In some patients with type 2 diabetes mellitus (DM) without hypertension, cardiac hypertrophy and attenuated cardiac function are observed, and this insult is termed diabetic cardiomyopathy. To date, ...microRNA (miRNAs or miR) functions in diabetic cardiomyopathy remain to be elucidated.
To clarify the functions of miRNAs involved in diabetic cardiomyopathy caused by type 2 DM.
C57BL/6 mice were fed a high-fat diet (HFD) for 20 weeks, which induced obesity and type 2 DM. miRNA microarray analyses and real-time polymerase chain reaction revealed that miR-451 levels were significantly increased in the type 2 DM mouse hearts. Because excess supply of saturated fatty acids is a cause of diabetic cardiomyopathy, we stimulated neonatal rat cardiac myocytes with palmitic acid and confirmed that miR-451 expression was increased in a dose- and time-dependent manner. Loss of miR-451 function ameliorated palmitate-induced lipotoxicity in neonatal rat cardiac myocytes. Calcium-binding protein 39 (Cab39) is a scaffold protein of liver kinase B1 (LKB1), an upstream kinase of AMP-activated protein kinase (AMPK). Cab39 was a direct target of miR-451 in neonatal rat cardiac myocytes and Cab39 overexpression rescued the lipotoxicity. To clarify miR-451 functions in vivo, we generated cardiomyocyte-specific miR-451 knockout mice. HFD-induced cardiac hypertrophy and contractile reserves were ameliorated in cardiomyocyte-specific miR-451 knockout mice compared with control mice. Protein levels of Cab39 and phosphorylated AMPK were increased and phosphorylated mammalian target of rapamycin (mTOR) was reduced in cardiomyocyte-specific miR-451 knockout mouse hearts compared with control mouse hearts.
Our results demonstrate that miR-451 is involved in diabetic cardiomyopathy through suppression of the LKB1/AMPK pathway.
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.
To clarify the role of miR-33 involved in heart failure.
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.
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.
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.
Considerable numbers of non‐coding RNAs have been identified by RNA sequencing and genome‐wide profiling. These include microRNAs studied for decades and other non‐coding RNAs (ncRNAs) such as long ncRNAs. 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 these ncRNAs.
MicroRNAs (miRs) are small non-protein-coding RNAs that bind to specific mRNAs and inhibit translation or promote mRNA degradation. Recent reports have indicated that miR-33, which is located within ...the intron of sterol regulatory element-binding protein (SREBP) 2, controls cholesterol homoeostasis and may be a potential therapeutic target for the treatment of atherosclerosis. Here we show that deletion of miR-33 results in marked worsening of high-fat diet-induced obesity and liver steatosis. Using miR-33(-/-)Srebf1(+/-) mice, we demonstrate that SREBP-1 is a target of miR-33 and that the mechanisms leading to obesity and liver steatosis in miR-33(-/-) mice involve enhanced expression of SREBP-1. These results elucidate a novel interaction between SREBP-1 and SREBP-2 mediated by miR-33 in vivo.
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.
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.
These data strongly suggest that inhibition of miR-33 will be effective as a novel strategy for treating AAA.
Familial hypercholesterolemia is an inherited disorder that remains underdiagnosed. Conventional genetic testing methods such as next-generation sequencing (NGS) or target PCR are based on the ...amplification process. Due to the efficiency limits of polymerase and ligase enzymes, these methods usually target short regions and do not detect large mutations straightforwardly. This study combined the long-read nanopore sequencing and CRISPR-Cas9 system to sequence the target DNA molecules without amplification. We originally designed and optimized the CRISPR-RNA panel to target the low-density lipoprotein receptor gene (LDLR) and proprotein convertase subtilisin/kexin type 9 gene (PCSK9) from human genomic DNA followed by nanopore sequencing. The average coverages for LDLR and PCSK9 were 106× and 420×, versus 1.2× for the background genome. Among them, continuous reads were 52x and 307x, respectively, and spanned the entire length of LDLR and PCSK9. We identified pathogenic mutations in both coding and splicing donor regions in LDLR. We also detected an 11,029 bp large deletion in another case. Furthermore, using continuous long reads generated from the benchmark experiment, we demonstrated how a false-positive 670 bp deletion caused by PCR amplification errors was easily eliminated.
Background
Cholesterol efflux from cells to apolipoprotein A‐I (apoA‐I) acceptors via the ATP‐binding cassette transporters ABCA1 and ABCG1 is thought to be central in the antiatherogenic mechanism. ...MicroRNA (miR)‐33 is known to target ABCA1 and ABCG1 in vivo.
