Significant Association Between Low Mitochondrial DNA Content in Peripheral Blood Leukocytes and Ischemic Stroke
Background Cumulative evidence has shown that low mitochondrial DNA (mtDNA) content is related to elevated oxidative stress and atherosclerosis, which play important roles in ischemic stroke. The objective of this study was to explore the association between mtDNA content in peripheral blood leukocytes and ischemic stroke.
Methods and Results A total of 350 patients with first‐ever ischemic stroke and 350 healthy controls were recruited in this case‐control study. The mtDNA content in peripheral blood leukocytes was determined by quantitative real‐time polymerase chain reaction. The levels of oxidized glutathione, reduced glutathione, and 8‐hydroxy‐2′‐deoxyguanosine were measured by ELISA kits. Multivariate logistic regression models were used to analyze the relationship between mtDNA content in peripheral blood leukocytes and ischemic stroke. Our results show that mtDNA content of patients with ischemic stroke was notably lower compared with controls. A significant association was found between low mtDNA content and ischemic stroke. Furthermore, significant interactions were identified between low mtDNA and proven risk factors in patients with ischemic stroke. The levels of oxidized glutathione and 8‐hydroxy‐2′‐deoxyguanosine were significantly greater in patients with ischemic stroke compared with controls.
Conclusions Our results demonstrate that low mtDNA content in peripheral blood leukocytes is associated with ischemic stroke. The relationship of low mtDNA content and ischemic stroke was particularly notable in individuals who had low mtDNA content combined with diabetes mellitus, metabolic syndrome, or cigarette smoking. Oxidative stress may be one of the contributory factors to decreased mtDNA content in patients with ischemic stroke.
What Is New?
Our study demonstrated the association between low mitochondrial DNA content in peripheral blood leukocytes and ischemic stroke.
Synergistic effects were observed between diabetes mellitus, metabolic syndrome, cigarette smoking, and low mitochondrial DNA content on ischemic stroke risk.
What Are the Clinical Implications?
Other than traditional cardiovascular risk factors, mitochondrial dysfunction may promote ischemic stroke risk.
Stroke is the third leading cause of death and an important cause of adult disability in Taiwan. The majority of stroke cases are ischemic stroke.1 Accumulating evidence shows that oxidative stress plays a key role in the development of ischemic stroke. Mitochondria are the major cellular source of reactive oxygen species (ROS). Oxidative damage to mitochondria is likely to lead to increased production of ROS through disruption of respiratory function.2 Excess mitochondria‐derived ROS from any cause can induce endothelial dysfunction,3 which is closely associated with the development of atherosclerosis and atherosclerotic plaque,4 which are critical etiologic factors of ischemic stroke.
Human mitochondrial DNA (mtDNA) is 16 569‐bp long and encodes 13 polypeptides of the electron transport chain and ATP synthase.5 Changes in the biogenesis and function of mitochondria have been documented in cardiovascular disease (CVD), atherosclerosis, metabolic syndrome (MS), and diabetes mellitus (DM).6, 7, 8, 9, 10, 11, 12, 13 For example, Karamanlidis and coworkers6 reported that the mtDNA content and mtDNA‐encoded proteins were significantly reduced in the failing human heart, indicative of impaired mitochondrial biogenesis. They also observed a strong correlation between mtDNA content and mRNA levels of mtDNA‐encoded genes. Another population‐based study indicated that higher mtDNA content was associated with smaller left ventricular diastolic and systolic diameters and volumes and improved left ventricular systolic and diastolic function.7 Chien et al found that atherogenesis was associated with decreased amounts of mtDNA among patients with DM.8 Kim et al showed that there was a negative association between leukocyte mtDNA and MS after adjustment for potential confounding variables.11 Significantly lower mtDNA content was observed in patients with type 2 DM compared with patients with normal glucose tolerance.8 Recently, a case‐control study by Chen et al13 revealed that low mtDNA content in peripheral blood leukocytes was associated with an increased risk of coronary heart disease.
Although several studies have demonstrated an association between mtDNA mutations and increased risk of ischemic stroke,10, 14, 15 there are few studies that have investigated the association between mtDNA content in peripheral blood leukocytes and the risk of ischemic stroke. Furthermore, cumulative evidence shows that low mtDNA content is related to elevated oxidative stress and atherosclerosis, which play important roles in ischemic stroke. Therefore, the aim of this study was to determine whether there is a relationship between mtDNA content in peripheral blood leukocytes and ischemic stroke risk using an epidemiologic study design.
