- Email: [email protected]
A novel chondroprotective property of TSG-6 has therapeutic potential for OA
Abstracts / Osteoarthritis and Cartilage 24 (2016) S8eS62
S19
molecules by a cell-based high-throughput screening (HTS) in human chondrocytes. Methods: To induce cellular senescence, immortalized human chondrocytes (TC28a2) were seeded (5000 cells/well) in 384 well plates, and treated with IL-6 (10 ng/ml) for 24 hours to induce cellular senescence or defective autophagy. Then, chondrocytes were incubated with Prestwick Chemical Library (1280 approved drugs with chemical and pharmacological diversity, as well as bioavailability and safety in humans) at 10 mM for 48 hours. Chondrocytes were incubated for 20 min with 4% formaldehyde and washed 3 times with PBS. Then, nuclei was stained with Hoechst 33342 (10 mM), while b-galactosidase subcellular structures and autophagy vacuoles were stained by using Imagene Green C12FDG substrate (10 mM) and Cyto-ID® (1 mM), respectively. Plates were imaged by using Operetta® High Content Screening (HCS) system in non-confocal mode using the 20x WD objective. For each well, 4 fields and 4 planes of bright field, Hoechst and fluorescein channels were obtained. Image analysis was performed by Harmony software. Relative intensity of C12FDG in cytoplasm and number of autophagosomes per area of cytoplasm were determined to quantitate b-galactosidase activity and autophagy flux respectively. Low signal of senescence was found for chloroquine (30 mM) and used as reference for anti-senescence activity. High signal of autophagy flux was found for Rapamycin (5 mM) and used as reference for proautophagy activity. Results: A primary screening was performed to identify anti-senescence compounds by measurement of senescence-associated bgalactosidase activity. From the total number of 1280 small molecules, 299 compounds with anti-senescence effects were identified by HTS. A secondary screening with the 299 compounds was performed for “cherry picking”. Finally, 216 compounds were confirmed to have antisenescence activity. The anti-senescence compounds were analyzed by monitoring autophagy flux. 38 compounds with both anti-senescence and pro-autophagy effects were selected. For compound validation, a reporter cell line was generated by lentiviral transfection of pBABE-mCherry-EGFP-LC3 plasmid in TC28a2 chondrocytes. LC3 reporter chondrocytes can be used to determine autophagy activation by color changes upon stimulation. The preliminary results indicate a subset of compounds selectively induced LC3-associated autophagy activation. Conclusions: These observations provide a unique opportunity to study cartilage aging with the objective to explore the therapeutic potential of pharmacological prevention of chondrocyte senescence and autophagy as a strategy to slow or reverse aging-associated changes, prevent the onset of OA and provide benefits for its clinical management.
surgery. The knee joints were harvested and processed for histopathological analysis. The healing tissue with a small amount of surrounding host tissue was harvested form the articular cartilage defect sites for RNA isolation and gene expression analysis by quantitative RT-PCR (qPCR). At least 10 mice per strain group were examined at each time point. Statistical analyses were performed with Student's t-test and ANOVA. Results: At 2e6 weeks after surgery, the migration of bone marrow stem cells and chondrocyte differentiation were more abundant in the osteochondral defects created in Nfat1/ mice than in WT mice. qPCR analyses showed up-regulated expression levels of the mRNAs for aggrecan, type-9 collagen, type-10 collagen (a marker of hypertrophic chondrocytes), and specific proinflammatory cytokines and matrix metalloproteinases (Mmps) in Nfat1/ healing tissue. At 12 weeks after surgery, oteochondral defects were filled with repair cartilage tissue in both WT and Nfat1/ mice; however, more hypertrophic chondrocytes and overgrowth of repair tissues were observed in Nfat1/ defects than in WT defects. The expression of mRNA for type10 collagen and specific proinflammatory cytokines and Mmps was continuously up-regulated in Nfat1/ defects. At 26 weeks after surgery, repaired subchondral bone and cartilage tissue was remodeled to partially fit the articular surface in WT mice, with mild to moderate degenerative changes. In contrast, more abundant endochondral ossification and segmentation of repair tissue were observed in the defect sites of Nfat1/ mice, leading to severe incongruity and destruction of the articular surface. No significant sex differences in severity of OA were detected at any time-points. Conclusions: NFAT1 deficiency provokes hypertrophic repair, instead of lack of healing tissue, during the repair of articular cartilage defects created in the trochlear grove of the lower femur. Although OA develops in the patellofemoral joints of both WT and Nfat1/ mice at 26 weeks after the surgery, incongruity and destruction of the articular surface is more severe in Nfat1/ patellofemoral joints, indicating that Nfat1 deficiency promotes the progression of posttraumatic OA in mice. These novel findings may provide new insights into the strategies for prevention and treatment of posttraumatic OA.
