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author:("Yan, dahuang")
1.  Increased Atherosclerotic Lesions in LDL Receptor Deficient Mice With Hematopoietic Nuclear Receptor Rev‐erbα Knock‐ Down 
Background
Nuclear receptor Rev‐erbα plays important roles in circadian clock timing, lipid metabolism, adipogenesis, and vascular inflammation. However, the role of Rev‐erbα in atherosclerotic lesion development has not been assessed in vivo.
Methods and Results
The nuclear receptor Rev‐erbα was knocked down in mouse haematopoietic cells by means of shRNA‐lentiviral transduction, followed by bone marrow transplantation into LDL receptor knockout mice. The Rev‐erbα protein in peripheral macrophage was reduced by 70% as compared to control mice injected with nontargeting shRNA lentivirus‐transduced bone marrow. A significant increase in atherosclerotic lesions was observed around the aorta valves as well as upon en face aorta analysis of Rev‐erbα knock‐down bone marrow recipients (P<0.01) as compared to the control mice, while plasma cholesterol, phospholipid, and triacylglycerol levels were not affected. Overexpression of Rev‐erbα in bone marrow mononuclear cells decreased inflammatory M1 while increasing M2 macrophage markers, while Rev‐erbα knock down increased the macrophage inflammatory phenotype in vitro and in vivo. Furthermore, treatment of differentiating macrophages with the Rev‐erbα ligand heme promoted expression of antiinflammatory M2 markers.
Conclusions
These observations identify hematopoietic cell Rev‐erbα as a new modulator of atherogenesis in mice.
doi:10.1161/JAHA.113.000235
PMCID: PMC3828791  PMID: 23963755
atherosclerosis; macrophages; Rev‐erbα
2.  Osbpl8 Deficiency in Mouse Causes an Elevation of High-Density Lipoproteins and Gender-Specific Alterations of Lipid Metabolism 
PLoS ONE  2013;8(3):e58856.
OSBP-related protein 8 (ORP8) encoded by Osbpl8 is an endoplasmic reticulum sterol sensor implicated in cellular lipid metabolism. We generated an Osbpl8−/− (KO) C57Bl/6 mouse strain. Wild-type and Osbpl8KO animals at the age of 13-weeks were fed for 5 weeks either chow or high-fat diet, and their plasma lipids/lipoproteins and hepatic lipids were analyzed. The chow-fed Osbpl8KO male mice showed a marked elevation of high-density lipoprotein (HDL) cholesterol (+79%) and phospholipids (+35%), while only minor increase of apolipoprotein A-I (apoA-I) was detected. In chow-fed female KO mice a less prominent increase of HDL cholesterol (+27%) was observed, while on western diet the HDL increment was prominent in both genders. The HDL increase was accompanied by an elevated level of HDL-associated apolipoprotein E in male, but not female KO animals. No differences between genotypes were observed in lecithin:cholesterol acyltransferase (LCAT) or hepatic lipase (HL) activity, or in the fractional catabolic rate of fluorescently labeled mouse HDL injected in chow-diet fed animals. The Osbpl8KO mice of both genders displayed reduced phospholipid transfer protein (PLTP) activity, but only on chow diet. These findings are consistent with a model in which Osbpl8 deficiency results in altered biosynthesis of HDL. Consistent with this hypothesis, ORP8 depleted mouse hepatocytes secreted an increased amount of nascent HDL into the culture medium. In addition to the HDL phenotype, distinct gender-specific alterations in lipid metabolism were detected: Female KO animals on chow diet showed reduced lipoprotein lipase (LPL) activity and increased plasma triglycerides, while the male KO mice displayed elevated plasma cholesterol biosynthetic markers cholestenol, desmosterol, and lathosterol. Moreover, modest gender-specific alterations in the hepatic expression of lipid homeostatic genes were observed. In conclusion, we report the first viable OsbplKO mouse model, demonstrating a HDL elevating effect of Osbpl8 knock-out and additional gender- and/or diet-dependent impacts on lipid metabolism.
doi:10.1371/journal.pone.0058856
PMCID: PMC3598917  PMID: 23554939
3.  OSBP-Related Protein 8 (ORP8) Regulates Plasma and Liver Tissue Lipid Levels and Interacts with the Nucleoporin Nup62 
PLoS ONE  2011;6(6):e21078.
