P5400CD34+ cells predict long-term cardiovascular mortality in people with critical limb ischemia: a possible pathogenic role of the microRNA-21/PDCD4 axis

Abstract

Abstract Background/Introduction The annual rate of restenosis, amputation, and death of subjects with type 2 diabetes (T2DM) and critical limb ischemia (CLI) is very high even after successful revascularization. We have recently demonstrated that the in-vitro migratory ability of proangiogenic bone marrow (BM)-derived CD45dimKDR+CXCR4+ CD34+ cells predicts cardiovascular mortality at 18 months after percutaneous angioplasty (PTA) in people with T2DM and CLI. Purpose To verify the predictor at a longer follow-up time and to investigate the underpinning mechanism. Methods We analyzed the rate of cardiovascular mortality in the previously studied cohort of 119 subjects at a six-year follow-up. In addition, we compared apoptosis and angiogenic capability of MAC-sorted BM-CD34+ cells in a newly-recruited series of patients with T2DM and CLI and control nondiabetic subjects (CTRL). Following the screening of a spectrum of microRNA (miR) using PCR-based Exiqon technology, we validated the modulated miRs in BM-CD34+ cells from 6 new donors per group. Finally, we focused on miR-21 and its inhibitory targets, by assessing the effect of miR-21 modulation of functional outcomes, including apoptosis (caspase assay and Annexin V positive staining) and the induction of in-vitro endothelial networking on Matrigel. Results Multivariable regression model analysis confirmed that CD34+ cell migration forecasts cardiovascular mortality after revascularization. BM-CD34+ cells isolated from T2DM-CLI donors undergo apoptosis in a higher percentage and are less proangiogenic in-vitro than cells from CTRL. Of the 56 microRNAs commonly expressed in CD34+ cells from the two studied groups, 6 miRs were differentially expressed. Biological validation confirmed a significant down-modulation of miR-21 in CD34+ cells from T2DM-CLI patients. The expression of the miR-21 inhibitory target, PDCD4, a regulator of apoptosis, was increased in CD34+ cells from T2DM-CLI patients compared to CTRL (1.6±0.6 vs. 74.4±14.9 average±SE 2ddCt via PCR). Silencing miR-21 in CD34+ cells from CTRL phenocopied the T2DM-CLI behavior, resulting in significantly increased apoptosis and inhibition of EC networking. Likewise, exposure of endothelial cells to patient-derived CD34+ CCM induced apoptosis and reduced the network formation ability on Matrigel, with these functional liabilities being associated with miR-21 inhibition and PDCD4 upregulation. Conclusions Migratory activity of CD34+ cells predicts cardiovascular mortality at 6 years follow-up after PTA in subjects with T2DM and CLI. Complicated T2DM is associated with a specific miR signature in BM CD34+ cells which could negatively impact on vascular cell survival and angiogenic capacity, thereby possibly contributing to adverse cardiovascular outcomes. The miR-21/ PDCD4 duo may represent a therapeutic target to rescue diabetes-associated complications. Acknowledgement/Funding British Heart Foundation grant RG/13/17/30545, Cariplo Foundation (code: 2016-0922)