseases, although this association was less strong in OA. Remarkably,
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
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even in the absence of synovitis there was increased number of mast cells present in OA synovium compared to RA (fig. 1). Interestingly, we observed a trend towards an association between the number of mast cells and an increased KL-grade (p-value 0.05) in OA patients, independently of synovitis. No associations were found with self-reported pain. Conclusions: Prevalence of mast cells in OA synovial tissue is relatively high, reflects only partially synovitis and associates with structural damage in OA patients, suggesting a role of mast cells in this disease. Further research is warranted to unravel the underlying pathophysiological mechanisms in OA.
synovial expression of leptin and adiponectin in OA rabbits. However, high levels of serum leptin, probably due to systemic synthesis such as that derived from the liver were found in HFD fed animals. Taken together our data indicate that synovial adipocytes and dyslipemia could play pivotal roles in OA joint deterioration in patients with MetS, and support the concept that the link between obesity and OA transcends mechanical loading.
99 SYNOVIAL LIPODYSTROPHY INDUCED BY HYPERCHOLESTEROLEMIA AGGRAVATES SYNOVITIS IN AN EXPERIMENTAL MODEL OF OSTEOARTHRITIS IN RABBITS ~ aga-Vera, S. Perez-Baos, I. Prieto-Potin, F. Lopez-Oliva, L. Pen ~ a, A. Larran G. Herrero-Beaumont, R. Largo. IIS-Fundacion Jimenez Diaz UAM, Madrid, Spain
J.T. Sieker y, U.M. Ayturk y, B.L. Proffen y, A.M. Kiapour y, M. Weissenberger z, M.M. Murray y. y Boston Children's Hosp., Harvard Med. Sch., Boston, MA, USA; z Dr. Senckenberg Inst. of Pathology, Goethe Univ., Frankfurt, Germany
Purpose: Metabolic syndrome (MetS) is a well-recognized risk factor for osteoarthritis (OA) initiation and progression. While obesity contribution is well characterized, less known is the effect of each component of this syndrome on its own. Particularly interesting is the role of hypercholesterolemia, given the high incidence of cardiovascular events and atherosclerosis in OA patients. In fact, obesity is characterized by alterations in adipose tissue metabolism and the high production of inflammatory mediators derived from adipose tissue, named adipokines, that seem to be associated to OA progression. In this study, our aim was to study the effect of hypercholesterolemia without any other component of MetS in the synovial inflammation in knee OA. Methods: 16 four month-old male New Zeland rabbits (3 - 3.5 kg body weight) were fed with a high fat diet (HFD) containing 0.5% cholesterol and 4% peanut oil ad libitum. Six weeks later, bilateral osteoarthritis (OA) was induced by anterior cruciate ligament transection (ACLT) and partial medial meniscectomy, in 10 of these animals (OAþHFD group), and in 10 regular chow fed rabbits (OA group). Additionally, 10 healthy rabbits fed with regular chow were simultaneously followed (Control group). Twelve weeks after OA induction, animals were euthanized SM isolated for histological and molecular biology studies. Results: HFD, OA and OAþHFD animals gain significantly less weight than control rabbits at the end of the study. We did not find significant differences in Systolic blood pressure, serum glucose or oral glucose tolerance between the groups studied. Rabbits receiving the HFD showed higher total serum cholesterol, triglycerides and C-Reactive Protein levels than rabbits fed a normal diet. Synovitis score (Krenn scale) was increased in HFD and OA rabbits, and particularly in OAþHFD group, where the more severe score was observed. Synovitis in OAþHFD was characterized by a massive infiltration of foam RAM11þ cells, with higher presence of multinucleated foam cells and stromal fibrosis than in OA. We then characterized the changes in adipocyte size and the percentage of adipose tissue area (%ATA) in the SM of the rabbits. A huge decrease in the %ATA together with a marked decrease in the adipocyte size was observed in OAþHFD in comparison to OA and control animals. Furthermore, perilipin1a synthesis was significantly decreased in OAþHFD in comparison to OA and control rabbits. In comparison to controls, synovial leptin and adiponectin gene expressions were markedly decreased in HFD and OA rabbits, and especially in OAþHFD animals (p<0.01 vs OA), and positively correlated with %ATA in these animals. By contrast, serum leptin was increased in HFD and OAþHFD in comparison to control rabbits. We did not find