Emma Yu

Mitochondrial DNA Damage Can Promote Atherosclerosis Independently of Reactive Oxygen Species Through Effects on Smooth Muscle Cells and Monocytes and Correlates With Higher-Risk Plaques in Humans

Background— Mitochondrial DNA (mtDNA) damage occurs in both circulating cells and the vessel wall in human atherosclerosis. However, it is unclear whether mtDNA damage directly promotes atherogenesis or is a consequence of tissue damage, which cell types are involved, and whether its effects are mediated only through reactive oxygen species. Methods and Results— mtDNA damage occurred early in the vessel wall in apolipoprotein E–null (ApoE−/−) mice, before significant atherosclerosis developed. mtDNA defects were also identified in circulating monocytes and liver and were associated with mitochondrial dysfunction. To determine whether mtDNA damage directly promotes atherosclerosis, we studied ApoE−/− mice deficient for mitochondrial polymerase-&ggr; proofreading activity (polG−/−/ApoE−/−). polG−/−/ApoE−/− mice showed extensive mtDNA damage and defects in oxidative phosphorylation but no increase in reactive oxygen species. polG−/−/ApoE−/− mice showed increased atherosclerosis, associated with impaired proliferation and apoptosis of vascular smooth muscle cells, and hyperlipidemia. Transplantation with polG−/−/ApoE−/− bone marrow increased the features of plaque vulnerability, and polG−/−/ApoE−/− monocytes showed increased apoptosis and inflammatory cytokine release. To examine mtDNA damage in human atherosclerosis, we assessed mtDNA adducts in plaques and in leukocytes from patients who had undergone virtual histology intravascular ultrasound characterization of coronary plaques. Human atherosclerotic plaques showed increased mtDNA damage compared with normal vessels; in contrast, leukocyte mtDNA damage was associated with higher-risk plaques but not plaque burden. Conclusions— We show that mtDNA damage in vessel wall and circulating cells is widespread and causative and indicates higher risk in atherosclerosis. Protection against mtDNA damage and improvement of mitochondrial function are potential areas for new therapeutics.

The mitochondria-targeted antioxidant MitoQ decreases features of the metabolic syndrome in ATM+/–/ApoE–/– mice

A number of recent studies suggest that mitochondrial oxidative damage may be associated with atherosclerosis and the metabolic syndrome. However, much of the evidence linking mitochondrial oxidative damage and excess reactive oxygen species (ROS) with these pathologies is circumstantial. Consequently the importance of mitochondrial ROS in the etiology of these disorders is unclear. Furthermore, the potential of decreasing mitochondrial ROS as a therapy for these indications is not known. We assessed the impact of decreasing mitochondrial oxidative damage and ROS with the mitochondria-targeted antioxidant MitoQ in models of atherosclerosis and the metabolic syndrome (fat-fed ApoE(-/-) mice and ATM(+/-)/ApoE(-/-) mice, which are also haploinsufficient for the protein kinase, ataxia telangiectasia mutated (ATM). MitoQ administered orally for 14weeks prevented the increased adiposity, hypercholesterolemia, and hypertriglyceridemia associated with the metabolic syndrome. MitoQ also corrected hyperglycemia and hepatic steatosis, induced changes in multiple metabolically relevant lipid species, and decreased DNA oxidative damage (8-oxo-G) in multiple organs. Although MitoQ did not affect overall atherosclerotic plaque area in fat-fed ATM(+/+)/ApoE(-/-) and ATM(+/-)/ApoE(-/-) mice, MitoQ reduced the macrophage content and cell proliferation within plaques and 8-oxo-G. MitoQ also significantly reduced mtDNA oxidative damage in the liver. Our data suggest that MitoQ inhibits the development of multiple features of the metabolic syndrome in these mice by affecting redox signaling pathways that depend on mitochondrial ROS such as hydrogen peroxide. These findings strengthen the growing view that elevated mitochondrial ROS contributes to the etiology of the metabolic syndrome and suggest a potential therapeutic role for mitochondria-targeted antioxidants.

Mitochondria in vascular disease.

