Human pericardial fluid exosomes regulate macrophage immunophenotype: new prospective for cardiovascular myocardium-epicardium crosstalk in coronary artery disease

Abstract

Abstract Background The incidence and severity of ischemic heart disease (IHD) is exacerbated by coronary artery disease (CAD). Monocytes and macrophages are central to atherosclerosis. Endogenous small extracellular vesicles (sEVs) can shuttle microRNAs and other molecular cargos from cell to cell, mediating expressional and functional response in the recipient cells. Recent evidence supports a role for sEVs in modulating macrophage phenotype. The pericardial fluid (PF) is in direct contact with the epicardium and contain sEVs of myocardial origin (C. Beltrami et.al 2017). We recently showed that human PF-sEVs are capable to modulate cardiovascular cells via microRNA shuttling (C.Beltrami et.al. 2017). Moreover, epicardial cells reportedly cross-talk with the myocardium to regulate immune response to IHD (V.Ramjee et.al 2017). Purpose This study sought to investigate whether PF-sEVs regulate macrophages, contributing to a specific immunophenotypes in CAD patients. Methods PF was collected from either CAD patients undergoing coronary bypass surgery (CABG) or non-atherosclerotic patients operated for mitral valve repair (non-CAD control group). sEVs were isolated using size exclusion chromatography and characterised for size (Nanosight tracking analysis; NTA) and microRNA content (Exiqon miR-array). Monocytes from healthy donors were isolated from buffy coats and differentiated into macrophages following established protocols. Macrophages were incubated with either CAD-sEVs or non-CAD sEVs for 24h at 37oC. PBS and sEV-free serum were used as negative controls and LPS as a proinflammatory control. The cells were collected and processed for mRNA analyses (qRT-PCR) and Flow cytometry (FACS). Results Twenty-four-hour exposure to CAD-sEVs induces a proinflammatory profile of human macrophages. While non-CAD-sEVs did not statistically differ from PBS nor Exo-free groups, CAD-sEVs increased the mRNA level of IL1a, NOS3, TNFa, CCL2 and IL6 (comparison to both non-CAD and negative controls). These data were not associated with changes in apoptosis. Bioinformatics analysis showed that 11 miRNAs where consistently increased, and 67 miRNAs decreased in the PF-sEVs from all CAD patients compared to non-CAD. miRNA targets prediction and pathway analyses (R-Project) revealed that the deregulated miRNAs could regulate macrophage motility and cytokine signalling. Conclusions We demonstrate, for the first time, that sEVs isolated from the PF of CAD patients induce a proinflammatory profile of human macrophages. These clinically relevant results could drive to decipher improved therapeutics able to modulate the epicardial/myocardial immune response in CAD patients. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Biritish Heart Fundation grant to Prof. Costanza Emanueli