Microparticles in systemic sclerosis: Potential pro-inflammatory mediators and pulmonary hypertension biomarkers

Abstract

Background and objective: Endothelial microparticles (EMP) are submicron vesicles released from endothelial cells. We aimed to determine the utility of EMP as biomarkers of pulmonary arterial hypertension (PAH) in systemic sclerosis (SSc) patients and the pathogenic role of microparticles (MP) in vascular inflammation.

Methods: Levels of EMP (CD144+, CD31+, CD62E+ and CD143+) were compared between three groups (10 SSc patients with PAH, 10 SSc patients without pulmonary hypertension (no-PH) and 10 healthy age- and sex-matched controls). Human pulmonary artery endothelial cells (HPAEC) were exposed in vitro to MP obtained from SSc patients or healthy controls, and levels of cytokines and inflammatory adhesion molecules were compared.

Results: CD144+ EMP were significantly higher in the SSc-PAH group compared to either the SSc-no PH or healthy controls (diagnostic accuracy 80%, P = 0.02). Compared to controls, SSc patients had higher CD31+/CD62E+ ratios, indicating larger contributions of apoptosis to EMP release (P = 0.04). Patients with limited SSc had significantly higher levels of CD143+ EMP compared to those with diffuse subtype (P = 0.008). When HPAEC were exposed to MP from SSc patients, there was a significant increase in inflammatory cytokines and adhesion molecules. Interestingly, exposure to healthy control MP caused a reduction in inflammatory markers.

Conclusion: EMP (particularly CD144+) are promising biomarkers of PAH in SSc but require further study. MP isolated from SSc patients induced an increase in endothelial cell inflammation and may be an important pathogenic factor in SSc.

Trial registration: ClinicalTrials.gov NCT02331225.

Keywords: biomarkers; cardiovascular; cytokines and inflammatory mediators; respiratory; scleroderma.

Figure 1

(A) CD144+ endothelial microparticles (EMP) from healthy controls versus patients with systemic sclerosis (SSc) with pulmonary arterial hypertension (PAH) and without pulmonary hypertension (PH). (B) Receiver operating characteristics (ROC) curve for CD144+ EMP levels to differentiate SSc patients with PAH from SSc patients without PH. A CD144+ EMP cut-off of 479 yielded 90% sensitivity and 50% specificity to detect SSc-PAH, whereas a cut-off of 1278 provided 60% sensitivity and 100% specificity (area under the curve (AUC): 0.80, 95% CI: 0.60–0.99, P = 0.02). (C) CD143 + EMP for SSc patients with diffuse versus limited sub-type. (D) ROC curve for CD143 + EMP to differentiate SSc patients with diffuse versus limited subtype. A CD143+ EMP cut-off of 42.5 yielded 92% sensitivity and 71% specificity, whereas a cut-off of 85 yielded 62% sensitivity and 100% specificity for limited versus diffuse SSc (AUC: 0.84, 95% CI: 0.65–1.00, P = 0.02).

Figure 2

Levels of macrophage inflammatory protein (MIP)-1α (A), G-CSF (B), interferon-gamma (IFN-γ) (C), IL-2 (D), IL-4 (E) and MIP-1β (F) under three in vitro conditions: human pulmonary artery endothelial cell (HPAEC) stimulated by TNF-α alone, TNF-α + microparticles (MP) isolated from healthy subjects and TNF-α + MP from systemic sclerosis (SSc) patients. Data are displayed as mean ± SEM. Overall P-value is shown in the upper right hand corner and intergroup comparisons are displayed as *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Reverse transcriptase PCR (RT-PCR) for VCAM-1 (A), ICAM-1 (B) and MCP-1 (C) after human pulmonary artery endothelial cell (HPAEC) were exposed to microparticles (MP) isolated from healthy subjects or systemic sclerosis (SSc) patients and stimulated for 3 h with TNF-α, and (D–F) after 24 h of TNF-α stimulation. Data are displayed as mean ± SEM. Overall P-value is shown in the upper right hand corner and intergroup comparisons are displayed as *P < 0.05; **P < 0.01; ***P < 0.001.