Article pubs.acs.org/molecularpharmaceutics
Prediction of Antiarthritic Drug Efficacies by Monitoring Active Matrix Metalloproteinase‑3 (MMP-3) Levels in Collagen-Induced Arthritic Mice Using the MMP‑3 Probe Aeju Lee,†,‡,# Kyeongsoon Park,§,# Sung-Jae Choi,∥ Dong-Hyun Seo,⊥ Kwangmeyung Kim,† Han Sung Kim,⊥ Kuiwon Choi,† Ick Chan Kwon,† Soo-Young Yoon,‡ and Inchan Youn*,† †
Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-Dong, Seongbuk-gu, Seoul 136-791, South Korea ‡ Department of Laboratory Medicine, College of Medicine, Korea University Guro Hospital, Seoul 152-703, South Korea § Division of Bio-imaging, Chuncheon Center, Korea Basic Science Institute, 192-1, Hyoja 2-dong, Chuncheon, Gangwon-do 200-701, South Korea ∥ Department of Internal Medicine, Division of Rheumatology, College of Medicine, Korea University Ansan Hospital, Ansan, Kyungki-do 425-707, South Korea ⊥ Department of Biomedical Engineering, Institute of Medical Engineering, and Yonsei-Fraunhofer Medical Device Lab, Yonsei University, Seoul 120-749, South Korea S Supporting Information *
ABSTRACT: Active matrix metalloproteinase-3 (MMP-3) is a prognostic marker of rheumatoid arthritis (RA). We recently developed an MMP-3 probe that can specifically detect the active form of MMP-3. The aim of this study was to investigate whether detection and monitoring of active MMP-3 could be useful to predict therapeutic drug responses in a collageninduced arthritis (CIA) model. During the period of treatment with drugs such as methotrexate (MTX) or infliximab (IFX), MMP-3 mRNA and protein levels were correlated with fluorescence signals in arthritic joint tissues and in the serum of CIA mice. Also, bone volume density and erosion in the knee joints and the paws of CIA mice were measured with microcomputed tomography (micro-CT), X-ray, and histology to confirm drug responses. In joint tissues and serum of CIA mice, strong fluorescence signals induced by the action of active MMP-3 were significantly decreased when drugs were applied. The decrease in RA scores in drug-treated CIA mice led to fluorescence reductions, mainly as a result of down-regulation of MMP-3 mRNA or protein. The micro-CT, X-ray, and histology results clearly showed marked decreases in bone and cartilage destruction, which were consistent with the reduction of fluorescence by down-regulation of active MMP-3 in drug-treated CIA mice. We suggest that the MMP-3 diagnostic kit could be used to detect and monitor the active form of MMP-3 in CIA mice serum during a treatment course and thereby used to predict the drug response or resistance to RA therapies at an earlier stage. We hope that monitoring of active MMP-3 levels in arthritis patients using the MMP-3 diagnostic kit will be a promising tool for drug discovery, drug development, and monitoring of drug responses in RA therapy. KEYWORDS: rheumatoid arthritis, active MMP-3, therapeutic response monitoring, methotrexate, infliximab
1. INTRODUCTION Biological changes in biomarkers at the molecular and cellular levels often occur before any signs of structural, functional, or anatomic changes become evident. Thus, imaging and detection methods for specific biomarkers are extremely interesting because biomarkers can provide additional information on disease status, thereby facilitating diagnosis, drug discovery and development, and prediction of drug responses. Rheumatoid arthritis (RA) is a systemic, chronic, and autoimmune inflammatory disease characterized by synovial inflammation, hyperplasia of synovial tissues, and destruction of bone and cartilage.1 As RA prognostic markers, C-reactive protein (CRP), rheumatoid © 2014 American Chemical Society
factor (RF), erythrocyte sedimentation rate (ESR), and anticyclic citrullinated peptide (Anti-CCP) are currently used.2,3 Even though the above markers are well-known as good parameters for prognostic purposes, they have limitations in RA prognosis. For example, detection of CRP and ESR is not necessarily reflective of RA because their expression levels may be affected by other stimuli of the acute phase response. Also, Received: Revised: Accepted: Published: 1450
October 24, 2013 January 17, 2014 March 27, 2014 March 27, 2014 dx.doi.org/10.1021/mp400622q | Mol. Pharmaceutics 2014, 11, 1450−1458
Molecular Pharmaceutics
Article