20 NFAT1 DEFICIENCY PROVOKES HYPERTROPHIC REPAIR ARTICULAR CARTILAGE DEFECTS AND PROGRESSION POSTTRAUMATIC OSTEOARTHRITIS
Purpose: TSG-6 is a protein that is not constitutively expressed in most adult tissues, but is secreted (by stromal and immune cells) in response to inflammatory mediators and is believed to play a role in protecting tissues from the damaging effects of inflammation. For example, many of the anti-inflammatory and tissue reparative properties of mesenchymal stromal cells (MSCs) have recently been attributed to TSG-6 production. Several mechanisms for this have been proposed/identified, including TSG-6's suppression of neutrophil migration; in this context the isolated recombinant Link module of human TSG-6 (Link_TSG6) is as potent as the full-length recombinant protein (rhTSG-6). Previously, TSG-6 treatment/overexpression has been found to be chondroprotective, reducing cartilage breakdown in mouse models of inflammatory arthritis, where this has been attributed, in large part, to its inhibitory effect on neutrophils. However, the effects of TSG-6 on chondrocyte function have not been studied. The aim here was to investigate whether TSG-6 can suppress the effects of pro-inflammatory cytokines on chondrocytes and test the activities of TSG-6 in models of osteoarthritis (OA). Methods: Chondrocytes in 3D pellets were treated with IL-1 or TNF with/without recombinant TSG-6 proteins (Link_TSG6 or rhTSG-6); expression of ADAMTS4, ADAMTS5 and MMP13 were quantified by qPCR. Human OA cartilage explants were cultured with IL-1 and oncostatin M with/without TSG-6 for 7 or 14 days and proteoglycan release measured using the dimethylene blue assay. A rat surgicallyinduced model of OA (ACLTpMx) was treated with intra-articular injections of Link_TSG6 protein at 7-, 14- and 21-days after surgery; joint damage was evaluated at the 4-week endpoint by macroscopy and histology, and pain was assessed by tactile allodynia with von Frey hairs. TSG-6 protein was immunolocalised in cartilage obtained from OA patients.
OF OF
J. Wang, K.L. Caldwell, Q. Lu, Y. Feng, N.C. Barnthouse, A.H. Miller. Univ. of Kansas Med. Ctr., Kansas City, KS, USA Purpose: We currently face major obstacles in our efforts to repair damaged articular cartilage due to limited capacity of the tissue to regenerate an appropriate articular surface. Human and animal studies have demonstrated that an osteochondral defect caused by joint trauma or by microfracture surgery can be repaired morphologically through the proliferation and differentiation of bone marrow mesenchymal stem cells into cartilage cells which synthesize a cartilage matrix. However, the repaired articular cartilage tissue degenerates during the late-stage of repair, and the joints with articular cartilage lesions eventually develop osteoarthritis (OA). The purpose of this study was to investigate whether deficiency of the NFAT1 transcription factor affects the repair of osteochondral defects and progression of posttraumatic OA in mice. Methods: Nfat1 knockout (Nfat1/) and wild-type (WT) mice with BALB/c background, both sexes, were used for creation of osteochondral defects and various tissue analyses. All animal procedures were approved by the Institutional Animal Care and Use Committee. The osteochondral defect procedure was performed on mice at the age of 2 to 3 months under general anesthesia and sterile conditions. Under a surgical microscope, a 0.3-mm wide x 1.5-mm long osteochondral defect was created in the center of the trochlear grove of the lower femur penetrating into the subchondral bone marrow cavities. Operated mice were euthanized at 2, 6, 12, and 26 weeks after
21 A NOVEL CHONDROPROTECTIVE THERAPEUTIC POTENTIAL FOR OA
PROPERTY
OF
TSG-6
HAS
A.J. Day y, S.P. Drummond y, S. Anand z, E. Bartnik x, C.M. Milner y. y Univ. of Manchester, Manchester, United Kingdom; z Stepping Hill Hosp., Stockport, United Kingdom; x Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany
S20
Abstracts / Osteoarthritis and Cartilage 24 (2016) S8eS62
Results: Treatment with Link_TSG6 and rhTSG-6 proteins suppressed the response of MSC-derived chondrocytes (in 3D pellet cultures) to inflammatory cytokines (IL-1 and TNF), inhibiting their expression of ADAMTS4, ADAMTS5 and MMP13; i.e. the aggrecanse and collagenase enzymes responsible for cartilage degradation in OA. Link_TSG6 was generally more potent than rhTSG-6 and, for example, was able to completely abolish the expression of MMP13 in response to IL-1b. Link_TSG6 significantly reduced proteoglycan loss from cytokinestimulated human cartilage explants obtained from OA patients (e.g. a reduction of 31.0 ± 6.3% over 7 days with a 1 mM dose when data are pooled for 7 donors); rhTSG-6 had a small (but not significant) inhibitory effect. Intra-articular administration of Link_TSG6 in a rat model of OA resulted in a dose-dependent (and significant) reduction in cartilage damage (e.g. fibrillation and ulceration). For example, at the highest dose tested only 1 animal (out of 18) had exposed bone and 5 had intact cartilage as compared to 5 and 1 animals, respectively, in the vehicle control group of 20 rats. Link_TSG6 treatment also significantly reduced pain. High levels of TSG-6 staining in OA cartilage were associated with the cells and matrix in the most damaged margins of the tissue, but staining was also detectable in a low percentage of cells within the deep and mid zones of macroscopically intact regions; this localisation pattern is consistent with TSG-6 expression being a response to tissue disruption/disease, which might serve to limit excessive matrix turnover. Conclusions: These results indicate that Link_TSG6 has the potential to be developed as a biological therapy for osteoarthritis. The chondroprotective activity of TSG-6 combined with its anti-inflammatory and anti-bone-resorptive properties make it a unique therapeutic target.
datasets. Based on the training data, two logistic regressions were fit: (i) using the clinical covariates only; (ii) using the clinical covariates plus the selected MRM and/or ELISA markers. The fitted scores were used to compute the area under the curve (AUC) of Receiver Operator Characteristic curves. Results: The sample was 82% female, mean age 64 (SD 10) years, mean BMI 28 (SD 6) kg/m2. The proportion of the sample meeting each definition of knee OA progression was 75% ANY, 52% JSN, 66% osteophyte and 29% KL progression. The number of individuals with complete observations that were analyzed in each group is provided in Table 1. Clinical parameters alone (age, gender and BMI) yielded areas under the curve (AUCs) of 0.68e0.73 (Table 1). These clinical parameters were only significant for predicting osteophyte progression. MRM or ELISAbased biomarkers in combination with clinical parameters significantly improved the prediction of OA progression (all definitions) over clinical variables alone achieving AUCs ranging from 0.84e0.97 (Table 1). Any progression was predicted by 10 MRM markers; osteophyte progression by 7 MRM markers; JSN progression by 6 ELISA markers; and KL progression by 1 ELISA marker. Conclusions: We developed a method of predicting OA progression based on a panel of mass spectrometry and ELISA based biomarkers. Markers predicting osteophyte progression were distinctly different from those predicting JSN progression. These results suggest that a select set of biomarkers identified from targeted and non-targeted analyses could significantly improve prediction of knee OA progression and could be useful for enriching knee OA clinical trials for progressors.
22 DEVELOPMENT OF A SERUM BIOMARKER PANEL PREDICTIVE OF KNEE OSTEOARTHRITIS PROGRESSION
Table 1 Analysis of clinical covariates and biomarkers for predicting knee OA progression.
