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

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.

The regulatory role of microRNAs in angiogenesis‐related diseases

“…In addition, serum miR‐124, miR‐9 and miR‐219 are down‐regulated in patients with acute ischaemic stroke 171, 172. And circulating miR‐15a and miR‐16 are enriched in critical limb ischaemia (CLI) patients and serum miR‐15a expression is also positively associated with post‐revascularization restenosis 173. A recent study reported that circulating miR‐363, miR‐487b, miR‐125b‐2, miR‐27a, miR‐422a, miR‐488, miR‐627, miR‐290, miR‐10a, miR‐182, miR‐200b, miR‐298, miR‐106b‐5p and miR‐4306 are increased while circulating miR‐122, miR‐148a, let‐7i, miR‐19a, miR‐320d, miR‐4429, miR‐30a, miR‐126, miR‐9, miR‐219, miR‐320e and miR‐320d are reduced following ischaemic stroke 174.…”

MicroRNAs; easy and potent targets in optimizing therapeutic methods in reparative angiogenesis

“…Consistently, the miR‐15 family has been reported to be increased in PCs and serum of patients with ischaemic conditions, which is associated with the impairment of human circulating pro‐angiogenic function of PCs 29 The family members overexpressed by muscles in response to ischaemic condition act to negatively regulate bone marrow‐derived PC response to chemotactic stimuli fibroblast growth factor (FGF), VEGF‐A and stromal cell‐derived factor‐1α (SDF‐1α) 29.…”

“…Notable in animal models and in patients with chronic ischaemia condition, increased levels of miR‐15s and ‐503 have been observed in plasma and circulating c‐Kit + cells 29.…”

“…In this context, antagonism of endogenous miRNA regulators such as miR‐15s, miR‐23s and miR‐222s that are induced under pathological conditions and also target ‘gatekeepers’ of endothelial activation may represent a powerful strategy to inhibit or activate angiogenesis in a wide range of pathological conditions 29, 45, 71.…”

“…Thus, EC dysfunction and ageing‐related degenerative problems lie, at least in part, within the tissue‐specific released miRNAs 6, 15, 29, 50, 51, 80.…”