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Association of plasma and synovial fluid periostin with radiographic knee osteoarthritis: Cross-sectional study
Joint Bone Spine 82 (2015) 352–355
Available online at
ScienceDirect www.sciencedirect.com
Original article
Association of plasma and synovial fluid periostin with radiographic knee osteoarthritis: Cross-sectional study Sittisak Honsawek a,b,∗ , Vajara Wilairatana b , Wanvisa Udomsinprasert a , Peerasit Sinlapavilawan b , Napaphat Jirathanathornnukul a a Department of Biochemistry and Orthopaedics, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, 1873, Rama IV road, Patumwan, 10330 Bangkok, Thailand b Department of Orthopaedics, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, 10330 Bangkok, Thailand
a r t i c l e
i n f o
Article history: Accepted 28 January 2015 Available online 13 April 2015 Keywords: Osteoarthritis Periostin Plasma Radiographic severity Synovial fluid
a b s t r a c t Objective: To investigate plasma and synovial fluid (SF) periostin of knee osteoarthritis (OA) patients and to determine the relationship between periostin levels and the radiographic severity. Methods: A total of 110 subjects (90 knee OA patients and 20 healthy controls) were enrolled in this study. Plasma and SF periostin were examined using an enzyme-linked immunosorbent assay. OA grading was performed using the Kellgren-Lawrence classification. Results: Although plasma periostin was greater in OA patients than in controls, the difference was not significant. Additionally, SF periostin was significantly higher with respect to paired plasma (P < 0.001). Moreover, plasma and SF periostin demonstrated significantly positive correlation with the radiographic severity of knee OA (r = 0.537, P < 0.001 and r = 0.427, P < 0.001, respectively). Subsequent analysis revealed that there was a positive correlation between plasma and SF periostin (r = 0.368, P < 0.001). Conclusions: Plasma and SF periostin levels were positively correlated with the radiographic severity of knee OA. © 2015 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.
1. Introduction Osteoarthritis (OA) is a prevalent, slowly progressive joint disease clinically manifested by pain, crepitation, stiffness, joint swelling, limited range of motion, and disability. The essential features of OA include articular cartilage degeneration, subchondral bone sclerosis, osteophyte formation at joint margins, and synovial membrane inflammation [1]. Although the pathophysiology of OA remains uncertain, a number of risk factors contributing to the OA development include ageing, obesity, genetic predisposition, and previous joint injury [2]. Several biochemical factors have been known as playing key roles in the perpetuation of OA. Periostin, also known as osteoblast-specific factor-2 (OSF-2), is a disulfide linked 90-kDa heparin-binding N terminus-glycosylated protein that belongs to the family of fasciclins based on its homology to fasciclin 1 (FAS1) superfamily of molecules [3]. It is a transforming growth factor-beta (TGF-) inducible molecule
∗ Corresponding author. Tel.: ++662 256 4482; fax: ++662 256 4482. E-mail addresses: [email protected], sittisak [email protected] (S. Honsawek).
that serves as both adhesion molecule and tumor suppressor [4]. Periostin, an 836 amino acid secreted protein, contains several putative sites for gamma carboxylation of their glutamic acid residues [5]. It was initially identified in periosteum and bone, and in bone fracture, periostin expression was upregulated and localized in preosteoblastic cells within the periosteum, as well as in undifferentiated mesenchymal stem cells proximity to the fracture site [6]. In bone, periostin is thought to be involved in osteoblast recruitment, proliferation, differentiation, attachment, and survival [7]. Periostin is found to be expressed in dark hypertrophic chondrocytes characterized by electron-dense cytoplasm with abundant rough endoplasmic reticulum and Golgi apparatus and condensed nuclear chromatin [8]. Furthermore, periostin expression is evident in cartilage and bone cells, suggesting a potential role of periostin in endochondral bone formation [9]. In recent years, global gene expression profiling studies on subchondral bone samples, mainly relying on cDNA microarray technology, resulted in the identification of periostin contributing to OA development [10,11]. Periostin was present in the articular cartilage matrix of the surgically induced mice after the induction of OA by destabilization of the medial meniscus using microarray and real-time polymerase chain reaction analysis [10]. In
http://dx.doi.org/10.1016/j.jbspin.2015.01.023 1297-319X/© 2015 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.
S. Honsawek et al. / Joint Bone Spine 82 (2015) 352–355
addition, it has been demonstrated that periostin could be evident in osteoblasts under the tidemark, in osteocytes, and in lining cells in osteoarthritic subchondral bone of OA patients at mRNA and protein levels [11]. These findings prompted us to consider the hypothesis that periostin may be responsible for the pathogenesis of OA. However, the circulating and synovial fluid (SF) periostin of patients with primary knee OA and its significance in the OA patients have received little attention. Therefore, the purposes of this study were to investigate plasma and SF periostin collected from OA patients and to determine the relationship between plasma and SF periostin and the radiographic severity in OA.
