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1.  MicroRNA-33b knock-in mice for an intron of sterol regulatory element-binding factor 1 (Srebf1) exhibit reduced HDL-C in vivo 
Scientific Reports  2014;4:5312.
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 treating atherosclerosis. Primates, but not rodents, express miR-33b from an intron of SREBF1. Therefore, humanized mice, in which a miR-33b transgene is inserted within a Srebf1 intron, are required to address its function in vivo. We successfully established miR-33b knock-in (KI) mice and found that protein levels of known miR-33a target genes, such as ABCA1, ABCG1, and SREBP-1, were reduced compared with those in wild-type mice. As a consequence, macrophages from the miR-33b KI mice had a reduced cholesterol efflux capacity via apoA-I and HDL-C. Moreover, HDL-C levels were reduced by almost 35% even in miR-33b KI hetero mice compared with the control mice. These results indicate that miR-33b may account for lower HDL-C levels in humans than those in mice and that miR-33b is possibly utilized for a feedback mechanism to regulate its host gene SREBF1. Our mice will also aid in elucidating the roles of miR-33a/b in different genetic disease models.
PMCID: PMC4058878  PMID: 24931346
2.  MicroRNA-33 Deficiency Reduces the Progression of Atherosclerotic Plaque in ApoE−/− Mice 
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.
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.
PMCID: PMC3540673  PMID: 23316322
ABCA1; ABCG1; atherosclerosis; HDL-C; microRNA
3.  Caloric Restriction, Aerobic Exercise Training, and Soluble Lectin-like Oxidized LDL Receptor-1 Levels in Overweight and Obese Postmenopausal Women 
Elevated circulating levels of soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) have been observed in obese persons and are reduced by weight loss. However, it is not known if combining caloric restriction (CR) with exercise training is better in reducing sLOX-1 levels than CR alone.
We examined whether the addition of aerobic exercise to a weight loss intervention differentially affects sLOX-1 levels in 61 abdominally obese postmenopausal women randomly assigned to a CR only (n=22), CR + moderate-intensity exercise (n=22), or CR + vigorous-intensity exercise (n=17) intervention for 20 weeks. The caloric deficit was ~2,800 kcal/week for all groups.
The intervention groups were similar at baseline with respect to body weight, body composition, lipids, and blood pressure. However, plasma sLOX-1 levels were higher in the CR only group (99.90 ± 8.23 pg/ml) compared to both the CR + moderate-intensity exercise (69.39 ± 8.23 pg/ml, p=0.01) and CR + vigorous-intensity exercise (72.83 ± 9.36 pg/ml, p=0.03) groups. All three interventions significantly reduced body weight (~14%), body fat, and waist and hip circumferences to a similar degree. These changes were accompanied by a 23% reduction in sLOX-1 levels overall (−19.00 ± 30.08 pg/ml, p<0.0001), which did not differ among intervention groups (p=0.13). Changes in body weight, body fat, and VO2 max were not correlated with changes in sLOX-1 levels. In multiple regression analyses in all women combined, baseline sLOX-1 levels (β = − 0.70 ± 0.06, p<0.0001), age (β = 0.92 ± 0.43, p=0.03) and baseline BMI (β = 1.88 ± 0.66, p=0.006) were independent predictors of the change in sLOX-1 with weight loss.
Weight loss interventions of equal energy deficit have similar effects on sLOX-1 levels in overweight and obese postmenopausal women, with the addition of aerobic exercise having no added benefit when performed in conjunction with CR.
PMCID: PMC3023845  PMID: 20856256
obesity; weight loss; caloric restriction; aerobic exercise; soluble receptor
4.  Variation in the Lectin-like Oxidized LDL Receptor 1 (LOX-1) Gene Is Associated With Plasma Soluble LOX-1 Levels 
Experimental physiology  2008;93(9):1085-1090.
