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Cited by 129Cholesterol and Lipid MetabolismMicroRNA in disease regulationAtherosclerosis and Cardiovascular Diseases

Genetic dissection of the impact of miR-33a and miR-33b during the progression of atherosclerosis

Open access
Price, Nathan L., Rotllan, Noemi, Canfrán-Duque, Alberto, Zhang, Xinbo, Pati, Paramita, Arias, Noemi, Moen, Jack, Mayr, Manuel +4 more·2017·Cell reports
DOI 10.1016/j.celrep.2017.10.023PMC PMC5687841
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Abstract

As an important regulator of macrophage cholesterol efflux and HDL biogenesis, miR-33 is a promising target for treatment of atherosclerosis, and numerous studies demonstrate that inhibition of miR-33 increases HDL levels and reduces plaque burden. However, important questions remain about how miR-33 impacts atherogenesis, including whether this protection is primarily due to direct effects on plaque macrophages or regulation of lipid metabolism in the liver. We demonstrate that miR-33 deficiency in Ldlr −/− mice promotes obesity, insulin resistance, and hyperlipidemia but does not impact plaque development. We further assess how loss of miR-33 or addition of miR-33b in macrophages and other hematopoietic cells impact atherogenesis. Macrophage-specific loss of miR-33 decreases lipid accumulation and inflammation under hyperlipidemic conditions, leading to reduced plaque burden. Therefore, the pro-atherogenic effects observed in miR-33-deficient mice are likely counterbalanced by protective effects in macrophages, which may be the primary mechanism through which anti-miR-33 therapies reduce atherosclerosis.

AI snapshot

Written from the paper’s full text.

TL;DR

This study uses new miR-33 knockout and miR-33b knock-in mouse models to dissect how miR-33 loss in whole body versus hematopoietic cells affects atherosclerosis in Ldlr−/− mice. It finds that macrophage/hematopoietic loss of miR-33 reduces plaque burden and improves reverse cholesterol transport, while whole-body miR-33 deficiency causes obesity, insulin resistance, and hyperlipidemia that offset plaque protection.

Key findings

  • Whole-body miR-33 deficiency in Ldlr−/− mice (DKO) increased body weight, impaired insulin sensitivity, and raised VLDL/LDL cholesterol and triglycerides after Western diet, but did not change total plaque area or lipid accumulation versus Ldlr−/− controls.
  • Hematopoietic-specific loss of miR-33 via bone marrow transplant into Ldlr−/− mice significantly reduced total plaque area, lipid accumulation, necrotic core size, CD68/TUNEL staining, and total and Ly-6Chigh macrophages after 12 weeks of Western diet, without altering plasma lipids or lipoprotein profile.
  • miR-33−/− macrophages showed increased ABCA1/ABCG1 expression, improved in vivo reverse cholesterol transport (increased [3H]-cholesterol in plasma and feces over 48 h), reduced cholesterol esters and phosphatidylethanolamines/phosphatidylserines, and increased efferocytosis with upregulated MERTK.
  • Bone marrow from miR-33b knock-in mice did not affect plaque size or lipid accumulation, likely because miR-33b expression in macrophages is much lower than miR-33a; global miR-33b knock-in assessment was prevented by poor survival and reduced size.

Methods

  • Generated a miR-33−/− mouse by CRISPR/Cas9 excision of roughly 200 bp of the Srebp-2 intronic region encoding miR-33, and a miR-33b knock-in mouse replacing mouse Srebp-1 with human sequence including miR-33b.
  • Used Ldlr−/− atherosclerosis model with Western diet (1.25% cholesterol) for 12 weeks, bone marrow transplants, metabolic tests, plaque histology, FACS, lipidomics, and in vivo reverse cholesterol transport using [3H]-cholesterol-loaded bone marrow-derived macrophages injected into WT mice.
  • RNA-seq was performed on an Illumina HiSeq 2000 (1 × 75 bp), aligned with TopHat2, analyzed with cuffdiff and pathway tools; data deposited in GEO GSE103133. Statistics used unpaired two-sided t-test or one-way ANOVA with Bonferroni correction, Kolmogorov-Smirnov normality check, Mann-Whitney when non-normal, and P<0.05.

