Cyclic phosphatidic acid relieves osteoarthritis symptoms
- Mari Gotoh†1,
- Aya Nagano†1,
- Ryoko Tsukahara1, 2, 3,
- Hiromu Murofushi2,
- Toshiro Morohoshi3,
- Kazuyuki Otsuka4 and
- Kimiko Murakami-Murofushi1Email author
© Gotoh et al.; licensee BioMed Central Ltd. 2014
Received: 31 January 2014
Accepted: 7 August 2014
Published: 14 August 2014
Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. Natural cPA and its chemically stabilized cPA derivative, 2-carba-cPA (2ccPA), inhibit chronic and acute inflammation, and 2ccPA attenuates neuropathic pain. Osteoarthritis (OA) is a degenerative disease frequently associated with symptoms such as inflammation and joint pain. Because 2ccPA has obvious antinociceptive activity, we hypothesized that 2ccPA might relieve the pain caused by OA. We aimed to characterize the effects of 2ccPA on the pathogenesis of OA induced by total meniscectomy in the rabbit knee joint.
Intra-articular injection of 2ccPA (twice a week for 42 days) significantly reduced pain and articular swelling. Histopathology showed that 2ccPA suppressed cartilage degeneration in OA. We also examined the effects of 2ccPA on the inflammatory and catabolic responses of human OA synoviocytes and chondrosarcoma SW1353 cells in vitro. 2ccPA stimulated synthesis of hyaluronic acid and suppressed production of the metalloproteinases MMP-1, -3, and -13. However, it had no effect on the production of interleukin (IL)-6, an inflammatory cytokine. The suppressive effect of 2ccPA on MMP-1 and -3 production in synoviocytes and on MMP-13 production in SW1353 cells was not mediated by the lysophosphatidic acid receptor, LPA1 receptor (LPA1R).
Our results suggest that 2ccPA significantly reduces the pain response to OA by inducing hyaluronic acid production and suppressing MMP-1, -3, and -13 production in synoviocytes and chondrocytes.
KeywordsCyclic phosphatidic acid 2-carba-cyclic phosphatidic acid Osteoarthritis Total meniscectomy Synoviocytes Chondrocytes Chonrosarcoma SW1353 cells Hyaluronic acid Metalloproteinase
Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator that was originally isolated from the myxoamoebae of a true slime mold, Physarum polycephalum, in 1992 . Later, cPA was found in mammalian tissues [2, 3]. cPA has distinct biological activities; it inhibits autotaxin [4–6], suppresses cancer cell invasion and metastasis [4, 7], attenuates ischemia-induced delayed neuronal cell death in rat hippocampal CA1 regions [8, 9], and inhibits chronic and acute inflammation-induced C-fiber stimulation, and attenuates neuropathic pain . Therefore, cPA is a promising candidate of therapeutic agent for pain.
Osteoarthritis (OA) is a degenerative disease frequently associated with inflammation, joint pain, swelling, and stiffness, leading to significant functional impairment and disability at articular joints . OA is caused by characteristic structural alterations of the joint, including focal degradation of articular cartilage, subchondral bone alterations, and synovitis [14–16]. OA joints are the biological site of inflammation and catabolism. Synovial inflammation likely contributes to the dysregulation of cartilage homeostasis, favoring an imbalance between the catabolic and anabolic activities of chondrocytes in remodeling the cartilage extracellular matrix (ECM) [17–19].
2ccPA inhibites chronic and acute inflammation-induced C-fiber stimulation and attenuates neuropathic pain ; we then assessed the ability of 2ccPA to relieve OA-related pain in a rabbit model in vivo. In addition, to investigate the molecular mechanisms of 2ccPA in OA-related cells, we examined the effects of 2ccPA on the inflammatory and catabolic responses of human OA synoviocytes and chondrosarcoma SW1353 cells in vitro.
Results and discussion
Inflammatory swelling assessment
Figure 2C shows the swelling ratio on Day 42 for the vehicle- and 2ccPA-treated groups. The swelling ratio was 11.87 ± 3.54% in the vehicle-treated group and 0.45 ± 1.78% in the 2ccPA-treated group. These results suggest 2ccPA exerts a constraining influence on the swelling resulting from OA inflammation in the rabbit meniscectomy model. Therefore, we believe 2ccPA is effective for relieving pain and reducing inflammation caused by OA.
HE-staining revealed that chondrocyte disorganization in the medial condyle of the femur was significant in the vehicle-treated group. However, chondrocytes were much more ordered in the 2ccPA-treated group than in the vehicle-treated group. Cluster formation was also attenuated in the 2ccPA-treated versus the vehicle-treated group. Saf-O staining showed a significant loss of stainable proteoglycan in the vehicle-treated group. However, proteoglycan loss was imperceptible in the 2ccPA-treated group. These results suggest 2ccPA relieved chondropathy and cartilage degeneration; in addition, the loss of stainable proteoglycan, chondrocyte disorganization, and cluster formation were considerably lower in the 2ccPA-treated group.
