Circulating endothelial progenitor cells (EPCs) are reduced and functionally impaired in diabetic patients, possibly contributing to the higher number and severity of ischemic events observed in this population. The molecular mechanisms underlying diabetes-associated EPC impairment are incompletely understood. microRNAs (miRs) post-transcriptionally inhibit the expression of their target genes. We aim to obtain mechanistic insights of diabetic EPCs impairment and to provide the first characterization of the EPC-associated miRNA with special respect to those potentially involved in the control of angiogenesis. Here we report results on miR-15a/-16.Methods
EPCs were prepared from the peripheral blood of type-2 diabetic patients undergoing revascularization for limb ischemia (LI) and from healthy individuals. The migratory activity of EPCs was measured in a transwell migration assay using SDF-1 (100ng/ml) as a chemo-attractant. A miR screening was performed (TaqMan PCR) in diabetic LI EPCs (n = 55) (controls: healthy donors n=17) to selected some differentially expressed miRs to be further analyzed. miR expression in EPCs was regulated using pre-miR, anti-miR and scramble control (all from Ambion).Results
As expected, EPC migration (modified Boyden chamber) to SDF was reduced by disease (migration index – ratio of SDF-1 to BSA migrated EPCs -: 1.08 + 0.02 vs 1.87 + 0.09 for healthy EPCs, P < 0.01, n=6 patients and n=7 controls). The miR-15a/-16 expression (relative to snRU6) was increased in "diseased" EPCs in comparison to controls (miR-15a: 5.19 + 0.85 vs 1.4 + 0.15, p < 0.05; miR-16: 4.91 + 0.91 vs 1.13 + 0.13, p < 0.05). To understand the effect of miR-15a/-16 upregulation on EPC capacities, we transfected pre-miR-15a and pre-miR-16 in healthy EPCs. miR-15a/-16 overexpression inhibited EPC migration to SDF-1 (migration index: 1.24 + 0.74 vs. 1.62 + 0.04 in scramble, p < 0.05). Next, to understand if increased endogenous miR-15a/-16 contributes to impaired EPC migration observed in patients, we transfected "diseased" EPCs with anti-miR-15a and anti-miR-16. This corrected the migratory capacity of patient-derived EPCs (migration index: 1.51 + 0.14 vs 1.1 + 0.09 in scramble, p < 0.05 and P=NS vs. healthy EPCs given scramble). Bioinformatic analyses predict BCL2, VEGFA, and AKT3 to be target genes of both miRNA-15a and miR-16, which additionally suggest anti-angiogenic and pro-apoptotic roles of these miRs.Conclusions
miR-15a/-16 are involved in SDF-1 migratory impairment of EPCs from diabetic patients with LI.