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(a). Western blotting for ABCA1 and ABCG1 proteins in peritoneal macrophages from WT and KI+/+ mice. Representative images are shown. β-actin was used as the loading control. (b). Cholesterol efflux to apoA-I and HDL-C in peritoneal macrophages from WT and KI+/+ mice (n = 6 each). Values are mean ± s.e.m. ***p < 0.001 (c). Mean plots of HPLC analysis for serum cholesterol in WT and KI+/+ mice (n = 4 and 5, respectively).
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MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports, including ours, indicated that miR-33a located within the intron of sterol regulatory element-binding protein (SREBP) 2 controls cholesterol homeostasis and can be a possible therapeutic target for trea...
Citations
... Although these findings are promising, their translational application is compromised given that mice only express the miR-33a isoform, whereas humans express both miR-33a and miR-33b. A miR-33b knock-in (KI) mouse model has therefore been generated, with the aim of analysing the impact of miR-33b on lipid metabolism; these genetically modified mice had decreased ABCA1 protein levels in the liver and peritoneal macrophages, and lower concentrations of plasma HDL cholesterol [46]. These observations have been strengthened by the use of an anti-miR oligonucleotide therapy to suppress miR-33a/b expression in African green monkeys, a model that circumvents the miR-33b deficiency found in mice. ...
MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally inhibit gene expression. These small molecules are involved in several biological conditions such as inflammation, cell growth and proliferation, and regulation of energy metabolism. In the context of metabolic and cardiovascular diseases, miR-33 is of particular interest as it has been implicated in the regulation of lipid and glucose metabolism. This miRNA is located in introns harboured in the genes encoding sterol regulatory element-binding protein (SREBP)-1 and SREBP-2, which are key transcription factors involved in lipid biosynthesis and cholesterol efflux. This review outlines the role of miR-33 in a range of metabolic and cardiovascular pathologies, such as dyslipidaemia, nonalcoholic fatty liver disease (NAFLD), obesity, diabetes, atherosclerosis, and abdominal aortic aneurysm (AAA), and it provides discussion about the effectiveness of miR-33 deficiency as a possible therapeutic strategy to prevent the development of these diseases.
... To clarify the role of miR-33b in vivo, we conducted experiments using mice with miR-33b knock-in (KI) in the intron of Srebf1 (miR-33b KI mice), similar to the condition in humans (Horie et al, 2014). These humanized miR-33b KI mice express miR-33b along with its host gene Srebf1 at the physiological level. ...
... There was no change in the body weight or food intake of the mice (Fig S1A and B), nor any change in their liver/body weight ratio ( Fig S1C). Blood sample analysis revealed a significant reduction in total cholesterol (T-Cho) and HDL-C levels in the blood of miR-33b KI mice, as reported previously (Horie et al, 2014). Low-density lipoprotein cholesterol (LDL-C) levels were also reduced in these blood samples. ...
... miR-33a is an intronic microRNA located within the SREBF2, a master regulator of cholesterol (Horie et al, 2010a). In contrast, miR-33b is present in large mammals, including humans, within an intron of SREBF1, a triglyceride regulator, but is absent in rodents (Horie et al, 2014). We previously generated miR-33b KI mice (a human model) carrying both miR-33a and miR-33b and showed that miR-33a/b, like the host genes, are important regulators of lipid metabolism (Horie et al, 2014). ...
Nonalcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma in their advanced stages; however, there are currently no approved therapies. Here, we show that microRNA (miR)-33b in hepatocytes is critical for the development of NASH. miR-33b is located in the intron of sterol regulatory element-binding transcription factor 1 and is abundantly expressed in humans, but absent in rodents. miR-33b knock-in (KI) mice, which have a miR-33b sequence in the same intron of sterol regulatory element-binding transcription factor 1 as humans and express miR-33b similar to humans, exhibit NASH under high-fat diet feeding. This condition is ameliorated by hepatocyte-specific miR-33b deficiency but unaffected by macrophage-specific miR-33b deficiency. Anti-miR-33b oligonucleotide improves the phenotype of NASH in miR-33b KI mice fed a Gubra Amylin NASH diet, which induces miR-33b and worsens NASH more than a high-fat diet. Anti-miR-33b treatment reduces hepatic free cholesterol and triglyceride accumulation through up-regulation of the lipid metabolism-related target genes. Furthermore, it decreases the expression of fibrosis marker genes in cultured hepatic stellate cells. Thus, inhibition of miR-33b using nucleic acid medicine is a promising treatment for NASH.
