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Notch1-antagonistic aptamer for chondrogenic differentiation of bone marrow stromal cells
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
Sections were stained with H-E, Safranin O, and expression of F4/80, IL1b, p-IKK a/b, MMP-13 were analyzed by immunohistochemistry. Results: Real time RT-PCR analysis: IL-1b increased the expressions levels of MMP-13 and MMP-3 mRNA in both WT and p21(-/-) mice group. When we compared the MMPs expressions, both MMP-3 and 13 expression levels were significantly elevated in p21(-/-) mice (Figure 1). ELISA: Serum levels of IL-1b were significantly elevated in p21(-/-) mice at each time points except control group(Fig. 2). Histrogical evaluation: Safranin O staining in joint cartilage tissues were evaluated by OARSI histopathology classification. P21(-/-) mice post DMM surgery 8 weeks showed irregular articular surface, superficial clefts and proteoglycan content loss. OARSI histopathology classification score was 13.6 in P21(-/-) mice and 8.5 in Wt mice, there were significance between two groups (Fig.3). We evaluated severity of synovitis with OARSI recommended scoring system in H-E stain section. p21(-/-) mice showed outgrowth of synovial membrane and evaluated higher severity score in both 1 and 8 weeks. (Fig 4). Immunohistochemical analysis showed that F4/80, IL-1b, p-IKK a/b and MMP-13 were more expressed in both synovium and cartilage of p21(-/-) mice. Number of positive cells were significantly increased in p21(-/-) mice compared with WT (Fig.5). Conclusions: Previous study reported that p21 reduce the secretion of IL-1beta by p21 knock out macrophages in vitro study. We demonstrated that p21deficiency increased the expression of IL-1b systemically in OA progression, and increased inflammation markers and catabolic factors such as MMPs in joint. Further, we confirmed that chondrocytes isolated from p21(-/-) mice were more susceptible to increase the expressions of IL-1b-induced MMPs. Therefore, we concluded that.
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Figure 3.
Figure 4.
Figure 1.
Figure 5.
643 NOTCH1-ANTAGONISTIC APTAMER FOR CHONDROGENIC DIFFERENTIATION OF BONE MARROW STROMAL CELLS J.-E. Kim, M.-L. Kang, G.-I. Im. Dongguk Univ. Int'l Hosp., Goyang, Republic of Korea
Figure 2.Ă
Purpose: During murine skeletal development, Notch-1 is strongly localized within the condensing mesenchyme during the early stages of chondrogenesis [1]. Notch signaling is implicated in the repression of chondrogenic differentiation of mesenchymal stem cell (MSC) [2]. Aptamers are oligonucleotide or peptide molecules that bind to a specific target molecule such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers exhibit significant advantages relative to protein therapeutics in terms of size, synthetic accessibility and modification by medicinal chemistry.
S388
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
The purpose of this study was to investigate the possibility of Notch1antagonistic aptamer as nucleic acid therapeutics to induce chondrogenic differentiation of human bone marrow stromal cells (hBMSCs). Methods: 1) Cytotoxicity test hBMSCs (1104 cells / well on 96 well plate) were treated with three Notch1 aptamer clone candidates from 1 to 1000 nM for 7 days. On day 1, 4 and 7, MTT assay was performed. 2) Validation of Notch1-antagonistic aptamer To select Notch1-antagonistic aptamer, expression of Notch1 downstream target genes including Hes and Hey were investigated by RT-qPCR on hBMSCs treated with each of Notch1 aptamer candidate in DMEM/F12. 3) Median effective dose (ED50) Pellets of hBMSCs (2.5105 cells, passage 3e5) were cultured in DMEM/F-12 supplemented with dexamethasone (10-7 M), BSA (7.5% w/v), L-proline (50 mM), ascorbate-2phosphate (50 mM), sodium pyruvate (1 mM) and ITS (1% v/v) containing different concentration of Notch1 aptamer (1, 10, 100 and 1000 nM). After 14 days of chondrogenic induction, RT-qPCR for chondrogenic markers of COL2, SOX9 and ACAN was performed. 