Mitochondrial DNA damage and vascular function in patients with diabetes mellitus and atherosclerotic cardiovascular disease

Fetterman et al. Cardiovasc Diabetol (2016) 15:53 DOI 10.1186/s12933-016-0372-y ORIGINAL INVESTIGATION Cardiovascular Diabetology Open Access Mitochondrial DNA damage and vascular function in patients with diabetes mellitus and atherosclerotic cardiovascular disease Jessica L. Fetterman1*, Monica Holbrook1, David G. Westbrook2, Jamelle A. Brown2, Kyle P. Feeley2, Rosa Bretón‑Romero1, Erika A. Linder1, Brittany D. Berk1, Robert M. Weisbrod1, Michael E. Widlansky3, Noyan Gokce1, Scott W. Ballinger2 and Naomi M. Hamburg1 Abstract Objective: Prior studies demonstrate mitochondrial dysfunction with increased reactive oxygen species generation in peripheral blood mononuclear cells in diabetes mellitus. Oxidative stress-mediated damage to mitochondrial DNA promotes atherosclerosis in animal models. Thus, we evaluated the relation of mitochondrial DNA damage in periph‑ eral blood mononuclear cells s with vascular function in patients with diabetes mellitus and with atherosclerotic cardiovascular disease. Approach and results: We assessed non-invasive vascular function and mitochondrial DNA damage in 275 patients (age 57 ± 9 years, 60 % women) with atherosclerotic cardiovascular disease alone (N = 55), diabetes mellitus alone (N = 74), combined atherosclerotic cardiovascular disease and diabetes mellitus (N = 48), and controls age >45 without diabetes mellitus or atherosclerotic cardiovascular disease (N = 98). Mitochondrial DNA damage measured by quantitative PCR in peripheral blood mononuclear cells was higher with clinical atherosclerosis alone (0.55 ± 0.65), diabetes mellitus alone (0.65 ± 1.0), and combined clinical atherosclerosis and diabetes mellitus (0.89 ± 1.32) as compared to control subjects (0.23 ± 0.64, P < 0.0001). In multivariable models adjusting for age, sex, and relevant cardiovascular risk factors, clinical atherosclerosis and diabetes mellitus remained associated with higher mitochon‑ drial DNA damage levels (β = 0.14 ± 0.13, P = 0.04 and β = 0.21 ± 0.13, P = 0.002, respectively). Higher mitochon‑ drial DNA damage was associated with higher baseline pulse amplitude, a measure of arterial pulsatility, but not with flow-mediated dilation or hyperemic response, measures of vasodilator function. Conclusions: We found greater mitochondrial DNA damage in patients with diabetes mellitus and clinical athero‑ sclerosis. The association of mitochondrial DNA damage and baseline pulse amplitude may suggest a link between mitochondrial dysfunction and excessive small artery pulsatility with potentially adverse microvascular impact. Background Type 2 diabetes mellitus affects an estimated 1 in 10 Americans and this number is expected climb with the current obesity epidemic [1]. Diabetes mellitus is a *Correspondence: 1 Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, 72 East Concord Street, E‑784, Boston, MA 02118, USA Full list of author information is available at the end of the article significant risk factor for cardiovascular disease; however, the mechanisms behind this increased risk are incompletely understood [1, 2]. Elevated oxidant levels have been shown to contribute to vascular dysfunction both in animal models and clinical studies [3–5]. Mitochondria are an important source and target of oxidants that may contribute to vascular disease in diabetes mellitus [6–8]. Mitochondrial DNA is more susceptible to oxidative damage compared to nuclear DNA due to multiple © 2016 Fetterman et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fetterman et al. Cardiovasc Diabetol (2016) 15:53 factors including a limited repair capacity and close proximity to the electron transport chain [9, 10]. Mitochondrial DNA damage has been closely associated with dysfunctional oxidative phosphorylation, which leads to further oxidative stress resulting in a positive-feedback cycle. In an animal model of atherosclerosis, excess mitochondrial DNA damage promoted atherosclerosis and plaque vulnerability through increased monocyte activation [11]. In a prior human study of patients with coronary artery disease, the extent of mitochondrial DNA damage in circulating white cells was associated with high risk plaque burden [12]. We have previously described altered mitochondrial oxidative phosphorylation, membrane potential and morphology in peripheral blood mononuclear cells which was associated with vascular dysfunction in patients with diabetes [7, 13]. The objective of the present study was to assess the relation of mitochondrial DNA damage in peripheral blood mononuclear cells to vascular function and the presence of diabetes mellitus and atherosclerotic cardiovascular disease. Methods Study participants We enrolled four groups of patients (N = 275): (1) clinically established atherosclerotic cardiovascular disease (Athero; coronary artery disease and/or peripheral artery disease); (2) diabetes mellitus (DM; fasting glucose levels >126 mg/dL or medication therapy); (3) diabetes mellitus and atherosclerosis (Athero + DM); (4) controls with no clinically established atherosclerosis, no diabetes mellitus (fasting glucose <100 mg/dL) and age >45 years. Patients with clinical atherosclerotic cardiovascular disease were enrolled from outpatient cardiology and vascular surgery practices. Coronary artery disease was defined based on angiography or documented history of myocardial infarction. Peripheral artery disease was defined as ankle brachial index ≤0.9 or prior peripheral revascularization. All participants gave written informed consent and all study protocols were approved by the Boston Medical Center Institutional Review Board. Study protocol Clinical history and relevant clinical covariates were compiled from participant interviews and medical records. Blood pressure was assessed with an automatic recorder (Dinamap; General Electric Healthcare) and body mass index (BMI) was calculated from measured height and weight. All studies were performed in the fasted state. Peripheral blood mononuclear cells were isolated by differential centrifugation of a blood sample. Briefly, venous blood was collected into a density gradient solution for the isolation of lymphocytes and monocytes Page 2 of 7 (BD Vacutainer CPT cell preparation tubes with sodium citrate; Becton, Dickinson, and Company) and the tubes were spun at 3000 rpm for 30 min at room temperature. Cell layers were collected, pelleted, and stored a (...truncated)