353
Table 1 Baseline clinical characteristics of knee OA patients and controls.
n Age (years) Gender (M/F) BMI (kg/m2 )
Knee OA patients
Controls
P
90 70.0 ± 0.8 16/74 26.8 ± 0.4
20 68.7 ± 1.3 5/15 26.4 ± 0.7
0.5 0.2 0.4
BMI: body mass index; OA: osteoarthritis.
of variation (CVs) were 2.5–3.5% and 5.4–8.9%, respectively. The sensitivity of this assay was 0.375 ng/ml.
2. Methods 2.1. Subjects
2.3. Statistical analysis
The current study was carried out in accordance with the guidelines of the Declaration of Helsinki, and written informed consent was obtained from all participants prior to their enrolment in the study. This study was approved by the Institutional Review Board on Human Research of our institute. Ninety patients aged 49–84 years diagnosed with primary knee osteoarthritis (16 males and 74 females; mean age 70.0 ± 0.8 years) according to the criteria of the American College of Rheumatology, and 20 age/gender-matched healthy controls with no clinical and radiological evidence of OA (5 males and 15 females; mean age 68.7 ± 1.3 years) were recruited. All patients were scheduled to undergo diagnostic or therapeutic arthroscopy or total knee arthroplasty in our institute. Clinical data were attentively reviewed to preclude any forms of secondary OA and inflammatory joint disorders. None of the participants had underlying diseases such as diabetes, advanced liver or renal diseases, histories of medication interfering with bone metabolism (such as corticosteroids or bisphosphonates), other forms of arthritis, cancer or other chronic inflammatory diseases. Knee radiography was taken when each participant was standing on both legs with fully extended knee and the X-ray beam was centred at the level of the joint. Radiographs were scored for Kellgren and Lawrence (KL) grade (0–4) [12], including joint space narrowing (JSN grade 0–3) and osteophytes (grade 0–3) [13]. All preoperative radiographs were assessed in a blinded manner to the patient’s clinical and laboratory data. The intrarater reliability kappa values were 0.91 for KL grade, 0.72 for JSN grade, and 0.79 for osteophyte grade. OA patients were defined as having radiographic knee OA of KL grade ≥ 2 in at least 1 knee. Controls were defined as having neither radiographic hip OA nor knee OA, as indicated by KL grades of 0 for both hips and both knees. The grading of more severely affected knee in each patient was used for data analysis.
Statistical analysis was conducted using the statistical package for social sciences (SPSS) software, version 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Demographic data between patients and controls were compared by Chi-square tests and unpaired Student’s t-tests, where appropriate. Comparisons between KL subgroups were performed using one-way analysis of variance. Pearson’s correlation coefficient was employed to determine correlations between periostin values and radiographic severity. Standardized -regression coefficients and their significance in a multivariate linear regression analysis were performed to identify the correlation between the highest quartile level of SF periostin and KL grade. Data were expressed as a mean ± standard error of the mean. P-values < 0.05 were considered significant.
3. Results 3.1. Baseline clinical characteristics The baseline clinical characteristics of the subjects are illustrated in Table 1. Data were normally distributed. There were no statistically significant differences in the ages or gender ratios (male/female) between OA patients and controls. Fig. 1 reveals that there has been a slight increase in plasma periostin of OA patients when compared with that of controls; however, no significant differences were observed (149.7 ± 8.9 ng/ml vs. 145.7 ± 4.8 ng/ml, P > 0.05). SF periostin was approximately two-fold greater than in paired plasma samples of OA patients (278.0 ± 23.3 ng/ml vs. 149.7 ± 8.9 ng/ml, P < 0.001). SF periostin was positively correlated with plasma periostin (r = 0.368, P < 0.001) (Fig. 2A).
2.2. Laboratory methods Following a 12-h overnight fast, venous blood samples were collected into ethylenediamine tetraacetic acid tubes, centrifuged, and stored immediately at −80 ◦ C for later measurement. SF was aspirated from the affected knee of OA patients using sterile knee puncture just prior to surgery when the arthroscopy or total knee arthroplasty was performed. The specimen was then centrifuged to remove cells and joint debris and then stored at −80 ◦ C until analysis. Plasma and SF periostin levels were analyzed using sandwich enzyme-linked immunosorbent assay kit according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA). The antibodies specific for periostin generated by the entire immunogen were utilized. Recombinant human periostin was used to generate a standard curve. The intra- and inter-assay coefficients
Fig. 1. Periostin levels in plasma and synovial fluid of OA patients and healthy controls.
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S. Honsawek et al. / Joint Bone Spine 82 (2015) 352–355
Fig. 2. Correlations between plasma and synovial fluid periostin and radiographic severity in osteoarthritis (OA) patients. A. Plasma periostin was correlated with synovial fluid periostin in OA patients. B. Plasma periostin was correlated with radiographic severity in OA patients. C. Synovial fluid periostin was correlated with radiographic severity in OA patients.