significant differences in the SM gene expression of the proinflammatory cytokines IL-1b, COX-2 or MCP-1, between OA and OAþHFD animals. However, TNF SM expression was significantly increased in OAþHFD vs. OA, and significantly correlated with the presence of RAM11þ cells in the SM of these rabbits. Conclusions: Our data show that hypercholesterolemia is an aggravating factor for osteoarthritic synovitis, inducing an increased infiltration of macrophages and the disappearance of adipose tissue in the SM. Hypercholesterolemia induced a profound decrease in the adipocyte content in the OA SM, together with a remarkable increase in synovial TNF expression. Furthermore, HFD induced a decrease in the
100 SYNOVIAL MEMBRANE MACROPHAGE MARKER GENE EXPRESSION IS ASSOCIATED WITH MARKERS OF COLLAGEN DEGRADATION IN EARLY POST-TRAUMATIC OSTEOARTHRITIS
Purpose: To identify synovial membrane monocyte, macrophage and lymphocyte markers associated with the extent of intraarticular collagen degradation after anterior cruciate ligament (ACL) injury. Methods: Data were obtained from a preclinical efficacy trial of intraarticular corticosteroid administration to prevent ACL-injury induced synovitis and collagen degradation. Eighteen minipigs (mean weight of 33.4 kg) were allocated to untreated unilateral ACL transection (ACLT), unilateral ACLT and immediate injection of 20mg triamcinolone acetonide (STEROID), or no surgery (INTACT, n ¼ 6 each group). At 14 days, synovial membrane and fluid were obtained. Synovial fluid concentration of COL-2 3/4C short was quantified by ELISA (IBEX, 60-1002001). Based on automated measurements using hematoxylin-eosin stained sections and a custom ImageJ script, synovial lining and stromal areas that demonstrated the highest nuclei density were selected. Cell types were differentiated into mononuclear leukocytes (MNL), fibroblast-like synoviocytes (FLS) and polymorphonuclear leukocytes (PMNL) and counted by a blinded reader. RNA-sequencing was used to quantify transcriptome wide gene expression in synovial membranes. Reads uniquely aligned to exons were used for differential expression analysis using custom R scripts and edgeR. P-values were corrected using a Benjamini & Hochberg approach to adjust for the transcriptome-wide analysis. Differential expression was considered significant when the adjusted p remained <0.05 during a leave-one-out validation and transcript abundance was >5 reads per kilobase of exon model per million mapped reads (RPKM) in at least one of the compared groups. Transcript abundance and differential expression of 50 genes associated with monocytes, macrophages, lymphocytes and 19 genes associated with macrophage function were evaluated in this analysis. Linear regression analysis was used to test the association of differentially expressed marker genes with synovial fluid COL-2 3/4C short level and considered significant when p<0.05. Results: Synovial fluid concentration of COL-2 3/4C short doubled after ACL transection from 0.24mg/ml (95% confidence interval of 0.08e0.39) to 0.49mg/ml (0.39e0.59) in INTACT and ACLT, respectively (p ¼ 0.02). Compared to ACLT, COL-2 3/4C short levels were significantly reduced to 0.29mg/ml (0.23e0.35, p ¼ 0.04) in STEROID. The latter was considered similar to INTACT (p ¼ 0.78). MNL were more abundant than FLS, and the latter more abundant than PMNL. The number of MNL/hpf was non-significantly increased from 680 (439e920) in INTACT to 939 (560e1317) in ACLT lining and from 431 (203e660) to 697 (332e1062) in the stroma (p ¼ 0.41 and 0.37, respectively). Compared to ACLT, MNL/hpf decreased significantly to 379 (217e540, p ¼ 0.03) in STEROID lining and decreased non-significantly to 380 (224e537) in the stroma (p ¼ 0.25). No significant differences were observed in the number of FLS and PMNL in synoval lining or stroma. While 11 of 16 monocyte markers (69%) and 15 of 24 macrophage markers (63%) were expressed at a substantial level, defined by >5 RPKM in at least one group, only 1 of 10 lymphocyte markers (10%) was expressed above this threshold (CD4). Eight of the substantially expressed marker genes and 5 of 19 genes associated with macrophage function were significantly differentially expressed in at least one comparison of the three groups. Of the 13 differentially expressed genes, MARCO (R^2 ¼ 0.35), SOCS3 (0.34), CCR1 (0.25), IL4R (0.25) and MMP2 (0.24) were significantly associated with the synovial fluid COL-2
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
