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Objective— Atherosclerosis is a common disease caused by a variety of metabolic and inflammatory disturbances. MicroRNA (miR)-33a within SREBF2 (sterol regulatory element-binding factor 2) is a potent target for treatment of atherosclerosis through regulating both aspects; however, the involvement of miR-33b within SREBF1 remains largely unknown. Although their host genes difference could lead to functional divergence of miR-33a/b, we cannot dissect the roles of miR-33a/b in vivo because of lack of miR-33b sequences in mice, unlike human. Approach and Results— Here, we analyzed the development of atherosclerosis using miR-33b knock-in humanized mice under apolipoprotein E–deficient background. MiR-33b is prominent both in human and mice on atheroprone condition. MiR-33b reduced serum high-density lipoprotein cholesterol levels and systemic reverse cholesterol transport. MiR-33b knock-in macrophages showed less cholesterol efflux capacity and higher inflammatory state via regulating lipid rafts. Thus, miR-33b promotes vulnerable atherosclerotic plaque formation. Furthermore, bone marrow transplantation experiments strengthen proatherogenic roles of macrophage miR-33b. Conclusions— Our data demonstrated critical roles of SREBF1 -miR-33b axis on both lipid profiles and macrophage phenotype remodeling and indicate that miR-33b is a promising target for treating atherosclerosis.

Objective— Abdominal aortic aneurysm (AAA) is an increasingly prevalent and ultimately fatal disease with no effective pharmacological treatment. Because matrix degradation induced by vascular inflammation is the major pathophysiology of AAA, attenuation of this inflammation may improve its outcome. Previous studies suggested that miR-33 (microRNA-33) inhibition and genetic ablation of miR-33 increased serum high-density lipoprotein cholesterol and attenuated atherosclerosis. Approach and Results— MiR-33a-5p expression in central zone of human AAA was higher than marginal zone. MiR-33 deletion attenuated AAA formation in both mouse models of angiotensin II– and calcium chloride–induced AAA. Reduced macrophage accumulation and monocyte chemotactic protein-1 expression were observed in calcium chloride–induced AAA walls in miR-33 −/− mice. In vitro experiments revealed that peritoneal macrophages from miR-33 −/− mice showed reduced matrix metalloproteinase 9 expression levels via c-Jun N-terminal kinase inactivation. Primary aortic vascular smooth muscle cells from miR-33 −/− mice showed reduced monocyte chemotactic protein-1 expression by p38 mitogen-activated protein kinase attenuation. Both of the inactivation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were possibly because of the increase of ATP-binding cassette transporter A1 that is a well-known target of miR-33. Moreover, high-density lipoprotein cholesterol derived from miR-33 −/− mice reduced expression of matrix metalloproteinase 9 in macrophages and monocyte chemotactic protein-1 in vascular smooth muscle cells. Bone marrow transplantation experiments indicated that miR-33–deficient bone marrow cells ameliorated AAA formation in wild-type recipients. MiR-33 deficiency in recipient mice was also shown to contribute the inhibition of AAA formation. Conclusions— These data strongly suggest that inhibition of miR-33 will be effective as a novel strategy for treating AAA.

Rationale: Heart failure and atherosclerosis share the underlying mechanisms of chronic inflammation followed by fibrosis. A highly conserved microRNA (miR), miR-33, is considered as a potential therapeutic target for atherosclerosis because it regulates lipid metabolism and inflammation. However, the role of miR-33 in heart failure remains to be elucidated. Objective: To clarify the role of miR-33 involved in heart failure. Methods and Results: We first investigated the expression levels of miR-33a/b in human cardiac tissue samples with dilated cardiomyopathy. Increased expression of miR-33a was associated with improving hemodynamic parameters. To clarify the role of miR-33 in remodeling hearts, we investigated the responses to pressure overload by transverse aortic constriction in miR-33–deficient (knockout [KO]) mice. When mice were subjected to transverse aortic constriction, miR-33 expression levels were significantly upregulated in wild-type left ventricles. There was no difference in hypertrophic responses between wild-type and miR-33KO hearts, whereas cardiac fibrosis was ameliorated in miR-33KO hearts compared with wild-type hearts. Despite the ameliorated cardiac fibrosis, miR-33KO mice showed impaired systolic function after transverse aortic constriction. We also found that cardiac fibroblasts were mainly responsible for miR-33 expression in the heart. Deficiency of miR-33 impaired cardiac fibroblast proliferation, which was considered to be caused by altered lipid raft cholesterol content. Moreover, cardiac fibroblast–specific miR-33–deficient mice also showed decreased cardiac fibrosis induced by transverse aortic constriction as systemic miR-33KO mice. Conclusion: Our results demonstrate that miR-33 is involved in cardiac remodeling, and it preserves lipid raft cholesterol content in fibroblasts and maintains adaptive fibrotic responses in the remodeling heart.

