| Background/Aims:Coronary heart disease (CHD) is an important disease that threatening people’s life and health. Acute coronary syndrome (ACS) is the most dreaded complications of CHD, with high morbidity and mortality. Although early diagnosis and reperfusion strategy greatly improved the ACS prognosis, it is difficult to predict this kind of events in the clinic. Platelets play an important role in ACS, and platelet hyper-activity is associated with ACS, but in vitro platelet function tests cannot fully reflect the platelet function in vivo. Therefore, plasma biomarker that can accurately reflect the platelet status in vivo may be a way to predict ACS. Few studies have reported plasma exosomes containing miRNAs as a marker of platelet activation in vivo and in ACS. Therefore, we aimed to investigate (1) develop atherothrombosis mouse model by fed ApoE knock out (ApoE-/-) mice with high cholesterol diet, and make tandem stenosis (TS) to the right carotid artery; (2) investigate the levels of miR-223, miR-339 and miR-21 in plasma exosomes before thrombosis in TS models; (3) thrombin-activated platelet-derived exosomal miR-223, miR-339 and miR-21 levels in human and mouse, and the consistence with atherothrombosis models; (4) the mechanisms underlying the platelet-derived exosomes in atherothrombosis and their function in vascular smooth muscle cells (SMCs).Methods:ApoE-/- mice (n=55) were fed with high cholesterol diet for 6 weeks then performed TS surgery (n=40) and sham surgery (n=15), and at post-operation 4 weeks TS mice were checked artery flow by ultrasound and veinous blood sample were collected then, and at post-operation 7 weeks carotid artery was collected for pathology analysis. Plasma samples were collected from TS group of our successfully developed atherothrombosis mouse models at post-operation 4 weeks, and pooled for isolation of exosomes via differential ultracentrifugation, and total RNAs were extracted and reverse transcripted to cDNA for quantitative real-time PCR (qRT-PCR). cel-miR-39 was added as internal control, △△CT was performed to compare miR-223, miR-339 and miR-21 expression difference between the atherothrombosis and sham groups. Blood was taken from healthy volunteers and wild type (WT) mice, platelets were isolated and purified, and then treated with thrombin (1 U/ml) and PBS for 1 hour at 37℃ respectly. Exosomes were isolated via differential ultracentrifugation, and the levels of miR-223, miR-339 and miR-21 were compared between these two groups. Primary arotic SMCs were isolated from WT mice (4-6). Thrombin-activated platelet-derived exosomes were labeled with PKH26 and incubated with SMCs for uptake experiments, confocal microscope was used to determine the SMCs uptake concentration-time manner. Regarding to the metabolic manner of exosomes in SMCs, miR-223, miR-339 and miR-21 levels were determined after SMCs uptake platelet-derived exosomes. DAVID Tools were used to analyze the pathways that were enriched among the miRNA target genes. qRT-PCR was performed to screen the mRNAs expression levels of platelet-derived exosome target genes in vascular SMCs and immune-fluorescence staining was performed to identify the protein expression levels in vitro and in vivo. Ki-67 immuno-fluorescence and TUNEL assay were performed to determine the proliferation and apoptosis effects of platelet-derived exosome on SMCs.Results:(1) The incidence of thrombosis in TS group was 37.5%(15/40), plaque erosion accounted for 35%(14/40) and plaque rupture for 2.5%(1/40); intima media ratio in sham group, TS proximal section, TS thrombosis section, and TS distal section were 0.119±0.029 vs 0.504±0.038 vs 1.078±0.072 vs 1.195± 0.116, p<0.001; Ki-67 immuo-fluorescence intensity in these four sections were 1.002±0.027 vs 3.102±0.357 vs 2.837±0.063 vs 3.120±0.403, p=0.001; TUNEL intensity in these four sections were 0.997±0.095 vs 0.945±0.091 vs 5.472±1.077 vs 1.727±0.242, p=0.001; SMCs in TS group showed increased proliferation and migration, but only thrombosis sections were associated with increased apoptosis. (2) No obstruction was observed under carotid ultrasound at post-operation 4 weeks, plasma exosomal miR-223, miR-339 and miR-21 levels in TS group that successfully developed thrombosis compared to sham group were 1.83±0.35 vs 0.97±0.15, p=0.017; 3.56±0.25 vs 1.03±0.15, p<0.001; 2.83±0.31 vs 0.98±0.18, p=0.01. (3) The miR-223, miR-339 and miR-21 levels in thrombin-activated and PBS treated human platelet-derived exosomes and debris were 2.05±0.48 vs 0.17±0.04, p=0.002; 1.43±0.31 vs 0.28 ±0.07, p=0.003; 1.40±0.17 vs 0.25±0.02, p<0.001; and these miRNAs levels in mouse platelet-derived exosomes and debris in thrombin compared to PBS treatment were 1.34±0.10 vs 0.45±0.02, p<0.001; 1.50±0.38 vs 0.40±0.05, p=0.007; 1.41±0.23 vs 0.47±0.12, p=0.003. (4) SMCs uptake platelet-derived exosomes increased with concentration, uptake time pattern followed parabola curve:f(x)=-0.1345x2+3.2978x-1.0153, x (h), R2=0.9906. According to this formula, SMCs began to uptake exosome at the incubation time of 0.5 hours, and reached a peak at 12.25 hours, and then vanished at 24.2 hours. The levels of cellular miR-223, miR-339 and miR-21 of SMCs incubated with platelet-derived exosomes compared to PBS for 24 hours were 3.780±0.085 vs 1.027±0.041, p<0.001; 3.607±0.110 vs 0.993±0.047, p<0.001; 5.620±0.269 vs 1.007±0.054, p<0.001. miR-223, miR-339 and miR-21 target genes enrichment KEGG pathway analysis shows that MAPK signaling pathway in the second rank,2.9%(p=5.8×10-5) of total genes in this pathway are regulated by these miRNAs. And Platelet-derived growth factor receptor-beta (PDGFRβ) which in MAPK signaling pathway was inhibited in mRNA level (0.67±0.19 vs 1.00±0.05, p=0.048) and protein level (0.68±0.09 vs 1.00±0.20, p=0.005). Furthermore, PDGFRβ expression in TS group withour thrombosis compared to sham group were 3.03±0.42 vs 1.00±0.10, p<0.001; but in thrombosis sections, the PDGFRβ expression was significantly decreased compared to proximal sections 2.15±0.10 vs 3.92±0.28, p=0.004. In vitro, PDGF-BB induced Ki-67 positive SMCs were decreased by platelet-derived exosome treatment (4.15±1.91% vs 8.40±2.83%, p=0.047), and for longer starvation the TUNEL positive SMCs were increased by platelet-derived exosome treatment (13.35±1.45% vs 5.85±1.57%, p=0.025).Conclusions:Our data indicate that increased levels of plasma exosomal miR-223, miR-339 and miR-21 may reflect thrombin induced platelet activation in atherothrombosis mouse models, and then predict thrombotic events. Thrombin-activated platelet-derived exosome inhibits PDGFRβ expression in SMCs in vitro, and the decreased PDGFRβ expression in thrombosis section SMCs may be caused by platelet-derived exosomes. But the function of platelet-derived exosome to atherothrombosis needs further study. Our study found a new signal transduction between platelet and SMCs, and platelet-derived exosomes may be biomarker for predicting atherothrombosis.
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