3.2. Association between plasma and SF periostin and OA severity On the basis of the radiographic KL classification, OA patients were categorized into 3 subgroups with regard to OA grading (KL grade 2: 28; KL grade 3: 28; KL grade 4: 34). Plasma and SF periostin in knee OA patients with different KL subgroups are demonstrated in Table 2. Knee OA patients with higher radiographic severity had significantly higher periostin values in both plasma and SF (P < 0.001). Furthermore, the relationships between plasma and SF periostin with the radiographic severity of knee OA were determined. Plasma periostin was directly correlated with the radiographic severity of knee OA (r = 0.537, P < 0.001) (Fig. 2B). SF periostin of OA patients were also positively associated with KL grade (r = 0.427, P < 0.001) (Fig. 2C). Subsequent analysis evidenced that SF periostin was significantly associated with osteophyte severity (r = 0.335, P = 0.005) and JSN severity (r = 0.311, P = 0.02). Particularly, when SF periostin was divided into quartiles with the highest quartile corresponding to level > 389.4 ng/ml, we found that the highest quartile level of SF periostin was correlated with KL grade of OA (r = 0.421, P < 0.001). Multivariate regression analysis demonstrated that the correlation between the highest quartile level of SF periostin and KL grade was still significant after adjusting for age ( = 0.461, P = 0.01). 4. Discussion Periostin is a vitamin K-dependent glutamate-containing matricellular protein, originally isolated from a mouse osteoblast cell line [3,4]. It is encoded by the Postn gene in humans, and to date, TGF-1, 2, and 3, bone morphogenetic protein (BMP)-2 and
4, vascular endothelial growth factor, connective growth factor 2, vitamin K, valsartan (an angiotensin II antagonist), and interleukin (IL)-3, 4, 6, and 13 have been documented to activate periostin expression in a cell-specific context [14]. Periostin is an extracellular matrix (ECM) protein involved in regulating intercellular adhesion via an interaction with other ECM proteins, including collagen, fibronectin, tenascin-C, integrin, and periostin itself [15,16]. Recently, periostin expression is found to be positively correlated with the fibrosis grade in bone marrow fibrosis [17]. Previous studies have shown that serum periostin was elevated in breast cancer patients with bone metastases [18] and positively correlated with bone periostin mRNA expression in breast cancer bone metastases [19]. Circulating periostin was also positively correlated with total alkaline phosphatase in post-menopausal women with low bone mass [20] and positively associated with incident fracture risk [21]. The present study has demonstrated, for the first time, that periostin was detectable in both plasma and SF derived from patients with primary knee OA. Plasma periostin in OA patients was insignificantly higher than that in healthy controls. In addition, SF periostin in OA patients was substantially increased compared to paired plasma periostin. Furthermore, both plasma and SF periostin were positively correlated with the radiographic severity in knee OA patients. In this study, we found that periostin levels of OA patients were significantly higher in SF than those observed in paired plasma samples. Moreover, advanced knee OA patients exhibited higher periostin levels compared with early knee OA patients. Our results suggest that there is enhanced local production of periostin in knee OA. In the light of these considerations, several plausible mechanisms could be responsible for the elevation of periostin in knee OA patients. The source of periostin in SF may stem from chondrocytes, osteocytes, and synovial cells in the local tissues (cartilage, meniscus, ligament, tendon, subchondral bone, and inflamed synovium) and extra-articular tissues. The findings observed in this study mirror those of the previous studies that have examined periostin tissue distribution of samples obtained from OA joints [10,11]. Loeser et al. reported that periostin was present in chondrocytes in articular cartilage, meniscus, and growth plate, as well as osteoblasts in the periosteum during the OA development [10]. This also accords with Chou’s findings which showed the presence of periostin expression in subchondral bone osteoblasts of OA knees [11]. Another possible explanation for high periostin might be attributed to the increased periostin synthesis, which exceeded its clearance. Our observation indicates a substantially increased production in the systemic and local expression of periostin in patients with advanced knee OA. Additionally, other extraskeletal tissues probably play a part in plasma periostin elevation. High expressions of periostin in adult skin, kidney, liver, and other gastro-intestinal organs have been previously reported [22]. It should be emphasized that there were significant positive correlations between plasma and SF periostin and the radiographic severity of OA. These findings are in line with our previous studies of BMP-7, basic fibroblast growth factor, and connective tissue growth factor in primary knee OA [23–25]. These observations
Table 2 Plasma and synovial fluid periostin in osteoarthritis patients. P-values for differences among Kellgren and Lawrence subgroups.
n Age (years) Gender (M/F) BMI (kg/m2 ) SF periostin (ng/ml) Plasma periostin (ng/ml)
Total
KL grade 2
KL grade 3
KL grade 4
P
90 70.0 ± 0.8 16/74 26.8 ± 0.4 278.0 ± 23.3 149.7 ± 8.9
28 68.8 ± 1.5 5/23 26.8 ± 0.6 171.1 ± 20.8 99.6 ± 7.7
28 68.8 ± 1.2 7/21 26.1 ± 0.7 242.1 ± 29.5 129.6 ± 11.3
34 71.9 ± 1.5 4/30 27.4 ± 0.7 395.7 ± 47.6 207.5 ± 16.4
0.2 0.5 0.4 < 0.001 < 0.001
BMI: body mass index; KL: Kellgren and Lawrence; SF: synovial fluid.
