title: 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.
abstract: BACKGROUND
  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.
  
  
  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.
authors.affiliation: MultiMedica, Milan, Italy.
authors.forename: Gaia
authors.initials: G
authors.lastname: Spinetti
authors.forename: Orazio
authors.initials: O
authors.lastname: Fortunato
authors.forename: Andrea
authors.initials: A
authors.lastname: Caporali
authors.forename: Saran
authors.initials: S
authors.lastname: Shantikumar
authors.forename: Micol
authors.initials: M
authors.lastname: Marchetti
authors.forename: Marco
authors.initials: M
authors.lastname: Meloni
authors.forename: Betty
authors.initials: B
authors.lastname: Descamps
authors.forename: Ilaria
authors.initials: I
authors.lastname: Floris
authors.forename: Elena
authors.initials: E
authors.lastname: Sangalli
authors.forename: Rosa
authors.initials: R
authors.lastname: Vono
authors.forename: Ezio
authors.initials: E
authors.lastname: Faglia
authors.forename: Claudia
authors.initials: C
authors.lastname: Specchia
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Pintus
authors.forename: Paolo
authors.initials: P
authors.lastname: Madeddu
authors.forename: Costanza
authors.initials: C
authors.lastname: Emanueli
mesh_terms: D000818:Animals; D002465:Cell Movement / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D017690:Cell Transplantation / Q000379:methods; D002478:Cells, Cultured; D048909:Diabetes Complications / Q000097:blood* / Q000235:genetics / Q000473:pathology; D003924:Diabetes Mellitus, Type 2 / Q000097:blood / Q000235:genetics / Q000473:pathology; D057809:HEK293 Cells; D006614:Hindlimb / Q000098:blood supply* / Q000473:pathology; D006801:Humans; D007511:Ischemia / Q000097:blood* / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D035683:MicroRNAs / Q000009:adverse effects* / Q000096:biosynthesis; D009389:Neovascularization, Pathologic / Q000097:blood* / Q000235:genetics
chemical_list: C505361:MIRN15 microRNA, human; C505362:MIRN16 microRNA, human; D035683:MicroRNAs