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

Results

Neutrophil Elastase and DPP-IV (Serine Proteases)

NE has been repeatedly implicated in the pathogenesis of COPD due to its potential role in the development of emphysema by degrading the extracellular matrix in the lungs.[17] Elevated NE in sputum of Asthma patients and its role in hypersecretion from goblet cells, have been reported in previous studies.[18,19] Asthma is another impotant inflammatory respiratory disease and its symptoms often overlap with COPD such as coughing, wheezing and shortness of breath. Therefore, any specific biomarker for COPD should be able to differentiate Asthma from COPD. In this study, we performed qualitative analysis of serum NE from equal number of subjects from three groups- controls, COPD and Asthma patients. The qualitative analysis revealed a less profound difference between serum NE from controls and COPD patients [p-0.0241; 95% CI] as compared to a significant elevation in serum NE between controls and Asthma patients [p = 0.0002; 95% CI] (Figure 1). Further, the quantitative analysis of serum NE in COPD patients estimated average concentration of (0.21 ± 0.018 μg/ml) as compared to controls (0.047 ± 0.014 μg/ml), represented in Table 1.

Figure 1.

Qualitative analysis of serum NE from controls vs COPD vs Asthma patients. The p-values are calculated by unpaired t-test with 95% confidence interval using Graphpad prism 5.0 software

DPP-IV or CD26 is antoher serine exopeptidase, which has recently been reported to have significantly lower concentration in COPD patients.[9] The decreased activity of the soluble DPP-IV has been shown to be an indicator of COPD.[20] However, a less profound decrease in serum concentration of DPP-IV in COPD patients as compared to the controls [p = 0.0010; 95% CI] (Figure 2) was observed in our study. Quantitative analysis estimated a range of (1200–1800 ng/ml) in controls group as compared to COPD patients (900–1100 ng/ml).

Figure 2.

Measurement of serum DPP-IV levels in COPD patients vs the controls. The p-values are calculated by unpaired t-test with 95% confidence interval using Graphpad prism 5.0 software

Caspases- [3 & 7] (Cysteine Proteases)

Different caspases have been shown to be the mediators of apoptotic processes in COPD, with probable activation by the extracellular signals or intrinsic pathways (mitochondrial and endoplasmic reticulum).[10] An approximate 3-fold higher caspase- 3/7 activity was observed in COPD patients vs controls [p < 0.0001; 95% CI] (Figure 3a). Further, the qualitative analysis of caspase- [3&7] in the sera samples of controls and COPD patients was performed. The serum caspase-3 was not found to be significantly different in COPD patients vs controls [p = 0.04; 95% CI] (Figure 3b). However, a significant elevation in serum caspase-7 was observed in CODP patients as compared to controls [p < 0.0001; 95% CI] (Figure 3c).

Figure 3.

Activity measurement and qualitative analysis of serum caspases- [3&7]. (a) estimation of caspase-3/7 activity; (b) qualitative estimation of serum caspase-3; and (c) caspase-7 from controls vs COPD patients. The p-values are calculated by unpaired t-test with 95% confidence interval using Graphpad prism 5.0 software

MMP- [2 & 9] (Matrix Metalloproteases)

MMPs are the zinc/calcium-dependent endopeptidases that play crucial role in the extracellular matrix remodelling.[21] MMPs are crucial in pathogenesis of both respiratory diseases, COPD and Asthma; therefore, we attempted to assess crucial MMPs, which are distinct for COPD only. In the present study, the qualitative analysis of serum MMP-2 from equal number of subjects from three groups- controls, COPD and Asthma patients, revealed a significant elevation of serum MMP-2 in COPD patients and controls group [p < 0.0001; 95% CI] (Figure 4a). Previously, the role of MMP-9 has been implicated in various cellular processes such as cellular migration and airway inflammatory responses in COPD[22] and Asthma.[23] However, no significant difference in serum MMP-9 was observed in controls and COPD patients [p = 0.6; 95% CI] (Figure 4b). The quantitative analysis of serum MMP-2 in COPD patients estimated a significant increase with an average concentration of (0.71 ± 0.0647 μg/ml) as compared to the controls (0.05 ± 0.0083 μg/ml) (Table 1).

Figure 4.

Qualitative analysis of serum MMP-2 in controls vs COPD vs Asthma patients and serum MMP-9 from controls and COPD patients. The p-values are calculated by unpaired t-test with 95% confidence interval using Graphpad prism 5.0 software

Increase in ROS Levels in COPD Patients

A key characteristic of COPD is the disruption of the oxidant/antioxidant balance due to generation of reactive oxygen species (ROS) from exogenous sources such as cigarette smoke, air pollutants or from endogenous sources viz. neutrophils and macrophages.[24] Therefore, the generation of ROS is a prominent indicator of the inflammatory reactions occurring in COPD. The present study estimated ROS from controls and COPD patients sera; a significantly elevated ROS in COPD patients vs controls indicated towards disruption of oxidant-antioxidanct balance [p < 0.0001; 95% CI] (Figure 5).

Figure 5.

Measurement of reactive oxygen species (ROS) in controls and COPD patients. The p-values are calculated by unpaired t-test with 95% confidence interval using Graphpad prism 5.0 software

Mass Spectrometric Analysis of COPD Proteome

The mass spectrometric analysis is an extremely sensitive technique and has become a method of choice for analysing the proteome of disease samples vs the controls. Signature proteins can be quickly identified from a relatively small sample volume. After the biochemical analysis of various serum proteases, we performed proteomics analysis of 7 COPD patients and 1 control. The proteomic analysis enabled us to identify differentially expressed proteins in COPD patients. Amongst, the differentially expressed proteins some of the proteins were in higher orders of expression as compared to the controls and vice-versa (Table 2). The major proteins which had a negative fold-change in COPD patients vs the controls were protease inhibitors- Preg. Zone protein, α-2 Macroglobulin (A2MG), Peptidase Inhibitor (PI16). The decreased levels of protease inhibitors strongly point towards an altered protease-antiprotease balance, as higher protease activities correlate well with decreased protease inhibitor concentrations in COPD. Another protein found to have negative fold change was Serotransferrin (TRFE_Human), which is also an important part of the defense against oxidative damage and also corroborated with the increased ROS levels in COPD patients. Interestingly, among the proteins with positive fold-change were proteases such as Carboxy peptidase B2 (CBPB2), Matrix Metalloprotease-2 (MMP-2) and Human Leukocyte Elastase (HLE). In our study, the positive fold-change represented the degradative processes as observed in COPD patients. Another protein (cytokine suppressor (SOCS-3)) was also identified with positive fold-change, which has been reported to be involved in the negative regulation of cytokines, correlating well with abrupt cytokine signaling in COPD.

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