key: cord-353600-5wo74ms4 authors: Tyrrell, Daniel J.; Goldstein, Daniel R. title: Ageing and atherosclerosis: vascular intrinsic and extrinsic factors and potential role of IL-6 date: 2020-09-11 journal: Nat Rev Cardiol DOI: 10.1038/s41569-020-0431-7 sha: doc_id: 353600 cord_uid: 5wo74ms4 The number of old people is rising worldwide, and advancing age is a major risk factor for atherosclerotic cardiovascular disease. However, the mechanisms underlying this phenomenon remain unclear. In this Review, we discuss vascular intrinsic and extrinsic mechanisms of how ageing influences the pathology of atherosclerosis. First, we focus on factors that are extrinsic to the vasculature. We discuss how ageing affects the development of myeloid cells leading to the expansion of certain myeloid cell clones and induces changes in myeloid cell functions that promote atherosclerosis via inflammation, including a potential role for IL-6. Next, we describe vascular intrinsic factors by which ageing promotes atherogenesis — in particular, the effects on mitochondrial function. Studies in mice and humans have shown that ageing leads to a decline in vascular mitochondrial function and impaired mitophagy. In mice, ageing is associated with an elevation in the levels of the inflammatory cytokine IL-6 in the aorta, which participates in a positive feedback loop with the impaired vascular mitochondrial function to accelerate atherogenesis. We speculate that vascular and myeloid cell ageing synergize, via IL-6 signalling, to accelerate atherosclerosis. Finally, we propose future avenues of clinical investigation and potential therapeutic approaches to reduce the burden of atherosclerosis in old people. The number of old people (aged >65 years) is rising worldwide, and cardiovascular diseases are the largest contributor to morbidity and mortality in this popu lation 1, 2 . Changes in diet and lifestyle contribute to the high cardiovascular morbidity and mortality in old indi viduals, but many biological processes that are altered with ageing also contribute to this increased cardio vascular risk. As a result, therapies for cardiovascular disease that are effective in young and middle aged peo ple might be less effective in older people. Additionally, novel therapies might be required to improve disease management specifically in old people. Deciphering the mechanisms by which ageing promotes atheroscle rotic cardiovascular disease will be fundamental for the development of novel therapies to reduce the burden of atherosclerosis with ageing. The development of new therapies is especially relevant with the coronavirus dis ease 2019 (COVID19) pandemic, because old people and particularly those with cardiovascular diseases are at a substantially higher risk of morbidity and death 3, 4 . In this Review, we describe two major areas by which ageing promotes atherosclerosis. First, we dis cuss age related factors that are extrinsic to the vascula ture, focusing on the effects of ageing on myeloid cells of the immune system. Age related effects in the bone marrow skew the differentiation of haematopoietic cells towards the myeloid cell lineage. Ageing also promotes the generation of clones of haematopoietic cells with out clear development of haematopoietic malignancy or other known clonal disorder, a phenomenon known as clonal haematopoiesis of indeterminate potential (CHIP) [5] [6] [7] [8] . Clinical studies from the past decade have revealed that the presence of CHIP increases the risk of cardiovascular diseases 6, 7 . Intriguingly, the increased risk of cardiovas cular disease associated with the presence of CHIP is abrogated in patients with a loss of function mutation in IL6 (ref. 9 ). However, the precise mechanisms by which CHIP promotes the development of cardiovascular diseases are yet to be fully clarified. We next address vascular intrinsic factors, discussing how ageing impairs vascular bioenergetics by compro mising mitochondrial function and how this alteration connects with inflammatory pathways within the vascu lature to promote atherosclerosis 10, 11 . We describe studies in mice and humans showing that, in the aorta, ageing impairs both mitochondrial function and the removal of damaged mitochondria (mitophagy) 10, 11 . We describe experimental evidence reported in 2020 demonstrating that the age mediated increase in the levels of the plei otropic cytokine IL6 in the aorta occurs in a positive Clonal haematopoiesis of indeterminate potential (CHIP) . Clonal expansion of haematopoietic stem cells that carry certain somatic mutations that confer a cell proliferation advantage. feedback loop with vascular mitochondrial dysfunction and that these alterations promote atherosclerosis 11 . The CANTOS study 12 demonstrated that IL1β block ade reduces the risk of recurrent cardiovascular events in patients aged >60 years. Importantly, the greatest bene fit of IL1β blockade was seen in patients who had low plasma IL6 levels 13 . This pivotal clinical study indicates that chronic inflammation, potentially via IL6 signal ling, is a major contributor to age related atherosclerosis. Given this observation, we speculate that increased ath erosclerosis with ageing could result from a synergy between myeloid cells of the immune system and the vasculature via IL6 signalling (fIg. 1 ). This mechanism is especially important because clinically approved agents targeting this pathway (such as anti IL6 therapies) are already available and could reduce the risk of cardio vascular disease in old people. Finally, we propose that future experimental and clinical investigation will be required to determine the contribution of this