The lectin-like ox-LDL receptor 1 (LOX-1) expressed on vascular cells plays a major role in atherogenesis by internalizing and degrading oxidized LDL. LOX-1 can be cleaved from the cell surface and released as soluble LOX-1 (sLOX-1), and elevated sLOX-1 levels may be indicative of atherosclerotic plaque instability. We examined associations between the LOX-1 3′UTR-C/T and G501C polymorphisms and plasma sLOX-1 levels in 97 healthy older men and women. The frequencies for the 3′UTR-T and 501C alleles were 46% and 10%, respectively. Plasma sLOX-1 levels were significantly higher in the 3′UTR CC genotype group compared to both the CT (p=0.02) and TT (p=0.002) genotype groups. Plasma sLOX-1 were also significantly higher in the 501GC genotype group compared to the GG genotype group (p=0.004). In univariate analyses, sLOX-1 levels were significantly associated with both the 3′UTR-C/T and G501 C polymorphisms. These associations remained significant after adjusting for age, gender, race, and BMI. In conclusion, variation in the LOX-1 gene is associated with plasma sLOX-1 levels in older men and women.
PMCID: PMC2652129  PMID: 18469066
receptor; cardiovascular; gene expression
5.  Elevated Soluble Lectin-like Oxidized LDL Receptor 1 (LOX-1) Levels in Obese Postmenopausal Women 
Obesity (Silver Spring, Md.)  2008;16(6):1454-1456.
We investigated the association between soluble lectin-like oxidized LDL receptor 1 (sLOX-1) levels and obesity in older women. Fifty-one (10 lean, 22 overweight, and 19 obese) postmenopausal women were included in this small retrospective analysis. Plasma sLOX-1 levels were measured using a chemiluminescent ELISA. Plasma levels of sLOX-1 were significantly higher in obese women (55.33±4.49 pg/mL) compared to lean (30.91±6.19 pg/mL, p=0.002) and overweight women (38.31±4.18 pg/mL, p=0.017). Plasma sLOX-1 levels were positively associated with body weight, BMI, total body fat, and trunk fat. The relationship between sLOX-1 and BMI was attenuated after adjustment for age, HRT, and body fat. In conclusion, obese women have higher sLOX-1 levels, which may reflect increased LOX-1 expression in adipose tissue.
PMCID: PMC2677801  PMID: 18388896
obesity; postmenopausal women; receptors
6.  Oxidized phospholipids in the macula increase with age and in eyes with age-related macular degeneration 
Molecular Vision  2007;13:772-778.
There is good evidence that oxidative stress is involved in the pathogenesis of age-related macular degeneration (AMD). Because AMD has risk factors and histopathology similar to with atherosclerosis, we hypothesized that oxidized phospholipids, which contribute to the pathogenesis of atherosclerosis, would accumulate in the eyes of AMD patients. To test this hypothesis, we investigated whether oxidized phospholipids were present in normal eyes and whether the level changed with increasing age. We then, we determined whether the levels of oxidized phospholipids were higher in eyes with AMD.
Twenty normal human donor eyes and six eyes with AMD were studied. Immunohistochemistry was performed on a tissue strip from the macular region using an antibody against oxidized phosphatidylcholine. Western blot analysis was also performed on proteins extracted from the posterior retina of donor eyes. The immunoreactivity of the specimens and the bands were quantified with NIH image software.
Immunohistochemistry showed oxidized phosphatidylcholine was present in the photoreceptors and retinal pigment epithelium of the normal human macular area, and their levels increased with age. Eyes with AMD showed more intense immunoreactivity for oxidized phospholipids than age-matched normal eyes.
These findings suggest that oxidative stress is involved in the pathogenesis of AMD possibly by oxidizing phospholipids in the photoreceptors as demonstrated in the arterial intima of patients with atherosclerosis. It is likely that controlling oxidation of phospholipids may be a potential treatment for AMD.
PMCID: PMC2768762  PMID: 17563727
7.  MicroRNA-33 regulates sterol regulatory element-binding protein 1 expression in mice 
Nature Communications  2013;4:2883.
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.
The micro-RNA miR-33 is encoded by an intron of the gene encoding sterol regulatory-binding protein 2 (SREBP-2) and controls cholesterol homoeostasis. Here, Horie et al. identify SREBP-1 as a target of miR-33 and show that deletion of miR-33 promotes diet-induced obesity and liver steatosis in mice.
PMCID: PMC3863899  PMID: 24300912

Results 1-7 (7)