Elevated Serum Matrix Metalloprotease (MMP-2) as a Candidate Biomarker for Stable COPD

Durga Mahor; Vandana Kumari; Kapil Vashisht; Ruma Galgalekar; Ravindra M. Samarth; Pradyumna K. Mishra; Nalok Banerjee; Rajnikant Dixit; Rohit Saluja; Sajal De; Kailash C. Pandey

Disclosures

BMC Pulm Med. 2020;20(302) 

In This Article

Methods

Sample Collection

The inclusion criteria for stable COPD patients was- symptoms (dyspnoea, chronic cough/sputum); exposure to risk factors such as smoking; ratio (FEV1:FVC) < 0.7 in the spirometry test and no exacerbation during the last 3 months. COPD patients with active pulmonary tuberculosis, heart diseases, kidney diseases and cancer were excluded from the study, which could interfere with the expression profile of various serum proteases. 10 asthmatic patients were enrolled in this study by following the Global Initiative for Asthma (GINA) 2018 guidelines. Written informed consent was obtained from all the patients before sample collection. 3 ml of venous blood was collected from (n = 35) COPD patients and (n = 15) controls under aseptic conditions. The blood samples were allowed to clot by leaving it undisturbed at room temperature and centrifuged at 2000×g for 1 min.; serum was collected, aliquoted and stored at − 80 °C till further use. For comparative analysis of specific proteases, a total of (n = 10) asthmatic patients were also enrolled in this study by following the Global Initiative for Asthma (GINA) 2018 guidelines.

Qualitative Measurement of Serum NE, DPP-IV, Caspases and MMPs

Direct-Enzyme-Linked Immunosorbent Assay (ELISA) was performed to measure the serum proteases- (NE, DPP-IV, caspases- [3 & 7] and MMP- [2 & 9]; in different COPD patients and the controls. The sera samples were diluted (1:100) in 1X PBS and coated in 96-well plates; incubated overnight at 4 °C. After coating of the antigen, well contents were aspirated and blocking buffer (0.5% BSA in 1X PBS) was added to each well, followed by incubation for 2 h at 25 °C. Primary antibodies [anti-elastase (1:1000); anti-caspase- [3 & 7] (1:2000)] and anti-MMP- [2 & 9] (11000)], were added and incubated for 2 h. at room temperature. After primary antibody incubation, the plates were thoroughly washed with wash buffer (0.05% Tween-20 in 1X PBS) and secondary antibody- anti-mice HRP (13000) (Santa Cruz Biotechnology) was added and plates were again washed (three times) and the peroxidase substrate solution (o-Phenylenediamine dihydrochloride (OPD)) in sodium citrate, pH -5.0 + H2O2) was added. As the peroxidase reacted with the OPD, a dark yellow product was formed; the intensity of the yellow colour was proportional to the amount of tested antigens in the sera samples. Stop solution was added to terminate the reaction followed by 30 min incubation and absorbance was recorded at 405 nm by using an ELISA plate reader.

Fluorometric Assays for Human Caspase-3/7 Activity

The activity of human caspases-3/7 in COPD patients and the controls sera were assessed by measuring the cleavage of a fluorogenic substrate [Z-DEVD-AMC] at excitation and emission wavelengths of 355 nm & 460 nm, respectively. Assay was performed using HEPES buffer (50 mM HEPES pH 7.5, 150 mM NaCl & 5 mM DTT). Purified recombinant human caspase-[3 & 7] were used as controls. All reactions were performed in duplicate and data was analysed using Graphpad Prism 5.0.

Recombinant Expression and Purification of MMP-2

The recombinant construct of MMP-2 was gifted by Raquel Gerlach, University of Sao Paulo, Ribeirao Preto, SP, Brazil and expressed as per the protocol described earlier.[16] Briefly, the recombinant construct was transformed into BL21(DE3)/pLysS E. coli cells. Single colonies were grown in LB media containing 100 μg/mL ampicillin and 34 μg/mL chloramphenicol and 20% glucose. The culture was allowed to grow overnight at 37 °C with shaking at 180 rpm. Further, secondary culture (500 ml) was inoculated with 1% of overnight grown culture, with appropriate antibiotics and grown at 37 °C until an OD 600 of 0.5–0.7 was reached. The secondary was induced with 0.5–1 mM IPTG and allowed to grow further for 18 h at 18[0] C. Cells were then harvested and resuspended into the phosphate buffer for lysis by sonication followed by centrifugation at 14000 RPM at 4 °C for 15–20 min. The supernatant was collected and purified by NI-NTA affinity chromatography using imidazole gradient. The purified fractions of protein was analysed by SDS-PAGE.

Quantitative Estimation of DPP-IV, NE and MMP-2 in COPD Patients and Controls

The quantiation of DPP-IV was performed using DPP-IV human ELISA kit (Thermo Fisher Scientific), as per the manufacturer's protocol. To estimate the concentration of serum NE and MMP-2 in COPD patients and the controls, recombinant NE and MMP-2 (1 mg/ml stock) were coated in 96-well plates with a range of (1.25–800 μg) protein per well. Direct ELISA was performed to generate a standard curve for the above proteins. The respective proteins were estimated by standard curves.

Measurement of Free Radicals

Intracellular reactive oxygen species (ROS levels) was measured in COPD patients and the controls sera. The sera samples were diluted (1:100) in 1X PBS followed by incubation with a cell-permeant dye [1X of H2DCFDA (dichloro-dihydrofluorescein diacetate) (Sigma)] for 30 min. in a 96-well plate. Fluorometric measurements (excitation at 510 nm and emission at 530 nm) were performed in duplicate, and the results were expressed as the mean fluorescence intensity.

Mass Spectrometry Analysis of COPD Patients and Controls

Proteomics analysis of stable COPD patients and control sera samples was peformed by resolving diluted sera samples (1:20) in 15% SDS-PAGE (20 × 18.3 cm). Protein bands with different expression were selected from SDS-PAGE and preserved for identifying the protein sequence identity. Mass spectrometry was performed at Central Instrumental facility (CIF) South campus, University of Delhi, India.

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