2. MATERIALS AND METHODS 2.1. Materials. The MMP-3 peptide substrate [Gly-ValPro-Leu-Ser(tBu)-Leu-Thr(tBu)-Met-Gly-Lys-(Boc)-Gly-Gly; Ser-Leu is the cleavage site] was synthesized using standard solid-phase peptide chemistry. The MMP-3-activatable darkquenched fluorogenic peptide Cy5.5-Gly-Val-Pro-Leu-Ser-LeuThr-Met-Gly-Lys(BHQ-3)-Gly-Gly (m/z 2326) was prepared by chemical conjugation of amine groups at the N-terminus and a lysine in the MMP-3 peptide with Cy5.5 NHS ester (GE Healthcare, Piscataway, NJ, USA) and BHQ-3 NHS ester (Biosearch Technologies, Inc., Novato, CA, USA), respectively, as previously described.14,17,18 The MMP-3 probe for detection of active MMP-3 was synthesized by covalently coupling glycol chitosan (MW = 250 kDa, Sigma, St. Louis, MO, USA) with the MMP-3 fluorogenic peptide in the presence of 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide hydrochloride (EDC) (Sigma) and N-hydroxysulfosuccinimide (Sulfo-NHS) (Pierce, Rockford, IL, USA).14,19,20 The determined molecular weight of the MMP-3 probe was 524 235 Da. Recombinant human MMPs such as MMP-2, -3, -7, -9, and -13 (R&D Systems, Minneapolis, MN, USA) were used to verify the enzyme responses of the MMP-3 probe. 2.2. Development and Scoring of Collagen-Induced Arthritic Mice. In vivo animal studies were performed according to the guidelines of the Institutional Animal Care Committee of the Korea Institute of Science and Technology (2011−01−017). The CIA model was established according to published protocols.21 Briefly, bovine type-II collagen (Sigma) was dissolved at a concentration of 2 mg/mL in 0.1 M acetic acid by stirring for 4 h at 4 °C. A 1:1 (v/v) emulsion consisting of collagen and complete Freund’s adjuvant (CFA) (Sigma) was prepared, and mice were immunized via intradermal injection of the emulsion (50 μL) into the tail. Two weeks after the primary immunization, a booster immunization was performed. The incidence of arthritis in the CIA-susceptible strain of mice was very high within 6 weeks of the first immunization, and 95% of the mice developed severe arthritis. The clinical severity of arthritis in each paw was quantified according to a graded scale from 0 to 4, as previously described.21 A mean arthritis score was determined by totaling the scores of the four paws of the mice and dividing the result by the total number of mice in each group.16,21 2.3. MTX and IFX Administration in CIA Mice. CIA mice were divided into three groups according to the treatments as follows: (1) normal saline (control group; n = 10); (2) 20 mg/kg MTX (JW Pharmaceutical, Korea) (n = 10); (3) 10 mg/kg IFX (Schering-Plough Labo NV, Brussels, Belgium) (n = 10). MTX and IFX were administered once per week (for a total of four injections) between 3 and 6 weeks after RA induction. 2.4. In Vivo NIRF Imaging of Active MMP-3 in CIA Mouse Knee Joints. CIA and normal mice were intravenously injected with the MMP-3 probe (10 μg/100 μL in PBS, pH 7.4) at 3, 5, and 7 weeks after the first immunization (n = 10/group). At 1 h postinjection with the MMP-3 probe, mice were anaesthetized with isoflurane (1% w/v in 2 L of oxygen), and near-IR fluorescence (NIRF) images of the whole body and the hind limbs were obtained using an eXplore Optix system [Advanced Research Technologies Inc. (ART), Montreal, Canada). The laser power and count time settings were optimized at 13 μW and 0.3 s per point, respectively. The total NIRF intensity in the hind limbs (360 mm2) was calculated
RF is nonspecific and may be present in healthy people or in those with other autoimmune diseases. Anti-CCP exhibits a high specificity for RA diagnosis but has a relatively low sensitivity. Therefore, clinicians are still looking for new methods that can accurately detect biomarkers with high sensitivity and specificity for the early diagnosis of RA. Matrix metalloproteinase-3 (MMP-3) is a proteolytic enzyme that plays a pivotal role in joint destruction. MMP-3 has increasingly gained interest as an important biomarker for RA diagnosis in clinics because elevated MMP-3 is closely involved in RA progression4,5 and is associated with the serum and synovium of RA patients.6−8 Recently, Mamehara et al.9 reported that MMP-3 in serum of RA patients can be a useful marker for the prediction of joint destruction during the course of treatment. Thus, MMP-3 detection techniques are necessarily required because MMP-3 is an important biomarker and predictor of diagnosis and drug response for RA. It was reported that MMP-3 is locally produced in the inflamed joint and released into the bloodstream, where serum levels of MMP-3 are lowered up to 300−500 times.10,11 In particular, the level of active MMP-3 is much lower than that of pro-MMP-3 in serum. Additionally, the high levels of α2-macroglubulin in serum completely enclose active MMP-3.12 For these reasons, detection of MMP-3, especially in its active form, in serum of RA patients is difficult. Currently, several MMP-3 detection methods are used, such as enzyme-linked immunosorbent assay (ELISA), Western blot, and zymography. However, these methods have limitations for detection of the active forms of MMPs. Although an ELISA kit can detect total MMP-3 in serum at nanomolar levels with high sensitivity, it cannot specifically differentiate between the pro and active forms of MMP-3. Western blotting is a