Cellular prion protein (PrPC) misfolds to form infectivity‐associated scrapie prion protein and generates C‐terminal fragments C1 and C2 in healthy and prion‐infected animals. C1 cleavage occurs N‐terminally of PrPC's hydrophobic domain, whereas the larger C2 fragment is generated by cleavage at the end of the octarepeat region. As the PrP‐like proteins Doppel and Shadoo (Sho) have been reported to inhabit similar membrane environments as PrPC, we investigated endoproteolysis by using a panel of mutant alleles. Doppel undergoes efficient in vivo cleavage at a C1 site mapped to the start of the globular domain, which is a structurally similar cleavage site to that in PrPC. Sho is processed to C1 and C2 fragments, and proved refractory to mutagenesis to inactivate C1 cleavage. As a reciprocal product of C1 cleavage, Sho also engenders a metabolically stable N1 fragment with a C‐terminus after its hydrophobic domain, an observation that may account for N1's association with membrane and/or cellular fractions in vitro and in vivo. Our data indicate that glycosylation status and yet to be identified proteases modulate internal C1 and C2 proteolysis events within the mammalian prion protein family.

Hepatic triacylglycerol levels are governed through synthesis, degradation and export of this lipid. Here we demonstrate that enforced expression of hepatic lipase in the endoplasmic reticulum in McArdle RH7777 hepatocytes resulted in a significant decrease in the incorporation of fatty acids into cellular triacylglycerol and cholesteryl ester accompanied by attenuation of secretion of apolipoprotein B-containing lipoproteins. Hepatic lipase-mediated depletion of intracellular lipid storage increased the expression of peroxisome proliferator-activated receptor α and its target genes and augmented oxidation of fatty acids. These data show that 1) hepatic lipase is active in the endoplasmic reticulum and 2) intracellular hepatic lipase modulates cellular lipid metabolism and lipoprotein secretion.

During prion infections of the central nervous system (CNS) the cellular prion protein, PrPC, is templated to a conformationally distinct form, PrPSc. Recent studies have demonstrated that the Sprn gene encodes a GPI-linked glycoprotein Shadoo (Sho), which localizes to a similar membrane environment as PrPC and is reduced in the brains of rodents with terminal prion disease. Here, analyses of prion-infected mice revealed that down-regulation of Sho protein was not related to Sprn mRNA abundance at any stage in prion infection. Down-regulation was robust upon propagation of a variety of prion strains in Prnp(a) and Prnp(b) mice, with the exception of the mouse-adapted BSE strain 301 V. In addition, Sho encoded by a TgSprn transgene was down-regulated to the same extent as endogenous Sho. Reduced Sho levels were not seen in a tauopathy, in chemically induced spongiform degeneration or in transgenic mice expressing the extracellular ADan amyloid peptide of familial Danish dementia. Insofar as prion-infected Prnp hemizygous mice exhibited accumulation of PrPSc and down-regulation of Sho hundreds of days prior to onset of neurologic symptoms, Sho depletion can be excluded as an important trigger for clinical disease or as a simple consequence of neuronal damage. These studies instead define a disease-specific effect, and we hypothesize that membrane-associated Sho comprises a bystander substrate for processes degrading PrPSc. Thus, while protease-resistant PrP detected by in vitro digestion allows post mortem diagnosis, decreased levels of endogenous Sho may trace an early response to PrPSc accumulation that operates in the CNS in vivo. This cellular response may offer new insights into the homeostatic mechanisms involved in detection and clearance of the misfolded proteins that drive prion disease pathogenesis.

A reduction in low density lipoprotein (LDL) cholesterol or an increase in high density lipoprotein (HDL) cholesterol can reduce the risk of development of atherosclerosis through overlapping or independent mechanisms. However, the clinical outcome of combined therapy remains in debate. In this study, we first characterized effects of various constructs of helper-dependent adenoviral vector (HDAd) expressing apolipoprotein E3 or LDL receptor (LDLR) in vivo on plasma cholesterol levels. Using this information, we designed experiments and compared the effects of long-term (28 weeks) LDL cholesterol lowering or raising HDL cholesterol, or a combination of both on advanced atherosclerosis in Ldlr(-/-) mice, a mouse model of familial hypercholesterolemia. Our major findings are: (i) various factors influence in vivo functional activity, which appear to be context dependent; (ii) apolipoprotein AI (APOAI) gene transfer, which raises HDL cholesterol, retards progression of atherosclerosis but does not induce regression; (iii) LDLR or LDLR and APOAI combination gene therapy induces lesion regression; however, LDLR gene transfer accounts for the majority of the effects of combined gene therapy; (iv) LDLR gene therapy reduces interleukin-7, which is a master regulator of T-cell homeostasis, but APOAI gene therapy does not. These results indicate that LDL cholesterol lowering is effective and sufficient in protection against atherosclerosis and induction of regression of pre-existing atherosclerosis.