HIGHLY
V.B. Kraus y, J. Catterall y, E. Soderblom z, M.A. Moseley z, S. Suchindran z. y Duke Univ. Sch. of Med., Durham, NC, USA; z Duke Univ., Durham, NC, USA Purpose: Osteoarthritis (OA) is acknowledged to be a phasic disease, with periods of quiescence and no more than 30% of individuals with knee OA progressing at any one time. The majority of OA clinical trials suffer from low power due to inefficient means of identifying progressors. Baseline age, sex, body mass index, knee pain, general bone mineral content, and joint space width are poor predictors of knee OA progression. The goal of this study was to discover serum biomarkers with high predictive capability for knee OA progression. Methods: Using a systematic, unbiased and iterative mass spectrometry approach, we created a Multiple Reaction Monitoring (MRM) panel for 146 peptides (99 proteins) in serum. The selection of proteins was based on results of discovery proteomic studies in synovial fluid (n ¼ 23), urine (n ¼ 45) and serum (n ¼ 40) from knee OA radiographic progressors and non-progressors. To identify the peptides most predictive of knee OA progression, we tested the MRM panel in the serum of additional subjects with symptomatic knee OA, with and without progression, from the Prediction of Osteoarthritis Progression (POP) cohort and the Genetics of Generalized OA (GOGO) study. Knee OA progression was defined as any knee OA progression, or on the basis of osteophyte, joint space narrowing (JSN) or Kellgren Lawrence (KL) grade progression over the mean 3e4 year follow-up. Quantitative LC/MS/MS was performed on 1 mg of protein digest per sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Synapt G2 HDMS high resolution accurate mass tandem mass spectrometer (Waters Corp) via a nanoelectrospray ionization source. Samples were assayed in singlicate along with a representative “QC Pool” sample created from equal portions of all individual samples which was run periodically throughout the total acquisition window. Samples were also spiked with a constant amount of yeast alcohol dehydrogenase (ADH). The mean and median technical variability of a measured protein intensity across eight QC Pool injections were 16.3% and 12.4%, respectively, with variations in measured ADH intensities of 10.4%. Samples were also used to quantify 19 candidate biomarkers for which ELISA kits were available. The optimal multimarker predictors were identified by LASSO regression using the R package “glmnet”. To evaluate the value of added MRM and/or ELISA markers for predicting OA progressor versus non-progressor status, the data were randomly split 50 times into half training and half testing
Clinical Outcome (number and type of marker used for prediction)
Complete Observations
Clinical Parameters (age, gender, BMI) (AUC ± SD)
Clinical Parameters plus Biomarkers (AUC ± SD)
Any Progression (10 MRM) Osteophyte Progression (7 MRM) JSN Progression (6 ELISA) KL Progression (1 ELISA)
83
0.725 ± 0.127
0.902 ± 0.078
76
0.720 ± 0.057
0.900 ± 0.052
52
0.681 ± 0.063
0.971 ± 0.040
63
0.700 ± 0.053
0.836 ± 0.076
23 PREDICTIVE VALUES OF SIGNS AND SYMPTOMS FOR ONSET OF STRUCTURAL KNEE OA WITHIN 5 YEARS. D. Schiphof, J.H. Waarsing, E.H. Oei, S.M. Bierma-Zeinstra. Erasmus MC, Univ. Med. Ctr., Rotterdam, Netherlands Purpose: To investigate novel preventive or early management strategies for OA, identification of populations at high risk and at earlier (pre-clinical) stages is essential. Symptoms that indicate early knee OA were found in a previous cross-sectional study. In the present study we estimated the predictive value of signs and symptoms (S&S) of history taking and physical examination for structural knee OA at 5 years follow-up. Methods: In a subset of 891 females (aged 45e60 years) of the Rotterdam Study, a population based study in the Netherlands, MRIs, radiographs, an physical examination and knee specific questionnaires were collected. After five years follow-up the same measurements were performed. Of 891 women who participated at baseline, 700 women participated at follow-up. Of these 700 women, 592 women did not have OA at baseline (defined at MRI), which were the women we used for this study. Baseline and follow-up MRIs were scored together with the semi-quantitative MRI OA knee score (MOAKS). Based on the MRI scores we defined TFOA and PFOA per knee, using a previously published MRI definition for OA. Radiographs were scored with Kellgren and Lawrence criteria (K&L). Radiographic OA was defined as K&L> ¼ 2.