inflamma tory pathway in age related atherosclerosis. We acknowl edge that other inflammatory pathways and cytokines could contribute to age related atherosclerosis, and the source of these cytokines (including IL6) could be senescent adipocytes. A detailed discussion of the con tribution of ageing to senescence and atherosclerosis has been published previously 14 . Ageing affects the immune system in complex ways (as reviewed previously [15] [16] [17] , and various components of the immune system contribute to atherosclerosis 18, 19 . This Review focuses on clones of myeloid cells that increase with ageing and how these clones contribute to athero sclerosis. We do not describe how ageing affects other cells of the immune system, which has been reviewed previously (for example, B cells 20 , T cells 21 , eosinophils or dendritic cells 22 ). In addition, we focus on vascular mito chondrial function and how mitochondrial dysfunction could influence inflammatory pathways within the vasculature. However, given that most of the available evidence indicates that oxidative stress is not a major driver of biological ageing [23] [24] [25] , and given the complex roles that oxidative stress has in atherosclerosis 26 , we do not describe in detail the contributions of oxidative stress in age related atherosclerosis. Neither do we describe in detail how ageing affects other processes within the arte rial wall, such as extracellular matrix remodelling or pro duction of pro fibrotic and pro calcific factors, which can promote atherosclerosis indirectly via increasing arterial stiffness and hypertension 27, 28 , and which have been previously reviewed 29, 30 . Effects of ageing on myeloid cell production. Numerous subpopulations of immune cells of various lineages have been implicated in atherosclerosis, including macrophages 31 , dendritic cells 32 , T helper 1 (T H 1) cells 33 and B cells 34 (reviewed previously 18, 35 ), all of which are affected by ageing. Immune cells are generated in the bone marrow via haematopoiesis from regenerative haematopoietic stem cells (HSCs) 36, 37 . Monocytes, mac rophages and neutrophils are derived from myeloid biased HSCs. With ageing, although the absolute number of HSCs increases 38 , HSCs lose their regener ative capacity 39, 40 . This loss of regenerative potential is accompanied by an expansion of the number of HSCs that are committed to the platelet (megakaryocytes) and myeloid lineages 38, 41 . Competitive bone marrow transplantation studies in mice have demonstrated that aged HSCs have a reduced repopulation capacity, with an imbalance towards myeloid cell differentiation, compared with young HSCs 38, 42 . Several major biological pathways contribute to ageing, including DNA damage, mitochondrial dysfunction, cell senescence, impaired autophagy, epigenetic alterations and gene transcrip tion dysregulation 25 . Transcriptomic studies in mice have shown that with ageing, HSCs upregulate stress responses and inflammatory pathways and downregu late the expression of genes related to genetic stability 43 . With ageing, HSCs exhibit an increase in epigenetic dysregulation, specifically downregulation in chroma tin remodelling and transcriptional silencing 43 , and increases in DNA methylation (as reviewed previously 44 ). These epigenetic alterations are accompanied by func tional defects in HSCs, including a reduction in HSC homing to the bone marrow and HSC proliferation 43, 45 . Importantly, mutations in genes such as IDH2, which alter epigenetic regulation, lead to impairments in hae matopoietic progenitors in mice 46 and are associated with T cell lymphomas in humans 47 , a malignancy that increases with ageing. Although whether HSCs, or stem cells in general, undergo senescence is questionable 48 , the clearance of senescent cells improves HSC engraft ment in bone marrow transplantation mouse models and reduces myeloid skewing 49 . Autophagy deficient young mice have increased mitochondrial content and metabolism that lead to mitochondrial stress in HSCs compared with wild type young mice 50 . These features are also observed in aged wild type mice and are asso ciated with a skewing towards the myeloid lineage and a reduced proliferative capacity of HSCs 50, 51 . Loss of microRNA146a (miR146a) in HSCs with ageing also promotes a myeloid bias 52 . Furthermore, myeloid cells derived from miR146a deficient HSCs have elevated levels of both IL6 and tumour necrosis factor (TNF) 52 , which connects altered regulation of transcription in HSCs to inflammation. Overall, these studies indicate that ageing has effects on HSCs via several complex pathways that lead to reduced HSC function. Ageing also alters haematopoiesis by influencing the bone marrow niche independently of the direct effects • Ageing-related alterations in the bone marrow increase the phenomenon of clonal haematopoiesis of indeterminate potential (CHIP) and promote a skewing towards myeloid cell differentiation, both of which can accelerate atherosclerosis. • The increased risk of atherosclerotic cardiovascular diseases associated with the presence of CHIP might be mediated by IL-6 signalling and/or inflammasome activation. • Ageing is associated with a decline in mitochondrial function and an increase in IL-6 levels in the vasculature, and both effects probably accelerate atherosclerosis independently of chronic hyperlipidaemia. • The role of the vasculature and myeloid cells of the immune system in promoting age-related atherosclerosis might be mediated by shared inflammatory pathways, in particular IL-6 signalling. Senescence A state of permanent replicative arrest in normally proliferative