Limitations

  • The paper states that whole-body metabolic changes in DKO mice—increased body weight, insulin resistance, and dyslipidemia—preclude direct examination of how miR-33 regulation of HDL biogenesis and bile acid metabolism contributes to atherosclerosis.
  • The paper states that global assessment of miR-33b addition was not possible because homozygous miR-33bKI mice had sub-Mendelian birth rates, poor survival, and reduced size; adult inducible models are needed.
  • The paper notes that in vitro macrophage polarization and apoptosis experiments may not properly reflect conditions within atherosclerotic plaques, and specific miR-33 targets mediating many effects require further work.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.celrep.2017.10.023

Cites

41 of 41 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

30 of 46 cited sources checked · 17 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Some studies of long-term anti-miR-33 treatment have reported deleterious effects including increased circulating levels of triglycerides and the development of hepatic steatosis.

    “Additionally, some studies of long-term anti-miR-33 treatment have reported deleterious effects including increased circulating levels of triglycerides (TAGs) and the development of hepatic steatosis (Allen et al., 2014; Goedeke et al., 2014).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Allen et al., 2014 source

      The Results section reports that 11 weeks of anti-miR-33 treatment raised circulating VLDL-TAG in mice. It also reports no increase in hepatic TAG. The Discussion mentions hepatic steatosis only in a different study of genetically miR-33-deficient mice fed a high-fat diet, not as an effect of long-term anti-miR-33 treatment. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • cites Goedeke et al., 2014 source
  • Premisecitation

    Antagonism of miR-33 can also increase circulating levels of HDL-C in non-human primates.

    “Importantly, studies performed in monkeys have demonstrated that antagonism of miR-33 can also increase circulating levels of HDL-C in non-human primates (Rayner et al., 2011a; Rottiers et al., 2013).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Rayner et al., 2011a

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Rottiers et al., 2013 source
  • Premisecitation

    Antagonism of miR-33 in mouse models of atherosclerosis can reduce plaque burden by either impeding the progression or promoting the regression of atherosclerotic plaques.

    “Numerous reports have demonstrated that antagonism of miR-33 in mouse models of atherosclerosis can reduce plaque burden by either impeding the progression or promoting the regression of atherosclerotic plaques (Rayner et al., 2011b; Rotllan et al., 2013).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Rayner et al., 2011b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Rotllan et al., 2013 source
  • Premisecitation

    miR-33a and miR-33b help prevent further loss of lipids by targeting genes involved in cholesterol trafficking and efflux and fatty acid β-oxidation.

    “miR-33a and miR-33b help prevent further loss of lipids by targeting genes involved in cholesterol trafficking and efflux”
    • Also stated in: § INTRODUCTION · plain
      “and fatty acid β-oxidation [carnitine palmitoyltransferase 1A (CPT1A), carnitine O-octanoyltransferase (CROT), and hydroxyacyl-CoA dehydrogenase/3-ketoacy-CoA thiolase/enoyl-CoA hydratase β subunit (HADHB) (Davalos et al., 2011; Gerin et al., 2010)].”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Marquart et al., 2010 source
    • cites Davalos et al., 2011 source
    • cites Gerin et al., 2010 source

      The Discussion says miR-33 suppresses ABCA1 to prevent cholesterol from leaving cells, and the results show that miR-33a reduces cholesterol export and targets genes involved in fatty acid β-oxidation. The paper also identifies a predicted cholesterol-distribution target, NPC1. However, it says the experiments focused on miR-33a; shared predicted targets and a mixed miR-33a/miR-33b antagomir experiment do not establish the full effect for miR-33b separately. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    Other similar studies did not observe any beneficial effects of anti-miR-33 therapy on atherogenesis.

