Smoking Status Impacts Mitochondrial Function in Peripheral Arterial Disease Patient-Derived Mesenchymal Stem Cells

Abstract

INTRODUCTION: Patient mesenchymal stem cells (MSCs) present a new regenerative medicine tool to treat peripheral arterial disease (PAD). Mitochondrial function, i.e., bioenergetics, and turnover via mitophagy are key regulators of “stemness” and may impact regenerative potential of MSCs. Patient smoking status is known to impact MSC proliferation, but the effect on mitochondrial function is unknown. We hypothesize that smoking status impacts mitophagy and mitochondrial bioenergetics in MSCs. METHODS: Non-PAD MSCs were purchased from ATCC. PAD MSCs were obtained after major amputation under our IRB-approved project, Learning from Failure. Mitochondria were stained with Mitotracker Green FM and lysosomes with Lysotracker Deep Red for co-localization in live cells using confocal microscopy. Co-localization was quantified using FIJI software and reported using Spearman’s Rank correlation coefficient. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XFe96 Analyzer. RESULTS: Two PAD patient lines were from smokers and two from non-smokers. Mitophagy (co-localization of mitochondria with lysosomes) did not differ significantly by smoking status in PAD MSCs. PAD MSCs also did not differ in mitophagy when compared with the non-PAD control. Maximal mitochondrial respiration (maximal OCR) differed significantly among the smoking PAD, non-smoking PAD, and non-PAD control MSCs (p < .0001). Glycolytic capacity (ECAR) also differed significantly between groups (p < .0001) (Figure). CONCLUSION: Active smoking status affected mitochondrial bioenergetics in patient MSCs, suggesting a potential impact on “stemness.” Further characterization of the impact of smoking on regenerative capacity of patient MSCs will inform optimal development of stem cell-based therapies to treat PAD.Figure