which allows determination of trabecular and cortical subchondral bone morphometric parameters as well as hyaline and calcified cartilage thickness This technic correlates very well with histological staining assessment (respectively R2 ¼ .94 and .82 for hyaline and calcified cartilage thickness). This technic also allows to assess a proteoglycan (PG) content estimation (inverse relationship between attenuation and PG content). The material level properties (indentation depth, Young’s modulus and working energy(area under the force displacement curve)) of the distal femur was evaluated using bioindentation with Bioindenter equipment (CSM Instrument, Switzerland). Indentation tests corresponding to around 75% of the whole cartilage thickness were performed with an accuracy lower than 9% for all investigated parameters. Results: Systemic IGF-I was decreased in LP group (18%, p<.001). As expected, in LP group, SB compartment was altered: trabecular SB mass was decreased (10%, p<.01) as well as SB cortical plate thickness of the medial condyle (12%, p<.05). Despite no morphologic changes of the cartilage of the medial condyle (thickness tended to decrease by 8% but not significantly), hyaline cartilage biomechanical properties (force, elastic modulus and working energy) were respectively decreased by 47, 58 and 41% (p<.01). We did not observed alterations of PG. Conclusions: We identified degradation of hyaline cartilage material level properties and a SB cortical thickening as well as trabecular SB deterioration with only mild alteration of hyaline cartilage in rats fed a low protein diet. Since these parameters have been documented to be altered in a similar way in early OA, we suggest that alteration of the somatotropic axis induced by protein malnutrition could predispose to OA. Deterioration of cartilage material level properties could be the result of cartilage collagen network disorganization as it has been already described in bone as well as alteration of pattern of joint loads distribution. The alteration of SB mass also reflects alteration of bone cellular activities which could also alter cartilage metabolism as already described in OA and SB interplay. Since protein malnutrition is frequent in elderly this mechanism could be relevant in human. 97 EFFECT OF THE OTAGO EXERCISES ON POSTURAL BALANCE AND FEAR OF FALLING AMONG OLDER FALLERS WITH KNEE OSTEOARTHRITIS S. Mat y, C.T. Ng z, M.P. Tan y. y Dept. of Med., Faculty of Med., Univ. of Malaya, Kuala Lumpur, Malaysia; z Dept. of Rheumatology and Immunology, Singapore, Singapore Purpose: This study evaluated the effectiveness of an individualized home-based exercise program to improve postural balance, falls risk and fear of falling in older fallers with knee osteoarthritis (OA). Methods: This was a substudy of the Malaysian Falls Assessment and Intervention Trial (MyFAIT), a randomized controlled study. A subanalysis was conducted for participants recruited to the study who had radiological OA, with evidence of gait and balance disorder determined by a timed-up and go score of over 13.5s. Participants randomized to the intervention arm received home-based, individualized Otago exercises, the control received conventional treatment. Posturagraphy test, Knee Injury and Osteoarthritis outcome score (KOOS) and short FES-I were completed at baseline and 6 months. Results: Results from 41 participants (exercise ¼ 17; control ¼ 24) were included. The exercise group had better significantly better directional control compared to control group (mean score: 67.26 vs 58.72, P ¼ 0.044) at 6 months. Within-group analysis indicated that postural control parameters results (mean change: rising index: 8.94; 95% confidence interval, 17.38e 0.49; P ¼ 0.039; reaction time : 0.15; 95% confidence interval, 0.29e 0.001; P ¼ 0.048, maximal excursion: 6.88; 95% confidence interval, 0.69e13.06; P ¼ 0.032, directional control: 10.13; 95% confidence interval, 2.55e17.70; P ¼ 0.012) and short FES-I score (mean change: short FES-I: 3.12 ; 95% confidence interval, 5.93e 0.30; P ¼ 0.032) improved significantly in intervention group. No significant difference in pre and post measurements were found among the control group. Conclusions: Home-based strengthening exercise benefited older fallers with OA and gait and balance disorders in improving their directional control. It also helped in reducing fear of falling and improving postural balance. Our study was not powered for falls outcomes. Future research should therefore consider evaluating targeted exercise interventions in fallers with OA and gait and balance disorders in a larger randomized-controlled study.