4) In vitro chondrogenic differentiation The hBMSCs (2.5105 cells, passage 3e5) were made by pellets and were cultured in chondrogenic medium containing Notch1-antagoistic aptamer (200 nM) from day 0 to 14. Chondrogenic differentiation was evaluated by RT-qPCR for COL2, SOX9 and ACAN, GAG/DNA content and safranin-O/alcian blue staining. 5) Gene expression profiling with oligonucleotide microarrays Total RNA was isolated from confluent cultures of four groups [negative control (chondrogenic media (CM) treated), Notch1 aptamer in CM, TGF-b in CM, Notch1 aptamer and TGF-b in CM]. Gene expression profiling was performed by Illumina Expression Beadchip Analysis Service using Illumina HumanHT-12 v4 Expression BeadChip (Illumina, Inc., San Diego, CA, USA). Hierarchical clustering was performed complete linkage algorithm using GeneSpring GX 7.3.1 software (Agilent, Santa Clara, CA, USA). Results: 1) Cytotoxicity test The MTT data after exposure to the Notch1antagonistic aptamer candidates from 1 to 1000 nM for 7 days showed no significant cytotoxicity. 2) Validation of Notch1-antagonistic aptamer The gene expression of hes1 and hey1 significantly decreased in hBMSCs treated with Notch1antagonistic aptamer candidates for 7 days compared to those of untreated hBMSCs. 3) Median effective dose The gene expression of COL2, SOX9 and ACAN significantly increased in hBMSCs treated with Notch1-antagonistic aptamer (100nM) for 14 days compared to those of untreated hBMSCs. 4) In vitro chondrogenic differentiation The gene expression of COL2, SOX9 and ACAN increased in hBMSC pellets treated with Notch1antagoistic aptamer for both of 7 (0 to 7) and 14 (0 to 14) days compared to those of untreated hBMSCs pellets. The pellets treated with Notch1antagonistic aptamer showed greater intensity of safranin-O staining than those of untreated BMSCs. However, there was no synergistic effect of Notch1-antagonistic aptamer with TGF- b in chondrogenic differentiation of hBMSCs pellets. 5) Gene expression profiling with oligonucleotide microarrays We selected 82 chondrogenesis-related genes from microarray data and then hierarchical clustering of selected probes was performed. Different patterns were detectable between Notch1-antagonistic aptamer and untreated control. Conclusions: We investigated the Notch1-antagoistic aptamer as a candidate of nucleic acid therapeutics to induce chondrogenic differentiation of hBMSCs. Selected Notch1-antagonistic aptamer induced complete chondrogenesis in hBMSCs pellets even though early treatment (0 to 7 days). This research was supported by the National Research Foundation of Korea (Grant no: 2013R1A1A2062978).
644 ENAMPT IS LOCALISED TO AREAS OF CARTILAGE DAMAGE IN PATIENTS WITH HIP OSTEOARTHRITIS AND DRIVES CARTILAGE CATABOLISM LEADING TO PROTEOGLYCAN LOSS AND INFLAMMATION A.M. Philp y, M.J. Pearson y, J.M. Lord y, E.T. Davis z, S.W. Jones y. y Univ. of Birmingham, Birmingham, United Kingdom; z Royal Orthopaedic Hosp., Birmingham, United Kingdom Purpose: To characterise the expression and functional role of visfatin in the joint tissue of patients with hip osteoarthritis (OA). Methods: Femoral head joint tissues (cartilage, bone, synovium, muscle, adipose) from hip OA patients (n¼9) were collected following joint replacement surgery. Cartilage explants were stimulated with recombinant human visfatin protein. The production of pro-inflammatory cytokines, chemokines and cartilage proteases were measured by ELISA and Luminex. Visfatin localisation within human OA hip cartilage tissue was determined by immunohistochemistry. Proteoglycan loss was measured by release of sulphated glycosaminoglycan. Results: Visfatin was expressed by all the tissues that