    “However, other similar studies did not observe any beneficial effects of anti-miR-33 therapy on atherogenesis (Marquart et al., 2013).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Marquart et al., 2013 source
  • Classified as probably not a substantial claim. 10
  • May combine two claims. 26

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Most of the beneficial effects of miR-33 on atherosclerosis have been attributed to its ability to increase circulating levels of HDL-C and/or promote macrophage cholesterol efflux.

    “Most of the beneficial effects of miR-33 on atherosclerosis have been attributed to its ability to increase circulating levels of HDL-C and/or promote Mθ cholesterol efflux (Rayner et al., 2011b; Rayner et al., 2010)”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Rayner et al., 2011b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Rayner et al., 2010 source

      The paper reports the opposite direction of effect: miR-33 reduces macrophage cholesterol efflux and circulating HDL, while antagonizing miR-33 increases both. It describes these pathways as relevant to atherosclerosis but does not establish beneficial effects of miR-33 on atherosclerosis. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    In vivo antagonism or genetic deletion of miR-33 enhances hepatic ABCA1 expression and increases circulating HDL-cholesterol.

    “in vivo antagonism or genetic deletion of miR-33 enhances hepatic ABCA1 expression and increases circulating HDL-cholesterol (HDL-C) (Marquart et al., 2010; Najafi-Shoushtari et al., 2010; Rayner et al., 2010).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Marquart et al., 2010 source
    • cites Najafi-Shoushtari et al., 2010 source

      The Results report that in vivo miR-33a antisense treatment significantly increased plasma HDL-cholesterol in mice. They describe only modest effects on hepatic ABCA1 mRNA and protein, rather than clearly establishing the claimed enhancement, and do not report genetic deletion of miR-33. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Rayner et al., 2010 source

      The in vivo mouse passage (indexed under Abstract; Fig. 4D–F, page unavailable) reports that anti-miR-33 increased hepatic ABCA1 protein by 50% and plasma HDL by 25% after six days. It describes lentiviral antagonism, not genetic deletion of miR-33, so the deletion part is not supported by this source. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Bone marrow transplant from miR-33 deficient animals into Apoe−/− mice reduces lipid content in atherosclerotic plaques but does not have a significant impact on plaque size.

    “Additionally, bone marrow transplant from miR-33 deficient animals into Apoe−/− mice, to selectively remove miR-33 from Mθs and other hematopoietic cells, was found to reduce lipid content in atherosclerotic plaques but did not have a significant impact on plaque size (Horie et al., 2012).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Horie et al., 2012 source

  • Classified as probably not a substantial claim.

    Premisecitation

    miR-33a and miR-33b are intronic miRNAs encoded within the SREBP-2 and SREBP-1 genes respectively.

    “miR-33a and miR-33b are intronic miRNAs encoded within the SREBP-2 and SREBP-1 genes respectively (Marquart et al., 2010; Najafi-Shoushtari et al., 2010; Rayner et al., 2010).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Marquart et al., 2010 source
    • cites Najafi-Shoushtari et al., 2010 source
    • cites Rayner et al., 2010 source

      The passage labeled Abstract (page not provided) states that hsa-miR-33a is encoded within intron 16 of SREBF2, supporting the miR-33a/SREBP-2 part of the claim. The passages from our copy do not establish that miR-33b is encoded within SREBP-1. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Whole body loss of miR-33 reduces atherosclerotic plaque size and lipid content in Apoe−/− mice.

    “By crossing these animals into the Apoe−/− mouse model of atherosclerosis they were able to demonstrate that whole body loss of miR-33 reduces atherosclerotic plaque size and lipid content (Horie et al., 2012).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Horie et al., 2012 source

  • May combine two claims.

    Premisecitation

    RCT is the only mechanism by which arterial macrophages can remove excess cholesterol, thereby reducing the formation of atherosclerotic plaques by limiting the accumulation of lipid-laden macrophages within the arterial wall.

