Lauren Baker

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.

BAFF Receptor Deficiency Reduces the Development of Atherosclerosis in Mice—Brief Report

Objective—The goal of this study was to assess the role of B-cell activating factor (BAFF) receptor in B-cell regulation of atherosclerosis. Methods and Results—Male LDL receptor-deficient mice (Ldlr−/−) were lethally irradiated and reconstituted with either wild type or BAFF receptor (BAFF-R)–deficient bone marrow. After 4 weeks of recovery, mice were put on a high-fat diet for 6 or 8 weeks. BAFF-R deficiency in bone marrow cells led to a marked reduction of conventional mature B2 cells but did not affect the B1a cell subtype. This was associated with a significant reduction of dendritic cell activation and T-cell proliferation along with a reduction of IgG antibodies against malondialdehyde-modified low-density lipoprotein. In contrast, serum IgM type antibodies were preserved. Interestingly, BAFF-R deficiency was associated with a significant reduction in atherosclerotic lesion development and reduced numbers of plaque T cells. Selective BAFF-R deficiency on B cells led to a similar reduction in lesion size and T-cell infiltration but in contrast did not affect dendritic cell activation. Conclusion—BAFF-R deficiency in mice selectively alters mature B2 cell-dependent cellular and humoral immune responses and limits the development of atherosclerosis.

MFGE8 inhibits inflammasome-induced IL-1β production and limits postischemic cerebral injury

Milk fat globule-EGF 8 (MFGE8) plays important, nonredundant roles in several biological processes, including apoptotic cell clearance, angiogenesis, and adaptive immunity. Several recent studies have reported a potential role for MFGE8 in regulation of the innate immune response; however, the precise mechanisms underlying this role are poorly understood. Here, we show that MFGE8 is an endogenous inhibitor of inflammasome-induced IL-1β production. MFGE8 inhibited necrotic cell-induced and ATP-dependent IL-1β production by macrophages through mediation of integrin β(3) and P2X7 receptor interactions in primed cells. Itgb3 deficiency in macrophages abrogated the inhibitory effect of MFGE8 on ATP-induced IL-1β production. In a setting of postischemic cerebral injury in mice, MFGE8 deficiency was associated with enhanced IL-1β production and larger infarct size; the latter was abolished after treatment with IL-1 receptor antagonist. MFGE8 supplementation significantly dampened caspase-1 activation and IL-1β production and reduced infarct size in wild-type mice, but did not limit cerebral necrosis in Il1b-, Itgb3-, or P2rx7-deficient animals. In conclusion, we demonstrated that MFGE8 regulates innate immunity through inhibition of inflammasome-induced IL-1β production.

Vascular Smooth Muscle Cell Senescence Promotes Atherosclerosis and Features of Plaque Vulnerability.

Background— Although vascular smooth muscle cell (VSMC) proliferation is implicated in atherogenesis, VSMCs in advanced plaques and cultured from plaques show evidence of VSMC senescence and DNA damage. In particular, plaque VSMCs show shortening of telomeres, which can directly induce senescence. Senescence can have multiple effects on plaque development and morphology; however, the consequences of VSMC senescence or the mechanisms underlying VSMC senescence in atherosclerosis are mostly unknown. Methods and Results— We examined the expression of proteins that protect telomeres in VSMCs derived from human plaques and normal vessels. Plaque VSMCs showed reduced expression and telomere binding of telomeric repeat-binding factor-2 (TRF2), associated with increased DNA damage. TRF2 expression was regulated by p53-dependent degradation of the TRF2 protein. To examine the functional consequences of loss of TRF2, we expressed TRF2 or a TRF2 functional mutant (T188A) as either gain- or loss-of-function studies in vitro and in apolipoprotein E–/– mice. TRF2 overexpression bypassed senescence, reduced DNA damage, and accelerated DNA repair, whereas TRF2188A showed opposite effects. Transgenic mice expressing VSMC-specific TRF2T188A showed increased atherosclerosis and necrotic core formation in vivo, whereas VSMC-specific TRF2 increased the relative fibrous cap and decreased necrotic core areas. TRF2 protected against atherosclerosis independent of secretion of senescence-associated cytokines. Conclusions— We conclude that plaque VSMC senescence in atherosclerosis is associated with loss of TRF2. VSMC senes cence promotes both atherosclerosis and features of plaque vulnerability, identifying prevention of senescence as a potential target for intervention.

