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Plasma high density lipoproteins: Therapeutic targeting and links to atherogenic inflammation

      Highlights

      • Plasma HDL has athero-protective functions, related to its ability to promote efflux of cholesterol from macrophage foam cells.
      • Cholesterol efflux pathways suppress excessive myelopoiesis, macrophage inflammation, inflammasome activation and NETosis.
      • Therapeutic targeting of beneficial HDL functions that increase cholesterol efflux has strong potential to reduce CHD.

      Abstract

      Plasma HDL levels have an inverse relationship to coronary artery disease (CAD) risk, which led to the idea that increasing HDL levels therapeutically would ameliorate atherosclerosis. Human genetic deficiency of CETP caused markedly elevated HDL and moderately reduced non-HDL cholesterol levels, suggesting that CETP inhibitors might produce cardiovascular benefit. The CETP inhibitor anacetrapib reproduced the phenotype of homozygous CETP deficiency and showed a highly significant benefit for CAD in the REVEAL trial. However, the magnitude of this effect was moderate, and the mechanism of benefit remains unclear. Insights into the mechanisms underlying macrophage cholesterol efflux and reverse cholesterol transport have come from monogenic human disorders and transgenic mouse studies. In particular, the importance of the ATP binding cassette transporters ABCA1 and ABCG1 in promoting cholesterol efflux from myeloid and other hematopoietic cells has been shown and linked to aberrant myelopoiesis and macrophage inflammation. Recent studies have shown that myeloid deficiency of ABCA1 and ABCG1 leads to macrophage and neutrophil inflammasome activation, which in turn promotes atherosclerotic plaque development and notably the formation of neutrophil extracellular traps (NETs) in plaques. In addition, clonal hematopoiesis has emerged as an important CAD risk factor, likely involving macrophage inflammation and inflammasome activation. Further elucidation of the mechanisms linking plaque accumulation of cholesterol and oxidized lipids to myeloid cell inflammation may lead to the development of new therapeutics specifically targeting atherogenic inflammation, with likely benefit for CAD.