S. Honsawek et al. / Joint Bone Spine 82 (2015) 352–355
could be applied for possibly using as biomarkers to determine the OA severity. A number of caveats need to be emphasized regarding the present study. First, the study is cross-sectional in design with relatively small numbers of patients and controls. As a result, cause-and-effect relationships cannot be concluded and require prospective longitudinal studies to elucidate any relationships. However, with a small sample size, caution must be applied, as the findings might not be transferable to other populations. Second, SF samples were neither collected from healthy controls nor obtained from inflammatory arthritis patients leading to no direct comparison between SF periostin of controls and that of OA patients. Instead, circulating periostin reflects the systemic nature of the condition. In future studies, this may be overcome by collecting SF samples from patients with traumatic knee and/or inflammatory arthritis as controls. Additional immunohistochemical analysis of osteoarthritic cartilage and synovial tissues could render valuable information on the pathophysiologic role of periostin in OA development. Taken together, the current study demonstrated that SF periostin was markedly higher with respect to paired plasma in knee OA patients. There was a significant positive correlation between plasma and SF periostin. Moreover, plasma and SF periostin was positively correlated with the radiographic severity in knee OA. Although underlining mechanisms of this association and their cause-and-effect relationships are not entirely elucidated, there is abundant room for further research regarding the potential role of periostin in the pathogenesis of chronic degenerative joint disorders. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This research has been supported by the Ratchadaphiseksomphot Endowment Fund 2013 of Chulalongkorn University (CU-56-341-AS) and the Ratchadapiseksompotch Fund (RA55/22). The authors commemorate the 100th Anniversary of the King Chulalongkorn Memorial Hospital. We would like to thank Natthaphon Saetan for technical assistance. References [1] Felson DT, Zhang Y, Hannan MT, et al. The incidence and natural history of knee osteoarthritis in the elderly. Arthritis Rheum 1995;38:1500–5. [2] Pelletier JP, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets. Arthritis Rheum 2001;44:1237–47.
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[3] Horiuchi K, Amizuka N, Takeshita S, et al. Identification and characterization of a novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor beta. J Bone Miner Res 1999;14:1239–49. [4] Takeshita S, Kikuno R, Tezuka K, et al. Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I. Biochem J 1993;294:271–8. [5] Coutu DL, Wu JH, Monette A, et al. Periostin, a member of a novel family of vitamin K-dependent proteins, is expressed by mesenchymal stromal cells. J Biol Chem 2008;283:17991–8001. [6] Nakazawa T, Nakajima A, Seki N, et al. Gene expression of periostin in the early stage of fracture healing detected by cDNA microarray analysis. J Orthop Res 2004;22:520–5. [7] Zhu S, Barbe MF, Liu C, et al. Periostin-like-factor in osteogenesis. J Cell Physiol 2009;218:584–92. [8] Chen KS, Tatarczuch L, Mirams M, et al. Periostin expression distinguishes between light and dark hypertrophic chondrocytes. Int J Biochem Cell Biol 2010;42:880–9. [9] Zhu S, Barbe MF, Amin N, et al. Immunolocalization of periostin-like factor and periostin during embryogenesis. J Histochem Cytochem 2008;56: 329–45. [10] Loeser RF, Olex AL, McNulty MA, et al. Microarray analysis reveals age-related differences in gene expression during the development of osteoarthritis in mice. Arthritis Rheum 2012;64:705–17. [11] Chou CH, Wu CC, Song IW, et al. Genome-wide expression profiles of subchondral bone in osteoarthritis. Arthritis Res Ther 2013;15:R190. [12] Kellgren JH, Lawrence JS. Radiological assessment of osteo-arthrosis. Ann Rheum Dis 1957;16:494–502. [13] Felson DT, McAlindon TE, Anderson JJ, et al. Defining radiographic osteoarthritis for the whole knee. Osteoarthritis Cartilage 1997;5:241–50. [14] Norris RA, Moreno-Rodriguez R, Hoffman S, et al. The many facets of the matricelluar