encompass the OA hip joint including cartilage, bone, adipose and synovium, and was elevated in the synovial fluid (1.5 fold, p¼0.2) and in isolated synovial fibroblasts (8.4 fold, p¼0.02) of obese hip OA patients compared to normal-weight patients (n¼3). Expression of visfatin in hip cartilage was localised to areas of cartilage damage and fibrillation, where it was found to be co-expressed with MMP13 in the pericellular zone of chondrocytes. Extracellular visfatin (eNAMPT) induced the production of pro-inflammatory cytokines/chemokines (IL-6, MCP-1 (4 fold), CCL20 (7 fold) and CCL4 (20 fold)), cartilage proteases (MMP-1 (4 fold), -2 (3 fold), -3 (3 fold), -7 (2.2 fold), -8 (1.3 fold), -9 (1.2 fold), -10 (1.5 fold) and MMP-13 (5 fold)), and mediated proteoglycan loss in hip OA cartilage explant tissue. Extracellular visfatin-induced production of IL-6 was greater in cartilage from obese hip OA patients, compared to cartilage from normal-weight patients (7 fold vs >1500 fold, p¼0.02). Conclusions: The localised expression of visfatin to areas of cartilage fibrillation and its effect on driving catabolic changes and proteoglycan degradation indicate a central role for extracellular visfatin in the loss of cartilage integrity in patients with hip OA. 645 EVIDENCE OF ABNORMAL TYPE I COLLAGEN COMPOSITION IN OBESE PATIENTS WITH OA A.M. Philp y, R.L. Collier z, D.B. Bartlett y, J.M. Lord y, E.T. Davis x, S.W. Jones y. y Univ. of Birmingham, Birmingham, United Kingdom; z Univ. of Liverpool, Liverpool, United Kingdom; x Royal Orthopaedic Hosp., Birmingham, United Kingdom Purpose: The subchondral bone may be a primary driver of the pathogenesis of OA, since several studies suggest that changes to bone may precede cartilage degeneration in OA. Given the association of obesity with OA, and the purported role of adipose-secreted cytokines in OA disease pathology, the aim of this study was (1) to determine the relationship between adiposity, biomarkers of bone remodelling and
Sections were stained with H-E, Safranin O, and expression of F4/80, IL1b, p-IKK a/b, MMP-13 were analyzed by immunohistochemistry. Results: Real time RT-PCR analysis: IL-1b increased the expressions levels of MMP-13 and MMP-3 mRNA in both WT and p21(-/-) mice group. When we compared the MMPs expressions, both MMP-3 and 13 expression levels were significantly elevated in p21(-/-) mice (Figure 1). ELISA: Serum levels of IL-1b were significantly elevated in p21(-/-) mice at each time points except control group(Fig. 2). Histrogical evaluation: Safranin O staining in joint cartilage tissues were evaluated by OARSI histopathology classification. P21(-/-) mice post DMM surgery 8 weeks showed irregular articular surface, superficial clefts and proteoglycan content loss. OARSI histopathology classification score was 13.6 in P21(-/-) mice and 8.5 in Wt mice, there were significance between two groups (Fig.3). We evaluated severity of synovitis with OARSI recommended scoring system in H-E stain section. p21(-/-) mice showed outgrowth of synovial membrane and evaluated higher severity score in both 1 and 8 weeks. (Fig 4). Immunohistochemical analysis showed that F4/80, IL-1b, p-IKK a/b and MMP-13 were more expressed in both synovium and cartilage of p21(-/-) mice. Number of positive cells were significantly increased in p21(-/-) mice compared with WT (Fig.5). Conclusions: Previous study reported that p21 reduce the secretion of IL-1beta by p21 knock out macrophages in vitro study. We demonstrated that p21deficiency increased the expression of IL-1b systemically in OA progression, and increased inflammation markers and catabolic factors such as MMPs in joint. Further, we confirmed that chondrocytes isolated from p21(-/-) mice were more susceptible to increase the expressions of IL-1b-induced MMPs. Therefore, we concluded that.
S387
Figure 3.
Figure 4.
Figure 1.
Figure 5.