      1. HDL and atherosclerosis

      In epidemiological studies, plasma HDL-cholesterol levels show a robust inverse relationship with coronary heart disease (CHD) independent of other risk factors [
      • Emerging Risk Factors C.
      • Di Angelantonio E.
      • Sarwar N.
      • et al.
      Major lipids, apolipoproteins, and risk of vascular disease.
      ]. Infusions of HDL or increased expression of the main HDL protein, apoA-1, consistently reduce atherosclerosis in animal models [
      • Rubin E.M.
      • Krauss R.M.
      • Spangler E.A.
      • Verstuyft J.G.
      • Clift S.M.
      Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI.
      ,
      • Plump A.S.
      • Scott C.J.
      • Breslow J.L.
      Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse.
      ,
      • Badimon J.J.
      • Badimon L.
      • Fuster V.
      Regression of atherosclerotic lesions by high density lipoprotein plasma fraction in the cholesterol-fed rabbit.
      ,
      • Feig J.E.
      • Hewing B.
      • Smith J.D.
      • Hazen S.L.
      • Fisher E.A.
      High-density lipoprotein and atherosclerosis regression: evidence from preclinical and clinical studies.
      ]. In most studies, the ability of HDL to promote cholesterol efflux from macrophage foam cells is inversely correlated with human coronary atheroma burden and with incident CHD [
      • Monette J.S.
      • Hutchins P.M.
      • Ronsein G.E.
      • et al.
      Patients with coronary endothelial dysfunction have impaired cholesterol efflux capacity and reduced HDL particle concentration.
      ,
      • Khera A.V.
      • Demler O.V.
      • Adelman S.J.
      • et al.
      Cholesterol efflux capacity, high-density lipoprotein particle number, and incident cardiovascular events: an analysis from the JUPITER trial (justification for the use of statins in prevention: an intervention trial evaluating rosuvastatin).
      ,
      • Saleheen D.
      • Scott R.
      • Javad S.
      • et al.
      Association of HDL cholesterol efflux capacity with incident coronary heart disease events: a prospective case-control study.
      ,
      • Khera A.V.
      • Cuchel M.
      • de la Llera-Moya M.
      • et al.
      Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.
      ,
      • Rohatgi A.
      • Khera A.
      • Berry J.D.
      • et al.
      HDL cholesterol efflux capacity and incident cardiovascular events.
      ,
      • Li X.M.
      • Tang W.H.
      • Mosior M.K.
      • et al.
      Paradoxical association of enhanced cholesterol efflux with increased incident cardiovascular risks.
      ]. In contrast, Mendelian Randomization studies have failed to find a relationship between SNPs that increase HDL cholesterol levels and coronary heart disease [
      • Voight B.F.
      • Peloso G.M.
      • Orho-Melander M.
      • et al.
      Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study.
      ]. Nonetheless, preclinical studies and studies of human HDL functionality suggest that some approaches to increasing HDL levels or beneficial functions might be a way to reduce the substantial burden of CHD that remains in subjects optimally treated by LDL lowering [
      • Sabatine M.S.
      • Giugliano R.P.
      • Keech A.C.
      • et al.
      Evolocumab and clinical outcomes in patients with cardiovascular disease.
      ].
      The idea that HDL might mediate protection from atherosclerosis by stimulating an overall process of reverse cholesterol transport was originally proposed by Glomset [
      • Glomset J.A.
      The metabolic role of lecithin: cholesterol acyltransferase: perspectives from pathology.