protein periostin during cardiac development, remodeling, and pathophysiology. J Cell Commun Signal 2009;3:275–86. [15] Takayama G, Arima K, Kanaji T, et al. Periostin: a novel component of subepithelial fibrosis of bronchial asthma downstream of IL-4 and IL-13 signals. J Allergy Clin Immunol 2006;118:98–104. [16] Norris RA, Borg TK, Butcher JT, et al. Neonatal and adult cardiovascular pathophysiological remodeling and repair: developmental role of periostin. Ann N Y Acad Sci 2008;1123:30–40. [17] Oku E, Kanaji T, Takata Y, et al. Periostin and bone marrow fibrosis. Int J Hematol 2008;88:57–63. [18] Sasaki H, Yu CY, Dai M, et al. Elevated serum periostin levels in patients with bone metastases from breast but not lung cancer. Breast Cancer Res Treat 2003;77:245–52. [19] Contié S, Voorzanger-Rousselot N, Litvin J, et al. Increased expression and serum levels of the stromal cell-secreted protein periostin in breast cancer bone metastases. Int J Cancer 2011;128:352–60. [20] Anastasilakis AD, Polyzos SA, Makras P, et al. Circulating periostin levels do not differ between postmenopausal women with normal and low bone mass and are not affected by zoledronic acid treatment. Horm Metab Res 2014;46: 145–9. [21] Rousseau J, Sornay-Rendu E, Bertholon C, et al. Serum periostin is associated with fracture risk in postmenopausal women: a 7-year prospective analysis of the OFELY study. J Clin Endocrinol Metab 2014;99:2533–9. [22] Tilman G, Mattiussi M, Brasseur F, et al. Human periostin gene expression in normal tissues, tumors and melanoma: evidences for periostin production by both stromal and melanoma cells. Mol Cancer 2007;6:80. [23] Honsawek S, Chayanupatkul M, Tanavalee A, et al. Relationship of plasma and synovial fluid BMP-7 with disease severity in knee osteoarthritis patients: a pilot study. Int Orthop 2009;33:1171–5. [24] Honsawek S, Yuktanandana P, Tanavalee A, et al. Correlation between plasma and synovial fluid basic fibroblast growth factor with radiographic severity in primary knee osteoarthritis. Int Orthop 2012;36:981–5. [25] Honsawek S, Yuktanandana P, Tanavalee A, et al. Plasma and synovial fluid connective tissue growth factor levels are correlated with disease severity in patients with knee osteoarthritis. Biomarkers 2012;17:303–8.
Available online at
ScienceDirect www.sciencedirect.com
Original article
Association of plasma and synovial fluid periostin with radiographic knee osteoarthritis: Cross-sectional study Sittisak Honsawek a,b,∗ , Vajara Wilairatana b , Wanvisa Udomsinprasert a , Peerasit Sinlapavilawan b , Napaphat Jirathanathornnukul a a Department of Biochemistry and Orthopaedics, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, 1873, Rama IV road, Patumwan, 10330 Bangkok, Thailand b Department of Orthopaedics, Faculty of Medicine, Chulalongkorn University, King Chulalongkorn Memorial Hospital, Thai Red Cross Society, 10330 Bangkok, Thailand
a r t i c l e
i n f o
Article history: Accepted 28 January 2015 Available online 13 April 2015 Keywords: Osteoarthritis Periostin Plasma Radiographic severity Synovial fluid
a b s t r a c t Objective: To investigate plasma and synovial fluid (SF) periostin of knee osteoarthritis (OA) patients and to determine the relationship between periostin levels and the radiographic severity. Methods: A total of 110 subjects (90 knee OA patients and 20 healthy controls) were enrolled in this study. Plasma and SF periostin were examined using an enzyme-linked immunosorbent assay. OA grading was performed using the Kellgren-Lawrence classification. Results: Although plasma periostin was greater in OA patients than in controls, the difference was not significant. Additionally, SF periostin was significantly higher with respect to paired plasma (P < 0.001). Moreover, plasma and SF periostin demonstrated significantly positive correlation with the radiographic severity of knee OA (r = 0.537, P < 0.001 and r = 0.427, P < 0.001, respectively). Subsequent analysis revealed that there was a positive correlation between plasma and SF periostin (r = 0.368, P < 0.001). Conclusions: Plasma and SF periostin levels were positively correlated with the radiographic severity of knee OA. © 2015 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.