643 NOTCH1-ANTAGONISTIC APTAMER FOR CHONDROGENIC DIFFERENTIATION OF BONE MARROW STROMAL CELLS J.-E. Kim, M.-L. Kang, G.-I. Im. Dongguk Univ. Int'l Hosp., Goyang, Republic of Korea
Figure 2.Ă
Purpose: During murine skeletal development, Notch-1 is strongly localized within the condensing mesenchyme during the early stages of chondrogenesis [1]. Notch signaling is implicated in the repression of chondrogenic differentiation of mesenchymal stem cell (MSC) [2]. Aptamers are oligonucleotide or peptide molecules that bind to a specific target molecule such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers exhibit significant advantages relative to protein therapeutics in terms of size, synthetic accessibility and modification by medicinal chemistry.
S388
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
The purpose of this study was to investigate the possibility of Notch1antagonistic aptamer as nucleic acid therapeutics to induce chondrogenic differentiation of human bone marrow stromal cells (hBMSCs). Methods: 1) Cytotoxicity test hBMSCs (1104 cells / well on 96 well plate) were treated with three Notch1 aptamer clone candidates from 1 to 1000 nM for 7 days. On day 1, 4 and 7, MTT assay was performed. 2) Validation of Notch1-antagonistic aptamer To select Notch1-antagonistic aptamer, expression of Notch1 downstream target genes including Hes and Hey were investigated by RT-qPCR on hBMSCs treated with each of Notch1 aptamer candidate in DMEM/F12. 3) Median effective dose (ED50) Pellets of hBMSCs (2.5105 cells, passage 3e5) were cultured in DMEM/F-12 supplemented with dexamethasone (10-7 M), BSA (7.5% w/v), L-proline (50 mM), ascorbate-2phosphate (50 mM), sodium pyruvate (1 mM) and ITS (1% v/v) containing different concentration of Notch1 aptamer (1, 10, 100 and 1000 nM). After 14 days of chondrogenic induction, RT-qPCR for chondrogenic markers of COL2, SOX9 and ACAN was performed. 4) In vitro chondrogenic differentiation The hBMSCs (2.5105 cells, passage 3e5) were made by pellets and were cultured in chondrogenic medium containing Notch1-antagoistic aptamer (200 nM) from day 0 to 14. Chondrogenic differentiation was evaluated by RT-qPCR for COL2, SOX9 and ACAN, GAG/DNA content and safranin-O/alcian blue staining. 5) Gene expression profiling with oligonucleotide microarrays Total RNA was isolated from confluent cultures of four groups [negative control (chondrogenic media (CM) treated), Notch1 aptamer in CM, TGF-b in CM, Notch1 aptamer and TGF-b in CM]. Gene expression profiling was performed by Illumina Expression Beadchip Analysis Service using Illumina HumanHT-12 v4 Expression BeadChip (Illumina, Inc., San Diego, CA, USA). Hierarchical clustering was performed complete linkage algorithm using GeneSpring GX 7.3.1 software (Agilent, Santa Clara, CA, USA). Results: 1) Cytotoxicity test The MTT data after exposure to the Notch1antagonistic aptamer candidates from 1 to 1000 nM for 7 days showed no significant cytotoxicity. 2) Validation of Notch1-antagonistic aptamer The gene expression of hes1 and hey1 significantly decreased in hBMSCs treated with Notch1antagonistic aptamer candidates for 7 days compared to those of untreated hBMSCs. 3) Median effective dose The gene expression of COL2, SOX9 and ACAN significantly increased in hBMSCs treated with Notch1-antagonistic aptamer (100nM) for 14 days compared to those of untreated hBMSCs. 4) In vitro chondrogenic differentiation The gene expression of COL2, SOX9 and ACAN increased in hBMSC pellets treated with Notch1antagoistic aptamer for both of 7 (0 to 7) and 14 (0 to 14) days compared to those of untreated hBMSCs pellets. The pellets treated with Notch1antagonistic aptamer showed greater intensity of safranin-O staining than those of untreated BMSCs. However, there was no synergistic effect of Notch1-antagonistic aptamer with TGF- b in chondrogenic differentiation of hBMSCs pellets. 5) Gene expression profiling with oligonucleotide microarrays We selected 82 chondrogenesis-related genes from microarray data and then hierarchical clustering of selected probes was performed. Different patterns were detectable between Notch1-antagonistic aptamer and untreated control. Conclusions: We investigated the Notch1-antagoistic aptamer as a candidate of nucleic acid therapeutics to induce chondrogenic differentiation of hBMSCs. Selected Notch1-antagonistic aptamer induced complete chondrogenesis in hBMSCs pellets even though early treatment (0 to 7 days). This research was supported by the National Research Foundation of Korea (Grant no: 2013R1A1A2062978).