      ]. Over the subsequent four decades, the factors controlling HDL metabolism and the molecular underpinnings of the reverse cholesterol transport pathway have been elucidated with contributions by many laboratories [
      • Rubin E.M.
      • Krauss R.M.
      • Spangler E.A.
      • Verstuyft J.G.
      • Clift S.M.
      Inhibition of early atherogenesis in transgenic mice by human apolipoprotein AI.
      ,
      • Plump A.S.
      • Scott C.J.
      • Breslow J.L.
      Human apolipoprotein A-I gene expression increases high density lipoprotein and suppresses atherosclerosis in the apolipoprotein E-deficient mouse.
      ,
      • Lewis G.F.
      • Rader D.J.
      New insights into the regulation of HDL metabolism and reverse cholesterol transport.
      ,
      • Rosenson R.S.
      • Brewer Jr., H.B.
      • Davidson W.S.
      • et al.
      Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport.
      ,
      • Brooks-Wilson A.
      • Marcil M.
      • Clee S.M.
      • et al.
      Mutations in ABC1 in Tangier disease and familial high-density lipoprotein deficiency.
      ,
      • Acton S.
      • Rigotti A.
      • Landschulz K.T.
      • Xu S.
      • Hobbs H.H.
      • Krieger M.
      Identification of scavenger receptor SR-BI as a high density lipoprotein receptor.
      ]. The most important insights have been gained by the elucidation of human genetic deficiency states affecting HDL and by transgenic mouse studies. Fig. 1 illustrates the overall process of reverse cholesterol transport. This is initiated in the arterial wall by the ATP binding cassette transporters ABCA1 and ABCG1, which are induced in arterial wall macrophage foam cells by LXR activation and promote the efflux of cholesterol onto lipid poor ApoA-1 and HDL particles. Cholesterol in HDL may be esterified by lecithin:cholesterol acyltransferase (LCAT) and directly taken up in the liver by a process of selective free and esterified cholesterol removal mediated by scavenger receptor B1 (SR-BI). In humans, cholesteryl ester transfer protein (CETP) mediates the exchange of cholesteryl esters (CE) in HDL with triglycerides in VLDL or chylomicrons, leading to a net transfer of CE from HDL to the triglyceride-rich lipoproteins and LDL and subsequent removal in the liver via the LDL receptor and other pathways. Research in my laboratory initially focused on elucidating the role of CETP in lipoprotein metabolism. More recent studies have used mouse models to investigate the role of ABCA1 and ABCG1 in cholesterol efflux and atherosclerosis.
      Fig. 1
      Fig. 1The role of HDL in macrophage cholesterol efflux and reverse cholesterol transport.
      Macrophage cholesterol efflux and reverse cholesterol transport are initiated in the arterial wall by the ATP binding cassette transporters ABCA1 and ABCG1, which are induced in arterial wall macrophage foam cells by LXR activation and promote the efflux of cholesterol onto lipid poor apoA-1 or HDL particles.
      Cholesterol in HDL may be esterified by lecithin:cholesterol acyltransferase (LCAT) and directly taken up in the liver by a process of selective cholesteryl ester removal mediated by scavenger receptor B1 (SR-BI). This may be followed by the excretion of cholesterol into bile involving ABCG5/8. In humans, cholesteryl ester transfer protein (CETP) mediates the exchange of cholesteryl esters (CE) in HDL with triglycerides in VLDL or chylomicrons leading to a net transfer of CE form HDL to the triglyceride-rich lipoproteins and LDL and subsequent removal in the liver via the LDL receptor and other pathways. ABCA1 also initiates the formation of HDL particles in the liver and small intestine (not shown) by binding ApoA-1 and promoting its lipidation. HL, hepatic lipase; EL, endothelial lipase.

