Abstract 10638: MicroRNA-15a and MicroRNA-16 Increase in Circulating Pro-Angiogenic Cells (PACs) of Patients with Critical Limb Ischemia and Impair PAC Functions in vitro and in vivo
Objectives: Circulating Pro-Angiogenic Cells (PACs) could help restoring blood flow (BF) in patients with critical limb ischemia (CLI). However, CLI-PACs are reportedly functionally compromised. Micro-RNAs (miRs) were recently discovered to mediate endothelial and stem cell functions. Here, we aimed to define: 1) miR expression in CLI-PACs; 2) miR-15a/-16 role in PACs.
Methods: Expression of 28 miRs was assessed (TaqMan PCR) in PACs culture-enriched from circulating mononuclear cells of CLI patients or healthy controls (C) (n=7/group). PAC expression of miR-15a and miR-16 was further measured in larger groups (C: n=17; CLI: n=72). To study miR-15a/16 function, we transfected C-PACs with pre-miR-mimics or negative control, and CLI-PACs with anti-miR inhibitors or negative control. PAC migration toward SDF-1, FBS, VEGF-A, bFGF or BSA (control) was studied (transwell chambers). Bioinformatics-predicted miR direct target genes were measured in PACs (q-RT-PCR and WB). Finally, C-PACs were ex-vivo transfected with either pre-miR-15a/16 or anti-miR-15a/6 and transplanted (0.5x106 cells/mouse) in the ischemic adductor of immunodeficient CD1-Foxn1nu mice (n=12/group). BF recovery (colour laser Doppler) and muscle microvascular density were measured after 1 and 2 weeks of surgical induction of limb ischemia.
Results: Several miRs were differently regulated in CLI-PACs, including miR-15a and miR-16, which resulted increased (p<0.05 for both comparisons vs. C). Pre-miR-15a/16 decreased C-PACs migration toward each tested chemoattractant (p<0.05 for all comparison vs. negative control). Anti-miR-15a/16 partially corrected the impaired migratory capacity of CLI-PACs. Expression of VEGF-A and AKT3, predicted miR-15a/16 targets, were reduced in CLI-PACs and by pre-miR15a/16 in C-PACs. Transplantation of anti-miR-15a/16-C-PACs increased BF recovery and muscular arteriole density (p<0.05 for both comparisons vs. negative control-PACs). Pre-miR-15a/16-C-PACs did not improve BF or angiogenesis.
Conclusions: miR-15a and miR-16 impair the pro-angiogenic potential of PACs in CLI patients and may represent new molecular target for ex-vivo manipulation of cells for autologous transplantation.
- © 2012 by American Heart Association, Inc.