S19
molecules by a cell-based high-throughput screening (HTS) in human chondrocytes. Methods: To induce cellular senescence, immortalized human chondrocytes (TC28a2) were seeded (5000 cells/well) in 384 well plates, and treated with IL-6 (10 ng/ml) for 24 hours to induce cellular senescence or defective autophagy. Then, chondrocytes were incubated with Prestwick Chemical Library (1280 approved drugs with chemical and pharmacological diversity, as well as bioavailability and safety in humans) at 10 mM for 48 hours. Chondrocytes were incubated for 20 min with 4% formaldehyde and washed 3 times with PBS. Then, nuclei was stained with Hoechst 33342 (10 mM), while b-galactosidase subcellular structures and autophagy vacuoles were stained by using Imagene Green C12FDG substrate (10 mM) and Cyto-ID® (1 mM), respectively. Plates were imaged by using Operetta® High Content Screening (HCS) system in non-confocal mode using the 20x WD objective. For each well, 4 fields and 4 planes of bright field, Hoechst and fluorescein channels were obtained. Image analysis was performed by Harmony software. Relative intensity of C12FDG in cytoplasm and number of autophagosomes per area of cytoplasm were determined to quantitate b-galactosidase activity and autophagy flux respectively. Low signal of senescence was found for chloroquine (30 mM) and used as reference for anti-senescence activity. High signal of autophagy flux was found for Rapamycin (5 mM) and used as reference for proautophagy activity. Results: A primary screening was performed to identify anti-senescence compounds by measurement of senescence-associated bgalactosidase activity. From the total number of 1280 small molecules, 299 compounds with anti-senescence effects were identified by HTS. A secondary screening with the 299 compounds was performed for “cherry picking”. Finally, 216 compounds were confirmed to have antisenescence activity. The anti-senescence compounds were analyzed by monitoring autophagy flux. 38 compounds with both anti-senescence and pro-autophagy effects were selected. For compound validation, a reporter cell line was generated by lentiviral transfection of pBABE-mCherry-EGFP-LC3 plasmid in TC28a2 chondrocytes. LC3 reporter chondrocytes can be used to determine autophagy activation by color changes upon stimulation. The preliminary results indicate a subset of compounds selectively induced LC3-associated autophagy activation. Conclusions: These observations provide a unique opportunity to study cartilage aging with the objective to explore the therapeutic potential of pharmacological prevention of chondrocyte senescence and autophagy as a strategy to slow or reverse aging-associated changes, prevent the onset of OA and provide benefits for its clinical management.
surgery. The knee joints were harvested and processed for histopathological analysis. The healing tissue with a small amount of surrounding host tissue was harvested form the articular cartilage defect sites for RNA isolation and gene expression analysis by quantitative RT-PCR (qPCR). At least 10 mice per strain group were examined at each time point. Statistical analyses were performed with Student's t-test and ANOVA. Results: At 2e6 weeks after surgery, the migration of bone marrow stem cells and chondrocyte differentiation were more abundant in the osteochondral defects created in Nfat1/ mice than in WT mice. qPCR analyses showed up-regulated expression levels of the mRNAs for aggrecan, type-9 collagen, type-10 collagen (a marker of hypertrophic chondrocytes), and specific proinflammatory cytokines and matrix metalloproteinases (Mmps) in Nfat1/ healing tissue. At 12 weeks after surgery, oteochondral defects were filled with repair cartilage tissue in both WT and Nfat1/ mice; however, more hypertrophic chondrocytes and overgrowth of repair tissues were observed in Nfat1/ defects than in WT defects. The expression of mRNA for type10 collagen and specific proinflammatory cytokines and Mmps was continuously up-regulated in Nfat1/ defects. At 26 weeks after surgery, repaired subchondral bone and cartilage tissue was remodeled to partially fit the articular surface in WT mice, with mild to moderate degenerative changes. In contrast, more abundant endochondral ossification and segmentation of repair tissue were observed in the defect sites of Nfat1/ mice, leading to severe incongruity and destruction of the articular surface. No significant sex differences in severity of OA were detected at any time-points. Conclusions: NFAT1 deficiency provokes hypertrophic repair, instead of lack of healing tissue, during the repair of articular cartilage defects created in the trochlear grove of the lower femur. Although OA develops in the patellofemoral joints of both WT and Nfat1/ mice at 26 weeks after the surgery, incongruity and destruction of the articular surface is more severe in Nfat1/ patellofemoral joints, indicating that Nfat1 deficiency promotes the progression of posttraumatic OA in mice. These novel findings may provide new insights into the strategies for prevention and treatment of posttraumatic OA.