644 ENAMPT IS LOCALISED TO AREAS OF CARTILAGE DAMAGE IN PATIENTS WITH HIP OSTEOARTHRITIS AND DRIVES CARTILAGE CATABOLISM LEADING TO PROTEOGLYCAN LOSS AND INFLAMMATION A.M. Philp y, M.J. Pearson y, J.M. Lord y, E.T. Davis z, S.W. Jones y. y Univ. of Birmingham, Birmingham, United Kingdom; z Royal Orthopaedic Hosp., Birmingham, United Kingdom Purpose: To characterise the expression and functional role of visfatin in the joint tissue of patients with hip osteoarthritis (OA). Methods: Femoral head joint tissues (cartilage, bone, synovium, muscle, adipose) from hip OA patients (n¼9) were collected following joint replacement surgery. Cartilage explants were stimulated with recombinant human visfatin protein. The production of pro-inflammatory cytokines, chemokines and cartilage proteases were measured by ELISA and Luminex. Visfatin localisation within human OA hip cartilage tissue was determined by immunohistochemistry. Proteoglycan loss was measured by release of sulphated glycosaminoglycan. Results: Visfatin was expressed by all the tissues that encompass the OA hip joint including cartilage, bone, adipose and synovium, and was elevated in the synovial fluid (1.5 fold, p¼0.2) and in isolated synovial fibroblasts (8.4 fold, p¼0.02) of obese hip OA patients compared to normal-weight patients (n¼3). Expression of visfatin in hip cartilage was localised to areas of cartilage damage and fibrillation, where it was found to be co-expressed with MMP13 in the pericellular zone of chondrocytes. Extracellular visfatin (eNAMPT) induced the production of pro-inflammatory cytokines/chemokines (IL-6, MCP-1 (4 fold), CCL20 (7 fold) and CCL4 (20 fold)), cartilage proteases (MMP-1 (4 fold), -2 (3 fold), -3 (3 fold), -7 (2.2 fold), -8 (1.3 fold), -9 (1.2 fold), -10 (1.5 fold) and MMP-13 (5 fold)), and mediated proteoglycan loss in hip OA cartilage explant tissue. Extracellular visfatin-induced production of IL-6 was greater in cartilage from obese hip OA patients, compared to cartilage from normal-weight patients (7 fold vs >1500 fold, p¼0.02). Conclusions: The localised expression of visfatin to areas of cartilage fibrillation and its effect on driving catabolic changes and proteoglycan degradation indicate a central role for extracellular visfatin in the loss of cartilage integrity in patients with hip OA. 645 EVIDENCE OF ABNORMAL TYPE I COLLAGEN COMPOSITION IN OBESE PATIENTS WITH OA A.M. Philp y, R.L. Collier z, D.B. Bartlett y, J.M. Lord y, E.T. Davis x, S.W. Jones y. y Univ. of Birmingham, Birmingham, United Kingdom; z Univ. of Liverpool, Liverpool, United Kingdom; x Royal Orthopaedic Hosp., Birmingham, United Kingdom Purpose: The subchondral bone may be a primary driver of the pathogenesis of OA, since several studies suggest that changes to bone may precede cartilage degeneration in OA. Given the association of obesity with OA, and the purported role of adipose-secreted cytokines in OA disease pathology, the aim of this study was (1) to determine the relationship between adiposity, biomarkers of bone remodelling and