    “RCT is the only mechanism by which arterial macrophages (Mθs) can remove excess cholesterol, thereby reducing the formation of atherosclerotic plaques by limiting the accumulation of lipid-laden Mθs within the arterial wall (Rosenson et al., 2012).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Rosenson et al., 2012 source

      The Cellular Cholesterol Efflux section (page unavailable) places removal of excess cholesterol from plaque macrophages within the concept of RCT and describes several efflux pathways. The Experimental Animal Models of RCT section links greater macrophage RCT with atheroprotection. Neither section establishes that RCT is the only mechanism by which arterial macrophages can remove excess cholesterol. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    miR-33a and miR-33b are co-transcribed along with SREBP-2 and SREBP-1 and work in concert with their host genes to help maintain proper lipid levels within the cell.

    “This work led to the demonstration that miR-33a and miR-33b are co-transcribed along with SREBP-2 and SREBP-1, and work in concert with their host genes to help maintain proper lipid levels within the cell (Davalos et al., 2011; Marquart et al., 2010; Najafi-Shoushtari et al., 2010; Rayner et al., 2010).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Davalos et al., 2011 source
    • cites Marquart et al., 2010 source
    • cites Najafi-Shoushtari et al., 2010 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Loss of miR-33 results in a predisposition to the development of obesity and insulin resistance.

    “Additionally, a later report by this same group has demonstrated that loss of miR-33 results in a predisposition to the development of obesity and insulin resistance (Horie et al., 2013)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Horie et al., 2013 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Another group reported similar changes in efferocytosis upon inhibition of miR-33, attributed to regulation of autophagy by miR-33.

    “another group has very recently reported similar changes in efferocytosis upon inhibition of miR-33, which were attributed to regulation of autophagy by miR-33 (Ouimet et al., 2017).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Ouimet et al., 2017 source

  • May combine two claims.

    Premisecitation

    These effects are contrary to what was previously reported in Apoe−/− mice lacking miR-33, where they did find reduced plaque size, did not observe any difference in circulating VLDL-C, LDL-C, or TAGs, and did not provide any data on BW or insulin sensitivity.

    “These effects are contrary to what was previously reported in Apoe−/−mice lacking miR-33, where they did find reduced plaque size, did not observe any difference in circulating VLDL-C, LDL-C, or TAGs, and did not provide any data on BW or insulin sensitivity (Horie et al., 2012).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Horie et al., 2012 source

  • May combine two claims.

    Interpretationcitation

    Differences in miRNA expression vary between cell types, suggesting that the role of miR-33b in the liver and other tissues may be considerably more important.

    “These differences in miRNA expression vary between cell types suggesting that the role of miR-33b in the liver, where miR-33 is most highly expressed both in our mouse model and in human tissues (Najafi-Shoushtari et al., 2010), and other tissues may be considerably more important.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Najafi-Shoushtari et al., 2010 source

      The Results (page unavailable) report miR-33a/b expression across human and mouse tissues, but do not state that expression is highest in human liver—the specific point to which this citation is attached. The citing authors’ mouse-model observation and inference about miR-33b’s importance are their own, rather than findings attributed to this source. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • Rests on an assumption: Humans contain a second miR-33 isoform, miR-33b.
    • Rests on an assumption: The miR-33bKI mouse model is a valid model for human miR-33b function.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A previously described miR-33b knockin model was reported to have decreased expression of ABCA1 and other miR-33 target genes in primary hepatocytes.

    “a previously described miR-33b knockin model was reported to have decreased expression of ABCA1 and other miR-33 target genes in primary hepatocytes (Horie et al., 2014).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Horie et al., 2014 source

      The Results section on miR-33b KI target proteins (page unavailable) reports lower ABCA1 and SREBP-1 protein levels in the livers of knock-in mice. The separate Results section on primary hepatocytes reports increased Srebf1 and miR-33b after stimulation, not decreased ABCA1 or other target-gene expression in those cells. The claimed primary-hepatocyte finding is therefore not supported. (LLM, conf=0.89)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Phosphatidylserine and phosphatidylethanolamine are known to play important roles in macrophage mobility, phagocytosis, and apoptosis.