Participants and Study Design
A total of 350 patients with first‐ever ischemic stroke and 350 controls without stroke were recruited in this case‐control study. The patients with ischemic stroke were randomly selected from the Formosa Stroke Genetic Consortium (FSGC), which enrolled nearly 1700 patients with ischemic stroke between 2005 and 2010. FSGC is a platform for hospital collaborations on studies related to the molecular biology of cerebrovascular diseases. All cases of ischemic stroke from the FSGC were confirmed with computed tomography and/or MRI. The subtypes of ischemic stroke were classified according to the TOAST (Trial of Org 10172 in Acute Stroke Treatment) criteria.16 A tube of blood sample was collected from each patient during 3 days to 1 month post stroke. The details of the FSGC are described in a previous study.17 Controls were recruited from those who underwent a physical examination program at Taipei Medical University Hospital and Taipei Medical University Shuang‐Ho Hospital. After excluding patients with stroke history and frequency matching with cases by age (±5 years) and sex in a ratio of 1:1, a total of 350 control patients were randomly selected from 1150 healthy candidates. This study was performed in accordance with the Declaration of Helsinki and approved by the institutional review board for human subjects from Taipei Medical University. A written informed consent for blood sampling and genetic analysis was obtained from each patient before participation in this study.
Briefly, a structured questionnaire including demographic data, major CVD risk factors, blood biochemistry data, and medical history was collected by FSGC‐trained study nurses or research assistants. Participants with a smoking habit were defined as individuals who had smoked >100 cigarettes during their lifetime. Alcohol drinking was defined as the consumption of at least one alcoholic drink per day for more than 1 year. Hypertension was defined as blood pressure (BP) >140/90 mm Hg, the presence of chronic hypertension as diagnosed by a physician, or the use of antihypertensive medication. DM was defined by a clinical diagnosis of DM, a fasting glucose level >126 mg/dL, or the taking of DM medication. Body mass index was calculated as weight in kilograms divided by the square of height in meters. According to the National Cholesterol Education Program's Adult Treatment Panel III definition for MS, a participant was deemed to have the disease when ≥3 of the following criteria were satisfied: (1) waist circumference >102 cm in men and >88 cm in women; (2) triglyceride level ≥1.7 mmol/L; (3) high‐density lipoprotein cholesterol <1.0 mmol/L in men and <1.3 mmol/L in women; (4) BP ≥130/85 mm Hg or known treatment for hypertension; and (5) fasting glucose level of ≥6.1 mmol/L or known treatment for DM.
Determination of mtDNA Content in Peripheral Blood Leukocytes
Genomic DNA used for mtDNA quantification was extracted from peripheral blood leukocytes by using a nonorganic purification method.18 The ratio of mtDNA to nuclear DNA is often used as an estimate for the number of mtDNA per cell.19, 20, 21, 22 For this purpose, 2 genes, a D‐loop segment of human mtDNA sequence and nuclear DNA β2‐microglobulin, were selected to quantify the mtDNA. The nuclear DNA β2‐microglobulin was used to normalize the mtDNA amount per cell. A comparative CT method (ΔΔCT method) was performed according to the Applied Biosystems protocol for relative quantitation. All samples were assayed in duplicate using the StepOne Real‐Time Polymerase Chain Reaction System (Applied Biosystems) and the TaqMan method. The details of the method are described in Data S1.
Determination of Oxidative Stress and Markers Related to Mitochondrial Function in Plasma
Because of the limited funding and the available plasma specimens, two hundreds of study subjects were randomly selected from the participants with available plasma samples. Measurement of total glutathione (GSH) and oxidized GSH (GSSG) in plasma was performed by an enzymatic recycling method, using GSH reductase, according to the instructions of a GSH assay kit (Cayman Chemical). In brief, all plasma samples were deproteinated before assaying. An equal volume of 10% metaphosphoric acid was added to the sample. After centrifuge at 10 000g for 10 minutes, 20 μL of 4 mol/L triethanolamine reagent was added for measurement of the total GSH levels in the sample. For quantification of GSSG, 4 μL of 1 mol/L 2‐vinylpyridine solution was added to the deproteinated sample. After incubating at room temperature for 60 minutes, the sample was used to measure the GSSG levels. Finally, the total GSH and GSSG concentration was then determined by the end point method according to the procedure from the assay kit. The concentration of reduced GSH was calculated as total GSH−GSSG.
