Faculty Bibliography

The molecular basis for primary hereditary hypertriglyceridemia has been identified in fewer than 5% of cases. Investigation of monogenic dyslipidemias has the potential to expose key metabolic pathways. We describe a hitherto unreported disease in ten individuals manifesting as moderate to severe transient childhood hypertriglyceridemia and fatty liver followed by hepatic fibrosis and the identification of the mutated gene responsible for this condition. We performed SNP array-based homozygosity mapping and found a single large continuous segment of homozygosity on chromosomal region 12q13.12. The candidate region contained 35 genes that are listed in Online Mendelian Inheritance in Man (OMIM) and 27 other genes. We performed candidate gene sequencing and screened both clinically affected individuals (children and adults with hypertriglyceridemia) and also a healthy cohort for mutations in GPD1, which encodes glycerol-3-phosphate dehydrogenase 1. Mutation analysis revealed a homozygous splicing mutation, c.361-1G>C, which resulted in an aberrantly spliced mRNA in the ten affected individuals. This mutation is predicted to result in a truncated protein lacking essential conserved residues, including a functional site responsible for initial substrate recognition. Functional consequences of the mutation were evaluated by measuring intracellular concentrations of cholesterol and triglyceride as well as triglyceride secretion in HepG2 (hepatocellular carcinoma) human cells lines overexpressing normal and mutant GPD1 cDNA. Overexpression of mutant GPD1 in HepG2 cells, in comparison to overexpression of wild-type GPD1, resulted in increased secretion of triglycerides (p = 0.01). This finding supports the pathogenicity of the identified mutation.

ER-associated degradation (ERAD) rids the early secretory pathway of misfolded or misprocessed proteins. Some members of the protein disulfide isomerase (PDI) family appear to facilitate ERAD substrate selection and retrotranslocation, but a thorough characterization of PDIs during the degradation of diverse substrates has not been undertaken, in part because there are 20 PDI family members in mammals. PDIs can also exhibit disulfide redox, isomerization, and/or chaperone activity, but which of these activities is required for the ERAD of different substrate classes is unknown. We therefore examined the fates of unique substrates in yeast, which expresses five PDIs. Through the use of a yeast expression system for apolipoprotein B (ApoB), which is disulfide rich, we discovered that Pdi1 interacts with ApoB and facilitates degradation through its chaperone activity. In contrast, Pdi1's redox activity was required for the ERAD of CPY* (a misfolded version of carboxypeptidase Y that has five disulfide bonds). The ERAD of another substrate, the alpha subunit of the epithelial sodium channel, was Pdi1 independent. Distinct effects of mammalian PDI homologues on ApoB degradation were then observed in hepatic cells. These data indicate that PDIs contribute to the ERAD of proteins through different mechanisms and that PDI diversity is critical to recognize the spectrum of potential ERAD substrates.

Movement of free cholesterol between the cellular compartment and acceptor is governed by cholesterol gradients that are determined by several enzymes and reverse cholesterol transport proteins. We have previously demonstrated that adenosine A(2A) receptors inhibit foam cell formation and stimulate production of cholesterol 27-hydroxylase (CYP27A1), an enzyme involved in the conversion of cholesterol to oxysterols. We therefore asked whether the effect of adenosine A(2A) receptors on foam cell formation in vitro is mediated by CYP27A1 or apoE, a carrier for cholesterol in the serum. We found that specific lentiviral siRNA infection markedly reduced apoE or 27-hydroxylase mRNA in THP-1 cells. Despite diminished apoE expression (p < 0.0002, interferon-gamma (IFNgamma) CGS vs. IFNgamma alone, n = 4), CGS-21680, an adenosine A(2A) receptor agonist, inhibits foam cell formation. In contrast, CGS-21680 had no effect on reducing foam cell formation in CYP27A1 KD cells (4 +/- 2%; p < 0.5113, inhibition vs. IFNgamma alone, n = 4). Previously, we reported the A(2A) agonist CGS-21680 increases apoAI-mediated cholesterol efflux nearly twofold in wild-type macrophages. Adenosine receptor activation had no effect on cholesterol efflux in CYP27A1 KD cells but reduced efflux in apoE KD cells. These results demonstrate that adenosine A(2A) receptor occupancy diminishes foam cell formation by increasing expression and function of CYP27A1

Atherosclerotic plaque formation is fueled by the persistence of lipid-laden macrophages in the artery wall. The mechanisms by which these cells become trapped, thereby establishing chronic inflammation, remain unknown. Here we found that netrin-1, a neuroimmune guidance cue, was secreted by macrophages in human and mouse atheroma, where it inactivated the migration of macrophages toward chemokines linked to their egress from plaques. Acting via its receptor, UNC5b, netrin-1 inhibited the migration of macrophages directed by the chemokines CCL2 and CCL19, activation of the actin-remodeling GTPase Rac1 and actin polymerization. Targeted deletion of netrin-1 in macrophages resulted in much less atherosclerosis in mice deficient in the receptor for low-density lipoprotein and promoted the emigration of macrophages from plaques. Thus, netrin-1 promoted atherosclerosis by retaining macrophages in the artery wall. Our results establish a causative role for negative regulators of leukocyte migration in chronic inflammation