      1.1 Human genetic CETP deficiency and the development of CETP inhibitors

      In collaboration with colleagues in Japan, we first defined human genetic deficiency of CETP, which was characterized by markedly elevated levels of HDL cholesterol (HDL-C), as well as reduced levels of LDL cholesterol (LDL-C) and ApoB, a profile that is typically associated with reduced atherosclerosis [
      • Inazu A.
      • Brown M.L.
      • Hesler C.B.
      • et al.
      Increased high-density lipoprotein levels caused by a common cholesteryl-ester transfer protein gene mutation.
      ]. This led to the development of CETP inhibitors. These were shown to raise HDL-C and ApoA-1 levels, and for the more potent CETP inhibitors, there was also a lowering of HDL cholesterol and ApoB levels. Based on epidemiological observations, it was expected that this dramatic increase in HDL would deliver a marked anti-atherogenic effect. However, this was not the case for CETP inhibitors that were initially developed. In fact, the first CETP inhibitor to enter human clinical trials, torcetrapib, caused an excess of deaths and cardiovascular disease [
      • Barter P.J.
      • Caulfield M.
      • Eriksson M.
      • et al.
      Effects of torcetrapib in patients at high risk for coronary events.
      ]. This led many experts to conclude that the HDL itself was dysfunctional or harmful. However, significant off-target side effects, involving hyperaldosteronism and substantial hypertension, were attributed to torcetrapib [
      • Barter P.J.
      • Caulfield M.
      • Eriksson M.
      • et al.
      Effects of torcetrapib in patients at high risk for coronary events.
      ]. The demonstration of an overall anti-atherogenic effect of CETP inhibition was suggested by the majority of animal studies showing a pro-atherogenic effect of CETP expression [
      • Barter P.J.
      • Brewer Jr., H.B.
      • Chapman M.J.
      • Hennekens C.H.
      • Rader D.J.
      • Tall A.R.
      Cholesteryl ester transfer protein: a novel target for raising HDL and inhibiting atherosclerosis.
      ]. Moreover, multiple large human genetic studies showed that SNPs in the CETP gene, that are associated with increased HDL and reduced LDL cholesterol, are associated with reduced CHD [
      • Johannsen T.H.
      • Frikke-Schmidt R.
      • Schou J.
      • Nordestgaard B.G.
      • Tybjaerg-Hansen A.
      Genetic inhibition of CETP, ischemic vascular disease and mortality, and possible adverse effects.
      ,
      • Nomura A.
      • Won H.H.
      • Khera A.V.
      • et al.
      Protein-truncating variants at the cholesteryl ester transfer protein gene and risk for coronary heart disease.
      ,
      • Webb T.R.
      • Erdmann J.
      • Stirrups K.E.
      • et al.
      Systematic evaluation of pleiotropy identifies 6 further loci associated with coronary artery disease.
      ]. This includes SNPs that likely reduce the function of the promoter region upstream of the CETP gene [
      • Webb T.R.
      • Erdmann J.
      • Stirrups K.E.
      • et al.
      Systematic evaluation of pleiotropy identifies 6 further loci associated with coronary artery disease.
      ], and most importantly, CETP protein truncating mutations that abrogate the function of CETP [
      • Nomura A.
      • Won H.H.
      • Khera A.V.
      • et al.
      Protein-truncating variants at the cholesteryl ester transfer protein gene and risk for coronary heart disease.
      ]. This has permitted further human clinical studies to be performed to evaluate other members of this class of drugs. Subsequent studies trials with the relatively weak CETP inhibitor dalcetrapib [
      • Schwartz G.G.
      • Olsson A.G.
      • Abt M.
      • et al.
      Effects of dalcetrapib in patients with a recent acute coronary syndrome.
      ] and with the potent inhibitor evacetrapib [
      • Lincoff A.M.
      • Nicholls S.J.
      • Riesmeyer J.S.
      • et al.
      Evacetrapib and cardiovascular outcomes in high- risk vascular disease.
      ] were halted prematurely because of projected lack of efficacy.
      Finally, in the largest study of a CETP inhibitor, and the first to go to completion, the potent CETP inhibitor anacetrapib was shown to significantly reduce major coronary events [
      • Group H.T.R.C.
      • Bowman L.
      • Hopewell J.C.
      • et al.
      Effects of anacetrapib in patients with atherosclerotic vascular disease.
      ]. This study involved 30,449 patients with atherosclerotic cardiovascular disease who were randomized to receive anacetrapib 100 mg daily or placebo on top of effective statin therapy and followed for a median of 4.1 years. The study showed a highly significant reduction (rate ratio = 0.91, p < 0.004) in the composite primary endpoint of coronary death, myocardial infarction or coronary revascularization [
      • Group H.T.R.C.
      • Bowman L.
      • Hopewell J.C.
      • et al.
      Effects of anacetrapib in patients with atherosclerotic vascular disease.
      ]. The modest degree of reduction in the primary endpoint likely reflected the fact that control patients, who were highly effectively treated with statins, had an LDL cholesterol of 61 mg/dl, making it a high hurdle to show a major incremental effect of CETP inhibition. Merck decided not to seek marketing approval for anacetrapib, seemingly based on a combination of its clinical profile, which included a modest effect on cardiovascular endpoints, prolonged accumulation in adipose tissue, as well as marketing considerations. As monotherapy, potent CETP inhibitors lower LDL cholesterol by about 40% and ApoB by about 30%, similar to homozygous CETP deficiency. Thus, other CETP inhibitors lacking adipose accumulation could potentially be used to treat CHD, either in combination with statins or as monotherapy. However, further clinical trials of efficacy would likely be required.

