MO012MIXED ON LINE HEMODIAFILTRATION MODULATES MICRORNA223 EXPRESSION IN CIRCULATING PLASMA EXTRACELLULAR VESICLES: PROTECTIVE ROLE ON ENDOTHELIAL DYSFUNCTION AND VASCULAR CALCIFICATION IN HEMODIALYSIS PATIENTS

Abstract

Introduction and Aims: On line haemodiafiltration (OL-HDF) may improve cardiovascular outcome in patients with end stage chronic kidney disease. This effect may be ascribed to an enhanced clearance of middle molecules involved in endothelial dysfunction and vascular calcification. Increasing evidences indicate that extracellular vesicles (EV) act as intercellular mediators for the exchange of extracellular RNAs, particularly microRNA (miRNA), small noncoding RNA able to regulate post-transcriptional expression of different gene products. The effect of dialysis on plasma EV concentration and miRNA content has not been yet evaluated. The aim of this study was to investigate whether mixed OL-HDF (mOL-HDF) may modulate the release and the miRNA phenotype of plasma EV, correlating these results with clinical data indicating an increased risk of cardiovascular events. Methods: Thirty bicarbonate haemodialysis (BHD) patients were randomized to continue BHD (n=15) or to switch to mOL-HDF (n=15). Plasma concentration, size, cellular origin and miRNA content of EVs was evaluated for 9 consecutive months and compared with EVs from healthy controls (n=10). EV concentration and miRNA content was also correlated with arterial stiffness (pulse wave velocity analysis) and diastolic dysfunction. In vitro, EV-induced angiogenesis and apoptosis of human endothelial cells and osteoblast mineralization of vascular smooth muscle cells (VSMC) was evaluated and the role of selected miRNA was studied. Results: Haemodialysis patients showed higher plasma levels of endothelial cell-derived EVs than healthy subjects. Of interest, BHD and mOL-HDF patients showed no difference in number and size of EVs. However, EV miRNA content revealed an increased expression of the anti-angiogenic miR-223 in BHD-derived EVs in respect to healthy subjects and mOL-HDF. EVs from hemodialysis patients reduced angiogenesis, increased endothelial apoptosis and VSMC calcification in respect to EVs from healthy subjects. However, EVs from mOL-HDF significantly increased angiogenesis and reduced endothelial apoptosis and VSMC calcification when compared with EVs from BHD. Cell transfection with miR-223 mimic or antagomiR suggested an involvement of this miRNA in endothelial cell and VSMC dysfunction. In vitro data were confirmed by a correlation between miR-223 content of plasma EVs and arterial stiffness or diastolic dysfunction. Conclusions: EVs derived from hemodialysis patients carry miRNAs involved in endothelial dysfunction and vascular calcification. Switching from BHD to mOL-HDF decreased miR-223 in EVs. In vitro experiments indicated that miR-223 reduction was sufficient to decrease EV-induced inhibition of angiogenesis and VSMC calcification. These results may explain the protective effects on endothelial dysfunction and vascular calcification observed in clinical trials using OL-HDF, independently from middle molecule clearance.