TIMP-1: A Circulating Biomarker for Pulmonary Hypertension Diagnosis Among Chronic Obstructive Pulmonary Disease Patients

Abstract

Pulmonary hypertension (PH) is a common complication of chronic obstructive pulmonary disease (COPD) and induces increased mortality among COPD patients. However, there are no blood biomarkers to identify PH in COPD. Here, we investigated whether circulating angiogenic factors and cytokines could serve as (a) biomarker (s) for COPD-PH patients. Using Angiogenesis and Cytokine proteome profile array assay, we measured the level of 36 cytokines and 55 angiogenesis-associated proteins in plasma from four COPD patients with PH (COPD-PH) and four COPD patients without PH (COPD), respectively, tissue inhibitor of metalloproteinase 1 (TIMP-1) and thrombospondin 1(TSP-1) were significantly different between the two groups. Enzyme-linked immunosorbent assay (ELISA) was applied to measured TIMP-1 and TSP-1 in a validation cohort (COPD-PH, n = 28; COPD, n = 18), and TIMP-1 was the only factor that was significantly different between COPD-PH and COPD patients (P < 0.01). Logistic regression analysis demonstrated that elevated TIMP-1 was an independent risk factor for COPD-PH [odds ratio (OR) = 1.258, 95% CI: 1.005-1.574, P < 0.05). Next, we explored the expression level and function of TIMP-1 in human pulmonary arterial smooth muscle cells (hPASMCs) exposed to cigarette smoking extract (CSE, a major etiological factor of COPD). In cultured hPASMCs, CSE treatment increased both TIMP-1 protein level and cell proliferation, and exogenous TIMP-1 (25 ng/mL) treatment inhibited CSE-induced hPASMCs proliferation. Overall, our results indicated that TIMP-1 elevation could serve as a circulating biomarker to diagnose PH among COPD patients, and TIMP-1 elevation in COPD-PH could be adaptive.

Keywords: TIMP-1; biomarker; chronic obstructive pulmonary disease; diagnosis; pulmonary hypertension.

Figure 1

Angiogenesis array data. (A) Representative image of angiogenesis array of the plasma from COPD patients without PH [COPD, (a)] and with PH [COPD-PH, (b)] array blots. The levels of angiogenic factors are determined based on their blotting intensity in duplicates. (B–E) Relative protein level of for angiogenesis-associated protein determined by angiogenesis assay (Data were normalized to three pairs of reference spots). (F,G) Based on two-sample independent Student's t-test analysis, TIMP-1 and TSP-1 were significantly higher in COPD-PH than COPD group. Data were present as mean ± SEM (n = 4). ^*P < 0.05 vs. COPD group.

Figure 2

The results of TIMP-1 (A) and TSP-1 (B) between COPD and COPD-PH group in validation cohort by ELISA. The P-value of each factor was obtained from unpaired Student's t-test analysis. **P < 0.01 vs. COPD group; ns, no significant statistical difference.

Figure 3

Forest plot of combined analysis on PH identification among COPD. OR, odds ratio; RVD, the right ventricular diameter measured by Doppler Echocardiography; RAD, the transverse diameter of right atrium measured by Doppler Echocardiography.

Figure 4

CSE increased TIMP-1 level in cultured hPASMCs, and TIMP-1 decreased hPASMCs proliferation under CSE. (A) Western Blotting indicates 0.5% CSE treatment for 36 h increased TIMP-1 expression in hPASMCs. (B) TIMP-1 attenuated CSE-stimulated hPASMCs proliferation. CSE, Cigarette Smoking Extract; hPASMCs, human Pulmonary Arterial Smooth Muscle Cell, **P < 0.01 vs. Control group. TIMP-1, tissue inhibitor of metalloproteinase-1, CSE “+,” with the treatment of CSE; CSE “−,” without treatment of CSE; TIMP-1 “+,” with the treatment of TIMP-1; TIMP-1 “−,” without treatment of TIMP-1; ^#P < 0.01 vs. GroupA, ^$P < 0.01 vs. Group B, *P < 0.05 vs. Group A, ⁺P < 0.05 vs. Group B, ^∧P < 0.01 vs. Group C, N = 6.

Figure 5

Summary figure of the schematic workflow of the current study. (A) Initial screening of angiogenic factors and cytokines. (B) ELISA verification in validation cohort and related statistical analysis. (C) Involvement of TIMP-1 in the hPASMCs mechanism. COPD, Chronic obstructive pulmonary disease; COPD-PH, Chronic obstructive pulmonary disease associated pulmonary hypertension; CSE, Cigarette smoke exposure; ELISA, Enzyme-Linked Immunosorbent Assay; hPASMCs, Human Pulmonary Artery Smooth Muscle Cells; TIMP-1, Tissue Inhibitor of Metalloproteinases-1. This figure was created using BioRender.