Even at low-density lipoprotein cholesterol (LDL-C) goal, patients with cardiometabolic abnormalities remain at high risk of cardiovascular events. This paper aims (i) to critically appraise evidence for elevated levels of triglyceride-rich lipoproteins (TRLs) and low levels of high-density lipoprotein cholesterol (HDL-C) as cardiovascular risk factors, and (ii) to advise on therapeutic strategies for management. Current evidence supports a causal association between elevated TRL and their remnants, low HDL-C, and cardiovascular risk. This interpretation is based on mechanistic and genetic studies for TRL and remnants, together with the epidemiological data suggestive of the association for circulating triglycerides and cardiovascular disease. For HDL, epidemiological, mechanistic, and clinical intervention data are consistent with the view that low HDL-C contributes to elevated cardiovascular risk; genetic evidence is unclear however, potentially reflecting the complexity of HDL metabolism. The Panel believes that therapeutic targeting of elevated triglycerides (>/= 1.7 mmol/L or 150 mg/dL), a marker of TRL and their remnants, and/or low HDL-C (<1.0 mmol/L or 40 mg/dL) may provide further benefit. The first step should be lifestyle interventions together with consideration of compliance with pharmacotherapy and secondary causes of dyslipidaemia. If inadequately corrected, adding niacin or a fibrate, or intensifying LDL-C lowering therapy may be considered. Treatment decisions regarding statin combination therapy should take into account relevant safety concerns, i.e. the risk of elevation of blood glucose, uric acid or liver enzymes with niacin, and myopathy, increased serum creatinine and cholelithiasis with fibrates. These recommendations will facilitate reduction in the substantial cardiovascular risk that persists in patients with cardiometabolic abnormalities at LDL-C goal.

Although short-term incubation of hepatocytes with oleic acid (OA) stimulates secretion of apolipoprotein B100 (apoB100), exposure to higher doses of OA for longer periods inhibits secretion in association with induction of endoplasmic reticulum (ER) stress. Palmitic acid (PA) induces ER stress, but its effects on apoB100 secretion are unclear. Docosahexaenoic acid (DHA) inhibits apoB100 secretion, but its effects on ER stress have not been studied. We compared the effects of each of these fatty acids on ER stress and apoB100 secretion in McArdle RH7777 (McA) cells: OA and PA induced ER stress and inhibited apoB100 secretion at higher doses; PA was more potent because it also increased the synthesis of ceramide. DHA did not induce ER stress but was the most potent inhibitor of apoB100 secretion, acting via stimulation of autophagy. These unique effects of each fatty acid were confirmed when they were infused into C57BL6J mice. Our results suggest that when both increased hepatic secretion of VLDL apoB100 and hepatic steatosis coexist, reducing ER stress might alleviate hepatic steatosis but at the expense of increased VLDL secretion. In contrast, increasing autophagy might reduce VLDL secretion without causing steatosis.

VLDL is produced by the liver. Its major protein is apoB100. Docosahexaenoic acid (DHA), a dietary polyunsaturated fatty acid (PUFA), reduces VLDL levels and is used therapeutically for hypertriglyceridemia. In model systems, DHA lowers VLDL secretion by inducing presecretory apoB100 degradation, a process dependent on PUFA-derived lipid peroxides. We hypothesized that superoxide (SO) was a major participant in DHA-induced apoB100 degradation, given its promotion of lipid peroxidation. SO levels in a model of VLDL metabolism, rat hepatoma McArdle cells, were either decreased by a mimetic of superoxide dismutase 1 (SOD1) or by overexpressing SOD1 or increased by SOD1 siRNA. ApoB100 recovery was assessed by immunoprecipitation, SO by 2-hydroxyethidine, and lipid peroxides by thiobarbituric acid reactive substances. The SOD1 mimetic or SOD1 overexpression reduced SO and inhibited apoB100 degradation in DHA-treated cells by up to 100%. Surprisingly, silencing SOD1 did not increase DHA-induced degradation, although levels of SO were higher (+44%); those of lipid peroxides were similar, and their reduction by alpha-tocopherol decreased degradation by 50%. SO is required for lipid peroxidation in DHA-induced apoB100 degradation, but it is the peroxide level that has a tighter relationship to the level of degradation and the regulation of VLDL production.-Andreo, U., Elkind, J., Blachford, C., Cederbaum, A. I., Fisher, E. A. Role of superoxide radical anion in the mechanism of apoB100 degradation induced by DHA in hepatic cells