Mitochondria are often regarded as the powerhouse of the cell by generating the ultimate energy transfer molecule, ATP, which is required for a multitude of cellular processes. However, the role of mitochondria goes beyond their capacity to create molecular fuel, to include the generation of reactive oxygen species, the regulation of calcium, and activation of cell death. Mitochondrial dysfunction is part of both normal and premature ageing, but can contribute to inflammation, cell senescence, and apoptosis. Cardiovascular disease, and in particular atherosclerosis, is characterized by DNA damage, inflammation, cell senescence, and apoptosis. Increasing evidence indicates that mitochondrial damage and dysfunction also occur in atherosclerosis and may contribute to the multiple pathological processes underlying the disease. This review summarizes the normal role of mitochondria, the causes and consequences of mitochondrial dysfunction, and the evidence for mitochondrial damage and dysfunction in vascular disease. Finally, we highlight areas of mitochondrial biology that may have therapeutic targets in vascular disease.

Detection of Atherosclerotic Inflammation by 68Ga-DOTATATE PET Compared to [18F]FDG PET Imaging

Background Inflammation drives atherosclerotic plaque rupture. Although inflammation can be measured using fluorine-18-labeled fluorodeoxyglucose positron emission tomography ([18F]FDG PET), [18F]FDG lacks cell specificity, and coronary imaging is unreliable because of myocardial spillover. Objectives This study tested the efficacy of gallium-68-labeled DOTATATE (68Ga-DOTATATE), a somatostatin receptor subtype-2 (SST2)-binding PET tracer, for imaging atherosclerotic inflammation. Methods We confirmed 68Ga-DOTATATE binding in macrophages and excised carotid plaques. 68Ga-DOTATATE PET imaging was compared to [18F]FDG PET imaging in 42 patients with atherosclerosis. Results Target SSTR2 gene expression occurred exclusively in “proinflammatory” M1 macrophages, specific 68Ga-DOTATATE ligand binding to SST2 receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and carotid SSTR2 mRNA was highly correlated with in vivo 68Ga-DOTATATE PET signals (r = 0.89; 95% confidence interval [CI]: 0.28 to 0.99; p = 0.02). 68Ga-DOTATATE mean of maximum tissue-to-blood ratios (mTBRmax) correctly identified culprit versus nonculprit arteries in patients with acute coronary syndrome (median difference: 0.69; interquartile range [IQR]: 0.22 to 1.15; p = 0.008) and transient ischemic attack/stroke (median difference: 0.13; IQR: 0.07 to 0.32; p = 0.003). 68Ga-DOTATATE mTBRmax predicted high-risk coronary computed tomography features (receiver operating characteristics area under the curve [ROC AUC]: 0.86; 95% CI: 0.80 to 0.92; p < 0.0001), and correlated with Framingham risk score (r = 0.53; 95% CI: 0.32 to 0.69; p <0.0001) and [18F]FDG uptake (r = 0.73; 95% CI: 0.64 to 0.81; p < 0.0001). [18F]FDG mTBRmax differentiated culprit from nonculprit carotid lesions (median difference: 0.12; IQR: 0.0 to 0.23; p = 0.008) and high-risk from lower-risk coronary arteries (ROC AUC: 0.76; 95% CI: 0.62 to 0.91; p = 0.002); however, myocardial [18F]FDG spillover rendered coronary [18F]FDG scans uninterpretable in 27 patients (64%). Coronary 68Ga-DOTATATE PET scans were readable in all patients. Conclusions We validated 68Ga-DOTATATE PET as a novel marker of atherosclerotic inflammation and confirmed that 68Ga-DOTATATE offers superior coronary imaging, excellent macrophage specificity, and better power to discriminate high-risk versus low-risk coronary lesions than [18F]FDG. (Vascular Inflammation Imaging Using Somatostatin Receptor Positron Emission Tomography [VISION]; NCT02021188)

Mitochondrial DNA damage and atherosclerosis

Mitochondria are often regarded as the cellular powerhouses through their ability to generate ATP, the universal fuel for metabolic processes. However, in recent years mitochondria have been recognised as critical regulators of cell death, inflammation, metabolism, and the generation of reactive oxygen species (ROS). Thus, mitochondrial dysfunction directly promotes cell death, inflammation, and oxidative stress and alters metabolism. These are key processes in atherosclerosis and there is now evidence that mitochondrial DNA (mtDNA) damage leads to mitochondrial dysfunction and promotes atherosclerosis directly. In this review we discuss the recent evidence for and mechanisms linking mtDNA defects and atherosclerosis and suggest areas of mitochondrial biology that are potential therapeutic targets.

The role of mitochondrial DNA damage in the development of atherosclerosis.