Measurement of 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) in plasma was performed by a competitive ELISA method using 8‐OHdG ELISA kits from BioVision Inc and determined the levels of peroxisome proliferator‐activated receptor γ coactivator 1α (PPARGC1A, also known as PGC1A) and mitochondrial uncoupling protein 2 (UCP2) in plasma were performed by a quantitative sandwich enzyme immunoassay technique using human PPARGC1A ELISA kits and human UCP2 ELISA kits from CUSABIO Biotech following manufacturer's protocol. Briefly, 8‐OHdG, PPARGC1A, and UCP2 were detected using a fluorescence microplate reader at 450 nm. For PPARGC1A and UCP2, wavelength correction was performed at subtract readings at 540 nm from the readings at 450 nm. The amounts of 8‐OHdG, PPARGC1A, and UCP2 were calculated using a standard curve generated from the standard solution.
Based on power calculations (assuming equal group size) for logistic regression, 266 to 776 study patients were needed to provide 80% power to detect a statistically significant (α=0.05) odds ratio (OR) of 2.0~1.5. Student t test was used to compare continuous variables between groups in Table 1. In the same table, chi‐square test was applied to test differences in categorical variables. In Table 2, univariate and multivariate logistic regression models were used to evaluate the association of mtDNA content with ischemic stroke. Age, sex, smoking, and alcohol drinking–corrected Spearman rank partial correlation coefficients were calculated to evaluate the relationship between mtDNA and clinical data in Table 3. A Kolmogorov–Smirnov test was used to check normality. Because the distribution of mtDNA content and oxidative stress markers did not meet the normality assumption, a Wilcoxon rank sum test and generalized linear models adjusting for age, sex, DM, hypertension, smoking, alcohol drinking, and body mass index were used in Table 4. A Wilcoxon rank sum test and a Kruskal–Wallis test were used to compare medians in Figure 1 and Figure S1, respectively. A Rothman's synergy index23 (S index) was used to measure the interaction on an additive scale in Figure 2. The confidence interval (CI) of the S index was estimated with the method described by Hosmer and Lemeshow.24 A synergistic interaction was defined as an S index >1. Spearman rank partial correlation coefficients with age, sex, smoking, and alcohol drinking‐adjusted were used to determine the correlations of mtDNA content with the markers related to mitochondrial function in Figure 3. P values for 2‐tailed tests were calculated, and statistical significance was set at P<0.05. Analyses were performed using SAS statistical software 9.1 (SAS Institute).
Characteristics of Study Participants
A total of 700 study patients including 350 patients with ischemic stroke and 350 controls were recruited in this study. The basic characteristics of patients with ischemic stroke and controls are shown in Table 1. The distribution of cigarette smoking, alcohol drinking, MS, hypertension, and DM were significantly different between patients and controls. These risk factors were more common in patients with ischemic stroke. Dyslipidemia and overweight were also more frequently observed in patients with ischemic stroke, but the difference did not reach statistical significance.
Association of mtDNA Content in Peripheral Blood Leukocytes With Ischemic Stroke
As shown in Figure 1, the median of mtDNA content from peripheral blood leukocytes in patients with ischemic stroke was significantly lower compared with controls. The relationships between traditional risk factors, mtDNA content, and ischemic stroke are shown in Table 2. A significantly higher risk of ischemic stroke was found in patients with cigarette smoking (OR, 2.31; 95% CI, 1.70–3.14), alcohol drinking (OR, 1.87; 95% CI, 1.24–2.80), MS (OR, 2.88; 95% CI, 2.11–3.91), hypertension (OR, 2.32; 95% CI, 1.70–3.18), and DM (OR, 3.99; 95% CI, 2.82–5.65). There was also a strong association between the lower mtDNA content and ischemic stroke (OR, 11.77; 95% CI, 8.26–16.77). The results remained mainly unchanged in a multivariate logistic regression model (OR, 11.73) that adjusted for age, sex, hypertension, DM, MS, body mass index, smoking, and alcohol drinking. We also compared the mtDNA content in different subtypes of ischemic stroke, and no significant difference was observed according to Kruskal–Wallis test (P=0.446) (Figure 1).