Like the medial condyle of the femur, chondrocyte disorganization and cluster formation in the medial condyle of the tibia were significant in the vehicle-treated group; however, these morphologic changes were reduced in the 2ccPA-treated group. Saf-O staining showed that the loss of stainable proteoglycan in the vehicle-treated group was attenuated by 2ccPA.
Other morphologic changes in the medial condyle of the femur and tibia, including loss of the superficial layer, cartilage erosion, and fibrillation and/or fissures were less substantial in the 2ccPA-treated group than in the vehicle-treated group. Due to mechanical friction during meniscectomy in the rabbit OA model, serious cartilage degeneration occurs within a week after surgery and progresses gradually [20, 21]. In this study, cartilage degradation induced by meniscectomy was suppressed in the 2ccPA-treated group, suggesting that in the rabbit OA model, 2ccPA may influence pain and catabolic regulation; therefore, 2ccPA provided chondroprotective effects during OA progression.
Measurement of hyaluronic acid in synoviocyte and chondrosarcoma SW1353 cells in vitro
Synovial inflammation likely contributes to dysregulation of cartilage homeostasis, favoring an imbalance between the catabolic and anabolic activities of the chondrocyte in remodeling cartilage extra cellular matrix (ECM). Cartilage tissue is altered and damaged by inflammatory mediators and degradative enzymes in the synovial fluid of OA [17–19] and the proportion of high-molecular-weight hyaluronic acid decrease while the inflammatory cytokines and ECM-degrading enzyme MMPs increase [17, 19, 20, 22]. The pain and swelling scores in this study demonstrated the antinociceptive and anti-inflammatory effects of 2ccPA administration. Therefore, we investigated the chondroprotective effects of 2ccPA in vitro by using synoviocytes and chondrosarcoma cell line SW1353, an in vitro model for primary chondrocytes in OA.
Inflammatory cytokine expression and secretion from IL-1β-stimulated synoviocytes and chondrosarcoma SW1353 cells
It has been reported that the production of inflammatory cytokine IL-6 increases in OA joints and triggers catabolic reactions [16, 17]. Then, we performed ELISA to measure the production of IL-6 by both cells in culture media. The amount of IL-6 produced by IL-1β–stimulated synoviocytes and SW1353 cells increased to 12.9 ± 0.2 ng/mL and 0.254 ± 0.011 ng/mL, respectively (Figure 5). On synoviocytes, NSAIDs treatment reduced the inductive effect while 10 μM 2ccPA had no effect on IL-6 production (Figure 5A). On the other hand, NSAIDs and 2ccPA showed negligible effects on IL-6 production in SW1353 cells (Figure 5B).
MMP-1, -3, and -13 expression and secretion from IL-1β-stimulated synoviocytes and chondrosarcoma SW1353 cells
MMP-1 production increased to 4.9 ± 0.2 ng/mL and 0.60 ± 0.06 ng/mL with IL-1β stimulation in synoviocytes and SW1353 cells, respectively. In synoviocytes, MMP-1 secretion was suppressed dose-dependently by 3 and 10 μM 2ccPA. In SW1353 cells, MMP-1 secretion was suppressed by 1 and 3 μM 2ccPA but dose-dependency was poor. These results suggest that to elicit suppressive function of 2ccPA, it is necessary to choose a certain dose depending on cell types.
MMP-3 production increased to 68.7 ± 4.8 ng/mL and 20.3 ± 1.3 ng/mL with IL-1β stimulation in synoviocytes and SW1353 cells, respectively. MMP-3 production was much higher than that of MMP-1 and -13. In synoviocytes, MMP-3 secretion was suppressed dose-dependently by 2ccPA. In contrast, MMP-3 secretion in SW1353 cells was not affected by 2ccPA treatment.MMP-13 production increased to 0.81 ± 0.18 ng/mL with IL-1β stimulation in SW1353 cells, and the MMP-13 secretion was suppressed by 2ccPA. Although expression of MMP-13 mRNA was observed in synoviocytes (Figure 6A), the amount of protein was not detected.
There are some reports that MMP-1 and -13 degrade collagen and are expressed in synoviocytes and chondrocytes, respectively [18, 19]. MMP-3 degrades non-collagen matrix components of the joint and contributes to proteoglycan loss [18, 19]; its expression is high in comparison to other MMPs . Our results are consistent with these reports. We suggest that the obvious suppressive effects of 2ccPA on MMP-1 and -3 production in synoviocytes, and on MMP-13 in chondrosarcoma SW1353 cells may offer the appropriate evidence to explain the chondroprotective effect we observed in vivo (Figure 3).