Effects of DNA Damage in Smooth Muscle Cells in Atherosclerosis

RATIONALE DNA damage and the DNA damage response have been identified in human atherosclerosis, including in vascular smooth muscle cells (VSMCs). However, although double-stranded breaks (DSBs) are hypothesized to promote plaque progression and instability, in part, by promoting cell senescence, apoptosis, and inflammation, the direct effects of DSBs in VSMCs seen in atherogenesis are unknown. OBJECTIVE To determine the presence and effect of endogenous levels of DSBs in VSMCs on atherosclerosis. METHODS AND RESULTS Human atherosclerotic plaque VSMCs showed increased expression of multiple DNA damage response proteins in vitro and in vivo, particularly the MRE11/RAD50/NBS1 complex that senses DSB repair. Oxidative stress-induced DSBs were increased in plaque VSMCs, but DSB repair was maintained. To determine the effect of DSBs on atherosclerosis, we generated 2 novel transgenic mice lines expressing NBS1 or C-terminal deleted NBS1 only in VSMCs, and crossed them with apolipoprotein E(-/-) mice. SM22α-NBS1/apolipoprotein E(-/-) VSMCs showed enhanced DSB repair and decreased growth arrest and apoptosis, whereas SM22α-(ΔC)NBS1/apolipoprotein E(-/-) VSMCs showed reduced DSB repair and increased growth arrest and apoptosis. Accelerating or retarding DSB repair did not affect atherosclerosis extent or composition. However, VSMC DNA damage reduced relative fibrous cap areas, whereas accelerating DSB repair increased cap area and VSMC content. CONCLUSIONS Human atherosclerotic plaque VSMCs show increased DNA damage, including DSBs and DNA damage response activation. VSMC DNA damage has minimal effects on atherogenesis, but alters plaque phenotype inhibiting fibrous cap areas in advanced lesions. Inhibiting DNA damage in atherosclerosis may be a novel target to promote plaque stability.

Necrotic Cell Sensor Clec4e Promotes a Proatherogenic Macrophage Phenotype Through Activation of the Unfolded Protein Response

Background: Atherosclerotic lesion expansion is characterized by the development of a lipid-rich necrotic core known to be associated with the occurrence of complications. Abnormal lipid handling, inflammation, and alteration of cell survival or proliferation contribute to necrotic core formation, but the molecular mechanisms involved in this process are not properly understood. C-type lectin receptor 4e (Clec4e) recognizes the cord factor of Mycobacterium tuberculosis but also senses molecular patterns released by necrotic cells and drives inflammation. Methods: We hypothesized that activation of Clec4e signaling by necrosis is causally involved in atherogenesis. We addressed the impact of Clec4e activation on macrophage functions in vitro and on the development of atherosclerosis using low-density lipoprotein receptor–deficient (Ldlr−/−) mice in vivo. Results: We show that Clec4e is expressed within human and mouse atherosclerotic lesions and is activated by necrotic lesion extracts. Clec4e signaling in macrophages inhibits cholesterol efflux and induces a Syk-mediated endoplasmic reticulum stress response, leading to the induction of proinflammatory mediators and growth factors. Chop and Ire1a deficiencies significantly limit Clec4e-dependent effects, whereas Atf3 deficiency aggravates Clec4e-mediated inflammation and alteration of cholesterol efflux. Repopulation of Ldlr−/− mice with Clec4e−/− bone marrow reduces lipid accumulation, endoplasmic reticulum stress, and macrophage inflammation and proliferation within the developing arterial lesions and significantly limits atherosclerosis. Conclusions: Our results identify a nonredundant role for Clec4e in coordinating major biological pathways involved in atherosclerosis and suggest that it may play similar roles in other chronic inflammatory diseases.