... In the present study, we investigated the effects of miR-33a and miR-33b on AAA in order to clarify the pathogenesis in humans. We previously generated miR-33b KI mice (miR-33a +/+ miR-33b +/+ ), which have human miR-33b in intron 16 of Srebf1 14 . miR-33b KI mice have both miR-33a and miR-33b as in humans. ...
... 1. In experiments using genetically modified mice, WT mice with only miR-33a, KOKI mice with only miR-33b 15 , and mice with both miR-33a and miR-33b 14 were subjected to aneurysm formation using the CaCl 2 method 16 , and the mice deteriorated progressively in proportion to the total amount of miR-33. 2. We created 12-base AMOs (anti-miR-33a and anti-miR-33b) specific for miR-33a and miR-33b, respectively, which were able to inhibit miR-33a and miR-33b individually in in vitro experiments. ...
... Animals. miR-33b knock-in (KI) mice were generated as described previously 14 . miR-33a knockout miR-33b knock-in mice (KOKI) were described previously 15 . ...
Abdominal aortic aneurysm (AAA) is a lethal disease, but no beneficial therapeutic agents have been established to date. Previously, we found that AAA formation is suppressed in microRNA (miR)-33-deficient mice compared with wild-type mice. Mice have only one miR-33, but humans have two miR-33 s, miR-33a and miR-33b. The data so far strongly support that inhibiting miR-33a or miR-33b will be a new strategy to treat AAA. We produced two specific anti-microRNA oligonucleotides (AMOs) that may inhibit miR-33a and miR-33b, respectively. In vitro studies showed that the AMO against miR-33b was more effective; therefore, we examined the in vivo effects of this AMO in a calcium chloride (CaCl2)-induced AAA model in humanized miR-33b knock-in mice. In this model, AAA was clearly improved by application of anti-miR-33b. To further elucidate the mechanism, we evaluated AAA 1 week after CaCl2 administration to examine the effect of anti-miR-33b. Histological examination revealed that the number of MMP-9-positive macrophages and the level of MCP-1 in the aorta of mice treated with anti-miR-33b was significantly reduced, and the serum lipid profile was improved compared with mice treated with control oligonucleotides. These results support that inhibition of miR-33b is effective in the treatment for AAA.
... However, miR-33a-5P suppresses the expression of ABCG1 in THP-1 macrophages [218]. Moreover, several reports identified miR-33b as a suppressor of ABCG1 expression [210,221,274,275]. Thus, miR-33b reduces the expression of ABCG1 and cholesterol efflux while no general opinion seems to exist on miR-33a influence on ABCG1 expression in humans. ...
Atheroprotective properties of human plasma high-density lipoproteins (HDLs) are determined by their involvement in reverse cholesterol transport (RCT) from the macrophage to the liver. ABCA1, ABCG1, and SR-BI cholesterol transporters are involved in cholesterol efflux from macrophages to lipid-free ApoA-I and HDL as a first RCT step. Molecular determinants of RCT efficiency that may possess diagnostic and therapeutic meaning remain largely unknown. This review summarizes the progress in studying the genomic variants of ABCA1, ABCG1, and SCARB1, and the regulation of their function at transcriptional and post-transcriptional levels in atherosclerosis. Defects in the structure and function of ABCA1, ABCG1, and SR-BI are caused by changes in the gene sequence, such as single nucleotide polymorphism or various mutations. In the transcription initiation of transporter genes, in addition to transcription factors, long noncoding RNA (lncRNA), transcription activators, and repressors are also involved. Furthermore, transcription is substantially influenced by the methylation of gene promoter regions. Post-transcriptional regulation involves microRNAs and lncRNAs, including circular RNAs. The potential biomarkers and targets for atheroprotection, based on molecular mechanisms of expression regulation for three transporter genes, are also discussed in this review.