Angiogenesis & Synovial Tissue Biology 98 CHARACTERIZATION OF SYNOVIAL MAST CELLS IN KNEE OSTEOARTHRITIS: ASSOCIATION WITH SYNOVITIS AND CLINICAL PARAMETERS B. de Lange-Brokaar y, M. Kloppenburg y, S. Andersen y, A. Dorjee y, E. Yusuf y, L. Herb-van Toorn y, H. Kroon y, A.-M. Zuurmond z, V. Stojanovic-Susulic x, J. Bloem y, R. Nelissen y, R. Toes y, A. Ioan-Facsinay y. y Leiden Univ. Med. Ctr., Leiden, Netherlands; z TNO, Leiden, Netherlands; x Janssen Res. & Dev., Spring House, PA, USA Purpose: Mast cells have been previously suggested to be more abundant in OA synovium compared to RA synovium, in contrast to all other synovial inflammatory cells investigated. Moreover, mast cell degranulation products have been identified in synovial fluid of OA patients, indicating a possible activation of these cells in OA. In this study, we sought evidence for a possible role of these cells in OA by quantifying and characterizing mast cells in the osteoarthritic (OA) synovium using immunofluorescence and investigating their association with clinical parameters in comparison with rheumatoid arthritis (RA) samples. Methods: Patients with knee OA, fulfilling the ACR criteria, and RA patients, fulfilling the ACR criteria, were included. Synovial tissues of 56 symptomatic OA and 49 RA patients were obtained. Of these, 22 OA and 23 RA patients underwent arthroscopy and had no indication for joint replacement and are therefore referred to as “established” patients. The rest of the patients underwent arthroplasty and are referred to as “endstage” patients. Two to three paraffin slides were used to quantify inflammation using haematoxylin and eosin staining (synovitis score 0e9), and numbers of (degranulated) mast cells (per 10 high-power fields) using double immunofluorescence for CD117 and tryptase. Average scores per patient were used for analysis. Degranulated mast cells were defined as CD117þ or CD117-tryptaseþ cells for which at least 2 tryptase granules next to the cells were readily detectable. All other tryptaseþ cells were defined as non-degranulated mast cells. Knee radiographs of OA patients were scored according to the Kellgren and Lawrence (KL) system and pain was determined in OA patients at baseline by visual analogue scale (VAS). Results: Mast cells were readily detectable in both OA and RA synovial tissues and were present mostly in the sublining layer. Median (range) of mast cells was significantly higher in OA samples (45 (1e168)) compared to RA samples (4 (1e47)) (p-value < 0.001), while no differences were observed between established and end-stage OA, or established and end-stage RA samples. OA synovium contained more mast cells than RA synovium both in established and end-stage disease samples. All samples contained approximately 17% degranulated mast cells, irrespective of disease and disease stage. As expected, a lower median (range) synovitis score was present in OA (2.5 (0e6.0)) compared to RA (4.6 (0e8.0)) and this held true when established OA and RA, or end-stage OA and RA were compared. The synovitis score was significantly correlated with the number of mast cells (in OA Spearman’s rho (p-value) 0.3 (0.023) and RA 0.5 (p-value < 0.001)) in both diseases, although this association was less strong in OA. Remarkably,
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
S67
even in the absence of synovitis there was increased number of mast cells present in OA synovium compared to RA (fig. 1). Interestingly, we observed a trend towards an association between the number of mast cells and an increased KL-grade (p-value 0.05) in OA patients, independently of synovitis. No associations were found with self-reported pain. Conclusions: Prevalence of mast cells in OA synovial tissue is relatively high, reflects only partially synovitis and associates with structural damage in OA patients, suggesting a role of mast cells in this disease. Further research is warranted to unravel the underlying pathophysiological mechanisms in OA.
synovial expression of leptin and adiponectin in OA rabbits. However, high levels of serum leptin, probably due to systemic synthesis such as that derived from the liver were found in HFD fed animals. Taken together our data indicate that synovial adipocytes and dyslipemia could play pivotal roles in OA joint deterioration in patients with MetS, and support the concept that the link between obesity and OA transcends mechanical loading.