Effect of mtDNA Content With Traditional Risk Factors of CVD on Ischemic Stroke
In order to further analyze the interaction between traditional risk factors and mtDNA content, 5 common traditional CVD risk factors (MS, hypertension, DM, smoking, and alcohol drinking) were selected to investigate their impacts with mtDNA on ischemic stroke. Study patients were stratified into 4 groups according to risk factor status and level of mtDNA content (Figure 2). Compared with the patients with high mtDNA content and without MS, the proportion of patients with ischemic stroke was increased in patients with only low mtDNA content or only MS, and the proportion of patients with ischemic stroke was further increased in patients with both low mtDNA content and MS (S index=2.52; 95% CI, 1.47–4.31). Similar interaction effects were observed between low mtDNA content and smoking (S index=2.07; 95% CI, 1.17–3.67) and low mtDNA content and DM (S index=3.50; 95% CI, 1.83–6.72). However, there were no synergistic effects between hypertension, alcohol drinking, and mtDNA content on ischemic stroke.
Correlations Between mtDNA Content and Clinical Data
In Spearman partial correlation analysis (Table 3), our data indicated that peripheral blood leukocytes mtDNA content was negatively correlated with age (ρ=−0.14), smoking (ρ=−0.15), systolic BP (ρ=−0.36), diastolic BP (ρ=−0.19), fasting glucose (ρ=−0.13), triglycerides (ρ=−0.16), and waist‐hip ratio (ρ=−0.19) after being adjusted for sex, age, smoking, and alcohol drinking. In contrast, peripheral blood mtDNA content was positively correlated with high‐density lipoprotein cholesterol (ρ=0.08).
Oxidative Stress Marker and mtDNA Content in Patients With Ischemic Stroke and Controls
The levels of GSSG, GSH/GSSG ratio, and 8‐OHdG in plasma were used to determine oxidative stress. As shown in Table 4, the medians of GSSG and 8‐OHdG were significantly greater in patients with ischemic stroke compared with controls. After adjusting for age, sex, DM, hypertension, smoking, alcohol drinking, and body mass index, the concentration of GSSG was still significantly greater in patients with ischemic stroke and the greater level of 8‐OHdG in patients with ischemic stroke turned to borderline significance. The results indicated that oxidative stress was significantly greater in patients with ischemic stroke compared with controls. However, mtDNA content was lower in patients with ischemic stroke compared with controls.
Association of mtDNA Content With Markers Related to Mitochondrial Function
To examine whether reduced mtDNA content indicated mitochondrial dysfunction, we measured the levels of PPARGC1A and UCP2, which involved mitochondrial biogenesis and mediated proton leak across the inner membrane of mitochondria as the indicators of mitochondria function. In Figure 3A, mtDNA content ≥1.09 was classified as high mtDNA content and <1.09 as low mtDNA content. Both PPARGC1A and UCP2 were negatively correlated with high mtDNA content. In Figure 3B, a significantly moderate negative correlation was found between mtDNA content (continuing value) and UCP2 (ρ=−0.31, P=0.005) after adjusting for age, sex, smoking, and alcohol drinking.