cells. www.nature.com/nrcardio on HSCs. The bone marrow niche provides a support ing environment for HSC function and includes mesen chymal cells and endothelial cells 53 . How ageing affects the bone marrow niche is not clear, but the presence of chronic systemic inflammation might contribute. Ageing leads to a chronic systemic low grade inflammatory state 54, 55 , which might be mediated by cellular senescence that leads to the production of inflammatory mediators (termed the senescence-associated secretory phenotype (SASP)) 56,57 . One source of senescent inflammatory cells is the adipose tissue, which typically increases in size with ageing 58, 59 . The number of adipocytes also increases in the bone marrow with ageing, accompanied by an elevation in the levels of pro inflammatory cytokines, including IL6 (refs 60,61 ). These cytokines promote a skewing towards myeloid cell differentiation and an increase in platelet production, the latter of which could contribute to thrombosis 60, 61 . Importantly, adipocytes arising from leptin receptor positive progenitors in the bone marrow, but not within other fat depots, synthesize stem cell factor, which promotes HSC regeneration 62, 63 . Senescent stromal cells in the bone marrow are another potential source of inflammation 64 . These cells can dif ferentiate into adipocytes in the ageing bone marrow 65 and further promote an inflammatory environment. The function of bone marrow endothelial cells also declines with ageing 66 . Furthermore, the number of vascular niches in the bone marrow that support HSC regenera tion decreases with ageing, but can be restored in aged mice by activating Notch signalling in endothelial cells 53 . Activation of the innate immune receptor Toll like receptor 4 (TLR4) induces myeloid differentiation in HSCs in mice 67 . Ageing is associated with alterations in gut microbiota 68 , which could act as a microbial source for TLR4 stimulation (for example, lipopolysaccharide (LPS) from Gram negative bacteria activates TLR4). TLR4 activation could then increase the imbalance of HSC differentiation towards the myeloid lineage. In a 2019 study in mice, β 2 adrenergic receptor signalling in the bone marrow niche was found to increase with ageing in association with increased generation of myeloid cells and platelets through an IL6 dependent mechanism 60 . This study also demonstrated that the bone marrow niche switches from an endosteal to a non endosteal mutation show an increased production of IL-6 and IL-1β, which can contribute to accelerated atherosclerosis. Ageing can also have pro-atherogenic effects directly on the vasculature. Ageing is associated with an increase in the levels of IL-6, possibly mediated by increased production by vascular smooth muscle cells (VSMCs), and mitochondrial genomic instability and with a decline in mitochondrial function in the vasculature. The reduced mitochondrial function alters mitophagy and increases IL-6 levels, creating a positive feedback loop that accelerates atherogenesis. Vascular ageing also leads to the production of chemoattractants that increase myeloid cell recruitment into the arterial wall, further promoting atherosclerosis. Impaired mitochondrial function combined with reduced mitophagy might lead to increased levels of reactive oxygen species (ROS). Senescence-associated secretory phenotype (sAsP). secretion of cytokines, chemokines, growth factors and proteases by senescent cells. Nature reviews | Cardiology niche with ageing, indicating that ageing shifts myeloid cell production away from the bone tissue to further within the bone marrow 60 . This study also found that a mouse model of Hutchinson-Gilford progeria syn drome, which is associated with accelerated ageing 69 , had an imbalance favouring myeloid cells over lymphoid cells in the peripheral blood 60 . This effect was mitigated by administration of a β 3 adrenergic receptor agonist 60 . Overall, clear evidence indicates that ageing alters the bone marrow niche via multiple mechanisms to impair HSC function and promote myeloid cell differentiation. Clonal haematopoiesis and cardiovascular disease: clinical correlation. The positive selection and expansion of clones of HSCs carrying certain somatic mutations, known as clonal haematopoiesis, occurs commonly with ageing. Approximately 10% of individuals aged >70 years carry mutations associated with clonal haemato poiesis, whereas these mutations are rare in individu als aged <40 years 7, 70, 71 . These clones of haematopoietic cells harbour single somatic mutations most commonly in genes associated with haematological malignan cies, such as DNMT3A, TET2 and ASXL1. Individuals with mutations in these genes have an increased risk of developing haematological malignancies (HR 11-12, depending on the study) 7, 70, 71 . All cause mortality is increased in individuals with any somatic mutation asso ciated with clonal haematopoiesis (HR 1-2) compared with those with no mutations 7 . Interestingly, the cause of the increased mortality in these individuals is not only the higher rate of haematological malignancies but also a higher rate of adverse cardiovascular events 7,70-72 . The association between clonal haematopoiesis and the risk of adverse cardiovascular events remained even after adjustment for traditional cardiovascular risk fac tors, such as diabetes mellitus, hypertension, smoking and BMI, in multivariate analyses 6 . As a result of these studies, the term CHIP was coined to distinguish the phenomenon of clonal haematopoiesis without clear development of haematopoietic malignancy or other known clonal disorder from the pre malignant clonal haematopoiesis