20 NFAT1 DEFICIENCY PROVOKES HYPERTROPHIC REPAIR ARTICULAR CARTILAGE DEFECTS AND PROGRESSION POSTTRAUMATIC OSTEOARTHRITIS
Purpose: TSG-6 is a protein that is not constitutively expressed in most adult tissues, but is secreted (by stromal and immune cells) in response to inflammatory mediators and is believed to play a role in protecting tissues from the damaging effects of inflammation. For example, many of the anti-inflammatory and tissue reparative properties of mesenchymal stromal cells (MSCs) have recently been attributed to TSG-6 production. Several mechanisms for this have been proposed/identified, including TSG-6's suppression of neutrophil migration; in this context the isolated recombinant Link module of human TSG-6 (Link_TSG6) is as potent as the full-length recombinant protein (rhTSG-6). Previously, TSG-6 treatment/overexpression has been found to be chondroprotective, reducing cartilage breakdown in mouse models of inflammatory arthritis, where this has been attributed, in large part, to its inhibitory effect on neutrophils. However, the effects of TSG-6 on chondrocyte function have not been studied. The aim here was to investigate whether TSG-6 can suppress the effects of pro-inflammatory cytokines on chondrocytes and test the activities of TSG-6 in models of osteoarthritis (OA). Methods: Chondrocytes in 3D pellets were treated with IL-1 or TNF with/without recombinant TSG-6 proteins (Link_TSG6 or rhTSG-6); expression of ADAMTS4, ADAMTS5 and MMP13 were quantified by qPCR. Human OA cartilage explants were cultured with IL-1 and oncostatin M with/without TSG-6 for 7 or 14 days and proteoglycan release measured using the dimethylene blue assay. A rat surgicallyinduced model of OA (ACLTpMx) was treated with intra-articular injections of Link_TSG6 protein at 7-, 14- and 21-days after surgery; joint damage was evaluated at the 4-week endpoint by macroscopy and histology, and pain was assessed by tactile allodynia with von Frey hairs. TSG-6 protein was immunolocalised in cartilage obtained from OA patients.
OF OF
J. Wang, K.L. Caldwell, Q. Lu, Y. Feng, N.C. Barnthouse, A.H. Miller. Univ. of Kansas Med. Ctr., Kansas City, KS, USA Purpose: We currently face major obstacles in our efforts to repair damaged articular cartilage due to limited capacity of the tissue to regenerate an appropriate articular surface. Human and animal studies have demonstrated that an osteochondral defect caused by joint trauma or by microfracture surgery can be repaired morphologically through the proliferation and differentiation of bone marrow mesenchymal stem cells into cartilage cells which synthesize a cartilage matrix. However, the repaired articular cartilage tissue degenerates during the late-stage of repair, and the joints with articular cartilage lesions eventually develop osteoarthritis (OA). The purpose of this study was to investigate whether deficiency of the NFAT1 transcription factor affects the repair of osteochondral defects and progression of posttraumatic OA in mice. Methods: Nfat1 knockout (Nfat1/) and wild-type (WT) mice with BALB/c background, both sexes, were used for creation of osteochondral defects and various tissue analyses. All animal procedures were approved by the Institutional Animal Care and Use Committee. The osteochondral defect procedure was performed on mice at the age of 2 to 3 months under general anesthesia and sterile conditions. Under a surgical microscope, a 0.3-mm wide x 1.5-mm long osteochondral defect was created in the center of the trochlear grove of the lower femur penetrating into the subchondral bone marrow cavities. Operated mice were euthanized at 2, 6, 12, and 26 weeks after
21 A NOVEL CHONDROPROTECTIVE THERAPEUTIC POTENTIAL FOR OA
PROPERTY
OF
TSG-6
HAS
A.J. Day y, S.P. Drummond y, S. Anand z, E. Bartnik x, C.M. Milner y. y Univ. of Manchester, Manchester, United Kingdom; z Stepping Hill Hosp., Stockport, United Kingdom; x Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany
S20
Abstracts / Osteoarthritis and Cartilage 24 (2016) S8eS62
Results: Treatment with Link_TSG6 and rhTSG-6 proteins suppressed the response of MSC-derived chondrocytes (in 3D pellet cultures) to inflammatory cytokines (IL-1 and TNF), inhibiting their expression of ADAMTS4, ADAMTS5 and MMP13; i.e. the aggrecanse and collagenase enzymes responsible for cartilage degradation in OA. Link_TSG6 was generally more potent than rhTSG-6 and, for example, was able to completely abolish the expression of MMP13 in response to IL-1b. Link_TSG6 significantly reduced proteoglycan loss from cytokinestimulated human cartilage explants obtained from OA patients (e.g. a reduction of 31.0 ± 6.3% over 7 days with a 1 mM dose when data are pooled for 7 donors); rhTSG-6 had a small (but not significant) inhibitory effect. Intra-articular administration of Link_TSG6 in a rat model of OA resulted in a dose-dependent (and significant) reduction in cartilage damage (e.g. fibrillation and ulceration). For example, at the highest dose tested only 1 animal (out of 18) had exposed bone and 5 had intact cartilage as compared to 5 and 1 animals, respectively, in the vehicle control group of 20 rats. Link_TSG6 treatment also significantly reduced pain. High levels of TSG-6 staining in OA cartilage were associated with the cells and matrix in the most damaged margins of the tissue, but staining was also detectable in a low percentage of cells within the deep and mid zones of macroscopically intact regions; this localisation pattern is consistent with TSG-6 expression being a response to tissue disruption/disease, which might serve to limit excessive matrix turnover. Conclusions: These results indicate that Link_TSG6 has the potential to be developed as a biological therapy for osteoarthritis. The chondroprotective activity of TSG-6 combined with its anti-inflammatory and anti-bone-resorptive properties make it a unique therapeutic target.
datasets. Based on the training data, two logistic regressions were fit: (i) using the clinical covariates only; (ii) using the clinical covariates plus the selected MRM and/or ELISA markers. The fitted scores were used to compute the area under the curve (AUC) of Receiver Operator Characteristic curves. Results: The sample was 82% female, mean age 64 (SD 10) years, mean BMI 28 (SD 6) kg/m2. The proportion of the sample meeting each definition of knee OA progression was 75% ANY, 52% JSN, 66% osteophyte and 29% KL progression. The number of individuals with complete observations that were analyzed in each group is provided in Table 1. Clinical parameters alone (age, gender and BMI) yielded areas under the curve (AUCs) of 0.68e0.73 (Table 1). These clinical parameters were only significant for predicting osteophyte progression. MRM or ELISAbased biomarkers in combination with clinical parameters significantly improved the prediction of OA progression (all definitions) over clinical variables alone achieving AUCs ranging from 0.84e0.97 (Table 1). Any progression was predicted by 10 MRM markers; osteophyte progression by 7 MRM markers; JSN progression by 6 ELISA markers; and KL progression by 1 ELISA marker. Conclusions: We developed a method of predicting OA progression based on a panel of mass spectrometry and ELISA based biomarkers. Markers predicting osteophyte progression were distinctly different from those predicting JSN progression. These results suggest that a select set of biomarkers identified from targeted and non-targeted analyses could significantly improve prediction of knee OA progression and could be useful for enriching knee OA clinical trials for progressors.
22 DEVELOPMENT OF A SERUM BIOMARKER PANEL PREDICTIVE OF KNEE OSTEOARTHRITIS PROGRESSION
Table 1 Analysis of clinical covariates and biomarkers for predicting knee OA progression.