Cholesterol homeostasis is a tightly regulated process. Previous work has been dominated by sterol-response element—related mechanisms, but the influence of micro-RNAs (miRs) is increasingly recognized, with miR-223 becoming a recent member of this group. Several micro-RNAs (miRNAs or miR-) have been reported to be involved in cholesterol homeostasis.1–4 In a recent study, Vickers et al5 presented miR-223 as an important new player in this arena, one that they propose to be a coordinator of cholesterol homeostasis.5 This miRNA had been previously found to be rich in myeloid cells, where it regulates differentiation and inflammation,6,7 but the authors found that its abundance in hepatic cells is at least comparable with that of many functionally validated miRNAs. A relationship between miR-223 and cholesterol metabolism was suggested by finding its expression in human hepatoma (Huh7) cells was positively associated with intracellular cholesterol levels. In a series of studies in Huh7 cells, it was then shown that miR-223 (1) inhibited high-density lipoprotein cholesterol (HDL-C) uptake, (2) promoted cholesterol efflux, and (3) suppressed cholesterol biosynthesis. The authors also showed increased cholesterol levels in liver and plasma of miR-223 knockout mice, extending its involvement in cholesterol homeostasis in vitro to the in vivo setting. The experimental approaches taken are well established in the miRNA field and were coupled to a productive use of bioinformatics. This included investigating whether the effects of miR-223 on a particular RNA were direct, by cloning into a luciferase reporter plasmid the putative target sequence in the 3′-UTR of that mRNA (or this sequence deleted in nucleotides required for miR-223 binding). The effect of exogenously supplied miR-223 on luciferase activity in transfected HEK293 cells was then measured. Thus, they determined that the molecular mechanisms for the functional reductions in HDL-C uptake and cholesterol biosynthesis …

MicroRNA (miRNA) is a small noncoding RNA that modulates posttranscriptional translation. Growing evidence indicates that miRNA is involved in basic cell functions and oncogenesis. In order to find a possible functional role of miRNA in the heart, we first suppressed the expression of Dicer and Ago2, which are required for miRNA biogenesis and function, by lenti-virus-mediated siRNA in neonatal rat cardiomyocytes. Knockdown of Dicer and Ago2 expression significantly increased their beating rate; therefore, we hypothesized that miRNA may regulate the expression of cardiomyocyte contractile protein since cardiac contractility depends on the expression of two myosin heavy chain (MHC) genes, α and β, which are regulated in an antithetical manner by developmental, physiological, and pathological signals. As siRNA against Dicer, which results in the loss of all miRNA function, upregulated α-MHC and downregulated β-MHC mRNA expression, we tried to identify functional miRNAs which have an opposite effect against Dicer siRNA on α/β-MHC expression in the heart. We first performed miRNA microarray analysis in the heart of Dahl salt-sensitive rats in which systemic hypertension caused compensated concentric left ventricular hypertrophy (LVH) at the age of 11 weeks, followed by congestive heart failure (CHF) at the age of 17 weeks. In this model, decreased α/β-MHC ratio is observed at LVH and CHF stages. Next we overexpressed miRNAs which are upregulated in LVH and CHF stages in neonatal rat cardiomyocytes by lenti-virus. After screening these miRNAs, miR-206 was found to decrease the α/β-MHC mRNA expression ratio. The computational miRNA target prediction algorithm showed one of the targets of miR-206 is RXR-α, which regulates α/β-MHC transcription. Forced expression of miR-206 reduced the translation of RXR-α. Moreover, when the expression of RXR-α was downregulated by siRNA, the α/β-MHC mRNA expression ratio decreased in the same manner as miR-206. In conclusion, miR-206 controls the gene expression of MHC isoforms by targeting RXR-α, and these results suggest that miR-206 regulates cardiac contractility.

The differentiation of preadiopocytes (preAdps) into mature adipocytes (Adps) is closely associated with the expression of adipocyte (Adp)-specific gene products that are deeply involved in insulin resistance; therefore, it is critical to identify genes required for Adp differentiation. We used retrovirus insertion-mediated random mutagenesis to generate 3T3-L1 preAdp cells which lose their ability to differentiate into mature Adps. Using this approach, we discovered that tryptophan hydroxylase-1 (TPH1), a rate-limiting enzyme for the biosynthesis of serotonin (Ser), was required for Adp differentiation. While differentiation was clearly reduced in a mutant 3T3-L1 cell line that lacked expression of the normal TPH1 gene, reconstitution of the TPH1 gene in this cell line restored its differentiation potential. The administration of Ser enhanced differentiation in 3T3-L1 cells and in cells of the stromal vascular fraction from adult mice. On the other hand, differentiation was inhibited by 5-hydroxy tryptophan type 2A (5HT2A) receptor antagonists as well as the Ser synthesis inhibitor. Differentiation into mature Adp was significantly impaired in preAdps prepared from TPH1−/− mice compared to those from wild-type mice. We observed no differences in the histology of adipose tissue between TPH1−/−and wild-type mice at the basal state; however, after obesity was induced by a high-fat diet, increases in body weight and mesenteric fat weight were more prominent in TPH1−/− mice than in wild-type mice. In obese groups, we observed extremely large (140μm∼) Adps without any small Adps in TPH1−/− mice, in contrast to usually hypertrophied (μ80∼m) Adps and small Adps in wild-type mice. Interestingly, Ser treatment of mature cultured Adps enhanced lipolysis, which was blocked by the 5HT7 receptor antagonist but not by the 5HT2A receptor antagonist. Oral administration of 5HT2A receptor antagonist in high fat diet-induced obese mice reduced mesenteric fat weight and the number of small adipocytes; however, it did not increase the number of extremely large adipocytes as seen in TPH1−/−mice. Thus, Ser in adipose tissue acts as an autocrine factor required for Adp differentiation and lipolysis. Distinct receptor subtypes appear to mediate these two processes.