of clinical importance 73 . A follow up clinical study provided further evidence of the association between cardiovascular disease and CHIP 6 . In particular, old individuals (aged 60-70 years) with CHIP had an approximately twofold higher risk of incident coronary artery disease, a fourfold higher risk of early onset myocardial infarction and a three fold higher coronary artery calcium score than similarly aged individuals without CHIP 6 . Importantly, the size of the CHIP clone, defined as the variant allele frequency (VAF), correlates with the risk of cardiovascular dis ease. Specifically, individuals with a CHIP clone with a VAF of >10% have a 12 fold increased risk of cardi ovascular disease compared with individuals with no mutations, whereas the risk of cardiovascular disease is not significantly increased in CHIP carriers with a VAF of <10% 6 . This study has established that CHIP is associated with the risk of cardiovascular diseases and has developed a potential new paradigm that certain clones of haematopoietic cells accelerate atherogenesis 6 . However, to date, the presence of CHIP can be used only as a biomarker of atherosclerosis and is not therapeutically actionable. Mutations in TET2 are the second most prevalent somatic mutations associated with CHIP after those in DNMT3A. Mouse models have been used to elucidate the mechanistic contributions of TET2 mutations to ath erogenesis. In irradiated, atheroprone Ldlr −/− mice, those reconstituted with either Tet2 −/− or Tet2 +/− bone marrow had increased atherosclerotic lesion size compared with mice receiving wild type bone marrow 6, 74 . These data imply that deletion of one copy of the Tet2 gene is sufficient to increase atherosclerosis in mice. Further studies in mice showed that myeloid cell specific TET2 deficiency increases atherosclerotic plaque size 74 . Interestingly, TET2 deficiency in bone marrow derived macrophages leads to an elevated secretion of IL6 and IL1β (a signature cytokine produced by inflammasome activation) 75 in response to various stimuli (such as LDL, LPS and IFNγ) in vitro 74 . Furthermore, the increased atherogenic potential of Tet2 −/− bone marrow cells is reduced when bone marrow transplantation recipients are treated with a small molecule inhibitor of the NLRP3 inflammasome 74 . The effect of TET2 deficiency might not be limited to vascular diseases, because experimen tal studies have demonstrated that transfer of Tet2 −/− bone marrow cells into non irradiated mice accelerates the development of age related cardiac hypertrophy and fibrosis 76 , and TET2 deficiency in myeloid cells worsens the development of heart failure in mice after acute injury 77 . The contribution of IL6 to the cardiovascular risk in individuals with large CHIP clones (VAF >10%) was evaluated in 35,416 individuals without prevalent cardi ovascular disease enrolled in the UK Biobank registry 9 . The investigators examined whether an IL6R coding mutation that leads to reduced IL6 signalling alters the association between CHIP and the risk of adverse cardiovascular events (myocardial infarction, coronary artery disease revascularization, stroke or death). The study revealed that the presence of the IL6R mutation mitigated the increased risk of adverse cardiovascular events in individuals with large CHIP clones but not in individuals without CHIP 9 . These data indicate that IL6 signalling is causally linked to the increased risk of cardiovascular disease associated with CHIP. IL6 is released following inflammasome activation 78 ; therefore, the observed link between IL6 and CHIP suggests that inflammasome activation is a mecha nism by which CHIP promotes the development of cardiovascular diseases. This concept is compatible with the study in mice discussed above, showing that the NLRP3 inflammasome contributes to the increased atherosclerotic burden induced by Tet2 −/− bone marrow transplantation 74 . The contribution of IL1β to cardio vascular disease in humans was demonstrated in the CANTOS study 12, 13 , which showed that a monoclonal antibody against IL1β reduces the risk of recurrent car diovascular events in patients with previous myocardial infarction. The effects of IL1β blockade in the CANTOS trial were greater in patients who had lower circulating Variant allele frequency (VAf) . The proportion of sequences that match a gene mutation divided by the overall coverage at that gene locus. www.nature.com/nrcardio IL6 levels after IL1β blockade than in those with higher circulating IL6 levels 13 . However, the role of IL1β and IL6 in experimental models of atherosclerosis is not completely clear because data indicating that each of these cytokines has atheroprotective effects have been reported 79, 80 . However, these studies were performed in young mice, so these cytokines might have increasingly pathogenic roles with ageing. Monocytes and macrophages contribute to both the initiation of the chronic inflammatory process of ath erosclerosis and the resolution of the chronic vascular inflammation 81 . Ageing directly influences the function of monocytes and macrophages 16 . Human monocytes have lower levels of TLRs and a reduction in TLR dependent pro inflammatory cytokine production with ageing 16 . A study comparing bone marrow derived monocytes from young and aged atheroprone Ldlr −/− mice showed that ageing leads to a downregulation in the expression of Tnf and Il1b but monocyte chemotaxis is preserved 82 . Aged (6 month old) atherosclerotic Apoe −/− mice have a reduction in the number of vascular progenitor cells in the bone marrow compared with 1 month old atheroscle rotic Apoe −/− mice 83 . Furthermore, administration