    “these lipid subspecies are known to play important roles in Mθ mobility, phagocytosis, and apoptosis (Greenberg et al., 2006; Marino and Kroemer, 2013; Shiratsuchi et al., 1998).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Greenberg et al., 2006 source

      The Abstract reports that oxidized phosphatidylserine helps macrophages recognize and engulf apoptotic cells, supporting the phagocytosis and apoptosis-related portion of the claim. The cited passages do not establish a role for phosphatidylethanolamine or for either lipid in macrophage mobility; they also distinguish oxidized from nonoxidized phosphatidylserine. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Marino and Kroemer, 2013 source
    • cites Shiratsuchi et al., 1998 source

  • May combine two claims.

    Findingown data

    The number of apoptotic cells and the size of necrotic cores are reduced in plaques from mice transplanted with miR-33−/− bone marrow.

    “We further demonstrate that both the number of apoptotic cells and the size of the necrotic cores are reduced in plaques from mice transplanted with miR-33−/− bone marrow indicative of increased plaque stability.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Whole body loss of miR-33 in Ldlr−/− mice results in increased body weight, impaired insulin sensitivity, and a pro-atherogenic lipid profile.

    “Alternatively, whole body loss of miR-33 in Ldlr−/− mice was found to result in increased body weight (BW), impaired insulin sensitivity, and a pro-atherogenic lipid profile.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The Ldlr−/− mouse model of atherosclerosis has HDL-C levels and RCT capacity similar to wild-type mice.

  • May combine two claims.

    Findingown data

    miR-33 deficiency in Ldlr−/− mice promotes obesity, insulin-resistance and hyperlipidemia.

    “We demonstrate that miR-33 deficiency in Ldlr−/− mice promotes obesity, insulin-resistance and hyperlipidemia”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Loss of miR-33 improves reverse cholesterol transport from macrophages in vivo, resulting in a significant increase in radioactivity counts in both plasma and feces.

    “By quantifying the amount of [H3]-Cholesterol incorporated into the blood and feces of these animals over 48h, we are able to demonstrate that loss of miR-33 improves RCT from Mθs in vivo, resulting in a significant increase in radioactivity counts in both the plasma and feces (Figure 4C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The effects observed in this study are considerably more robust than previous work, with significant reductions also observed in total plaque size, necrotic core area, and number of apoptotic cells.

    “However, the effects observed in our study are considerably more robust, with significant reductions also observed in total plaque size, necrotic core area, and the number of apoptotic cells.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mouse models of atherosclerosis and bone marrow transplantation are informative for human atherosclerosis and the therapeutic potential of miR-33 inhibition.
    • Rests on an assumption: Bone marrow transplantation from miR-33−/− donors selectively affects hematopoietic cells without confounding effects on other tissues.

  • May combine two claims.

    Findingown data

    This predisposition to diet-induced obesity is accompanied by decreased insulin sensitivity and increased circulating levels of VLDL-C, LDL-C, and TAGs.

    “This predisposition to diet induced obesity is also accompanied by decreased insulin sensitivity and increased circulating levels of VLDL-C and LDL-C and TAGs.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mouse models of atherosclerosis and bone marrow transplantation are informative for human atherosclerosis and the therapeutic potential of miR-33 inhibition.

  • May combine two claims.

    Findingown data

    Neither the specific autophagic genes reported to be targeted by miR-33 nor most other genes related to autophagic function were significantly altered in our RNA-seq analysis.

    “neither the specific autophagic genes reported to be targeted by miR-33, nor most other genes related to autophagic function were found to be significantly altered in our RNA-seq analysis.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RNA-seq gene expression changes in macrophages reflect functional changes relevant to macrophage biology and atherosclerotic plaque formation.
    • Rests on an assumption: The observed gene expression changes are due to loss of miR-33 rather than secondary effects or other differences.

  • May combine two claims.

    Findingreasoning

    Macrophage miR-33b was not found to have a major impact on atherosclerosis, plaque formation, or lipid accumulation.

    “Alternatively, Mθ miR-33b was not found to have a major impact on atherosclerosis”
    • Also stated in: § DISCUSSION · hedged
      “Addition of miR-33b to Mθs does not appear to have any impact on plaque formation or lipid accumulation.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Humans contain a second miR-33 isoform, miR-33b.
    • Rests on an assumption: The miR-33bKI mouse model is a valid model for human miR-33b function.