... Remarkably, the study by Price et al. was performed in mice, where only the miR-33a isoform is expressed; thus, in humans, the contribution of miR-33b isoform in the liver could still be important in atherosclerosis, as long as its regulation is subjected to SREBP1, which is not downregulated under hyperlipidemia conditions. Thus, given the shared targets between miR-33a/b isoforms, and the ability of miR-33b to regulate HDL levels [79], it is possible that hepatic miR-33 in humans could to regulate lipid and lipoprotein metabolism impacting hyperlipidemia and atherosclerosis. In fact, studies using human miR-33b isoform knock-in mice, which regulates cholesterol metabolism in mice [79] and promotes atherosclerosis [80], have shown miR-33b expression is increased in response to high-cholesterol diet feeding [74]. ...
... Thus, given the shared targets between miR-33a/b isoforms, and the ability of miR-33b to regulate HDL levels [79], it is possible that hepatic miR-33 in humans could to regulate lipid and lipoprotein metabolism impacting hyperlipidemia and atherosclerosis. In fact, studies using human miR-33b isoform knock-in mice, which regulates cholesterol metabolism in mice [79] and promotes atherosclerosis [80], have shown miR-33b expression is increased in response to high-cholesterol diet feeding [74]. Moreover, higher levels of miR-33b in the liver suggest this is the dominant miR-33 isoform expressed in the liver, especially under atherosclerosis conditions [74]. ...
Purpose of Review
Non-coding RNAs (ncRNAs) including microRNAs (miRNAs) and circular RNAs (circRNAs) are pivotal regulators of mRNA and protein expression that critically contribute to cardiovascular pathophysiology. Although little is known about the origin and function of such ncRNAs, they have been suggested as promising biomarkers with powerful therapeutic value in cardiovascular disease (CVD). In this review, we summarize the most recent findings on ncRNAs biology and their implication on cholesterol homeostasis and lipoprotein metabolism that highlight novel therapeutic avenues for treating dyslipidemia and atherosclerosis.
Recent Findings
Clinical and experimental studies have elucidated the underlying effects that specific miRNAs impose both directly and indirectly regulating circulating high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) metabolism and cardiovascular risk. Some of these relevant miRNAs include miR-148a, miR-128-1, miR-483, miR-520d, miR-224, miR-30c, miR-122, miR-33, miR-144, and miR-34. circRNAs are known to participate in a variety of physiological and pathological processes due to their abundance in tissues and their stage-specific expression activation. Recent studies have proven that circRNAs may be considered targets of CVD as well. Some of these cirRNAs are circ-0092317, circ_0003546, circ_0028198, and cirFASN that have been suggested to be strongly involved in lipoprotein metabolism; however, their relevance in CVD is still unknown.
Summary
MicroRNA and cirRNAs have been proposed as powerful therapeutic targets for treating cardiometabolic disorders including atherosclerosis. Here, we discuss the recent findings in the field of lipid and lipoprotein metabolism underscoring the novel mechanisms by which some of these ncRNAs influence lipoprotein metabolism and CVD.
... Together these studies demonstrate that miR-33 is an important regulator of multiple steps in the RCT pathway. Indeed, miR-33b knock-in mice (Horie et al, 2014) were shown to have lower HDL-C levels compared to wild-type controls, while antagonism of miR-33 results in increased hepatic ABCA1 expression and elevated HDL-C levels in mice and non-human primates ( Ouimet et al, 2016a), which can impact the availability of cholesterol for cellular efflux. While it remains to be determined whether miR-33-mediated silencing of OSBPL6 affects cholesterol efflux to HDL-C levels in vivo, OSBPL6 mRNA levels positively correlate with ApoA1 and HDL-C levels in humans and OSBPL6 levels are decreased in the carotid arteries of patients with atherosclerosis (Ouimet et al, 2016a). ...
miRNAs have emerged as critical regulators of nearly all biologic processes and important therapeutic targets for numerous diseases. However, despite the tremendous progress that has been made in this field, many misconceptions remain among much of the broader scientific community about the manner in which miRNAs function. In this review, we focus on miR-33, one of the most extensively studied miRNAs, as an example, to highlight many of the advances that have been made in the miRNA field and the hurdles that must be cleared to promote the development of miRNA-based therapies. We discuss how the generation of novel animal models and newly developed experimental techniques helped to elucidate the specialized roles of miR-33 within different tissues and begin to define the specific mechanisms by which miR-33 contributes to cardiometabolic diseases including obesity and atherosclerosis. This review will summarize what is known about miR-33 and highlight common obstacles in the miRNA field and then describe recent advances and approaches that have allowed researchers to provide a more complete picture of the specific functions of this miRNA.