of bone marrow derived HSCs from young non atherosclerotic mice to non irradiated 6 month old Apoe −/− mice reduced atherogenesis after feeding a high fat diet 83 . This finding suggests that ageing is accompanied by a reduc tion in the number of atheroprotective progenitor cells in the bone marrow. Aged (18-21 month old) mice with chronic or induced acute hyperlipidaemia have more macrophage infiltration into atherosclerotic lesions than young mice 11, 82 . Furthermore, the aortas of aged athero sclerotic mice (12 months old) and rats (30 months old) have higher levels of macrophage attracting chemokines and IL6 than the aortas of young atherosclerotic mice (2 months old) and rats(10 months old) 82, 84 . Although macrophages and monocytes can have an increased basal secretion of inflammatory cytokines, such as IL1β, IL6 and IL8, with ageing 85 (possibly owing to senescence) 57 , whether these cells are the major contributors to the increased vascular production of IL6 with ageing during atherogenesis is unclear. Vascular cells such as vascular smooth muscle cells (VSMCs) have been shown in animal models to have an elevated IL6 production with ageing before any signs of atherosclerosis development 86, 87 . efferocytosis is a crucial mechanism for resolving plaque inflammation and reducing atherosclerosis progression 88 . In vivo and in vitro assays have indi cated that the phagocytic function of tissue alveolar macrophages to take up apoptotic neutrophils declines with ageing 89 and is associated with reduced expres sion of scavenger receptor CD204 (ref. 90 ). In a mouse model of peritonitis, ageing led to reduced resolution of acute inflammation and was associated with reduced levels of pro resolution lipid mediators, specifically resolvins 91 . Resolution of inflammation was also delayed with ageing in a human model of skin blistering 92 . This phenotype is related to reduced expression of the effe rocytotic receptor TIM4 in macrophages. Reduced TIM4 expression with ageing was caused by elevations in p38 mitogen activated protein kinase activity in macrophages, and treatment with an oral p38 inhibitor increased the resolution of blister inflammation in old individuals 92 . Overall, macrophages show impaired inflammation resolution properties with ageing; how ever, whether this impaired macrophage function contributes to increased atherosclerosis is not yet clear. Vascular mitochondrial dysfunction with ageing before atherogenesis initiation. Ageing affects the vasculature before the development of atherosclerosis. Generally, ageing is associated with remodelling of the arterial wall, with evidence of reduced endothelial cell function, increased collagen deposition, fibrosis and functionally stiffer vessels 28, 93, 94 . In addition, VSMCs acquire a more proliferative and synthetic function with ageing 86 . VSMCs also show an increased generation of reactive oxygen spe cies (ROS) and high oxidative damage 95 . Endothelial cells also have a dysregulated antioxidant capacity with ageing (mediated by the disruption of nuclear factor erythroid 2 related factor 2 signalling), thereby contributing to vas cular ageing 96, 97 . All these effects of ageing can contribute to the development of hypertension, a major risk factor for cardiovascular disease. Most studies on vascular ageing in rodent models have been performed in normolipidaemic animals. These studies provide evidence that mitochondrial dysfunction, a known hallmark of ageing 25 , contrib utes to vascular ageing before the initiation of athero genesis. Disease free, normolipidaemic mice develop mitochondrial dysfunction in the aorta as they age, first detected at 11 months of age (measured as a decline in oxygen consumption rate (OCR)) and becoming more evident as the mice reach 18 months of age 98 . The reduction in OCR is accompanied by an increase in mitochondrial DNA (mtDNA) damage 98 , a sign of mito chondrial genomic instability, which is another hallmark of ageing 25 . Furthermore, reduced vascular mitochon drial function with ageing is accompanied by a decrease in the expression of the mtDNA helicase Twinkle 98 , an enzyme involved in preserving mtDNA integrity. Aged transgenic mice expressing high levels of Twinkle show delayed vascular ageing; in particular, the decrease in aortic compliance and the increase in aortic stiffness are delayed in these mice compared with aged wild type mice 98 . Overall, experimental evidence indicates that mitochondrial dysfunction and mitochondrial genomic instability contribute to vascular ageing. In humans, atherosclerotic plaques show evidence of damage to mtDNA, which is associated with reduced mitochondrial function, specifically lower OCR in the fibrous cap and core regions of the atherosclerotic plaque than in the shoulder region of the plaque or in non overtly diseased regions of the aorta 10 . These findings are compatible with those of previous experimental work indicating that Apoe −/− mice fed a low fat, standard chow diet have increased vascular mtDNA damage but not nuclear DNA damage as the mice age 99, 100 . Furthermore, human atherosclerotic plaques have lower levels of mitochondrial complex I and complex II than non diseased aortic regions 10 . Similar findings are noted in Efferocytosis Phagocytosis of apoptotic cells by phagocytic cells. atherosclerotic Apoe −/− mice fed a high fat diet 10 . Apoe −/− mice overexpressing Twinkle have a reduced necrotic core area in atherosclerotic plaques compared with con trol Apoe −/− mice 10 . Mitochondrial dysfunction probably has a central role in ROS generation but the interaction between these two factors is complex. For