HIGHLY
V.B. Kraus y, J. Catterall y, E. Soderblom z, M.A. Moseley z, S. Suchindran z. y Duke Univ. Sch. of Med., Durham, NC, USA; z Duke Univ., Durham, NC, USA Purpose: Osteoarthritis (OA) is acknowledged to be a phasic disease, with periods of quiescence and no more than 30% of individuals with knee OA progressing at any one time. The majority of OA clinical trials suffer from low power due to inefficient means of identifying progressors. Baseline age, sex, body mass index, knee pain, general bone mineral content, and joint space width are poor predictors of knee OA progression. The goal of this study was to discover serum biomarkers with high predictive capability for knee OA progression. Methods: Using a systematic, unbiased and iterative mass spectrometry approach, we created a Multiple Reaction Monitoring (MRM) panel for 146 peptides (99 proteins) in serum. The selection of proteins was based on results of discovery proteomic studies in synovial fluid (n ¼ 23), urine (n ¼ 45) and serum (n ¼ 40) from knee OA radiographic progressors and non-progressors. To identify the peptides most predictive of knee OA progression, we tested the MRM panel in the serum of additional subjects with symptomatic knee OA, with and without progression, from the Prediction of Osteoarthritis Progression (POP) cohort and the Genetics of Generalized OA (GOGO) study. Knee OA progression was defined as any knee OA progression, or on the basis of osteophyte, joint space narrowing (JSN) or Kellgren Lawrence (KL) grade progression over the mean 3e4 year follow-up. Quantitative LC/MS/MS was performed on 1 mg of protein digest per sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Synapt G2 HDMS high resolution accurate mass tandem mass spectrometer (Waters Corp) via a nanoelectrospray ionization source. Samples were assayed in singlicate along with a representative “QC Pool” sample created from equal portions of all individual samples which was run periodically throughout the total acquisition window. Samples were also spiked with a constant amount of yeast alcohol dehydrogenase (ADH). The mean and median technical variability of a measured protein intensity across eight QC Pool injections were 16.3% and 12.4%, respectively, with variations in measured ADH intensities of 10.4%. Samples were also used to quantify 19 candidate biomarkers for which ELISA kits were available. The optimal multimarker predictors were identified by LASSO regression using the R package “glmnet”. To evaluate the value of added MRM and/or ELISA markers for predicting OA progressor versus non-progressor status, the data were randomly split 50 times into half training and half testing
Clinical Outcome (number and type of marker used for prediction)
Complete Observations
Clinical Parameters (age, gender, BMI) (AUC ± SD)
Clinical Parameters plus Biomarkers (AUC ± SD)
Any Progression (10 MRM) Osteophyte Progression (7 MRM) JSN Progression (6 ELISA) KL Progression (1 ELISA)
83
0.725 ± 0.127
0.902 ± 0.078
76
0.720 ± 0.057
0.900 ± 0.052
52
0.681 ± 0.063
0.971 ± 0.040
63
0.700 ± 0.053
0.836 ± 0.076
23 PREDICTIVE VALUES OF SIGNS AND SYMPTOMS FOR ONSET OF STRUCTURAL KNEE OA WITHIN 5 YEARS. D. Schiphof, J.H. Waarsing, E.H. Oei, S.M. Bierma-Zeinstra. Erasmus MC, Univ. Med. Ctr., Rotterdam, Netherlands Purpose: To investigate novel preventive or early management strategies for OA, identification of populations at high risk and at earlier (pre-clinical) stages is essential. Symptoms that indicate early knee OA were found in a previous cross-sectional study. In the present study we estimated the predictive value of signs and symptoms (S&S) of history taking and physical examination for structural knee OA at 5 years follow-up. Methods: In a subset of 891 females (aged 45e60 years) of the Rotterdam Study, a population based study in the Netherlands, MRIs, radiographs, an physical examination and knee specific questionnaires were collected. After five years follow-up the same measurements were performed. Of 891 women who participated at baseline, 700 women participated at follow-up. Of these 700 women, 592 women did not have OA at baseline (defined at MRI), which were the women we used for this study. Baseline and follow-up MRIs were scored together with the semi-quantitative MRI OA knee score (MOAKS). Based on the MRI scores we defined TFOA and PFOA per knee, using a previously published MRI definition for OA. Radiographs were scored with Kellgren and Lawrence criteria (K&L). Radiographic OA was defined as K&L> ¼ 2.