Matrix Metalloproteinase-10: A Novel Biomarker for Idiopathic Pulmonary Fibrosis

Akihiko Sokai; Tomohiro Handa; Kiminobu Tanizawa; Toru Oga; Kazuko Uno; Tatsuaki Tsuruyama; Takeshi Kubo; Kohei Ikezoe; Yoshinari Nakatsuka; Kazuya Tanimura; Shigeo Muro; Toyohiro Hirai; Sonoko Nagai; Kazuo Chin; Michiaki Mishima


Respiratory Research. 2015;16(120) 

In This Article


Study Population

This was an observational cohort study that enrolled 57 consecutive patients with IPF who visited the Department of Respiratory Medicine at Kyoto University Hospital. The diagnosis of IPF was made according to the diagnostic criteria for IPF.[1] Patients who had active malignant disease or whose forced expiratory volume in one second (FEV1)/FVC was < 70 % were excluded. The day of serum sampling was set as the baseline, and the patients were prospectively evaluated for clinical deterioration every 6 months from the baseline. Fifteen age- and sex-matched healthy volunteers without lung disease were recruited as healthy controls. Twenty patients with chronic obstructive pulmonary disease (COPD) were also recruited as disease controls. A diagnosis of COPD was made according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria.[19] The present study was approved by the Ethics Committee of Kyoto University, and informed consent was obtained from all patients.

Physiological Measurements and Bronchoalveolar Lavage

The pulmonary function test (PFT),[20] the 6-minute walk test (6MWT),[21] and the arterial blood gas (ABG) while breathing room air and bronchoalveolar lavage (BAL)[22,23] were performed according to published guidelines.

MMP Analysis

Serum and BALF samples were centrifuged immediately following sampling, and the supernatants were stored at -80 °C until needed for analysis. Serum MMP-1,-2, -3, -7, -8, -9, -10, -12, and -13 and BALF MMP-7 and -10 were analyzed using the Bio-Plex Pro Human MMP Panel (Bio-Rad Laboratories, Hercules, CA, USA). Serum samples were quantified according to the manufacturer's instructions using the Bio-Plex 200, a multiplex cytokine array system. BALF samples and their standards were diluted using bovine serum albumin-phosphate-buffered saline.

Outcome Evaluations

The outcome evaluations included clinical deterioration within 6 months from baseline and overall survival. Clinical deterioration was defined as a composite outcome consisting of admission due to respiratory failure, death, ≥ 10 % decline in the percentage of predicted FVC (%FVC), or ≥ 15 % decline in the percentage of predicted DLCO (%DLCO). We analyzed whether MMPs were associated with clinical deterioration and overall survival in patients with IPF.

MMP-10 Immunostaining

Lung tissue specimens obtained from 4 patients with IPF were used for immunohistochemical staining, which was performed on 5-μm paraffin-embedded sections of lung tissue to identify MMP-10-expressing sites using the standard streptavidin-biotin-peroxidase complex method. For antigen retrieval, slides were immersed in citrate buffer and heated in a microwave oven. A rabbit anti-human MMP-10 antibody (Abcam, Cambridge, MA, USA) was applied as a primary monoclonal antibody (1:500 dilution). Positive staining was visualized using 3,3'-Diaminobenzidine. A pathologist evaluated the localization of MMP-10 expression in IPF lung tissue and compared the results with control lung specimens, which were obtained from normal areas distant from the lesions caused by surgically diagnosed organizing pneumonia (OP).

Statistical Analyses

Statistical analyses were performed using JMP 10.0 (SAS Institute Inc., Cary, NC, USA). Comparisons were performed using Fisher's test, the Mann-Whitney U test, or the Steel-Dwass test, where appropriate. Correlations between variables were evaluated using Spearman's rank correlation coefficient. A logistic analysis was used to predict clinical deterioration within 6 months. To identify the factors predictive of mortality, we used a Cox proportional hazards model. A receiver operating characteristic (ROC) analysis of serum MMP-10 was performed to determine the threshold for predicting clinical deterioration. All analyses were considered statistically significant when p < 0.05.