LPA1 receptor function and the suppressive effect of 2ccPA in OA
Previous studies have suggested that hyaluronic acid reduces MMPs expression in synovial fluid [21, 23–25]. Therefore, MMP-1, -3, and -13 suppression by 2ccPA might be due to a 2ccPA-mediated increase in hyaluronic acid synthesis. Further studies are expected to clarify how 2ccPA modulates MMPs expression.
OA causes morbidity, activity limitation, physical disability, excess health care utilization, and reduces health-related quality of life (QOL), especially in people over 60 years old. However, OA management is now limited to the symptomatic treatment of pain and inflammation without reducing joint destruction, which leads to inevitable referral for total joint replacement. Given this unresolved therapeutic need, many challenges remain in the discovery and development of disease-modifying OA drugs (DMOADs) aimed at slowing, halting, or reversing the progression of structural damage of the articular cartilage [29, 30]. We believe 2ccPA is a promising DMOAD candidate. The pain-relieving mechanisms of 2ccPA in the pathogenesis of OA are now under investigation.
Our results suggest 2ccPA significantly reduces the pain response to OA by inducing hyaluronic acid production and suppressing MMP-1, -3, and -13 production in synoviocytes and chondrocytes. These activities might protect chondrocytes from destruction. As a result, pain and inflammatory swelling are relieved. It is strongly suggested that 2ccPA is a promising candidate of DMOAD.
Materials and methods
We used chemically synthesized 2-carba-cPA 18:1 (2ccPA) [4, 7]. In the in vivo experiments, 2ccPA was dissolved in saline, and saline was used as vehicle. In the in vitro experiments, 2ccPA was dissolved in phosphate-buffered saline (PBS) containing 0.1% fatty acid-free bovine serum albumin (BSA), and PBS containing 0.1% BSA solution was used as vehicle.
A rabbit model was used to investigate the effects of 2ccPA on the pathogenesis of OA. The design of this animal study was approved by the ethics committee of KAC Corporation (Ethics approval number: 12–0218), a contract research organization (Shiga Japan). All animals were purchased from KITAYAMA LABES Co., Ltd. (Japan), and all animal experiments were performed by KAC Corporation using 11- or 12-week-old male SPF New Zealand white rabbits (n = 12, body weight 2.1–2.3 kg). Animals were anesthetized with intravenous (i.v.) pentobarbital (32.4 mg/kg) prior to 1–4% isoflurane followed by subcutaneous infusion of lidocaine (approx. 3 mL) during surgery.
The meniscus of the right leg was totally removed. Briefly, the boundary between the patellar ligament and articular capsule of the right hind leg and the lateral-collateral ligament were dissected. Then, the articular capsule was removed to expose the interior meniscus, and the meniscus was completely removed. Following total meniscectomy of the right knee joint, the rabbits were randomly divided into vehicle- or 2ccPA-treated group. Intra-articular treatment was initiated 7 days after surgery. Vehicle (200 μL saline; Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) or 50 μg/mL 2ccPA (200 μL) was injected into the joint cavity twice per week over five consecutive weeks (at 7, 11, 14, 18, 21, 25, 28, 32, 35, and 39 days after surgery). The animals were individually caged (48.5 × 30 × 35 cm3), received tap water ad libitum, and were fed a standard diet (CR-3, 150 g/day; CLEA, Japan Inc.) throughout the trials. During the experiments, the SPF room temperature was 18°C with unidirectional airflow systems; lighting was provided for 12 h daily (7:00 AM–19:00 PM). The experimental schedule is shown in Figure 2A.
Measurement of pain
Measurement of swelling
Histopathological assessment of OA
At 42 days after meniscectomy, the rabbits were euthanized by exsanguination immediately after pentobarbital (32.4 mg/kg) administration. The femoral condyle and tibial plateau were resected and immediately fixed in 10% formalin buffer. After decalcification with ethylenediaminetetraacetic acid (EDTA), the samples were cut into 4-μm sections, then stained with HE for general morphology, or Saf-O for proteoglycan, and were observed using a 2.0 MP microscope (H-Micron, Hyogo, Japan, Figure 3A) and an optical microscope BX51TF (OLYMPUS, Tokyo, Japan, Figure 3B, 3C1 and 3C2). Images of 3–6 microscopic fields were incorporated into one image for Figure 3A.