Methods and Results
We assessed the impact of the genetic loss of miR‐33 in a mouse model of atherosclerosis. MiR‐33 and apoE double‐knockout mice (miR‐33−/−Apoe−/−) showed an increase in circulating HDL‐C levels with enhanced cholesterol efflux capacity compared with miR‐33+/+Apoe−/− mice. Peritoneal macrophages from miR‐33−/−Apoe−/− mice showed enhanced cholesterol efflux to apoA‐I and HDL‐C compared with miR‐33+/+Apoe−/− macrophages. Consistent with these results, miR‐33−/−Apoe−/− mice showed reductions in plaque size and lipid content. To elucidate the roles of miR‐33 in blood cells, bone marrow transplantation was performed in these mice. Mice transplanted with miR‐33−/−Apoe−/− bone marrow showed a significant reduction in lipid content in atherosclerotic plaque compared with mice transplanted with miR‐33+/+Apoe−/− bone marrow, without an elevation of HDL‐C. Some of the validated targets of miR‐33 such as RIP140 (NRIP1) and CROT were upregulated in miR‐33−/−Apoe−/− mice compared with miR‐33+/+Apoe−/− mice, whereas CPT1a and AMPKα were not.
Conclusions
These data demonstrate that miR‐33 deficiency serves to raise HDL‐C, increase cholesterol efflux from macrophages via ABCA1 and ABCG1, and prevent the progression of atherosclerosis. Many genes are altered in miR‐33‐deficient mice, and detailed experiments are required to establish miR‐33 targeting therapy in humans.
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-33
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)
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-33
DBH-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.
Background The cardio-ankle vascular index (CAVI) has been recently reported as a new index of aortic stiffness, which is less influenced by blood pressure than pulse wave velocity (PWV). The present ...study investigated the relationship between the levels of CAVI and carotid and coronary arteriosclerosis. Methods and Results The 443 consecutive patients who underwent CAVI, carotid sonography, and coronary angiography in hospital were examined. Intima - media thickness (IMT) and carotid plaque were evaluated by ultrasonography. The severity of coronary artery disease (CAD) was evaluated by coronary angiography and the subjects were divided into 4 groups (0, no significant organic stenosis: 1, 1-vessel disease: 2, 2-vessel disease: 3, 3-vessel disease). Univariate analyses showed that both CAVI and brachial-ankle PWV (baPWV) were associated with IMT and the presence of carotid plaque. Multiple stepwise regression analyses revealed that CAVI (p=0.0427), but not baPWV, was associated with the IMT. Both CAVI (p<0.0001) and baPWV (p=0.0140) were significantly associated with the severity of CAD. Multiple logistic analyses revealed that CAVI (p=0.0342), but not baPWV (p=0.8027), was associated with the presence of multivessel disease. Conclusion High CAVI implies progression of carotid and coronary arteriosclerosis. CAVI may be more closely linked with arteriosclerosis than baPWV. (Circ J 2008; 72: 1762 - 1767)
Ambient temperature is a risk factor for cardiovascular disease. Cold weather increases cardiovascular events, but paradoxically, cold exposure is metabolically protective because of UCP1 (uncoupling ...protein 1)-dependent thermogenesis.
We sought to determine the differential effects of ambient environmental temperature challenge and UCP1 activation in relation to cardiovascular disease progression.
Using mouse models of atherosclerosis housed at 3 different ambient temperatures, we observed that cold temperature enhanced, whereas thermoneutral housing temperature inhibited atherosclerotic plaque growth, as did deficiency in UCP1. However, whereas UCP1 deficiency promoted poor glucose tolerance, thermoneutral housing enhanced glucose tolerance, and this effect held even in the context of UCP1 deficiency. In conditions of thermoneutrality, but not UCP1 deficiency, circulating monocyte counts were reduced, likely accounting for fewer monocytes entering plaques. Reductions in circulating blood monocytes were also found in a large human cohort in correlation with environmental temperature. By contrast, reduced plaque growth in mice lacking UCP1 was linked to lower cholesterol. Through application of a positron emission tomographic tracer to track CCR2
cell localization and intravital 2-photon imaging of bone marrow, we associated thermoneutrality with an increased monocyte retention in bone marrow. Pharmacological activation of β3-adrenergic receptors applied to mice housed at thermoneutrality induced UCP1 in beige fat pads but failed to promote monocyte egress from the marrow.
Warm ambient temperature is, like UCP1 deficiency, atheroprotective, but the mechanisms of action differ. Thermoneutrality associates with reduced monocyte egress from the bone marrow in a UCP1-dependent manner in mice and likewise may also suppress blood monocyte counts in man.
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