1. Introduction Osteoarthritis (OA) is a prevalent, slowly progressive joint disease clinically manifested by pain, crepitation, stiffness, joint swelling, limited range of motion, and disability. The essential features of OA include articular cartilage degeneration, subchondral bone sclerosis, osteophyte formation at joint margins, and synovial membrane inflammation [1]. Although the pathophysiology of OA remains uncertain, a number of risk factors contributing to the OA development include ageing, obesity, genetic predisposition, and previous joint injury [2]. Several biochemical factors have been known as playing key roles in the perpetuation of OA. Periostin, also known as osteoblast-specific factor-2 (OSF-2), is a disulfide linked 90-kDa heparin-binding N terminus-glycosylated protein that belongs to the family of fasciclins based on its homology to fasciclin 1 (FAS1) superfamily of molecules [3]. It is a transforming growth factor-beta (TGF-) inducible molecule
∗ Corresponding author. Tel.: ++662 256 4482; fax: ++662 256 4482. E-mail addresses: [email protected], sittisak [email protected] (S. Honsawek).
that serves as both adhesion molecule and tumor suppressor [4]. Periostin, an 836 amino acid secreted protein, contains several putative sites for gamma carboxylation of their glutamic acid residues [5]. It was initially identified in periosteum and bone, and in bone fracture, periostin expression was upregulated and localized in preosteoblastic cells within the periosteum, as well as in undifferentiated mesenchymal stem cells proximity to the fracture site [6]. In bone, periostin is thought to be involved in osteoblast recruitment, proliferation, differentiation, attachment, and survival [7]. Periostin is found to be expressed in dark hypertrophic chondrocytes characterized by electron-dense cytoplasm with abundant rough endoplasmic reticulum and Golgi apparatus and condensed nuclear chromatin [8]. Furthermore, periostin expression is evident in cartilage and bone cells, suggesting a potential role of periostin in endochondral bone formation [9]. In recent years, global gene expression profiling studies on subchondral bone samples, mainly relying on cDNA microarray technology, resulted in the identification of periostin contributing to OA development [10,11]. Periostin was present in the articular cartilage matrix of the surgically induced mice after the induction of OA by destabilization of the medial meniscus using microarray and real-time polymerase chain reaction analysis [10]. In
http://dx.doi.org/10.1016/j.jbspin.2015.01.023 1297-319X/© 2015 Société franc¸aise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.
S. Honsawek et al. / Joint Bone Spine 82 (2015) 352–355
addition, it has been demonstrated that periostin could be evident in osteoblasts under the tidemark, in osteocytes, and in lining cells in osteoarthritic subchondral bone of OA patients at mRNA and protein levels [11]. These findings prompted us to consider the hypothesis that periostin may be responsible for the pathogenesis of OA. However, the circulating and synovial fluid (SF) periostin of patients with primary knee OA and its significance in the OA patients have received little attention. Therefore, the purposes of this study were to investigate plasma and SF periostin collected from OA patients and to determine the relationship between plasma and SF periostin and the radiographic severity in OA.
353
Table 1 Baseline clinical characteristics of knee OA patients and controls.
n Age (years) Gender (M/F) BMI (kg/m2 )
Knee OA patients
Controls
P
90 70.0 ± 0.8 16/74 26.8 ± 0.4
20 68.7 ± 1.3 5/15 26.4 ± 0.7
0.5 0.2 0.4
BMI: body mass index; OA: osteoarthritis.
of variation (CVs) were 2.5–3.5% and 5.4–8.9%, respectively. The sensitivity of this assay was 0.375 ng/ml.
2. Methods 2.1. Subjects
2.3. Statistical analysis
The current study was carried out in accordance with the guidelines of the Declaration of Helsinki, and written informed consent was obtained from all participants prior to their enrolment in the study. This study was approved by the Institutional Review Board on Human Research of our institute. Ninety patients aged 49–84 years diagnosed with primary knee osteoarthritis (16 males and 74 females; mean age 70.0 ± 0.8 years) according to the criteria of the American College of Rheumatology, and 20 age/gender-matched healthy controls with no clinical and radiological evidence of OA (5 males and 15 females; mean age 68.7 ± 1.3 years) were recruited. All patients were scheduled to undergo diagnostic or therapeutic arthroscopy or total knee arthroplasty in our institute. Clinical data were attentively reviewed to preclude any forms of secondary OA and inflammatory joint disorders. None of the participants had underlying diseases such as diabetes, advanced liver or renal diseases, histories of medication interfering with bone metabolism (such as corticosteroids or bisphosphonates), other forms of arthritis, cancer or other chronic inflammatory diseases. Knee radiography was taken when each participant was standing on both legs with fully extended knee and the X-ray beam was centred at the level of the joint. Radiographs were scored for Kellgren and Lawrence (KL) grade (0–4) [12], including joint space narrowing (JSN grade 0–3) and osteophytes (grade 0–3) [13]. All preoperative radiographs were assessed in a blinded manner to the patient’s clinical and laboratory data. The intrarater reliability kappa values were 0.91 for KL grade, 0.72 for JSN grade, and 0.79 for osteophyte grade. OA patients were defined as having radiographic knee OA of KL grade ≥ 2 in at least 1 knee. Controls were defined as having neither radiographic hip OA nor knee OA, as indicated by KL grades of 0 for both hips and both knees. The grading of more severely affected knee in each patient was used for data analysis.
