Abstract
Transplantion of bone marrow-derived endothelial progenitor cells (EPCs) may be a novel treatment for deep venous thrombosis (DVT). The present study probed into the role of microRNA (miR)-361-5p in EPCs and DVT recanalization. EPCs were isolated from male Sprague–Dawley (SD) rats and identified using confocal microscopy and flow cytometry. The viability, migration and tube formation of EPCs were examined using MTT assay, wound-healing assay and tube formation assay, respectively. Target gene and potential binding sites between miR-361-5p and fibroblast growth factor 1 (FGF1) were predicted by StarBase and confirmed by dual-luciferase reporter assay. Relative expressions of miR-361-5p and FGF1 were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot as needed. A DVT model in SD rats was established to investigate the role of EPC with miR-361-5p antagomir in DVT by Hematoxylin–Eosin (H&E) staining. EPC was identified as 87.1% positive for cluster of difference (CD)31, 2.17% positive for CD133, 85.6% positive for von Willebrand factor (vWF) and 94.8% positive for vascular endothelial growth factor receptor-2 (VEGFR2). MiR-361-5p antagomir promoted proliferation, migration and tube formation of EPCs and up-regulated FGF1 expression, thereby dissolving thrombus in the vein of DVT rats. FGF1 was the target of miR-361-5p, and overexpressed FGF1 reversed the effects of up-regulating miR-361-5p on suppressing EPCs. Down-regulation of miR-361-5p enhanced thrombus resolution in vivo and promoted EPC viability, migration and angiogenesis in vitro through targeting FGF1. Therefore, miR-361-5p may be a potential therapeutic target for DVT recanalization.
Highlights
Deep venous thrombosis (DVT) refers to the formation of a blood clot within a deep vein in contrast with venous thromboembolism, which includes superficial thrombophlebitis and pulmonary embolism [1]
The isolated Peripheral blood mononuclear cells (PBMC) were identified by confocal microscopy, and the double staining with functional marker fluorescein isothiocyanate (FITC)-UEA-I and Dil-Ac-LDL suggested that the isolated PBMCs were endothelial progenitor cell (EPC) (Figure 1B)
CD31, CD133, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR2) were markers of EPCs [20], and their expressions were measured using flow cytometry in order to confirm the identity of EPCs
Summary
Deep venous thrombosis (DVT) refers to the formation of a blood clot within a deep vein in contrast with venous thromboembolism, which includes superficial thrombophlebitis and pulmonary embolism [1]. Anti-coagulation is a major therapeutic strategy for DVT, but it cannot dissolve thrombus and restore the function of valves, at the same time, thrombosis may occur with post-thrombotic syndrome (PTS) [3]. Recent discoveries showed that successful DVT-related thrombi resolution plays a key role in DVT treatment [4], detailed mechanisms remained obscure. EPCs have the ability to differentiate into mature endothelial cells and play a major role in vascular integrity maintenance and endothelial damage as well as the resolution of thrombus in vivo [5,6,7]. EPCs can be homed and integrated into the injured blood vessel and thrombus to secrete angiogenesis factors, increasing the formation
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