Thrombin Stimulated Platelet-Derived Exosomes Inhibit Platelet-Derived Growth Factor Receptor-Beta Expression in Vascular Smooth Muscle Cells

Abstract

Background/Aims: It is difficult to predict acute thrombotic cardiovascular events in the clinic. Few studies have reported the presence of plasma exosomes containing microRNAs (miRNAs) in cardiovascular events. Therefore, we aimed to investigate the levels of miR-223, miR-339 and miR-21 in plasma exosomes before thrombosis in mouse models of carotid tandem stenosis, as well as the mechanisms underlying the origin and function of these exosomal miRNAs. Methods: Plasma samples were collected from the carotid tandem stenosis and sham control groups of our successfully developed atherothrombosis mouse models before thrombosis. Platelets from healthy volunteers and mice were purified to obtain thrombin stimulated platelet-derived exosomes. Exosomes were isolated via differential ultracentrifugation, and western blotting and transmission electron microscopy were used for their identification. The total RNA was extracted, and quantitative real-time PCR was performed to determine the expression levels of miR-223, miR-339 and miR-21. DAVID Tools were used to analyze the pathways that were enriched among the miRNA target genes. Immuno-fluorescence staining was performed to identify the protein expression levels of platelet-derived exosome target genes in vascular smooth muscle cells (SMCs) in vitro and in vivo. Results: The levels of miR-223, miR-339 and miR-21, which are associated with platelet activation, were elevated in pooled mouse plasma exosomes before thrombosis and enriched in thrombin-stimulated platelet-derived exosomes in vitro. Platelet-derived growth factor receptor-beta (PDGFRβ) was a target of these miRNAs, and PDGFRβ expression in vascular smooth muscle cells (SMCs) was inhibited following incubation with platelet-derived exosomes. Platelet-derived exosomes could also inhibit PDGF-stimulated SMC proliferation. Furthermore, a decrease in PDGFRβ expression was observed in vascular SMCs around thrombotic areas in vivo. Conclusions: Our data indicate that activated platelet-derived exosomes containing miR-223, miR-339 and miR-21 could be transferred into SMCs and inhibit PDGFRβ expression; these exosomal miRNAs may be a biomarker for predicting atherothrombosis.