We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum oxysterol-binding protein implicated in cellular lipid homeostasis. We now investigated its action in hepatic cells in vivo and in vitro. Adenoviral overexpression of ORP8 in mouse liver induced a decrease of cholesterol, phospholipids, and triglycerides in serum (−34%, −26%, −37%, respectively) and liver tissue (−40%, −12%, −24%), coinciding with reduction of nuclear (n)SREBP-1 and -2 and mRNA levels of their target genes. Consistently, excess ORP8 reduced nSREBPs in HuH7 cells, and ORP8 overexpression or silencing by RNA interference moderately suppressed or induced the expression of SREBP-1 and SREBP-2 target genes, respectively. In accordance, cholesterol biosynthesis was reduced by ORP8 overexpression and enhanced by ORP8 silencing in [3H]acetate pulse-labeling experiments. ORP8, previously shown to bind 25-hydroxycholesterol, was now shown to bind also cholesterol in vitro. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation analyses revealed the nuclear pore component Nup62 as an interaction partner of ORP8. Co-localization of ORP8 and Nup62 at the nuclear envelope was demonstrated by BiFC and confocal immunofluorescence microscopy. Furthermore, the impact of overexpressed ORP8 on nSREBPs and their target mRNAs was inhibited in cells depleted of Nup62. Our results reveal that ORP8 has the capacity to modulate lipid homeostasis and SREBP activity, probably through an indirect mechanism, and provide clues of an entirely new mode of ORP action.
doi:10.1371/journal.pone.0021078
PMCID: PMC3115989  PMID: 21698267
4.  AMPKα2 deletion Causes Aberrant Expression and Activation of NAD(P)H Oxidase and Consequent Endothelial Dysfunction in vivo: Role of 26S Proteasomes 
Circulation research  2010;106(6):1117-1128.
Rational
AMP-activated protein kinase (AMPK) is an energy sensor and ubiquitously expressed in vascular cells. Recent studies suggest that AMPK activation improves endothelial function by counteracting oxidative stress in endothelial cells. How AMPK suppresses oxidative stress remains to be established.
Objective
The aim of this study is to examine the effects of AMPK in regulating NAD(P)H oxidase, oxidative stress and endothelial function.
Methods and Results
AMPK activity, the markers of oxidative stress, NAD(P)H oxidase subunit expression (gp91phox, p47phox, p67phox, NOX1-4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of NF-κB (p50 and p65) were examined in cultured human umbilical vein endothelial cells (HUVEC) and mouse aortas isolated from AMPKα2 deficient mice. Compared to the wild type, acetylcholine (Ach)-induced endothelium-dependent relaxation was significantly impaired in parallel with increased production of oxidants in AMPKα2−/− mice. Further, pretreatment of aorta with either superoxide dismutase or Tempol or apocynin significantly improved Ach-induced endothelium-dependent relaxation in AMPKα2−/− mice. Analysis of aortic endothelial cells from AMPKα2−/− mice and human umbilical vein endothelial cells (HUVECs) expressing dominant negative AMPK or AMPK α2-specific siRNA revealed that loss of AMPK activity increased NAD(P)H oxidase subunit expression (gp91phox, p47phox, p67phox, NOX1-4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of NF-κB (p50 and p65), whereas AMPK activation by AICAR or over-expression of constitutively active AMPK had the opposite effect. Consistently, we found that genetic deletion of AMPKα2 in LDL receptor knockout (LDLr−/−) strain markedly increased 26S proteasome activity, IκB degradation, NF-κB transactivation, NAD(P)H oxidase subunit overexpression, oxidative stress, endothelial dysfunction, and atherosclerosis, all of which were largely suppressed by chronic administration of MG132, a potent cell permeable proteasome inhibitor..