99 SYNOVIAL LIPODYSTROPHY INDUCED BY HYPERCHOLESTEROLEMIA AGGRAVATES SYNOVITIS IN AN EXPERIMENTAL MODEL OF OSTEOARTHRITIS IN RABBITS ~ aga-Vera, S. Perez-Baos, I. Prieto-Potin, F. Lopez-Oliva, L. Pen ~ a, A. Larran G. Herrero-Beaumont, R. Largo. IIS-Fundacion Jimenez Diaz UAM, Madrid, Spain
J.T. Sieker y, U.M. Ayturk y, B.L. Proffen y, A.M. Kiapour y, M. Weissenberger z, M.M. Murray y. y Boston Children's Hosp., Harvard Med. Sch., Boston, MA, USA; z Dr. Senckenberg Inst. of Pathology, Goethe Univ., Frankfurt, Germany
Purpose: Metabolic syndrome (MetS) is a well-recognized risk factor for osteoarthritis (OA) initiation and progression. While obesity contribution is well characterized, less known is the effect of each component of this syndrome on its own. Particularly interesting is the role of hypercholesterolemia, given the high incidence of cardiovascular events and atherosclerosis in OA patients. In fact, obesity is characterized by alterations in adipose tissue metabolism and the high production of inflammatory mediators derived from adipose tissue, named adipokines, that seem to be associated to OA progression. In this study, our aim was to study the effect of hypercholesterolemia without any other component of MetS in the synovial inflammation in knee OA. Methods: 16 four month-old male New Zeland rabbits (3 - 3.5 kg body weight) were fed with a high fat diet (HFD) containing 0.5% cholesterol and 4% peanut oil ad libitum. Six weeks later, bilateral osteoarthritis (OA) was induced by anterior cruciate ligament transection (ACLT) and partial medial meniscectomy, in 10 of these animals (OAþHFD group), and in 10 regular chow fed rabbits (OA group). Additionally, 10 healthy rabbits fed with regular chow were simultaneously followed (Control group). Twelve weeks after OA induction, animals were euthanized SM isolated for histological and molecular biology studies. Results: HFD, OA and OAþHFD animals gain significantly less weight than control rabbits at the end of the study. We did not find significant differences in Systolic blood pressure, serum glucose or oral glucose tolerance between the groups studied. Rabbits receiving the HFD showed higher total serum cholesterol, triglycerides and C-Reactive Protein levels than rabbits fed a normal diet. Synovitis score (Krenn scale) was increased in HFD and OA rabbits, and particularly in OAþHFD group, where the more severe score was observed. Synovitis in OAþHFD was characterized by a massive infiltration of foam RAM11þ cells, with higher presence of multinucleated foam cells and stromal fibrosis than in OA. We then characterized the changes in adipocyte size and the percentage of adipose tissue area (%ATA) in the SM of the rabbits. A huge decrease in the %ATA together with a marked decrease in the adipocyte size was observed in OAþHFD in comparison to OA and control animals. Furthermore, perilipin1a synthesis was significantly decreased in OAþHFD in comparison to OA and control rabbits. In comparison to controls, synovial leptin and adiponectin gene expressions were markedly decreased in HFD and OA rabbits, and especially in OAþHFD animals (p<0.01 vs OA), and positively correlated with %ATA in these animals. By contrast, serum leptin was increased in HFD and OAþHFD in comparison to control rabbits. We did not find significant differences in the SM gene expression of the proinflammatory cytokines IL-1b, COX-2 or MCP-1, between OA and OAþHFD animals. However, TNF SM expression was significantly increased in OAþHFD vs. OA, and significantly correlated with the presence of RAM11þ cells in the SM of these rabbits. Conclusions: Our data show that hypercholesterolemia is an aggravating factor for osteoarthritic synovitis, inducing an increased infiltration of macrophages and the disappearance of adipose tissue in the SM. Hypercholesterolemia induced a profound decrease in the adipocyte content in the OA SM, together with a remarkable increase in synovial TNF expression. Furthermore, HFD induced a decrease in the