  • May combine two claims.

    Findingown data

    Loss of miR-33 in Ldlr−/− mice results in a significant increase in body weight, exacerbated by Western diet feeding.

    “we find that loss of miR-33 in Ldlr−/− mice results in a significant increase in BW, which is exacerbated by feeding of a WD.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mouse models of atherosclerosis and bone marrow transplantation are informative for human atherosclerosis and the therapeutic potential of miR-33 inhibition.

  • May combine two claims.

    Interpretationown data

    The pro-atherogenic effects observed in miR-33 deficient mice are likely counterbalanced by protective effects in macrophages, which may be the primary mechanism through which anti-miR-33 therapies reduce atherosclerosis.

    “Therefore, the pro-atherogenic effects observed in miR-33 deficient mice are likely counterbalanced by protective effects in macrophages, which may be the primary mechanism through which anti-miR-33 therapies reduce atherosclerosis.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The Ldlr−/− mouse model of atherosclerosis has HDL-C levels and RCT capacity similar to wild-type mice.

  • May combine two claims.

    Interpretationreasoning

    These changes likely offset the beneficial effects occurring in plaque macrophages, as there were no differences in plaque size or lipid accumulation in Ldlr−/− mice lacking miR-33.

    “Together these changes likely offset the beneficial effects occurring in plaque Mθs, as there were no differences in plaque size or lipid accumulation in Ldlr−/− mice lacking miR-33.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mouse models of atherosclerosis and bone marrow transplantation are informative for human atherosclerosis and the therapeutic potential of miR-33 inhibition.

  • May combine two claims.

    Interpretationreasoning

    These same pathways were largely repressed in Ldlr−/− mice compared to WT, suggesting that reduced lipid accumulation in DKO animals is likely the primary factor responsible for altered expression of these genes.

    “These same pathways were largely repressed in Ldlr−/− mice compared to WT, suggesting that the reduced lipid accumulation in DKO animals is likely the primary factor responsible for the altered expression of these genes.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RNA-seq gene expression changes in macrophages reflect functional changes relevant to macrophage biology and atherosclerotic plaque formation.
    • Rests on an assumption: The observed gene expression changes are due to loss of miR-33 rather than secondary effects or other differences.

  • May combine two claims.

    Interpretationreasoning

    These changes would be predicted to result in diminished inflammatory signaling through NFκB and TLR4 mediated pathways, resulting in reduced macrophage activation and polarization in vivo.

    “Indeed, these changes would be predicted to result in diminished inflammatory signaling through the NFκB and TLR4 mediated pathways resulting in a reduction in Mθ activation and polarization in vivo (Figure 6A and S7).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RNA-seq gene expression changes in macrophages reflect functional changes relevant to macrophage biology and atherosclerotic plaque formation.
    • Rests on an assumption: The observed gene expression changes are due to loss of miR-33 rather than secondary effects or other differences.

  • May combine two claims.

    Interpretationreasoning

    Because effects are independent of changes in circulating HDL-C levels, anti-miR-33 therapies have potential therapeutic value for promoting removal of lipids from atherosclerotic plaques and limiting plaque development.

    “Because these effects are independent of any changes in circulating HDL-C levels, these findings demonstrate the potential therapeutic value of anti-miR-33 therapies for promoting the removal of lipids from atherosclerotic plaques and thereby limiting plaque development.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mouse models of atherosclerosis and bone marrow transplantation are informative for human atherosclerosis and the therapeutic potential of miR-33 inhibition.

  • May combine two claims.

    Interpretationreasoning

    Efficient clearance of apoptotic cells limits prolonged inflammation and may contribute to the reduced recruitment and activation of macrophages lacking miR-33.

    “These findings are important, as efficient clearance of apoptotic cells limits prolonged inflammation and may contribute to the reduced recruitment and activation of Mθs lacking miR-33.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Efficient clearance of apoptotic cells limits prolonged inflammation.