example, low levels of ROS might improve cell fitness and pro mote survival, a concept known as mitohormesis 25, 101 . However, higher levels of ROS might contribute to age related chronic vascular diseases. The complex inter action between mitochondrial dysfunction and ROS might explain why disruption of some mitochondrial enzymatic pathways (such as NADPH oxidase 1 (NOX1) and NOX2 signalling) in atherosclerotic mice has no effect on age related atherosclerosis 102 , whereas partial deficiency of ROS scavenging enzymes (such as super oxide dismutase) 103 in atherosclerotic mice contributes to atherosclerosis 99 . However, one study found that mtDNA damage occurs in both VSMCs and monocytes and cor relates with atherosclerotic burden in humans but with out evidence of alterations in ROS levels 100 . Furthermore, clinical trials on antioxidants have yet to reveal a benefi cial effect in patients with atherosclerotic cardiovascular disease 104, 105 . Overall, mitochondrial dysfunction occurs during chronic hyperlipidaemia and atherogenesis, and this mitochondrial dysfunction promotes atherosclero sis. However, the precise role of ROS in this context is complex and requires further investigation. Part of the challenge of using stand ard mouse models of atherosclerosis (such as Ldlr −/− or Apoe −/− mice) to understand the role of ageing on atherogenesis is that even when fed a standard low fat diet, these mice age with chronic hyperlipidaemia. Therefore, the effects of ageing cannot be dissected from the effects of chronic hyperlipidaemia. A study in mice published in 2020 circumvented this issue by first examining mitochondrial function in the aortas of young and aged wild type mice without hyperlipidaemia or vascular diseases 11 . Consistent with previous studies, aged mice had evidence of reduced OCR in the aortas compared with young mice 11 . This OCR reduction in the vasculature from aged mice was accompanied by increased expression of the mitophagy protein Parkin and increased basal mitophagy (Box 1), a macroau tophagy process to remove damaged mitochondria. Altered mitochondrial quality control in the ageing vas culature without hyperlipidaemia is linked to arterial stiffening in mice 106 . The mitochondrial dysfunction and elevated Parkin levels with ageing in the mouse aorta are accompanied by an increase in TLR9, MYD88 and IL6 levels 11 . Importantly, blocking IL6 in aged mouse aortas in vitro increased the OCR and reduced Parkin levels. This study identified a positive feedback loop in which mitochondrial dysfunction and elevated IL6 levels coexist and positively influence each other 11 . However, the exact identity of the IL6 producing and IL6 responsive cell(s) has yet to be identified, although evidence suggests that VSMCs secrete more IL6 with ageing 87 . To study the link between the changes occurring with normolipidaemia in the aged aorta and atherogen esis, young and aged wild type mice were made acutely hyperlipidaemic by inducing a decrease in LDL recep tor levels with adeno associated virus vector mediated During homeostasis, damaged mitochondria are recycled via mitophagy, which is a specialized subset of macroautophagy (see the figure) . Mitophagy reduces the production of mitochondrial damage-associated molecular patterns (mtDAMPs) and limits inflammation. Mitochondrial depolarization results in the accumulation of the serine/threonine protein kinase PINK1 at the outer mitochondrial membrane, leading to the recruitment of Parkin, an E3 ubiquitin ligase that ubiquitylates mitochondrial membrane proteins including mitofusin 1 (MFN1), MFN2 and voltage-dependent anion-selective channel protein 1 (VDAC1). This ubiquitylation primes the mitochondria for targeting by the autophagy machinery, including sequestosome 1 (p62) and microtubule-associated protein 1 light chain 3 (LC3), to package mitochondria in autophagosomes and deliver them to lysosomes for degradation. Other mitophagy mechanisms involve the apoptotic BCL-2 family proteins BCL-2/ adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and NIP3-like protein (NIX; also known as BNIP3L), which dimerize and bind directly to LC3 and function as adaptors between mitochondria and autophagosomes. BNIP3 and NIX can also facilitate apoptosis and cell death by participating in the release of mitochondrial cytochrome c and opening of the mitochondrial permeability transition pore. PGC1α, proliferator-activated receptorγ co-activator 1α; TF, transcription factor. Release of mtDAMPs www.nature.com/nrcardio delivery of Pcsk9 and by feeding the mice a high fat diet for 10 weeks 11 , which is an established technique 107 . With this protocol, young and aged mice had similar and durable levels of hyperlipidaemia; however, aged hyperlipidaemic mice had larger atherosclerotic lesions with larger necrotic cores than young hyperlipidaemic mice 11 . Importantly, administering spermidine, an agent that increases macroautophagy and mitophagy, to aged hyperlipidaemic mice reduced the levels of both IL6 and Parkin in the aorta and reduced the size of ather osclerotic plaques 11 . This finding is consistent with pre vious studies showing that treatment with spermidine or trehalose, an agent that increases mitophagy, reduces stiffness in the aged vasculature in normolipidaemic, non atherosclerotic aged mice 106, 108 . The CANTOS study 12 showed that in old patients (aged >60 years) with cardiovascular disease, IL1β blockade reduces the risk of recurrent cardiovascular events, indi cating that chronic inflammation is a major contribu tor to age related atherosclerosis. As described above, mitochondrial dysfunction might coexist in a positive