good method for the detection of both pro- and active MMP-3, but the analysis range is limited to several micrograms.13,14 Zymography cannot accurately detect active MMP-3 because pro-MMP-3 can be transformed during the procedures of zymography and electrophoresis, and the MMP-2 transformed from pro- to active form could be detected.15 Our group recently developed an MMP-3 probe that can detect RA activity in vivo as early as 2 weeks after RA induction, before any signs of structural or anatomic changes are detectable with microcomputed tomography (micro-CT), histology, or X-ray.14,16 Also, we demonstrated that the MMP-3 probe can be used as a plate-based diagnostic kit for detection of active MMP-3 levels in mouse serum and can also detect active MMP-3 expression in fibroblast-like synoviocytes in a collagen-induced arthritis (CIA) model. More importantly, the MMP-3 probe and diagnostic kit could differentiate between the pro and active forms of MMP-3 and selectively detect the active form at nanomolar levels with high sensitivity in the serum of CIA mice.14 In this study, we investigated whether the MMP-3 diagnostic kit can predict the disease therapeutic response in a CIA model by monitoring of active MMP-3 levels in the serum of CIA mice for high-throughout drug screening, as the MMP-3 diagnostic kit can more easily, accurately, selectively, and specifically detect active MMP-3 levels in the serum of CIA mice. In order to evaluate whether the MMP-3 probe can be used as a promising tool for the evaluation of RA therapy, active MMP-3 levels were monitored using the MMP-3 probe and optical imaging modalities and correlated with therapeutic responses of antiarthritic drugs such as methotrexate (MTX) and infliximab (IFX) in CIA mice. 1451
dx.doi.org/10.1021/mp400622q | Mol. Pharmaceutics 2014, 11, 1450−1458
Molecular Pharmaceutics
Article
GGC CAT GAG GTC CAC CA-3′. The PCR products were separated by electrophoresis on a 2% agarose gel and visualized by Loading Star (Darwin Bio, Korea). All experiments were performed in triplicate. 2.7. MMP-3 Levels in Knee Joints of CIA Mice Measured by Western Blot. To identify the MMP-3 protein expression and tumor necrosis factor-α (TNF-α) as proinflammatory cytokine with or without drug treatment, CIA mice knee joint specimens (n = 3/group) were dipped in liquid nitrogen. The tissue powder was incubated for 1 h in ice with protein extraction buffer (pH 7.5, 0.2 M NaCl, 5 mM CaCl2, 1% Triton X-100) and then centrifuged at 13 000 rpm for 5 min at 4 °C. The supernatant was saved and assayed for protein concentration using NanoDrop 3300 (Thermo Scientific, Wilmington, DE, USA). Next, 20 μg of protein for each sample was mixed with sample buffer and heated to 100 °C for 10 min. Samples were then loaded onto a 7% SDS-PAGE gel. Gels were transferred to nitrocellulose membranes (Invitrogen, Grand Island, NY, USA), and the membranes were blocked with 5% skim milk in 1× Tris-buffered saline containing Tween 20 (TBST). The membranes were incubated with anti-MMP-3 (1:2000), anti-TNF-α (1:1000), and β-actin (1:5000) antibodies. The membranes were then washed three times with TBST and incubated with a goat anti-mouse horseradish peroxidase (HRP)-linked immunoglobulin G (IgG) antibody (1:5000) for 1 h at room temperature. Bands were detected using enhanced chemiluminescence and developed with X-ray film. 2.8. Micro-CT and X-ray. Micro-CT images of the knee joints of the mice with CIA (n = 5/group) were acquired at 3, 5, and 7 weeks after the first immunization using a SkyScan 1076 micro-CT scanner (SkyScan NV, Kontich, Belgium) at a resolution of 18 μm with the following parameters: 60 kV, 170 mA current, 2360 ms exposure time, and a 0.7° rotation step. For verification of bone destruction, three-dimensional models of the knee joints were reconstructed using SkyScan CT Analyzer version 1.8. The beam-hardening errors were corrected to improve the quality of the micro-CT images. Additionally, flat-field correction before scanning and beamhardening correction during reconstruction were performed to improve the quality of the micro-CT images. The volume of subchondral bone in the knees was also measured from reconstructed images. Also, knee joints of CIA mice (n = 5/group) were radiologically diagnosed at 5 weeks after RA induction. 2.9. Histology. After optical imaging, the CIA mice were sacrificed, and their knee joint tissues were prepared for histological evaluation. The knee joint tissues (n = 3/group) were placed in fixative/decalcifier (Thermo Fisher Scientific) for 7 days, dehydrated with a graded ethanol series, and embedded in paraffin. The specimens were then cut into 7 μm thick sections and stained using the Safranin O/Fast Green method. 2.10. Statistical Analysis. Statistical analyses were performed using ANOVA. P values of