Mitochondria are the cellular powerhouses, fuelling metabolic processes through their generation of ATP. However we now recognise that these organelles also have pivotal roles in producing reactive oxygen species (ROS) and in regulating cell death, inflammation and metabolism. Mitochondrial dysfunction therefore leads to oxidative stress, cell death, metabolic dysfunction and inflammation, which can all promote atherosclerosis. Recent evidence indicates that mitochondrial DNA (mtDNA) damage is present and promotes atherosclerosis through mitochondrial dysfunction. We will review the mechanisms that link mtDNA damage with atherosclerotic disease, and identify mitochondrial processes that may have therapeutic benefit.

Mitochondrial Respiration Is Reduced in Atherosclerosis, Promoting Necrotic Core Formation and Reducing Relative Fibrous Cap ThicknessHighlights

Objective— Mitochondrial DNA (mtDNA) damage is present in murine and human atherosclerotic plaques. However, whether endogenous levels of mtDNA damage are sufficient to cause mitochondrial dysfunction and whether decreasing mtDNA damage and improving mitochondrial respiration affects plaque burden or composition are unclear. We examined mitochondrial respiration in human atherosclerotic plaques and whether augmenting mitochondrial respiration affects atherogenesis. Approach and Results— Human atherosclerotic plaques showed marked mitochondrial dysfunction, manifested as reduced mtDNA copy number and oxygen consumption rate in fibrous cap and core regions. Vascular smooth muscle cells derived from plaques showed impaired mitochondrial respiration, reduced complex I expression, and increased mitophagy, which was induced by oxidized low-density lipoprotein. Apolipoprotein E–deficient (ApoE−/−) mice showed decreased mtDNA integrity and mitochondrial respiration, associated with increased mitochondrial reactive oxygen species. To determine whether alleviating mtDNA damage and increasing mitochondrial respiration affects atherogenesis, we studied ApoE−/− mice overexpressing the mitochondrial helicase Twinkle (Tw+/ApoE−/−). Tw+/ApoE−/− mice showed increased mtDNA integrity, copy number, respiratory complex abundance, and respiration. Tw+/ApoE−/− mice had decreased necrotic core and increased fibrous cap areas, and Tw+/ApoE−/− bone marrow transplantation also reduced core areas. Twinkle increased vascular smooth muscle cell mtDNA integrity and respiration. Twinkle also promoted vascular smooth muscle cell proliferation and protected both vascular smooth muscle cells and macrophages from oxidative stress–induced apoptosis. Conclusions— Endogenous mtDNA damage in mouse and human atherosclerosis is associated with significantly reduced mitochondrial respiration. Reducing mtDNA damage and increasing mitochondrial respiration decrease necrotic core and increase fibrous cap areas independently of changes in reactive oxygen species and may be a promising therapeutic strategy in atherosclerosis.

Restoring mitochondrial DNA copy number preserves mitochondrial function and delays vascular aging in mice.

Aging is the largest risk factor for cardiovascular disease, yet the molecular mechanisms underlying vascular aging remain unclear. Mitochondrial DNA (mtDNA) damage is linked to aging, but whether mtDNA damage or mitochondrial dysfunction is present and directly promotes vascular aging is unknown. Furthermore, mechanistic studies in mice are severely hampered by long study times and lack of sensitive, repeatable and reproducible parameters of arterial aging at standardized early time points. We examined the time course of multiple invasive and noninvasive arterial physiological parameters and structural changes of arterial aging in mice, how aging affects vessel mitochondrial function, and the effects of gain or loss of mitochondrial function on vascular aging. Vascular aging was first detected by 44 weeks (wk) of age, with reduced carotid compliance and distensibility, increased β‐stiffness index and increased aortic pulse wave velocity (PWV). Aortic collagen content and elastin breaks also increased at 44 wk. Arterial mtDNA copy number (mtCN) and the mtCN‐regulatory proteins TFAM, PGC1α and Twinkle were reduced by 44 wk, associated with reduced mitochondrial respiration. Overexpression of the mitochondrial helicase Twinkle (Tw+) increased mtCN and improved mitochondrial respiration in arteries, and delayed physiological and structural aging in all parameters studied. Conversely, mice with defective mitochondrial polymerase‐gamma (PolG) and reduced mtDNA integrity demonstrated accelerated vascular aging. Our study identifies multiple early and reproducible parameters for assessing vascular aging in mice. Arterial mitochondrial respiration reduces markedly with age, and reduced mtDNA integrity and mitochondrial function directly promote vascular aging.