Deletion of Chromosome 9p21 Noncoding Cardiovascular Risk Interval in Mice Alters Smad2 Signaling and Promotes Vascular Aneurysm

Background—Vascular aneurysm is an abnormal local dilatation of an artery that can lead to vessel rupture and sudden death. The only treatment involves surgical or endovascular repair or exclusion. There is currently no approved medical therapy for this condition. Recent data established a strong association between genetic variants in the 9p21 chromosomal region in humans and the presence of cardiovascular diseases, including aneurysms. However, the mechanisms linking this 9p21 DNA variant to cardiovascular risk are still unknown. Methods and Results—Here, we show that deletion of the orthologous 70-kb noncoding interval on mouse chromosome 4 (chr4&Dgr;70kb/&Dgr;70kb mice) is associated with reduced aortic expression of cyclin-dependent kinase inhibitor genes p19Arf and p15Inkb. Vascular smooth muscle cells from chr4&Dgr;70kb/&Dgr;70kb mice show reduced transforming growth factor-&bgr;–dependent canonical Smad2 signaling but increased cyclin-dependent kinase–dependent Smad2 phosphorylation at linker sites, a phenotype previously associated with tumor growth and consistent with the mechanistic link between reduced canonical transforming growth factor-&bgr; signaling and susceptibility to vascular diseases. We also show that targeted deletion of the 9p21 risk interval promotes susceptibility to aneurysm development and rupture when mice are subjected to a validated model of aneurysm formation. The vascular disease of chr4&Dgr;70kb/&Dgr;70kb mice is prevented by treatment with a cyclin-dependent kinase inhibitor. Conclusions—The results establish a direct mechanistic link between 9p21 noncoding risk interval and susceptibility to aneurysm and may have important implications for the understanding and treatment of vascular diseases.

Optimising experimental design for high-throughput phenotyping in mice: a case study.

To further the functional annotation of the mammalian genome, the Sanger Mouse Genetics Programme aims to generate and characterise knockout mice in a high-throughput manner. Annually, approximately 200 lines of knockout mice will be characterised using a standardised battery of phenotyping tests covering key disease indications ranging from obesity to sensory acuity. From these findings secondary centres will select putative mutants of interest for more in-depth, confirmatory experiments. Optimising experimental design and data analysis is essential to maximise output using the resources with greatest efficiency, thereby attaining our biological objective of understanding the role of genes in normal development and disease. This study uses the example of the noninvasive blood pressure test to demonstrate how statistical investigation is important for generating meaningful, reliable results and assessing the design for the defined research objectives. The analysis adjusts for the multiple-testing problem by applying the false discovery rate, which controls the number of false calls within those highlighted as significant. A variance analysis finds that the variation between mice dominates this assay. These variance measures were used to examine the interplay between days, readings, and number of mice on power, the ability to detect change. If an experiment is underpowered, we cannot conclude whether failure to detect a biological difference arises from low power or lack of a distinct phenotype, hence the mice are subjected to testing without gain. Consequently, in confirmatory studies, a power analysis along with the 3Rs can provide justification to increase the number of mice used.

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.

Response to Letter Regarding Article, “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”

We welcome the opportunity to respond to comments from Drs Stocker and Maghzal on our article.1 Increased reactive oxygen species (ROS) occur in human atherosclerosis in many cell types, and multiple experimental manipulations (predominantly in mice) suggest that ROS promote atherosclerosis. Because mitochondria are an important source of ROS that could be amplified by mitochondrial DNA (mtDNA) damage, the concept has arisen that mtDNA damage promotes atherosclerosis by elevating ROS. However, the polymerase-γ proofreading activity (polG) mouse model of mtDNA damage has been extensively characterized using multiple methods and does not show increased ROS by the age atherosclerosis was studied. Our use of the mitochondria-targeted mass spectrometry probe MitoB (Figure 2C) supported this interpretation. We also showed no difference in …