This is the first molecular epidemiologic study to demonstrate the negative association between peripheral blood mtDNA content and ischemic stroke. Previously, most neurologists focused on the role of mitochondria in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke syndrome, which are progressive neurodegenerative disorders.25 Only a few studies investigated the relationship between mtDNA mutations and the risk of ischemic stroke either using a genome‐wide association study or a haplogroup association study.10, 14, 15 In the present study, we found that patients with ischemic stroke exhibited strikingly lower peripheral blood mtDNA content compared with healthy controls. Similar results have been reported from studies of CVDs. Chen et al observed that low mtDNA content in peripheral blood leukocytes was linked to an increased risk of coronary heart disease.13 Ide et al26 reported that the mtDNA content was preferentially decreased by 44% in a murine model of myocardial infarction. In addition, previous studies also demonstrated that decline of leukocyte mtDNA content was involved in atherogenesis,27, 28 which played an important role in the development of ischemic stroke.29, 30 A recent study reported a significantly positive correlation between mtDNA content in peripheral blood mononuclear cells and mtDNA content in atherosclerotic plaque tissue.13 This finding indicates that leukocyte mtDNA content could be used as a surrogate of atherosclerotic plaque mtDNA content. Notably, measuring mtDNA content from peripheral blood samples is more acceptable to most patients. In our study, no significant difference was observed for mtDNA content in different subtypes of ischemic stroke. Although the mechanisms for different types of ischemic stroke may be different, atherosclerosis may account for the majority of these ischemic stroke subtypes. For patients with cardioembolism, many had atrial fibrillation and coronary artery disease. Atrial fibrillation is often considered a prothrombotic state. Therefore, the mtDNA level in different types of ischemic stroke may be similar.
Mitochondrial dysfunction can be caused by impaired mitochondrial biogenesis, which can be revealed by a decrease of mtDNA content. Increased oxidative stress may contribute to alterations in the copy number and integrity of mtDNA in human cells in pathological conditions.31, 32 Decreased mtDNA content was reported to result in excess mitochondrial ROS production.33 Increased production of ROS, in turn, leads to oxidative damage to lipids, proteins, and mtDNA.34 Furthermore, mtDNA damage may lead to decreased expression of constituent polypeptides of respiratory enzymes or assembly of defective respiratory enzymes that produce more ROS.35 ROS‐induced ROS release may amplify and target ROS signals under physiological conditions; however, when excessive, may contribute to the pathogenesis of endothelial dysfunction, atherosclerosis36, 37 and the evolution of CVD.38 Consistent with these basic research findings, our results showed that patients with ischemic stroke exhibited significantly lower mtDNA content compared with controls, suggesting a marked relationship between low mtDNA content in peripheral blood leukocytes and ischemic stroke.
The results of our study demonstrated interaction effects between MS, DM, smoking, and mtDNA content, in the modulation of ischemic stroke risk. Previously, several studies have reported high oxidative stress levels in patients with MS,39, 40 patients with DM,41, 42 and in smokers.43, 44 We also found significantly negative correlations between mtDNA content with BP, blood glucose, triglyceride, and waist‐hip ratio, which were in line with findings from previous studies.45, 46 Increased ROS was a common feature in MS, DM, smoking, and mitochondrial dysfunction. Cumulative oxidative stress may eventually accelerate ischemic stroke process. The underlying mechanisms of interaction effect between mtDNA and host variables on the risk of ischemic stroke need further study.
In our study, we found greater oxidative stress in patients with ischemic stroke compared with controls. However, mtDNA content was lower in patients with ischemic stroke compared with controls. One possible explanation may be that oxidative damage to mtDNA polymerase gamma declined mtDNA replicaiton and eventually led to loss of mitochondrial function.18 Oxidative stress may be one of the contributory factors to decreased mtDNA content in patients with ischemic stroke. We also found a significantly moderate negative correlation between mtDNA content and UCP2 and a weak negative correlation with PPARGC1A. Previous studies indicate that mitochondrial dysfunction induced by oxidative stress may lead to UCP2 and PPARGC1A expression.47, 48 Therefore, according to our results, we speculate that the decreased mtDNA is associated with mitochondrial dysfunction.
There are several potential limitations in this study. First, a case‐control study design was employed and mtDNA content was measured after disease onset. Thus, a reverse causality on the correlation between mtDNA content and ischemic stroke is possible. Previous reports indicate that mtDNA content appear to have high heritability49 and negative correlations with increased ROS, endothelial cells dysfunction, and atherosclerosis. All current knowledge of mtDNA supports the view that low mtDNA content is more likely to be a risk factor for ischemic stroke. Second, only Han Chinese patients were selected in this study. It is necessary to confirm this association in other populations. Third, there was a lack of a direct connection between changes in peripheral blood leukocytes mtDNA and incidence of stroke. Further research using a cohort study design is needed to clarify the relationship between reduced mtDNA and incidence of stroke. Finally, our study was observational in design, thus we cannot rule out the presence of unknown confounding variables, not accounted for in the final analysis.