Mitochondrial DNA damage promotes atherosclerosis and is associated with vulnerable plaque

Abstract Mitochondrial DNA (mtDNA) damage is associated with atherosclerotic disease in man. However, when mtDNA damage occurs, whether it promotes atherogenesis and whether the damage is associated with plaque volume or vulnerability are unknown. To assess the role of mtDNA defects in atherosclerosis, we first performed a time-course study in apolipoprotein E deficient (ApoE −/− ) mice. MtDNA damage was present at the earliest stages of atherogenesis, before histological evidence of disease, with mitochondrial dysfunction occurring in advanced disease. We then studied ApoE −/− mice that were doubly deficient for a proof reading deficiency of mitochondrial DNA polymerase (PolG −/− ApoE −/− mice). PolG −/− ApoE −/− mice had increased plaque burden and hypercholesterolaemia, despite a marked reduction in adiposity and no increase of reactive oxygen species. PolG −/− ApoE −/− mice had increased aortic mtDNA damage and decreased expression and respiration of complexes that have mtDNA-encoded subunits. PolG −/− ApoE −/− smooth muscle cells showed reduced ATP content, impaired proliferation, and increased apoptosis. To determine whether MtDNA damage correlates with human disease we studied 1096 plaques in 170 patients who had undergone three-vessel virtual histology intravascular ultrasound of their coronary arteries at Papworth Hospital. mtDNA damage correlated strongly with the number of vulnerable lesions but not plaque volume. Our results indicate that mtDNA damage occurs early in atherosclerosis and leads to respiratory dysfunction without increased oxidative stress. mtDNA damage causes impaired bioenergetics, changes cell proliferation and apoptosis, and promotes hypercholesterolaemia and atherosclerosis. mtDNA damage may also be a novel marker for unstable atherosclerosis in man. Funding British Heart Foundation.

D Mitochondrial DNA Damage can Promote Atherosclerosis Independently of Reactive Oxygen Species and Correlates with Higher Risk Plaques in Humans

Introduction Mitochondrial DNA (mtDNA) damage occurs in both the vessel wall and in circulating cells in human atherosclerosis. However, whether mtDNA damage promotes atherogenesis or is a consequence of tissue damage is unknown. We assessed the hypothesis that mtDNA damage is present, and can directly promote atherosclerosis and affect plaque composition. Methods To assess whether mtDNA damage may contribute to atherogenesis we examined apolipoprotein E null mice (ApoE-/-). We characterised the development of atherosclerotic plaques, and concomitantly assessed for mtDNA damage and mitochondrial dysfunction. We then studied ApoE-/- mice, also deficient for mtDNA polymerase γ proof reading activity (polG-/-/ApoE-/-), to determine whether mtDNA defects directly promote atherosclerosis. The mice were assessed for levels of atherosclerosis, mtDNA damage and mitochondrial respiratory function. We characterised phenotypic changes in vascular smooth muscle cells (VSMCs) and monocytes. We also examined the association between mtDNA damage and human disease. We used qPCR to quantify the levels of mtDNA damage in human plaques and normal aortic samples. Furthermore, we examined whether leukocyte mtDNA damage correlates with atherosclerosis extent, or plaque vulnerability. Results MtDNA damage occurred early in the vessel wall in ApoE-/- mice, before significant atherosclerosis developed. MtDNA defects were also identified in circulating monocytes and liver, and were associated with reduced respiratory complex activity. PolG-/-/ApoE-/- mice showed extensive mtDNA damage, impaired mitochondrial respiration and increased atherosclerosis in the absence of increased ROS. PolG-/-/ApoE-/- VSMCs had decreased oxygen consumption rate, and ATP content was reduced despite an increased in basal glycolysis. The bioenergetic impairment was associated with altered VSMC phenotype, with reduced proliferation and increased apoptosis. Furthermore polG-/-/ApoE-/- monocytes showed increased inflammatory cytokine release, and transplantation with polG-/-/ApoE-/- bone marrow induced plaque vulnerability. Consistent with these findings, leukocyte mtDNA damage in humans was associated with thin cap fibroatheromas- the lesions with the highest risk of cardiovascular events on subsequent follow up. Conclusions MtDNA defects promote atherosclerosis and plaque vulnerability, independently of ROS, through effects on VSMCs and monocytes. MtDNA damage is therefore not only causative, but also indicates higher risk in atherosclerosis. Protection against mtDNA damage, and improvement of mitochondrial function, are potential areas for new therapeutics.