008 Microrna Expression in Blood Derived Mononuclear Cells During Maturation From Early to Late Outgrowth Endothelial Progenitor Cells

Abstract

Endothelial Progenitor Cells (EPCs) circulate in the peripheral blood mononuclear cell (PBMC) fraction and can be selected under culture conditions which include appropriate matrix components and endothelial growth factors. Early outgrowth EPCs appear after the first 3 to 5 days of selective culture, and these nonproliferative cells still express MNC markers (i.e. CD14 and CD45), but also express some endothelial cell (EC) markers including CD31 and VEGFR2. Late outgrowth EPCs appear after 2 weeks, and exhibit a strong EC phenotype, and this highly proliferative population has lost all leukocyte markers. MicroRNAs (miRNAs) are small noncoding RNAs that have emerged as important regulators of gene expression. The aim of this study was to define the changes in miRNA expression profiles in MNC cultures during evolution from early to late outgrowth EPC. Changes in the expression of specific miRNAs contribute to the endothelial specification of cultured MNCs and the emergence of early or late outgrowth EPCs. Mononuclear cells (MNCs) were isolated from healthy participants (n=4) by leukaphesis and cultured for 1, 3, 5, 7 and 9 days under conditions promoting endothelial differentiation. Late outgrowth EPCs were derived from the same cultures and appeared after 14-21 days (n=3). MiRNA expression was assessed by (q)RT-PCR (Taqman), using a panel of 13 miRNAs previously identified in PBMCs, EPCs or mature ECs. MiRNA expression profiles were very similar in cells between 1 and 3 days (cultured MNCs); and 5, 7 and 9 days (early outgrowth EPCs), and therefore these were grouped for further analysis. MiR-92a was highly dominant in day 1-3 MNC cultures and remained relatively constant throughout the culture period. In early outgrowth EPCs (day 5-9), the expression of miR-146a increased 2.9±0.04 fold, (p<0.05). In contrast, late outgrowth EPCs exhibited marked increases in expression of miRs-126 , -222 and -221 compared to early EPCs (by 453-, 14- and 8.4-folds respectively; p<0.05). The ratio of miR-126 to miR-146 provided the strongest indicator of endothelial differentiation. These data demonstrate that MNCs exhibit specific patterns of miRNA expression tightly associated with the progression from early to late outgrowth EPCs. MiRNAs may play a critical role in regulating the maturation of MNCs from early to late outgrowth EPCs, and enhancing miR-126 or suppressing miR-146 may facilitate their differentiation to an endothelial phenotype.