  • May combine two claims.

    Interpretationown data

    A large proportion of genes dysregulated in response to loss of miR-33 were not altered in response to dyslipidemia, suggesting they could be directly or indirectly regulated by miR-33 independent of its effects on lipid content.

    “however a large proportion of the genes dysregulated in response to loss of miR-33 were not found to be altered in response to dyslipidemia, suggesting that these could be either directly or indirectly regulated by miR-33 independent of its effects on lipid content.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RNA-seq gene expression changes in macrophages reflect functional changes relevant to macrophage biology and atherosclerotic plaque formation.
    • Rests on an assumption: The observed gene expression changes are due to loss of miR-33 rather than secondary effects or other differences.

  • May combine two claims.

    Interpretationreasoning

    Pathways related to stress response are downregulated in both comparisons, indicating that these changes are likely regulated by factors independent of lipid content.

    “while pathways related to stress response are downregulated in both comparisons, indicating that these changes are likely regulated by factors independent of lipid content.”
    • Also stated in: § DISCUSSION · hedged
      “Therefore, the alterations in these stress response pathways is likely independent of the overall changes in lipid content.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: RNA-seq gene expression changes in macrophages reflect functional changes relevant to macrophage biology and atherosclerotic plaque formation.
    • Rests on an assumption: The observed gene expression changes are due to loss of miR-33 rather than secondary effects or other differences.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Lai L and colleagues reported that absence of miR-33 in macrophages alters membrane microdomains, influencing innate immune response.

    “Lai L and colleagues have recently reported that absence of miR-33 in Mθs alters membrane microdomains, influencing the innate immune response (Lai et al., 2016).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lai et al., 2016 source

  • May combine two claims.

    Interpretationown data

    This work supports promising therapeutic potential of miR-33 inhibitors for directly promoting efflux of cholesterol from arterial macrophages, thereby limiting macrophage lipid accumulation and preventing further macrophage recruitment and activation.

    “Overall, this work supports the promising therapeutic potential of miR-33 inhibitors for directly promoting the efflux of cholesterol from arterial Mθs, thereby limiting Mθ lipid accumulation and preventing further Mθ recruitment and activation.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Mouse models of atherosclerosis and bone marrow transplantation are informative for human atherosclerosis and the therapeutic potential of miR-33 inhibition.

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Bone marrow transplant experiments, plaque characterization and measurements of metabolic function, circulating and cellular lipids and reverse cholesterol transport were carried out using established techniques.

    “Bone marrow transplant experiments, plaque characterization and measurements of metabolic function, circulating and cellular lipids and reverse cholesterol transport were carried out using established techniques.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Animal studies were approved by the Institutional Animal Care and Use committee of Yale University School of Medicine.

    “Animal studies were approved by the Institutional Animal Care and Use committee of Yale University School of Medicine.”
    Quote in the paperNot a substantial claim
34 of 34

  • The paper relies on this without support.

    The intronic region of the SREBP-1 gene encoding miR-33b is disrupted in rodents and other small mammals.

    “These findings are especially relevant, as the intronic region of the SREBP-1 gene encoding miR-33b is disrupted in rodents and other small mammals.”

  • The paper relies on this without support.

    The Ldlr−/− mouse model of atherosclerosis has HDL-C levels and RCT capacity similar to wild-type mice.

    “in which HDL-C levels and RCT capacity are similar to wild-type (WT) mice”

  • The paper relies on this without support.

    Efficient clearance of apoptotic cells limits prolonged inflammation.

    “efficient clearance of apoptotic cells limits prolonged inflammation”

  • The paper relies on this without support.

    Humans contain a second miR-33 isoform, miR-33b.

    “humans contain a second miR-33 isoform, miR-33b, which may also play an important role in atherosclerosis.”

  • The paper relies on this without support.

    Mouse models of atherosclerosis and bone marrow transplantation are informative for human atherosclerosis and the therapeutic potential of miR-33 inhibition.