      1.2 Cholesterol efflux pathways and macrophage inflammation

      We and others showed that the ATP binding cassette transporters ABCA1 and ABCG1 promote cholesterol efflux from macrophages to lipid-poor apoA-1 and HDL particles, respectively [
      • Wang N.
      • Lan D.
      • Chen W.
      • Matsuura F.
      • Tall A.R.
      ATP-binding cassette transporters G1 and G4 mediate cellular cholesterol efflux to high-density lipoproteins.
      ,
      • Kennedy M.A.
      • Barrera G.C.
      • Nakamura K.
      • et al.
      ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation.
      ]. Abca1 and Abcg1 are induced in cholesterol loaded macrophages as a result of direct promoter activation by LXRs [
      • Costet P.
      • Luo Y.
      • Wang N.
      • Tall A.R.
      Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor.
      ,
      • Kennedy M.A.
      • Venkateswaran A.
      • Tarr P.T.
      • et al.
      Characterization of the human ABCG1 gene: liver X receptor activates an internal promoter that produces a novel transcript encoding an alternative form of the protein.
      ]. Cholesterol efflux pathways mediated by these transporters exert anti-atherogenic effects by suppressing inflammatory responses in myeloid cells [
      • Tall A.R.
      • Yvan-Charvet L.
      Cholesterol, inflammation and innate immunity.
      ]. Abca1/g1 deficient macrophages show heightened inflammatory responses to lipopolysaccharide, oxidized phospholipids and apoptotic cells in part related to increased cell surface expression of TLR4, increased signaling via MyD88 and TRIF dependent pathways and NADPH oxidase activation; these effects are dependent on increased cellular cholesterol content [
      • Yvan-Charvet L.
      • Welch C.
      • Pagler T.A.
      • et al.
      Increased inflammatory gene expression in ABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling via toll-like receptors, and neutrophil infiltration of atherosclerotic lesions.
      ,
      • Yvan-Charvet L.
      • Pagler T.A.
      • Seimon T.A.
      • et al.
      ABCA1 and ABCG1 protect against oxidative stress- induced macrophage apoptosis during efferocytosis.
      ]. ABCA1/G1 suppress excessive proliferation of hematopoietic stem cells, monocytosis, neutrophilia and macrophage accumulation in atherosclerotic plaques of hypercholesterolemic mice [
      • Murphy A.J.
      • Akhtari M.
      • Tolani S.
      • et al.
      ApoE regulates hematopoietic stem cell proliferation, monocytosis, and monocyte accumulation in atherosclerotic lesions in mice.
      ,
      • Westerterp M.
      • G-AS
      • Murphy A.J.
      • Shih A.
      • Cremers S.
      • Levine R.L.
      • Tall A.R.
      • Yvan-Charvet L.
      Regulation of hematopoietic stem and progenitor cell mobilization by cholesterol efflux pathways.
      ,
      • Yvan-Charvet L.
      • Pagler T.
      • Gautier E.L.
      • et al.
      ATP-binding cassette transporters and HDL suppress hematopoietic stem cell proliferation.
      ,
      • Wang M.
      • Subramanian M.
      • Abramowicz S.
      • et al.
      Interleukin-3/Granulocyte macrophage colony- stimulating factor receptor promotes stem cell expansion, monocytosis, and atheroma macrophage burden in mice with hematopoietic ApoE deficiency.
      ,
      • Westerterp M.
      • Bochem A.E.
      • Yvan-Charvet L.
      • Murphy A.J.
      • Wang N.
      • Tall A.R.
      ATP-binding cassette transporters, atherosclerosis, and inflammation.
      ]. We developed Abca1fl/flAbcg1fl/fl mice and showed increased atherosclerosis with myeloid (Myl) or endothelial knockout of these transporters [
      • Westerterp M.
      • Murphy A.J.
      • Wang M.
      • et al.
      Deficiency of ATP-binding cassette transporters A1 and G1 in macrophages increases inflammation and accelerates atherosclerosis in mice.
      ,
      • Westerterp M.
      • Tsuchiya K.
      • Tattersall I.W.
      • et al.
      Deficiency of ATP-binding cassette transporters A1 and G1 in endothelial cells accelerates atherosclerosis in mice.
      ].