Cell culture and measurement of hyaluronic acid, IL-6, and MMP-1, -3, and -13 produced by synoviocytes and chondrosarcoma SW1353 cells
All procedures were specifically approved by the ethics committee of Ochanomizu University (Ethics approval number: 24–12) and the National Institute of Biomedical Innovation, Japanese Collection of Research Bioresources Cell Bank (previously Health Science Research Resources Bank, Ethics approval number: 36); the patient gave full written informed consent for tissue donation. Synovial tissue was excised from the knee joint of a 60-year-old female patient with OA during replacement surgery. The patient-derived synoviocytes (Japanese Collection of Research Bioresources Cell Bank, HT91989516, Lot. 07042011) were plated at 1.5 × 104 cells/well with Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS, Life Technologies Corporation, CB) on 12-well plates and incubated overnight at 37°C in humidified 95% air and 5% CO2 atmosphere. Chondrosarcoma SW1353 cells were obtained from American Type Culture Collection (ATCC) (no. HTB-94) and were plated at 1.5 × 104 cells/well with DMEM containing 10% FBS on 12-well plates and incubated overnight as well as synoviocytes. The medium was replaced with serum-free DMEM, and the cells were serum-starved for 16 h. To measure hyaluronic acid, 2ccPA was added after serum starvation at final concentrations of 1, 3, and 10 μM and incubated. The culture media were collected at 24, 48, and 72 h, then the concentration of hyaluronic acid was measured by ELISA (R&D Systems, Inc. MN).
To measure IL-6 and MMPs, the cells were treated with either 10 μM of ibuprofen (IBF), diclofenac sodium (DCF) (Wako Pure Chemical Industries, Ltd. Osaka, Japan), or various concentrations of 2ccPA for 24 h in the presence of 10 ng/mL of IL-1β (R&D Systems, Inc.). Culture media were collected at 24 h and the concentrations of IL-6 and MMPs were measured by ELISA kit according to the manufacturer’s instructions (RayBiotech, Inc., GA). For treatment with Ki16425 (Cayman Chemicals, MI), the selective antagonist for LPA1R and LPA3R, the cells were plated at 1.5 × 104 cells/well on 12-well plates and incubated for 30 min with 10 μM of Ki16425 before adding 2ccPA (1, 3, or 10 μM) in the presence of 10 ng/mL IL-1β. Culture media were collected at 24 h and the concentration of each MMP was measured by ELISA kit.
Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR)
To quantitate the mRNA levels of inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α), MMPs (MMP-1, -3, -13), and LPA receptors, real-time RT-PCR was performed with SYBR Premix Ex Taq (Takara Bio, Inc., Shiga, Japan). Total RNA was extracted from cultured synoviocytes and SW1353 cells using ISOGEN reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. cDNA was synthesized with the PrimeScript RT reagent kit (Takara Bio, Inc.). mRNA levels were quantified on a lightCycler 96 system (Roche) instrument. Gene-specific primer sets for IL-1β, IL-6, IL-8, MMP-3, and -13 were described in . Gene-specific primer sets for TNF-α and MMP-1 were described in . Gene-specific primer sets for LPA1R, LPA4R, LPA5R, LPA6R, and p2y10 were described in . The following primer sets were used: GAPDH, 5′-GTGAAGGTCGGAGTCAACG-3′ (F) and 5′-TGAGGTCAATGAAGGGGTC-3′ (R); LPA2R, 5′-GAGGCCAACTCACTGGTCA-3′ (F) and 5′-GGCGCATCTCAGCATCTC-3′ (R); LPA3R, 5′-GAAGCTAATGAAGACGGTGATGA-3′ (F) and 5′-AGCAGGAACCACCTTTTCAC-3′ (R); and GPR87, 5′-AAATCCAGCAGGCAATTCAT-3′ (F) and 5′-CCCTGATGCTCTGGTTATGTT-3′ (R).
The data were calculated based on the Cq values, and the expression of each gene was normalized to GAPDH.
All values are reported as means ± standard error. The data were analyzed using the Student’s t-test. A P value less than 0.05 was considered statistically significant.
Bovine serum albumin
Cyclic phosphatidic acid
Dulbecco’s modified Eagle medium
Disease-modifying osteoarthritis drugs
Enzyme-linked immunosorbent assay
Fetal bovine serum
Hematoxylin and eosin
Liquid chromatography-mass spectroscopy
Lysophosphatidic acid receptor
2-carba-cyclic phosphatidic acid.
We are very grateful to Dr. Hisako AKIYAMA (Brain Science Institute, RIKEN) for experimental support and to Mr. Noboru YAMAWAKI (Kyocera Medical Corporation) for kind and helpful advice.
This work was supported in part by the Princess Takamatsu Cancer Research Fund, the Rational Evolutionary Design of Advanced Biomolecules (REDS3) Project, the Central Saitama Area in the Program for Fostering Regional Innovation (City Area Type), and a Grant-in-Aid for Scientific Research (KAKENHI, No. 26860144) from the Ministry of Education, Culture, Sports, Science and Technology.
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