... Although rodents have only one miR-33 in an intron of the Srebf2 gene, humans have another, miR-33b, in an intron of the SREBF1 gene. In studies with humanized miR-33b knock-in (miR-33b +/+ ) mice, miR-33b was found to accelerate atherosclerosis 16,17 . In addition, inhibition of miR-33 has been shown to improve dyslipidemia in non-human primates 18,19 . ...
... Consequently, miR-33a and miR-33b were most abundant in the brain compared with other organs, which was accompanied by the expression of their host genes, SREBF2 and SREBF1, respectively ( Supplementary Fig. 4a). We previously generated humanized miR-33b knock-in mice (miR-33b +/+ mice), in which the human miR-33b sequence is inserted within the same intron of Srebf1 as in human 16 . In these mice, miR-33b is physiologically co-expressed with its host gene, Srebf1. ...
... Rodents have only one miR-33 in an intron of the Srebf2 gene, while humans have another, miR-33b, in an intron of the SREBF1 gene. We previously generated miR-33b +/+ mice, which have the human miR-33b sequence in the same intron of Srebf1 as in humans 16 , and we found the acceleration of atherosclerosis in these mice 17 . In contrast to miR-33 −/− mice, these humanized mice exhibited reductions of Gabrb2 and Gabra4 in the hypothalamus, higher body temperature and metabolic rate, and higher sympathetic nerve activity even at room temperature. ...
Adaptive thermogenesis is essential for survival, and therefore is tightly regulated by a central neural circuit. Here, we show that microRNA (miR)-33 in the brain is indispensable for adaptive thermogenesis. Cold stress increases miR-33 levels in the hypothalamus and miR-33−/− mice are unable to maintain body temperature in cold environments due to reduced sympathetic nerve activity and impaired brown adipose tissue (BAT) thermogenesis. Analysis of miR-33f/f dopamine-β-hydroxylase (DBH)-Cre mice indicates the importance of miR-33 in Dbh-positive cells. Mechanistically, miR-33 deficiency upregulates gamma-aminobutyric acid (GABA)A receptor subunit genes such as Gabrb2 and Gabra4. Knock-down of these genes in Dbh-positive neurons rescues the impaired cold-induced thermogenesis in miR-33f/fDBH-Cre mice. Conversely, increased gene dosage of miR-33 in mice enhances thermogenesis. Thus, miR-33 in the brain contributes to maintenance of BAT thermogenesis and whole-body metabolism via enhanced sympathetic nerve tone through suppressing GABAergic inhibitory neurotransmission. This miR-33-mediated neural mechanism may serve as a physiological adaptive defense mechanism for several stresses including cold stress.
... Functionally, miR-33a and miR-33b decrease cholesterol efflux to ApoA1 (via ABCA1) and HDL (via ABCG1) in mice, but only ApoA1 in humans; this is matched by a regulation of HDL biogenesis in the mouse liver and RCT. [23][24][25][26][27]35,75 Given these data, it seems logical that miR-33 should potently regulate circulating HDL-C levels in vivo. For example, miR-33 loss-of-function mice, either with or without knockout of Ldlr or Apoe, have an increase in plasma HDL when fed chow or WD/HFD, while miR-33b KI mice have decreased plasma HDL-C levels. ...
... 122 Complementarily, miR-33b KI mice have reduced hepatic expression of Abca1 and Srebp1 as well as reduced cholesterol efflux by macrophages. 75 However, the work from Karunakaran et al. and Goedeke et al. does not validate Srebf1 as a miR-33 target, and they did not see a difference in weight gain in anti-miR-33-treated mice, a result that may be due to the different models used for miR-33 loss-of-function. 32,33 Additionally, while Price et al. recapitulated the metabolic dysfunction in miR-33 knockout mice, the authors speculate that this is likely due to SREBP-1-independent mechanisms. ...
MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. Since many microRNAs have multiple mRNA targets, they are uniquely positioned to regulate the expression of several molecules and pathways simultaneously. For example, the multiple stages of cholesterol metabolism are heavily influenced by microRNA activity. Understanding the scope of microRNAs that control this pathway is highly relevant to diseases of perturbed cholesterol metabolism, most notably cardiovascular disease (CVD). Atherosclerosis is a common cause of CVD that involves inflammation and the accumulation of cholesterol‐laden cells in the arterial wall. However, several different cell types participate in atherosclerosis, and perturbations in cholesterol homeostasis may have unique effects on the specialized functions of these various cell types. Therefore, our review discusses the current knowledge of microRNA‐mediated control of cholesterol homeostasis, followed by speculation as to how these microRNA–mRNA target interactions might have distinctive effects on different cell types that participate in atherosclerosis.
... Similarly, miR-33a-deficient mice demonstrated a 25-40% increase in HDL [18] and showed reduced atherosclerosis formation in an Apoe-deficient background [21]. On the other hand, miR-33b knock-in mice, in which miR-33b is inserted in the same intron as in humans, have levels of HDL-cholesterol that are reduced by almost 35%, in addition to severe atherosclerosis, when they are crossed with Apoe-deficient mice [22,23]. Moreover, miR-33a deficiency ameliorates aortic aneurysm both in Ca 2+ -and angiotensin II-induced mice models, which suggested that inhibition of miR-33 may be a novel therapeutic strategy for abdominal aortic aneurysm [24]. ...
Recently, advances in genomic technology such as RNA sequencing and genome‐wide profiling have enabled the identification of considerable numbers of non‐coding RNAs (ncRNAs). MicroRNAs have been studied for decades, leading to the identification of those with disease‐causing and/or protective effects in vascular disease. Although other ncRNAs such as long ncRNAs have not been fully described yet, recent studies have indicated their important functions in the development of vascular diseases. Here, we summarize the current understanding of the mechanisms and functions of ncRNAs, focusing on microRNAs, circular RNAs and long ncRNAs in vascular diseases.
... miR-33a and -33b (miR-33a/b) bind to miR response elements (MREs) within the 3= untranslated regions (UTR) of ABCA1 and suppress gene expression (7). miR-33b knock-in mice have reduced ABCA1 expression and a 35% reduction in HDL levels (8). Mice treated with anti-miR-33a show a 50% increase in HDL (9)(10)(11), while miR-33a knockout mice have increased ABCA1 expression and a concomitant 30 to 50% increase in HDL (12). ...
GWAS have linked IGF2BP2 SNPs with type 2 diabetes (T2D). Mice overexpressing mIGF2BP2 have elevated cholesterol levels when fed a diet that induces hepatic steatosis. These and other studies suggest an important role for IGF2BP2 in the initiation and progression of several metabolic disorders. The ATPase binding cassette protein, ABCA1, initiates nascent high-density apolipoprotein (HDL) biogenesis by transferring phospholipid and cholesterol to de-lipidated Apo-AI. Individuals with mutational ablation of ABCA1 have Tangier disease, which is characterized by a complete loss of HDL. microRNA-33a/b (miR-33a/b) bind to the 3′ UTR of ABCA1 and repress its post-transcriptional gene expression. Here, we show that IGF2BP2 works together with miR-33a/b in repressing ABCA1 expression. Our data suggests IGF2BP2 is an accessory protein of the argonaute (AGO2)/miR33a/b-RISC complex, as it directly binds to miR-33a/b, AGO2, and the 3′ UTR region of ABCA1 . Finally, we show that mice overexpressing human IGF2BP2 have decreased ABCA1 expression, increased LDL-C and cholesterol blood levels, and elevated SREBP-dependent signaling. Our data support the hypothesis that IGF2BP2 has an important role in maintaining lipid homeostasis through its modulation of ABCA1 expression, as its overexpression or loss leads to dyslipidemia.