Statistical analysis was conducted using the statistical package for social sciences (SPSS) software, version 16.0 for Windows (SPSS Inc., Chicago, IL, USA). Demographic data between patients and controls were compared by Chi-square tests and unpaired Student’s t-tests, where appropriate. Comparisons between KL subgroups were performed using one-way analysis of variance. Pearson’s correlation coefficient was employed to determine correlations between periostin values and radiographic severity. Standardized -regression coefficients and their significance in a multivariate linear regression analysis were performed to identify the correlation between the highest quartile level of SF periostin and KL grade. Data were expressed as a mean ± standard error of the mean. P-values < 0.05 were considered significant.
3. Results 3.1. Baseline clinical characteristics The baseline clinical characteristics of the subjects are illustrated in Table 1. Data were normally distributed. There were no statistically significant differences in the ages or gender ratios (male/female) between OA patients and controls. Fig. 1 reveals that there has been a slight increase in plasma periostin of OA patients when compared with that of controls; however, no significant differences were observed (149.7 ± 8.9 ng/ml vs. 145.7 ± 4.8 ng/ml, P > 0.05). SF periostin was approximately two-fold greater than in paired plasma samples of OA patients (278.0 ± 23.3 ng/ml vs. 149.7 ± 8.9 ng/ml, P < 0.001). SF periostin was positively correlated with plasma periostin (r = 0.368, P < 0.001) (Fig. 2A).
2.2. Laboratory methods Following a 12-h overnight fast, venous blood samples were collected into ethylenediamine tetraacetic acid tubes, centrifuged, and stored immediately at −80 ◦ C for later measurement. SF was aspirated from the affected knee of OA patients using sterile knee puncture just prior to surgery when the arthroscopy or total knee arthroplasty was performed. The specimen was then centrifuged to remove cells and joint debris and then stored at −80 ◦ C until analysis. Plasma and SF periostin levels were analyzed using sandwich enzyme-linked immunosorbent assay kit according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA). The antibodies specific for periostin generated by the entire immunogen were utilized. Recombinant human periostin was used to generate a standard curve. The intra- and inter-assay coefficients
Fig. 1. Periostin levels in plasma and synovial fluid of OA patients and healthy controls.
354
S. Honsawek et al. / Joint Bone Spine 82 (2015) 352–355
Fig. 2. Correlations between plasma and synovial fluid periostin and radiographic severity in osteoarthritis (OA) patients. A. Plasma periostin was correlated with synovial fluid periostin in OA patients. B. Plasma periostin was correlated with radiographic severity in OA patients. C. Synovial fluid periostin was correlated with radiographic severity in OA patients.
3.2. Association between plasma and SF periostin and OA severity On the basis of the radiographic KL classification, OA patients were categorized into 3 subgroups with regard to OA grading (KL grade 2: 28; KL grade 3: 28; KL grade 4: 34). Plasma and SF periostin in knee OA patients with different KL subgroups are demonstrated in Table 2. Knee OA patients with higher radiographic severity had significantly higher periostin values in both plasma and SF (P < 0.001). Furthermore, the relationships between plasma and SF periostin with the radiographic severity of knee OA were determined. Plasma periostin was directly correlated with the radiographic severity of knee OA (r = 0.537, P < 0.001) (Fig. 2B). SF periostin of OA patients were also positively associated with KL grade (r = 0.427, P < 0.001) (Fig. 2C). Subsequent analysis evidenced that SF periostin was significantly associated with osteophyte severity (r = 0.335, P = 0.005) and JSN severity (r = 0.311, P = 0.02). Particularly, when SF periostin was divided into quartiles with the highest quartile corresponding to level > 389.4 ng/ml, we found that the highest quartile level of SF periostin was correlated with KL grade of OA (r = 0.421, P < 0.001). Multivariate regression analysis demonstrated that the correlation between the highest quartile level of SF periostin and KL grade was still significant after adjusting for age ( = 0.461, P = 0.01). 4. Discussion Periostin is a vitamin K-dependent glutamate-containing matricellular protein, originally isolated from a mouse osteoblast cell line [3,4]. It is encoded by the Postn gene in humans, and to date, TGF-1, 2, and 3, bone morphogenetic protein (BMP)-2 and
4, vascular endothelial growth factor, connective growth factor 2, vitamin K, valsartan (an angiotensin II antagonist), and interleukin (IL)-3, 4, 6, and 13 have been documented to activate periostin expression in a cell-specific context [14]. Periostin is an extracellular matrix (ECM) protein involved in regulating intercellular adhesion via an interaction with other ECM proteins, including collagen, fibronectin, tenascin-C, integrin, and periostin itself [15,16]. Recently, periostin expression is found to be positively correlated with the fibrosis grade in bone marrow fibrosis [17]. Previous studies have shown that serum periostin was elevated in breast cancer patients with bone metastases [18] and positively correlated with bone periostin mRNA expression in breast cancer bone metastases [19]. Circulating periostin was also positively correlated with total alkaline phosphatase in post-menopausal women with low bone mass [20] and positively