    “Overall, this work supports the promising therapeutic potential of miR-33 inhibitors for directly promoting the efflux of cholesterol from arterial Mθs, thereby limiting Mθ lipid accumulation and preventing further Mθ recruitment and activation.”

  • The paper relies on this without support.

    RNA-seq gene expression changes in macrophages reflect functional changes relevant to macrophage biology and atherosclerotic plaque formation.

    “RNA-seq analysis has revealed a number of pathways and specific genes that may be involved in mediating the effects of miR-33 on Mθ function and atherosclerotic plaque formation.”

  • The paper relies on this without support.

    Bone marrow transplantation from miR-33−/− donors selectively affects hematopoietic cells without confounding effects on other tissues.

    “Overall this work demonstrates that loss of miR-33 selectively in hematopoietic cells is sufficient to reduce Mθ recruitment and lipid accumulation in atherosclerotic plaques.”

  • The paper relies on this without support.

    The miR-33bKI mouse model is a valid model for human miR-33b function.

    “In this work, we begin to assess the impact of miR-33 on atherogenesis by performing bone marrow transplants from miR-33bKI mice into the Ldlr−/− background.”

  • The paper relies on this without support.

    The observed gene expression changes are due to loss of miR-33 rather than secondary effects or other differences.

    “Our RNA-seq analysis in Mθs has revealed a number of genes that are differentially up and down regulated in response to loss of miR-33.”
9 of 9

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • MERTK expression is described as both increased and reduced

    Minor

    The Results state that MERTK is upregulated in miR-33-deficient macrophages, while the Discussion states that its protein levels are reduced in those cells.

1 of 1

Cites

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Authors
Price, N. L., Rotllan, N., Canfrán-Duque, A., Zhang, X., Pati, P., Arias, N., Moen, J., Mayr, M., Ford, D. A., Baldán, Á., Suárez, Y., & Fernández-Hernando, C.
Affiliations
  • Yale University (US)
  • Saint Louis University (US)
  • King's College London (GB)
Venue
Cell reports
Publisher
Elsevier BV
Published
2017-10
Volume
21
Issue
5
Pages
1317-1330
ISSN
2211-1247 (ISSN-L)
DOI
10.1016/j.celrep.2017.10.023
PMC
PMC5687841
OpenAlex
W2765749432
Semantic Scholar
d67a63bedaa9799f93d9875cb14bf863fb2855f2
Cited by
129
References
41
Citations by year
2026: 9 · 2025: 12 · 2024: 11 · 2023: 16 · 2022: 10 · 2021: 18 · 2020: 17 · 2019: 20 · 2018: 15 · 2017: 1
FWCI
13.38
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Research Portal (King's College London) (submittedVersion, cc-by-nc-nd)
Funders
  • NIH (R35HL135820, R01HL105945, R01HL135012, R01HL107794, F32DK10348902)
  • American Heart Association (16EIA27550005, 16GRNT26420047, 17SDG33110002)
  • American Diabetes Association (1-16-PMF-002)
  • Foundation Leducq Transatlantic Network of Excellence in Cardiovascular Research, MIRVAD
Topics
  • Health Sciences › Medicine › Surgery › Cholesterol and Lipid Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Life Sciences › Immunology and Microbiology › Immunology › Atherosclerosis and Cardiovascular Diseases
Keywords
miR-33a, miR-33b, atherosclerosis, atherogenesis, macrophage cholesterol efflux, HDL biogenesis, Ldlr-deficient mice, obesity, insulin resistance, hyperlipidemia, hepatic lipid metabolism, atherosclerotic plaque, macrophage lipid accumulation, inflammation
MeSH terms
Animals, Aorta, Blood Glucose, Cells, Cultured, Cholesterol, Cholesterol, HDL, Male, Mice, Inbred C57BL, Myocardium, Receptors, LDL, Macrophages, Peritoneal, Mice, Knockout, Disease Progression, MicroRNAs, Atherosclerosis, Mice, Gene Regulatory Networks, Mitochondrial Trifunctional Protein, beta Subunit, ATP Binding Cassette Transporter 1

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-20.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-20.

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