feedback loop with IL6 signalling 11 to increase chronic inflammation in vascular ageing. Furthermore, mito chondrial components that are released to the cyto sol after mitochondrial damage can stimulate innate immune responses 109 . Mitochondrial injury in turn can be induced by TLR stimulation leading to the activa tion of caspase 4 and caspase 5 in humans or caspase 11 in mice 110 . These inflammatory caspases cleave gasder min D, which enables gasdermin D to form pores in the outer mitochondrial membrane, leading to impaired mitochondrial membrane potential and further increas ing mitochondrial injury 110 . Whether this pathway is activated during ageing and in particular vascular age ing is unclear. Nevertheless, the involvement of such a pathway could explain why chronic TLR activation, via either microbial products or sterile inflammatory medi ators, could lead to a chronic basal inflammatory state and mitochondrial dysfunction in the vasculature with ageing. Mitochondrial injury leads to the release of mito chondrial components, known a s m it oc ho nd rial damageassociated molecular patterns (mtDAMPs), including mtDNA, that when in the cytosol, can activate intracel lular innate immune signalling pathways, such as the DNA sensing receptor cyclic GMP-AMP synthase and the inflammasome 75, [110] [111] [112] . Transfer of mitochondrial components into endosomes also activates the TLR9 inflammatory pathway 111 , but the detailed mechanisms are not fully elucidated. Mitochondria also contain N formylated peptides that induce inflammation via engaging the N formyl peptide receptor 1 to increase neutrophil chemoattraction 113 , arterial injury and ROS release 114 . Cardiolipin, a component of mitochondrial membranes, can directly bind to NLRP3 and activate the NLRP3 inflammasome 115 . If chronically activated, all these pathways could promote vascular ageing and also diminish mitochondrial function, although ascertain ing the definitive contributions of each pathway requires future investigation. Age related atherosclerosis might be mediated by alterations in the vasculature and mye loid cells via a shared inflammatory pathway. A potential candidate pathway is IL6 signalling because available evidence indicates that the level of IL6 is elevated with ageing in both the immune system and the vasculature. In the bone marrow niche in mice, IL6 levels increase with ageing, which is probably mediated by increased β 2 adrenergic receptor signalling and increased num bers of adipocytes 60 (fIg. 1) . IL6 directly acts on HSCs to promote a bias towards myeloid cell differentiation 60 . In mouse macrophages, TET2 deficiency, which is one of the most common genetic alterations found in the age related condition CHIP, increases IL6 secretion in vitro 74 . Importantly, the atherosclerosis promoting effects of CHIP seem to be abrogated in individuals with a loss of function IL6 genetic polymorphism 9 . In the vasculature, the level of IL6 increases with ageing, which is at least in part mediated by IL6 production by VSMCs 87 . IL6 is associated with ageing in general and is part of the 'inflammageing' phenotype 15, 16, 116 . Why ageing leads to elevated basal secretion of inflammatory cytokines (not solely IL6 but also other inflammatory mediators such as TNF) is not clear but might be caused by alter ations in the microbiota 117 , increased adiposity 118 , and changes in the immune system 17 and the vasculature 29, 30 . Elevated cytokine levels with ageing could also be a manifestation of chronic, latent infections such as with herpesviruses 119 , of cellular senescence 57, 86, 87 or, potentially, of mitochondrial dysfunction. The role of IL6 in young animal models of ath erosclerosis remains unclear and might relate to the complexities of IL6 signalling (Box 2). Specifically, signalling via the classic IL6 pathway occurs in a restricted number of cells (such as hepatocytes and some immune cells) and involves IL6 binding to the membrane bound IL6 receptor (IL6R), with subse quent association with the signal transducing IL6R subunit β (also known as gp130). Evidence indicates that classic IL6 signalling is important for tissue home ostasis, regeneration and host defence (as reviewed previously 120 ). Soluble IL6R can also engage IL6 in the circulation and activate a broader range of cells than the classic pathway, via membrane activation of gp130. This pathway is termed IL6 trans signalling (Box 2) and can result in chronic inflammation 120 . These different IL6 signalling pathways might explain the pleiotropic effects of IL6 in different tissues and cellular compartments and also the divergent role of IL6 in experimental atherosclerotic models. For instance, one study in Apoe −/− mice showed that admin istration of exogenous IL6 worsens atherosclerosis 121 . By contrast, another study in Apoe +/− mice showed that IL6 deficiency worsens atherosclerosis 80 Nature reviews | Cardiology atherosclerotic Ldlr −/− mice 122 . The study found that inhib iting IL6 trans signalling reduced atherosclerosis 122 , indicating that IL6 trans signalling might have a path ogenic role in atherosclerosis. Therefore, clinical ther apeutics to reduce atherosclerosis should focus on this IL6 pathway. Whether IL6 has a causal role in age related ather osclerosis is not known yet. The contribution of IL6 to age related atherosclerosis should be investigated in the future and should determine the main IL6 producing and IL6 responding cells. Furthermore, the iden tification of the major IL6 producing cells (fIg. 2) and whether IL6 activation occurs via the classic or trans signalling pathway with ageing could lead to more