Table 2.

Mitochondrial DNA in peripheral circulating cells and cardiovascular disease.

Population Characteristics	Study Design	Mitochondrial Function/mtDNA Copy Number	Oxidative Stress	Cell Viability/Apoptosis	Results	Reference
Ischemic stroke patients Total n = 350 Age: 60.9 ± 9.1 Male n = 246 Female n = 104 Control group N = 350 Age:60.4 ± 9.1 Male n = 246 Female n = 104	mtDNA in Peripheral Blood Leukocyte	-mtDNA content (rt PCR) -The ratio of mtDNA to NuclearDNA is used to estimate the number of mtDNA per cell	-oxidized glutathione (GSSG), and reduced glutathione (GSH), (enzymatic (method) -8-hydroxy-2’-deoxyguanosine (biomarker of oxidative DNA damage, ELISA)	NA	mtDNA content in peripheral leukocyte for ischemic stroke patients was significantly lower than the control group. P < 0.0001 mtDNA content evaluated for 150 ischemic stroke patients = 0.90, while in 50 control individuals = 1.20 -The level of GSSG and 8-hydroxy-2’-deoxyguanosine were higher in patients with ischemic stroke than on the control group. GSSG Ischemic stroke = 1.83 Control = 0.79 8-hydroxy-2´-deoxyguanosine ischemic stroke = 6.33 Control = 4.87 These results exhibited that oxidative stress was higher in patients with ischemic stroke than in control group	Lien et al., 2017, Journal of American Heart Association [91]
3 cohort study with a risk factor of CVD 1st: Cardiovascular Health Study (CHS) n = 4830 Age: >65 years 2nd: Atherosclerosis Risk in Communities (ARIC) n = 11153 Age: Between 45 to 65 years 3rd: Multiethnic Study of Atherosclerosis (MESA) n = 5887 Age: 45 to 85 years Control: NA	In CHS: DNA was extracted from the buffy coat using salt precipitation following proteinase K digestion In ARIC: DNA was extracted from the buffy coat of whole blood using (Qiagen) In MESA: DNA was extracted from leukocyte using (Qiagen)	In ARIC and MESA, mtDNA copy number was measured by using prob intensities of mitochondrial single nucleotide polymorphisms (SNP) on the Affymetrix Genome-Wide Human SNP Array 6.0 IN CHS: mtDNA was calculated using multiplexed TaqMan-based PCR	NA	NA	-The effect of mtDNA copy number on the incidence of coronary heart disease was higher than in stroke and in other CVDs In all 3 cohort groups, the mtDNA copy number was inversely associated with CVD events	Ashar et al., 2017, JAMA Cardiology [79]
Coronary Heart Disease (CHD) classified in 4 groups according to Gensini score 1-Gensini score: 0-–22 n = 99, Male 72, Age: 57.3 2-Gensini score: 22–55 n = 98, Male 73, Age:57.9 3-Gensini score: 55–96 n = 102, Male 79, Age: 58.3 4-Gensini score:96–254 n = 101, Male 86, Age: 58.8 -Control groupn = 110Age: 58.1	mtDNA of Leukocytes for CHF categorized by Gensini score	-genomic DNA was isolated from peripheral blood cells by E.Z.N.A blood DNA Midi Kit. -mtDNA quantification (Quantitative real time PCR).	NA	NA	mtDNA content of PBMCs was lower in CHD patients than in the control group. -mtDNA was reduced significantly, while Gensini score was increased suggesting the level of circulating mtDNA correlates with presence and severity of CHD.	Liu et al. 2017, Atherosclerosis [82]
Acute coronary syndrome (ACS) Total n = 14 Divided into 2 groups 1st group: (Survivor) who survive during 30 day of hospitalization n = 11, male 9, female 2 Age: 53 2nd group: (deceased) who died due to ACS during time of analysis n = 3 female Age: 87	Blood samples were collected from platelet poor plasma	-To evaluate mtDNA. Isolation performed with PROBA-NK reagent kit. -quantitation of mtDNA was performed by PCR	NA	NA	-Deceased group: the level of mtDNA level was higher (5900 copies/mL) than the survived group (36 copies/mL) p = 0.049 -increased level of mtDNA in plasma suggest a probability of death of 50% for ACS patients	Sudakov et al., 2017, European Journal of Medical Research [87]
Patients from the Atherosclerosis Risk in Communities (ARIC) n = 11093 male n = 4971 female n = 6122 Age: 57.9 ± 6.0	mtDNA in peripheral blood buffy coat	mtDNA copy number was measured by using prob intensities of mitochondrial single nucleotide polymorphisms (SNP) on the Affymetrix Genome-Wide Human SNP Array 6.0	NA		-Inverse association between mtDNA copy number and sudden cardiac death	Zhang et al., 2017, Eur Heart Journal [92]
Acute myocardial infarction patient undergoing primary angioplasty n = 55 male n = 47 female n = 8 Age: 57.4 ± 11.4 years Control group: n = 54 male n = 44 female n = 10 age: 55.3 ± 7.4	Peripheral blood leukocyte	Leukocyte mitochondrial DNA copy number (MCN) was measured from venous blood using PCR -AMI patients were divided into two groups according to median baseline leukocyte mtDNA copy number = 82/cell 1st group MCN ≥ 82 2nd group MCN < 82	NA	NA	-Baseline characteristics: In AMI patients the plasma leukocyte mtDNA copy number was significantly lower than in the control group. 122.7 ± 109.3 vs. 194.9 ± 119.5/cell p = 0.003 -AMI patients with lower MCN, had higher left ventricle shape sphericity index (SI), at 1,3,6 months after angioplasty and higher left ventricle diastolic and systolic volume at 6 months after angioplasty.	Huang et al., 2017, Circulatiog Journal, [90]