Low mtDNA content in peripheral blood leukocytes was significantly correlated with ischemic stroke. Synergistic effects were observed between MS, DM, smoking, and low mtDNA content on ischemic stroke risk. Oxidative stress may be one of the contributing factors to decreased mtDNA content in patients with ischemic stroke. Further studies are warranted to explore the underlying mechanism of the effect of mtDNA content on ischemic stroke. Stroke is among the most frequent causes of death and adult disability in highly developed countries. However, treatment options remain limited. Mitochondria‐targeted antioxidants may be a future option for treatment of ischemic stroke.
Sources of Funding
This work was supported in part by grants (MOST104‐2627‐M‐715‐002, MOST103‐2314‐B‐038‐020, and NSC100‐2314‐B‐038‐030‐MY3) from the Ministry of Science and Technology of Taiwan Government, the Ministry of Health and Welfare (MOHW105‐TDU‐B‐212‐133018), Taipei Medical University (SKH‐TMU‐102‐01), and the Dr. Chi‐Chin Huang Stroke Research Center.
Data S1. Supplemental Methods.
Figure S1. Box plot analysis illustrating the distribution of mitochondrial DNA (mtDNA) content in peripheral blood leukocytes among patients with different ischemic stroke subtypes.
We would like to thank the participants of the Formosa Stroke Genetic Consortium (FSGC) for their important contributions.
- ↵Hsieh FI, Lien LM, Chen ST, Bai CH, Sun MC, Tseng HP, Chen YW, Chen CH, Jeng JS, Tsai SY, Lin HJ, Liu CH, Lo YK, Chen HJ, Chiu HC, Lai ML, Lin RT, Sun MH, Yip BS, Chiou HY, Hsu CY; Taiwan Stroke Registry I . Get with the guidelines‐stroke performance indicators: surveillance of stroke care in the Taiwan Stroke Registry: Get With the Guidelines‐Stroke in Taiwan. Circulation. 2010;122:1116–1123.
- ↵Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT. The MIQE guidelines: minimum information for publication of quantitative real‐time PCR experiments. Clin Chem. 2009;55:611–622.
- ↵Karamanlidis G, Nascimben L, Couper GS, Shekar PS, del Monte F, Tian R. Defective DNA replication impairs mitochondrial biogenesis in human failing hearts. Circ Res. 2010;106:1541–1548.
- ↵Knez J, Cauwenberghs N, Thijs L, Winckelmans E, Brguljan‐Hitij J, Yang WY, Staessen JA, Nawrot TS, Kuznetsova T. Association of left ventricular structure and function with peripheral blood mitochondrial DNA content in a general population. Int J Cardiol. 2016;214:180–188.
- ↵Thorp E, Li Y, Bao L, Yao PM, Kuriakose G, Rong J, Fisher EA, Tabas I. Brief report: increased apoptosis in advanced atherosclerotic lesions of Apoe‐/‐ mice lacking macrophage Bcl‐2. Arterioscler Thromb Vasc Biol. 2009;29:169–172.
- ↵Rosa A, Fonseca BV, Krug T, Manso H, Gouveia L, Albergaria I, Gaspar G, Correia M, Viana‐Baptista M, Simoes RM, Pinto AN, Taipa R, Ferreira C, Fontes JR, Silva MR, Gabriel JP, Matos I, Lopes G, Ferro JM, Vicente AM, Oliveira SA. Mitochondrial haplogroup H1 is protective for ischemic stroke in Portuguese patients. BMC Med Genet. 2008;9:57.
- ↵Anderson CD, Biffi A, Rahman R, Ross OA, Jagiella JM, Kissela B, Cole JW, Cortellini L, Rost NS, Cheng YC, Greenberg SM, de Bakker PI, Brown RD Jr., Brott TG, Mitchell BD, Broderick JP, Worrall BB, Furie KL, Kittner SJ, Woo D, Slowik A, Meschia JF, Saxena R, Rosand J; International Stroke Genetics C . Common mitochondrial sequence variants in ischemic stroke. Ann Neurol. 2011;69:471–480.
- ↵Adams HP Jr., Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, Marsh EE III.. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke. 1993;24:35–41.