      2. Cholesterol efflux pathways suppress inflammasomes and NETosis

      Our recent studies in MylABCDKO mice have revealed a major role of ABCA1/G1-mediated cholesterol efflux pathways in suppressing the inflammasome [
      • Westerterp M.
      • Fotakis P.
      • Ouimet M.
      • et al.
      Cholesterol efflux pathways suppress inflammasome activation, netosis and atherogenesis.
      ]. MylABCDKO mice showed inflammasome activation in macrophages and neutrophils, involving both Nlrp3/caspase-1 and noncanonical/caspase-11. Unexpectedly, MylABCDKO mice showed prominent neutrophil extracellular traps (NETs) in atherosclerotic lesions, while deficiency of Nlrp3 or Caspase- 1/11 abolished NETosis and reduced lesion area, suggesting a novel role for ABCA1/G1 in suppressing inflammation associated with atherosclerosis. Recent studies in the CANTOS trial have highlighted the importance of inflammasome activation and IL-1b production in human coronary heart disease (CHD) [
      • Ridker P.M.
      • Everett B.M.
      • Thuren T.
      • et al.
      Antiinflammatory therapy with canakinumab for atherosclerotic disease.
      ], while other studies have suggested a role of NETosis in atherogenesis [
      • Knight J.S.
      • Luo W.
      • O'Dell A.A.
      • et al.
      Peptidylarginine deiminase inhibition reduces vascular damage and modulates innate immune responses in murine models of atherosclerosis.
      ] and plaque instability [
      • Franck G.
      • Mawson T.L.
      • Folco E.J.
      • et al.
      Roles of PAD4 and NETosis in experimental atherosclerosis and arterial injury: implications for superficial erosion.
      ,
      • Doring Y.
      • Soehnlein O.
      • Weber C.
      Neutrophil extracellular traps in atherosclerosis and atherothrombosis.
      ]. Thus, our studies showing that HDL and cholesterol efflux pathways can suppress these processes have significant potential translational relevance, especially in conditions where ABCA1/G1 are suppressed and HDL levels are low, which may include Type 2 diabetes, chronic kidney disease and ageing [
      • Sene A.
      • Khan A.A.
      • Cox D.
      • et al.
      Impaired cholesterol efflux in senescent macrophages promotes age-related macular degeneration.
      ,
      • Ganda A.
      • Yvan-Charvet L.
      • Zhang Y.
      • et al.
      Plasma metabolite profiles, cellular cholesterol efflux, and non-traditional cardiovascular risk in patients with CKD.
      ,
      • Mauldin J.P.
      • Nagelin M.H.
      • Wojcik A.J.
      • et al.
      Reduced expression of ATP-binding cassette transporter G1 increases cholesterol accumulation in macrophages of patients with type 2 diabetes mellitus.
      ,
      • Tang C.
      • Kanter J.E.
      • Bornfeldt K.E.
      • Leboeuf R.C.
      • Oram J.F.
      Diabetes reduces the cholesterol exporter ABCA1 in mouse macrophages and kidneys.
      ,
      • Daffu G.
      • Shen X.
      • Senatus L.
      • et al.
      RAGE suppresses ABCG1-mediated macrophage cholesterol efflux in diabetes.
      ,
      • Patel D.C.
      • Albrecht C.
      • Pavitt D.
      • et al.
      Type 2 diabetes is associated with reduced ATP-binding cassette transporter A1 gene expression, protein and function.
      ,
      • Kashyap S.R.
      • Osme A.
      • Ilchenko S.
      • et al.
      Glycation reduces the stability of ApoAI and increases HDL dysfunction in diet-controlled type 2 diabetes.
      ]. In these conditions, treatment of CHD by infusions of cholesterol-poor reconstituted HDL particles, which mediate cholesterol efflux by non-transporter dependent mechanisms, may be particularly beneficial.
      The role of the NLRP3 inflammasome in atherosclerosis was first explored by Latz in Ldlr−/- mice [
      • Duewell P.
      • Kono H.
      • Rayner K.J.
      • et al.
      NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals.
      ] and by Tschopp in Apoe−/− mice [
      • Menu P.
      • Pellegrin M.
      • Aubert J.F.
      • et al.
      Atherosclerosis in ApoE-deficient mice progresses independently of the NLRP3 inflammasome.
      ]. Latz et al. found a significant impact on lesion development and related this to cholesterol crystal formation even in early foam cell lesions, while Tschopp et al. reported no effects on atherogenesis. While some subsequent studies appeared to confirm the initial reports [
      • Usui F.
      • Shirasuna K.
      • Kimura H.
      • et al.
      Critical role of caspase-1 in vascular inflammation and development of atherosclerosis in Western diet-fed apolipoprotein E-deficient mice.
      ,
      • Zheng F.
      • Xing S.
      • Gong Z.
      • Mu W.
      • Xing Q.
      Silence of NLRP3 suppresses atherosclerosis and stabilizes plaques in apolipoprotein E-deficient mice.
      ], our own [
      • Westerterp M.
      • Fotakis P.
      • Ouimet M.
      • et al.
      Cholesterol efflux pathways suppress inflammasome activation, netosis and atherogenesis.
      ] and other studies [
      • Tumurkhuu G.
      • Shimada K.
      • Dagvadorj J.
      • et al.
      Ogg1-Dependent DNA repair regulates NLRP3 inflammasome and prevents atherosclerosis.
      ] found no impact of deletion of key inflammasome components, Nlrp3 or Caspase1/11, on lesion area or morphology in WD-fed Ldlr_/_ mice. However, when additional mutations that led to macrophage inflammasome activation were introduced into the Ldlr_/_ model, joint deficiencies with Nlrp3 or Caspase 1/11 showed that the NLRP3 inflammasome does contribute to atherogenesis [
      • Westerterp M.
      • Fotakis P.
      • Ouimet M.
      • et al.
      Cholesterol efflux pathways suppress inflammasome activation, netosis and atherogenesis.
      ,
      • Tumurkhuu G.
      • Shimada K.
      • Dagvadorj J.
      • et al.
      Ogg1-Dependent DNA repair regulates NLRP3 inflammasome and prevents atherosclerosis.
      ].