associated with incident fracture risk [21]. The present study has demonstrated, for the first time, that periostin was detectable in both plasma and SF derived from patients with primary knee OA. Plasma periostin in OA patients was insignificantly higher than that in healthy controls. In addition, SF periostin in OA patients was substantially increased compared to paired plasma periostin. Furthermore, both plasma and SF periostin were positively correlated with the radiographic severity in knee OA patients. In this study, we found that periostin levels of OA patients were significantly higher in SF than those observed in paired plasma samples. Moreover, advanced knee OA patients exhibited higher periostin levels compared with early knee OA patients. Our results suggest that there is enhanced local production of periostin in knee OA. In the light of these considerations, several plausible mechanisms could be responsible for the elevation of periostin in knee OA patients. The source of periostin in SF may stem from chondrocytes, osteocytes, and synovial cells in the local tissues (cartilage, meniscus, ligament, tendon, subchondral bone, and inflamed synovium) and extra-articular tissues. The findings observed in this study mirror those of the previous studies that have examined periostin tissue distribution of samples obtained from OA joints [10,11]. Loeser et al. reported that periostin was present in chondrocytes in articular cartilage, meniscus, and growth plate, as well as osteoblasts in the periosteum during the OA development [10]. This also accords with Chou’s findings which showed the presence of periostin expression in subchondral bone osteoblasts of OA knees [11]. Another possible explanation for high periostin might be attributed to the increased periostin synthesis, which exceeded its clearance. Our observation indicates a substantially increased production in the systemic and local expression of periostin in patients with advanced knee OA. Additionally, other extraskeletal tissues probably play a part in plasma periostin elevation. High expressions of periostin in adult skin, kidney, liver, and other gastro-intestinal organs have been previously reported [22]. It should be emphasized that there were significant positive correlations between plasma and SF periostin and the radiographic severity of OA. These findings are in line with our previous studies of BMP-7, basic fibroblast growth factor, and connective tissue growth factor in primary knee OA [23–25]. These observations
Table 2 Plasma and synovial fluid periostin in osteoarthritis patients. P-values for differences among Kellgren and Lawrence subgroups.
n Age (years) Gender (M/F) BMI (kg/m2 ) SF periostin (ng/ml) Plasma periostin (ng/ml)
Total
KL grade 2
KL grade 3
KL grade 4
P
90 70.0 ± 0.8 16/74 26.8 ± 0.4 278.0 ± 23.3 149.7 ± 8.9
28 68.8 ± 1.5 5/23 26.8 ± 0.6 171.1 ± 20.8 99.6 ± 7.7
28 68.8 ± 1.2 7/21 26.1 ± 0.7 242.1 ± 29.5 129.6 ± 11.3
34 71.9 ± 1.5 4/30 27.4 ± 0.7 395.7 ± 47.6 207.5 ± 16.4
0.2 0.5 0.4 < 0.001 < 0.001
BMI: body mass index; KL: Kellgren and Lawrence; SF: synovial fluid.
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could be applied for possibly using as biomarkers to determine the OA severity. A number of caveats need to be emphasized regarding the present study. First, the study is cross-sectional in design with relatively small numbers of patients and controls. As a result, cause-and-effect relationships cannot be concluded and require prospective longitudinal studies to elucidate any relationships. However, with a small sample size, caution must be applied, as the findings might not be transferable to other populations. Second, SF samples were neither collected from healthy controls nor obtained from inflammatory arthritis patients leading to no direct comparison between SF periostin of controls and that of OA patients. Instead, circulating periostin reflects the systemic nature of the condition. In future studies, this may be overcome by collecting SF samples from patients with traumatic knee and/or inflammatory arthritis as controls. Additional immunohistochemical analysis of osteoarthritic cartilage and synovial tissues could render valuable information on the pathophysiologic role of periostin in OA development. Taken together, the current study demonstrated that SF periostin was markedly higher with respect to paired plasma in knee OA patients. There was a significant positive correlation between plasma and SF periostin. Moreover, plasma and SF periostin was positively correlated with the radiographic severity in knee OA. Although underlining mechanisms of this association and their cause-and-effect relationships are not entirely elucidated, there is abundant room for further research regarding the potential role of periostin in the pathogenesis of chronic degenerative joint disorders. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This research has been supported by the Ratchadaphiseksomphot Endowment Fund 2013 of Chulalongkorn University (CU-56-341-AS) and the Ratchadapiseksompotch Fund (RA55/22). The authors commemorate the 100th Anniversary of the King Chulalongkorn Memorial Hospital. We would like to thank Natthaphon Saetan for technical assistance. References [1] Felson DT, Zhang Y, Hannan MT, et al. The incidence and natural history of knee osteoarthritis in the elderly. Arthritis Rheum 1995;38:1500–5. [2] Pelletier JP, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets. Arthritis Rheum 2001;44:1237–47.
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