100 SYNOVIAL MEMBRANE MACROPHAGE MARKER GENE EXPRESSION IS ASSOCIATED WITH MARKERS OF COLLAGEN DEGRADATION IN EARLY POST-TRAUMATIC OSTEOARTHRITIS
Purpose: To identify synovial membrane monocyte, macrophage and lymphocyte markers associated with the extent of intraarticular collagen degradation after anterior cruciate ligament (ACL) injury. Methods: Data were obtained from a preclinical efficacy trial of intraarticular corticosteroid administration to prevent ACL-injury induced synovitis and collagen degradation. Eighteen minipigs (mean weight of 33.4 kg) were allocated to untreated unilateral ACL transection (ACLT), unilateral ACLT and immediate injection of 20mg triamcinolone acetonide (STEROID), or no surgery (INTACT, n ¼ 6 each group). At 14 days, synovial membrane and fluid were obtained. Synovial fluid concentration of COL-2 3/4C short was quantified by ELISA (IBEX, 60-1002001). Based on automated measurements using hematoxylin-eosin stained sections and a custom ImageJ script, synovial lining and stromal areas that demonstrated the highest nuclei density were selected. Cell types were differentiated into mononuclear leukocytes (MNL), fibroblast-like synoviocytes (FLS) and polymorphonuclear leukocytes (PMNL) and counted by a blinded reader. RNA-sequencing was used to quantify transcriptome wide gene expression in synovial membranes. Reads uniquely aligned to exons were used for differential expression analysis using custom R scripts and edgeR. P-values were corrected using a Benjamini & Hochberg approach to adjust for the transcriptome-wide analysis. Differential expression was considered significant when the adjusted p remained <0.05 during a leave-one-out validation and transcript abundance was >5 reads per kilobase of exon model per million mapped reads (RPKM) in at least one of the compared groups. Transcript abundance and differential expression of 50 genes associated with monocytes, macrophages, lymphocytes and 19 genes associated with macrophage function were evaluated in this analysis. Linear regression analysis was used to test the association of differentially expressed marker genes with synovial fluid COL-2 3/4C short level and considered significant when p<0.05. Results: Synovial fluid concentration of COL-2 3/4C short doubled after ACL transection from 0.24mg/ml (95% confidence interval of 0.08e0.39) to 0.49mg/ml (0.39e0.59) in INTACT and ACLT, respectively (p ¼ 0.02). Compared to ACLT, COL-2 3/4C short levels were significantly reduced to 0.29mg/ml (0.23e0.35, p ¼ 0.04) in STEROID. The latter was considered similar to INTACT (p ¼ 0.78). MNL were more abundant than FLS, and the latter more abundant than PMNL. The number of MNL/hpf was non-significantly increased from 680 (439e920) in INTACT to 939 (560e1317) in ACLT lining and from 431 (203e660) to 697 (332e1062) in the stroma (p ¼ 0.41 and 0.37, respectively). Compared to ACLT, MNL/hpf decreased significantly to 379 (217e540, p ¼ 0.03) in STEROID lining and decreased non-significantly to 380 (224e537) in the stroma (p ¼ 0.25). No significant differences were observed in the number of FLS and PMNL in synoval lining or stroma. While 11 of 16 monocyte markers (69%) and 15 of 24 macrophage markers (63%) were expressed at a substantial level, defined by >5 RPKM in at least one group, only 1 of 10 lymphocyte markers (10%) was expressed above this threshold (CD4). Eight of the substantially expressed marker genes and 5 of 19 genes associated with macrophage function were significantly differentially expressed in at least one comparison of the three groups. Of the 13 differentially expressed genes, MARCO (R^2 ¼ 0.35), SOCS3 (0.34), CCR1 (0.25), IL4R (0.25) and MMP2 (0.24) were significantly associated with the synovial fluid COL-2