      3. Inflammasome activation in human atherosclerosis

      Our mouse studies showed that whole body Abca1 deficiency on the Ldlr−/− background induced inflammasome activation, and this effect was echoed by elevated IL-1 and IL-18 plasma levels (markers of inflammasome activation) in Tangier disease patients, indicating human relevance. This suggests that low HDL (decreasing ABCG1-mediated cholesterol efflux), defective apoA-1 and reduced expression of ABCA1/G1 in monocyte/macrophages may be sufficient to induce inflammasome activation in humans. A variety of studies suggest that these conditions may be commonly found in patients with Type 2 diabetes, chronic kidney disease and with ageing [
      • Sene A.
      • Khan A.A.
      • Cox D.
      • et al.
      Impaired cholesterol efflux in senescent macrophages promotes age-related macular degeneration.
      ,
      • Ganda A.
      • Yvan-Charvet L.
      • Zhang Y.
      • et al.
      Plasma metabolite profiles, cellular cholesterol efflux, and non-traditional cardiovascular risk in patients with CKD.
      ,
      • Mauldin J.P.
      • Nagelin M.H.
      • Wojcik A.J.
      • et al.
      Reduced expression of ATP-binding cassette transporter G1 increases cholesterol accumulation in macrophages of patients with type 2 diabetes mellitus.
      ,
      • Tang C.
      • Kanter J.E.
      • Bornfeldt K.E.
      • Leboeuf R.C.
      • Oram J.F.
      Diabetes reduces the cholesterol exporter ABCA1 in mouse macrophages and kidneys.
      ,
      • Daffu G.
      • Shen X.
      • Senatus L.
      • et al.
      RAGE suppresses ABCG1-mediated macrophage cholesterol efflux in diabetes.
      ,
      • Patel D.C.
      • Albrecht C.
      • Pavitt D.
      • et al.
      Type 2 diabetes is associated with reduced ATP-binding cassette transporter A1 gene expression, protein and function.
      ,
      • Kashyap S.R.
      • Osme A.
      • Ilchenko S.
      • et al.
      Glycation reduces the stability of ApoAI and increases HDL dysfunction in diet-controlled type 2 diabetes.
      ]. Clonal hematopoiesis involving common variants in hematopoietic genes (TET2, JAK2, ASXL1 and DNMT3a) that predispose to hematological malignancy has recently emerged as an important novel risk factor for CHD especially in the elderly [
      • Jaiswal S.
      • Fontanillas P.
      • Flannick J.
      • et al.
      Age-related clonal hematopoiesis associated with adverse outcomes.
      ,
      • Jaiswal S.
      • Natarajan P.
      • Silver A.J.
      • et al.
      Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease.
      ]. Recent studies in mice with myeloid Tet2 deficiency, modeling clonal hematopoiesis, have shown macrophage inflammasome activation, leading to increased IL-1b production and accelerated atherosclerosis [
      • Jaiswal S.
      • Natarajan P.
      • Silver A.J.
      • et al.
      Clonal hematopoiesis and risk of atherosclerotic cardiovascular disease.
      ,
      • Fuster J.J.
      • MacLauchlan S.
      • Zuriaga M.A.
      • et al.
      Clonal hematopoiesis associated with Tet2 deficiency accelerates atherosclerosis development in mice.
      ]. Along with the positive outcome of the CANTOS trial [
      • Ridker P.M.
      • Everett B.M.
      • Thuren T.
      • et al.
      Antiinflammatory therapy with canakinumab for atherosclerotic disease.
      ], involving IL 1b neutralization, this suggests that inflammasome activation is an important contributor to human CHD. However, the conditions promoting inflammasome activation in atherosclerosis and the links between the inflammasome and NETosis need to be more clearly delineated.

      4. Perspective

      Athero-thrombotic disease remains the major cause of disability and death in the industrialized world. Despite the success of LDL lowering therapies in treatment, there remains a large burden of residual risk. Attempts to treat this residual risk with CETP inhibitors that dramatically raise HDL have only met with modest success. Other approaches that more effectively stimulate cholesterol efflux pathways may be more successful in the future. There is a great deal of excitement about new targets for lowering levels of triglyceride and cholesterol rich remnants that have emerged from human genetic studies. The CANTOS trial, in which IL-b inhibition was found to reduce CVD, strongly supports the concept of anti-inflammatory therapies in the treatment of athero-thrombotic disease. However, an excess of deaths from infections may limit the clinical impact of this or other broadly immunosuppressive therapies. This highlights the need to understand more clearly the inflammatory mechanisms that are specific to atherogenesis. Perhaps the most obvious candidates for future studies are inflammatory mechanisms that are linked to oxidized phospholipids and to cholesterol uptake and removal from macrophages. Inflammasome activation and neutrophil NETosis, promoted by macrophage and neutrophil cholesterol accumulation, are emerging mechanisms underlying atherogenic inflammation. Clonal hematopoiesis has recently been discovered as a major non-traditional risk factor for CVD, and likely interacts with hyperlipidemia to promote atherogenic inflammation. In summary, many recent studies have linked hyperlipidemia, defective cholesterol efflux pathways and aberrant hematopoiesis to atherogenic inflammation. However, this area remains poorly understood, therapy is challenging, and there is a tremendous need for further research.

      Conflicts of interest

      The author declared he does not have anything to disclose regarding conflict of interest with respect to this manuscript.

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