Patients with diabetes mellitus and atherosclerosis cardiovascular disease Total n = 275 -only Atherosclerosis: N = 55 Female 18 Age:60 ± 10 -only DM: N = 74 Female 47 Age: 55 ± 10 -Atherosclerosis and DM N = 48 Female 31 Age: 62 ± 8 Control group n = 98 Female 49 Age: 55 ± 7	PBMCs	Measuring mitochondrial DNA damage in PBMCs by PCR. -Total DNA was separated using QIAmp DNA mini kit and quantification determined by using Pico-green assay kit	Oxidative stress of arterial pulsatility (increased baseline pulse amplitude p = 0.009)	NA	Mitochondrial DNA damage was higher in all 3 diseased group, as compared with controls, with the highest in the group combining atherosclerosis and diabetes. -mtDNA measured in DM alone (0.65 ± 1.0) -mtDNA measured in atherosclerosis alone (0.55 ± 0.65) -mtDNA measured in both atherosclerosis and DM (0.89 ± 1.32) p < 0.05 mtDNA damage correlated with baseline pulse amplitude	Fetterman et al., 2016, (Cardiovascular Diabetology) [86]
General population Total n = 701 Divided by 3 tertiles of mtDNA content -Tertile 1 mtDNA content 0.39–0.86 N = 233 Female 103 Age: 51.6 ± 16.8 EF% 61.3 ± 7.0 -Tertile 2 mtDNA content 0.86–1.10 N = 234 Female126 Age:53.5 ± 14.7 EF%: 63.3 ± 6.56 -Tertile 3 mtDNA content 1.11–3.06 N = 234 Female 128 Age:54.3 ± 14.2 EF%: 62.9 ± 6.65	Peripheral blood cells	To assess the circulating mtDNA content, PCR was used. Total DNA was extracted from peripheral blood sample using QIAmp DNA Mini Kit.	NA	NA	There is a relation between peripheral blood mtDNA copy number and left ventricular function. Higher mtDNA content was associated with better systolic and diastolic left ventricular function	Knez et al. 2016, International Journal of Cardiology [77]
Chronic Heart Failure Total N = 1700 -Ischemic HF N = 790 Male 543 Age: 62.6 ± 10.4 EF% 57 -Nonischemic HF N = 910 Male 572 Age: 53.8 ± 14.3 EF% 40 Control group n = 1700 male 1115 Age: 57.7 ± 11.0 EF%: NA	Circulating Leukocyte -Blood sample were drawn, and leukocyte were isolated in K2-EDTA tubes.	Total DNA was extracted by using QG-Mini80 workflow with a DB-S kit. And DNAs of cardiac tissues were isolated by using QIAmp DNA Mini Kit. And copy number ratio was evaluated.	ROS were quantified in heart tissues using Dihydroethidium (DHE) staining. -In lymphocyte intracellular ROS was analyzed by flow cytometry using DCFH-DA -LDL was detected		HF patients presented a low mtDNA content compared to control group. Median 0.83, IQR: 0.60–1.16 vs. median 1.00, IQR: 0.47–2.20)P < 0.001. Ischemic HF patients are more susceptible to lower mt DNA copy number(Median 0.77, IQR: 0.56–1.08) than non-ischemic HF median 0.91, IQR 0.63–1.22 -mtDNA content of leukocyte was not correlated with LV diameter p = 0.988 -in HF group, LDL was associated with the mtDNA copy number p = 0.007 -Lower circulating mtDNA was correlated with increased risk of HF, p < 0.001 -In HF patients, the level of ROS was higher than in control group in heart tissues and in lymphocytes.	Huang et al., 2016, Medicine [89]
Coronary heart Disease Patients N = 378 Male 279 Female 99 Age: 57.9 -Control group n = 378 male 279 female 99 Age: 58.9	Peripheral Blood Leukocytes -5 mL of venous blood was drawn from each individual and anticoagulated into sodium citrate tube.	-DNA was separated from peripheral blood leukocyte using E.Z.N.A blood DNA Midi Kit. -DNA content was measured using PCR	NA	NA	-mtDNA content was inversely related to increased risk of CHD -CHD group shows marked lower mtDNA content, compared to controls, p < 0.001, -CHF had higher neutrophils counts compared to controls (5.10 ± 1.66 vs. 4.50 ± 1.51) but no difference in WBC count p = 0.154	Chen et al. 2014 (Atherosclerosis) [88]
Myocardial infarction ST segment elevation MI (STEMI) n = 20, 5 femaleStable angina pectoris (SAP) n = 10, 1 female Both undergoing percutaneous coronary intervention (PCI) and categorized as transmural or non-transmural Age: between 30 and 75 years	Platelet poor plasma	Venous blood sample were gathered, and DNA was extracted from platelet poor plasma using QIAmp DNA blood Mini Kit -Quantification of mtDNA using real time PCR	NA	NA	-Baseline characteristics: Both groups were similar except SAP group which received more PCI treatment than the other group. -After PCI: 3 h later, mtDNA plasma level of NADH dehydrogenase subunit 1 (ND1) were increased in STEMI compared to SAP. p = 0.01 -patients with transmural: ND1 levels were greater in STEMI patients n = 10, than STEMI patients with non-transmural n = 6 -positive correlation between the severity of myocardial damage and the level of mtDNA, mtDNA being increased in myocardial infarction.	Bliksøen et al., 2012, [83]