MicroRNA-15a and MicroRNA-16 Impair Human Circulating Proangiogenic Cell Functions and Are Increased in the Proangiogenic Cells and Serum of Patients With Critical Limb Ischemia

• Published in: Circulation research 2013-01, Vol.112 (2), p.335-346
• Main Authors: Spinetti, Gaia, Fortunato, Orazio, Caporali, Andrea, Shantikumar, Saran, Marchetti, Micol, Meloni, Marco, Descamps, Betty, Floris, Ilaria, Sangalli, Elena, Vono, Rosa, Faglia, Ezio, Specchia, Claudia, Pintus, Gianfranco, Madeddu, Paolo, Emanueli, Costanza
• Format: Article
• Language: English
• Subjects: Animals; Cell Movement - genetics; Cell Survival - genetics; Cell Transplantation - methods; Cells, Cultured; Diabetes Complications - blood; Diabetes Complications - genetics; Diabetes Complications - pathology; Diabetes Mellitus, Type 2 - blood; Diabetes Mellitus, Type 2 - genetics; Diabetes Mellitus, Type 2 - pathology; HEK293 Cells; Hindlimb - blood supply; Hindlimb - pathology; Humans; Ischemia - blood; Ischemia - genetics; Mice; Mice, Nude; MicroRNAs - adverse effects; MicroRNAs - biosynthesis; Neovascularization, Pathologic - blood; Neovascularization, Pathologic - genetics
• ISSN: 0009-7330; 1524-4571; 1524-4571
• DOI: 10.1161/CIRCRESAHA.111.300418

RATIONALE:Circulating proangiogenic cells (PACs) support postischemic neovascularization. Cardiovascular disease and diabetes mellitus impair PAC regenerative capacities via molecular mechanisms that are not fully known. We hypothesize a role for microRNAs (miRs). Circulating miRs are currently investigated as potential diagnostic and prognostic biomarkers. OBJECTIVE:The objectives were the following(1) to profile miR expression in PACs from critical limb ischemia (CLI) patients; (2) to demonstrate that miR-15a and miR-16 regulate PAC functions; and (3) to characterize circulating miR-15a and miR-16 and to investigate their potential biomarker value. METHODS AND RESULTS:Twenty-eight miRs potentially able to modulate angiogenesis were measured in PACs from CLI patients with and without diabetes mellitus and controls. miR-15a and miR-16 were further analyzed. CLI-PACs expressed higher level of mature miR-15a and miR-16 and of the primary transcript pri–miR-15a/16-1. miR-15a/16 overexpression impaired healthy PAC survival and migration. Conversely, miR-15a/16 inhibition improved CLI-PAC–defective migration. Vascular endothelial growth factor-A and AKT-3 were validated as direct targets of the 2 miRs, and their protein levels were reduced in miR-15a/16–overexpressing healthy PACs and in CLI-PACs. Transplantation of healthy PACs ex vivo–engineered with anti–miR-15a/16 improved postischemic blood flow recovery and muscular arteriole density in immunodeficient mice. miR-15a and miR-16 were present in human blood, including conjugated to argonaute-2 and in exosomes. Both miRs were increased in the serum of CLI patients and positively correlated with amputation after restenosis at 12 months postrevascularization of CLI type 2 diabetes mellitus patients. Serum miR-15a additionally correlated with restenosis at follow-up. CONCLUSIONS:Ex vivo miR-15a/16 inhibition enhances PAC therapeutic potential, and circulating miR-15a and miR-16 deserves further investigation as a prognostic biomarker in CLI patients undergoing revascularization.