Conclusion
We conclude that AMPKα2 functions as a physiological suppressor of NAD(P)H oxidase and ROS production in endothelial cells. In this way AMPK maintains the non-atherogenic and non-inflammatory phenotype of endothelial cells.
doi:10.1161/CIRCRESAHA.109.212530
PMCID: PMC2920052  PMID: 20167927
AMPK; NAD(P)H Oxidase; NF-κB; Proteasome
5.  Accelerated lipid absorption in mice overexpressing intestinal SR-BI 
The Journal of Biological Chemistry  2006;281(11):7214-7219.
Dietary cholesterol absorption contributes to a large part of the circulating cholesterol. However, the mechanism of sterol intestinal uptake is not clearly elucidated. Scavenger receptor class B type I (SR-BI), major component in the control of cholesterol homeostasis, is expressed in the intestine, but its role in this organ remains unclear. We have generated transgenic mice over-expressing SR-BI primarily in the intestine by using the mouse SR-BI gene under the control of intestinal specific “apolipoprotein (apo) C-III enhancer coupled with apo A-IV promoter”. We found SR-BI overexpression with respect to the natural protein along the intestine and at the top of the villosities. After feeding a meal containing [14C] cholesterol and [3H] triolein, SR-BI transgenic mice presented a rise of intestinal absorption of both lipids that was not due to a defect in chylomicron clearance nor to a change in the bile flow or the bile acid content. Nevertheless, SR-BI transgenic mice showed a decrease of total cholesterol, but an increase of triglyceride content in plasma without any change in the HDL apo A-I level. Thus, we describe for the first time a functional role in vivo for SR-BI in cholesterol but also in triglyceride intestinal absorption.
doi:10.1074/jbc.M508868200
PMCID: PMC2034750  PMID: 16421100
Absorption; Animals; Apolipoproteins; chemistry; Bile Acids and Salts; metabolism; Cell Membrane; metabolism; Cholesterol; chemistry; metabolism; Chylomicrons; chemistry; DNA, Complementary; metabolism; Homeostasis; Immunohistochemistry; Intestines; chemistry; metabolism; Lipids; chemistry; Lipoproteins; chemistry; Liver; metabolism; Mice; Mice, Transgenic; Promoter Regions (Genetics); Receptors, Lipoprotein; metabolism; Receptors, Scavenger; chemistry; Scavenger Receptors, Class B; metabolism; Tissue Distribution; Triglycerides; metabolism; Triolein; chemistry; SR-BI; intestinal absorption; transgenic mice; cholesterol; triglycerides
6.  Further evaluation of plasma sphingomyelin levels as a risk factor for coronary artery disease 
Background
Sphingomyelin (SM) is the major phospholipid in cell membranes and in lipoproteins. In human plasma, SM is mainly found in atherogenic lipoproteins; thus, high levels of SM may promote atherogenesis.
Methods
We investigated in a median follow up of 6.0 years the association of SM with the incidence of a combined endpoint (myocardial infarction and cardiovascular death) in stable and unstable patients, and its relation to other marker of atherosclerosis in 1,102 patients with angiographically documented CAD and 444 healthy controls.
Results and discussion
Logistic regression analysis showed that SM categorized by median was associated with an elevated risk for CAD (HR 3.2, 95%CI 2.5–4.0, p < 0.05). SM levels were correlated with apoB (r = 0.34) and triglyceride levels (r = 0.31). In patients with stable angina (n = 614), SM categorized by median was not related to incidence of a combined endpoint (cardiovascular death and myocardial infarction) (p = 0.844 by Log-rank test). However, in patients with acute coronary syndrome (n = 488), elevated SM was related to the combined endpoint (p < 0.05 by Log-rank test), also in a multivariate Cox regression analysis including potential confounders (HR 1.8, 95%CI 1.0–3.3, p < 0.05).
Conclusion
The results of our study reveal that 1) human plasma SM levels are a risk factor for CAD; 2) the pro-atherogenic property of plasma SM might be related to metabolism of apoB-containing or triglyceride-rich lipoproteins; and 3) plasma SM levels are a predictor for outcome of patients with acute coronary syndrome.
doi:10.1186/1743-7075-3-5
PMCID: PMC1360085  PMID: 16396678

Results 1-6 (6)