Rationale: Activation of liver X receptors (LXRs) inhibits the progression of atherosclerosis and promotes regression of existing lesions. In addition, LXRalpha levels are high in regressive plaques. Macrophage arginase 1 (Arg1) expression is inversely correlated with atherosclerosis progression and is markedly decreased in foam cells within the lesion. Objective: To investigate LXRalpha regulation of Arg1 expression in cultured macrophages and atherosclerotic regressive lesions. Methods and Results: We found that Arg1 expression is enhanced in CD68+ cells from regressive versus progressive lesions in a murine aortic arch transplant model. In cultured macrophages, ligand-activated LXRalpha markedly enhances basal and interleukin-4-induced Arg1 mRNA and protein expression as well as promoter activity. This LXRalpha-enhanced Arg1 expression correlates with a reduction in nitric oxide levels. Moreover, Arg1 expression within regressive atherosclerotic plaques is LXRalpha-dependent, as enhanced expression of Arg1 in regressive lesions is impaired in LXRalpha-deficient CD68+ cells. LXRalpha does not bind to the Arg1 promoter but instead promotes the interaction between PU.1 and interferon regulatory factor (IRF)8 transcription factors and induces their binding of a novel composite element. Accordingly, knockdown of either IRF8 or PU.1 strongly impairs LXRalpha regulation of Arg1 expression in macrophage cells. Finally, we demonstrate that LXRalpha binds the IRF8 locus and its activation increases IRF8 mRNA and protein levels in these cells. Conclusions: This work implicates Arg1 in atherosclerosis regression and identifies LXRalpha as a novel regulator of Arg1 and IRF8 in macrophages. Furthermore, it provides a unique molecular mechanism by which LXRalpha regulates macrophage target gene expression through PU.1 and IRF8

OBJECTIVE: Patients with diabetes have increased cardiovascular risk. Atherosclerosis in these patients is often associated with increased plaque macrophages and dyslipidemia. We hypothesized that diabetic atherosclerosis involves processes that impair favorable effects of lipid reduction on plaque macrophages. RESEARCH DESIGN AND METHODS: Reversa mice are LDL receptor-deficient mice that develop atherosclerosis. Their elevated plasma LDL levels are lowered after conditional knockout of the gene encoding microsomal triglyceride transfer protein. We examined the morphologic and molecular changes in atherosclerotic plaques in control and streptozotocin-induced diabetic Reversa mice after LDL lowering. Bone marrow-derived macrophages were also used to study changes mediated by hyperglycemia. RESULTS: Reversa mice were fed a western diet for 16 weeks to develop plaques (baseline). Four weeks after lipid normalization, control (nondiabetic) mice had reduced plasma cholesterol (-77%), plaque cholesterol (-53%), and plaque cells positive for macrophage marker CD68+ (-73%), but increased plaque collagen (+116%) compared with baseline mice. Diabetic mice had similarly reduced plasma cholesterol, but collagen content increased by only 34% compared with baseline; compared with control mice, there were lower reductions in plaque cholesterol (-30%) and CD68+ cells (-41%). Diabetic (vs. control) plaque CD68+ cells also exhibited more oxidant stress and inflammatory gene expression and less polarization toward the anti-inflammatory M2 macrophage state. Many of the findings in vivo were recapitulated by hyperglycemia in mouse bone marrow-derived macrophages. CONCLUSIONS: Diabetes hindered plaque regression in atherosclerotic mice (based on CD68+ plaque content) and favorable changes in plaque macrophage characteristics after the reduction of elevated plasma LDL

Lipoproteins are a family of plasma nanoparticles responsible for the transportation of lipids throughout the body. High-density lipoprotein (HDL), the smallest of the lipoprotein family, measures 7-13 nm in diameter and consists of a cholesteryl ester and triglyceride core that is covered with a monolayer of phospholipids and apolipoproteins. We have developed an iron oxide core HDL nanoparticle (FeO-HDL), which has a lipid based fluorophore incorporated in the phospholipid layer. This nanoparticle provides contrast for optical imaging, magnetic resonance imaging (MRI) and transmission electron microscopy (TEM). Consequently, FeO-HDL can be visualized on the anatomical, cellular and sub-cellular level. In the current study we show that the biophysical features of FeO-HDL closely resemble those of native HDL and that FeO-HDL possess the ability to mimic HDL characteristics both in vitro as well as in vivo. We demonstrate that FeO-HDL can be applied to image HDL interactions and to investigate disease settings where HDL plays a key function. More generally, we have demonstrated a multimodal approach to study the behavior of biomaterials in vitro as well as in vivo. The approach allowed us to study nanoparticle dynamics in circulation, as well as nanoparticle targeting and uptake by tissues and cells of interest. Moreover, we were able to qualitatively assess nanoparticle excretion, critical for translating nanotechnologies to the clinic