- ↵Hsieh YC, Seshadri S, Chung WT, Hsieh FI, Hsu YH, Lin HJ, Tseng HP, Lien LM, Bai CH, Hu CJ, Jeng JS, Tang SC, Chen CI, Yu CC, Chiou HY; Formosa Stroke Genetic C . Association between genetic variant on chromosome 12p13 and stroke survival and recurrence: a one year prospective study in Taiwan. J Biomed Sci. 2012;19:1.
- ↵Bogacka I, Xie H, Bray GA, Smith SR. Pioglitazone induces mitochondrial biogenesis in human subcutaneous adipose tissue in vivo. Diabetes. 2005;54:1392–1399.
- ↵Rothman KJ. Interactions between causes. Modern Epidemiology. Michigan: Diaz de Santos Publishers. 1986. pp. 311–326.
- ↵Hanna MG, Nelson IP, Morgan‐Hughes JA, Wood NW. MELAS: a new disease associated mitochondrial DNA mutation and evidence for further genetic heterogeneity. J Neurol Neurosurg Psychiatry. 1998;65:512–517.
- ↵Ide T, Tsutsui H, Hayashidani S, Kang D, Suematsu N, Nakamura K, Utsumi H, Hamasaki N, Takeshita A. Mitochondrial DNA damage and dysfunction associated with oxidative stress in failing hearts after myocardial infarction. Circ Res. 2001;88:529–535.
- ↵Ballinger SW, Patterson C, Knight‐Lozano CA, Burow DL, Conklin CA, Hu Z, Reuf J, Horaist C, Lebovitz R, Hunter GC, McIntyre K, Runge MS. Mitochondrial integrity and function in atherogenesis. Circulation. 2002;106:544–549.
- ↵Madamanchi NR, Runge MS. Mitochondrial dysfunction in atherosclerosis. Circ Res. 2007;100:460–473.
- ↵Hollander M, Bots ML, Del Sol AI, Koudstaal PJ, Witteman JC, Grobbee DE, Hofman A, Breteler MM. Carotid plaques increase the risk of stroke and subtypes of cerebral infarction in asymptomatic elderly: the Rotterdam study. Circulation. 2002;105:2872–2877.
- ↵Ballinger SW, Patterson C, Yan CN, Doan R, Burow DL, Young CG, Yakes FM, Van Houten B, Ballinger CA, Freeman BA, Runge MS. Hydrogen peroxide‐ and peroxynitrite‐induced mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells. Circ Res. 2000;86:960–966.
- ↵Doughan AK, Harrison DG, Dikalov SI. Molecular mechanisms of angiotensin II‐mediated mitochondrial dysfunction: linking mitochondrial oxidative damage and vascular endothelial dysfunction. Circ Res. 2008;102:488–496.
- ↵Shenouda SM, Widlansky ME, Chen K, Xu G, Holbrook M, Tabit CE, Hamburg NM, Frame AA, Caiano TL, Kluge MA, Duess MA, Levit A, Kim B, Hartman ML, Joseph L, Shirihai OS, Vita JA. Altered mitochondrial dynamics contributes to endothelial dysfunction in diabetes mellitus. Circulation. 2011;124:444–453.
- ↵Yubero‐Serrano EM, Delgado‐Lista J, Pena‐Orihuela P, Perez‐Martinez P, Fuentes F, Marin C, Tunez I, Tinahones FJ, Perez‐Jimenez F, Roche HM, Lopez‐Miranda J. Oxidative stress is associated with the number of components of metabolic syndrome: LIPGENE study. Exp Mol Med. 2013;45:e28.
- ↵Njajou OT, Kanaya AM, Holvoet P, Connelly S, Strotmeyer ES, Harris TB, Cummings SR, Hsueh WC; Health ABCS . Association between oxidized LDL, obesity and type 2 diabetes in a population‐based cohort, the Health, Aging and Body Composition Study. Diabetes Metab Res Rev. 2009;25:733–739.
- ↵Mons U, Muscat JE, Modesto J, Richie JP Jr., Brenner H. Effect of smoking reduction and cessation on the plasma levels of the oxidative stress biomarker glutathione—post‐hoc analysis of data from a smoking cessation trial. Free Radic Biol Med. 2016;91:172–177.
- ↵Jornayvaz FR, Shulman GI. Regulation of mitochondrial biogenesis. Essays Biochem. 2010;47:69–84.