targeted therapeutics for atherosclerosis, especially given the availability of clinically approved agents to target IL6 (refs 123,124 ). Importantly, the risk-benefit balance of targeting IL6 in atherosclerosis will need to be deter mined, given that anti IL6 therapies in human studies increased the risk of infections 120 , similar to other bio logical agents (such as anti IL1β antibodies) that have been used to reduce atherosclerosis 12, 13 . However, other biological agents such as TNF inhibitors 125 might be ben eficial for the treatment of atherosclerotic cardiovascular disease and should be investigated in age related ather osclerosis. Finally, other inflammatory cytokines (such as TNF, C C motif chemokine 2 and IL18, which are all part of the SASP) 56 might have a pathogenic role in age related atherosclerosis and should be assessed in future studies. Therapies that can mitigate some of the detrimental biological effects of ageing, such as removing senescent cells (including senescent adipocytes) 126, 127 , improving mitochondrial function (for example, with metformin therapy) 128 , or augmenting macroautophagy (for exam ple, with rapamycin therapy) 129 or mitophagy, might reduce the burden of atherosclerosis in old people and should be investigated in future clinical studies. Agents that increase mitophagy, such as spermidine, have been shown in experimental studies to reduce atherosclero sis in both young 130 and aged 11 mice. Some or all these agents might have pleiotropic effects, which could reduce inflammation. Furthermore, these agents might synergize with specific anti inflammatory therapies to reduce atherosclerosis with ageing, which will require future clinical investigation. Ageing influences atherogenesis via multiple complex pathways, and one sole factor is unlikely to be a domi nant pathophysiological mechanism. In this Review, we provide an overview of how ageing affects two systems, myeloid cell haematopoiesis and the vasculature, to pro mote atherosclerosis. We lay a framework of a poten tial shared inflammatory pathway, mediated by IL6 signalling, that connects the role of the two systems in Box 2 | il-6 signalling IL-6 can signal via a classic signalling pathway and a trans-signalling pathway (see the figure) . In the classic IL-6 signalling pathway, IL-6 engages the membrane-bound IL-6 receptor (IL-6R) and subsequently interacts with the IL-6R subunitβ (also known as gp130). Intracellular signalling mainly involves activation of the Janus kinase (JAK) and the signal transducer and activator of transcription 3 (STAT3). The classic pathway is generally restricted to hepatocytes and immune cells such as myeloid cells and lymphocytes. The trans-signalling pathway is activated by IL-6 binding to soluble IL-6R (sIL-6R) in the circulation and then binding of the IL-6-sIL-6R complex to membrane-bound gp130 on a broad range of cells. sIL-6R is released by enzymatic cleavage of membrane-bound IL-6R by disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Activation of the IL-6 trans-signalling pathway generally leads to chronic inflammation, whereas the IL-6 classic signalling pathway is involved in cell growth, regeneration and host defence. age related atherosclerosis and propose future avenues of investigation to determine whether IL6 and/or other inflammatory pathways are feasible and effective ther apeutic targets to reduce the burden of atherosclerosis in old people. Anti inflammatory strategies should be considered in the context of other therapies that aim to reduce many of the detrimental biological effects of ageing. Overall, we hope that with the pursuit of further clinical investigation and trials, therapeutic options will be available in the future to reduce the burden of ath erosclerosis in the increasing number of old people in our society. Fig. 2 | il-6 as a potential therapeutic target in age-related atherosclerosis. IL-6 is upregulated in multiple tissues that have important roles in the increase in atherogenesis with ageing. Therefore, blockade of IL-6 might be an effective therapeutic strategy to reduce atherosclerosis development and progression during ageing. Blocking IL-6 might interfere with the increased IL-6 signalling in bone marrow adipocytes that occurs with ageing (which promotes a skewing towards myeloid cell differentiation), thereby reducing the risk of clonal haematopoiesis of indeterminate potential (CHIP). IL-6 blockade might also reduce the inflammatory potential of clones of myeloid cells associated with CHIP. IL-6 blockade might reduce atherosclerosis burden, although direct comparisons of efficacy and safety with IL-1β inhibition requires future investigation. IL-6 blockade might increase mitochondrial function and reduce the expression of Parkin, a mitochondrial stress protein, which might also contribute to reducing atherogenesis during ageing. HSC, haematopoietic stem cell; VSMC, vascular smooth muscle cell. 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Rapamycin, but not resveratrol or simvastatin, extends life span of genetically heterogeneous mice Spermidine reduces lipid accumulation and necrotic core formation in atherosclerotic plaques via induction of autophagy Acknowledgements D.J.T. is supported by NIH award F32-HL1400728, and D.R.G. is supported by NIH awards R01-HL127687, R01-AI138347 and K07-AG050096. Both authors researched data for the article, discussed its content, wrote the manuscript, and reviewed and edited it before submission. The authors declare no competing interests. Nature Reviews Cardiology thanks C. Leeuwenburgh, H. Oliveira, A. Tedgui and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.