AIMSInterleukin-7 (IL-7) is a master regulator of T-cell development and homoeostasis. Increased IL-7 levels are associated with inflammatory diseases. The aims of this study were to determine whether IL-7 is a biomarker for inflammatory conditions or an active participant in atherogenesis.METHODS AND RESULTSAdvanced atherosclerotic lesions in Apoe(-/-) mice were regressed by long-term cholesterol lowering through treatment with a helper-dependent adenovirus expressing apolipoprotein E (n= 6-10). Using this model, gene expression patterns in the aorta were analysed at an early phase of regression by microarray. After stringent statistical analysis, we found that IL-7 expression is significantly reduced in response to lowering of cholesterol (n= 6). To understand the importance of IL-7 down-regulation for atherosclerotic regression, we studied the effects and mechanisms of action of IL-7 on endothelial cells (ECs) in vitro as well as in vivo. Our major findings are: (i) IL-7 up-regulates cell adhesion molecules and monocyte chemoattractant protein-1 in ECs and promotes monocyte adhesion to ECs; (ii) this regulation is mediated by phosphatidylinositol 3-kinase (PI3K)/AKT-dependent and -independent activation of NF-κB; (iii) elevation of plasma IL-7 induces recruitment of monocytes/macrophages to endothelium without affecting plasma cholesterol (n= 5, 6); and (4) lack of IL-7 in bone marrow-derived cells reduces migration of monocytes/macrophages to the lesions (n= 5, 6).CONCLUSIONThese results suggest that IL-7 inflames endothelium via PI3K/AKT-dependent and -independent activation of NF-κB and recruits monocytes/macrophages to the endothelium, thus playing an active role in atherogenesis.

BACKGROUND:Myocardial energy metabolism is a strong predictor of postoperative cardiac function. This study profiled the metabolites and metabolic changes in the myocardium exposed to sevoflurane, propofol, and Intralipid and investigated the underlying molecular mechanisms.METHODS:Sevoflurane (2 vol%) and propofol (10 and 100 microM) in the formulation of 1% Diprivan (AstraZeneca Inc., Mississauga, ON, Canada) were compared for their effects on oxidative energy metabolism and contractility in the isolated working rat heart model. Intralipid served as a control. Substrate flux through the major pathways for adenosine triphosphate generation in the heart, that is, fatty acid and glucose oxidation, was measured using [H]palmitate and [C]glucose. Biochemical analyses of nucleotides, acyl-CoAs, ceramides, and 32 acylcarnitine species were used to profile individual metabolites. Lipid rafts were isolated and used for Western blotting of the plasma membrane transporters CD36 and glucose transporter 4.RESULTS:Metabolic profiling of the hearts exposed to sevoflurane and propofol revealed distinct regulation of fatty acid and glucose oxidation. Sevoflurane selectively decreased fatty acid oxidation, which was closely related to a marked reduction in left ventricular work. In contrast, propofol at 100 microM but not 10 microM increased glucose oxidation without affecting cardiac work. Sevoflurane decreased fatty acid transporter CD36 in lipid rafts/caveolae, whereas high propofol increased pyruvate dehydrogenase activity without affecting glucose transporter 4, providing mechanisms for the fuel shifts in energy metabolism. Propofol increased ceramide formation, and Intralipid increased hydroxy acylcarnitine species.CONCLUSIONS:Anesthetics and their solvents elicit distinct metabolic profiles in the myocardium, which may have clinical implications for the already jeopardized diseased heart.

Mobilization of hepatic triacylglycerol stores provides substrates for mitochondrial β-oxidation and assembly of VLDLs; however, the identity of lipolytic enzymes involved in the regulation of this process remains largely unknown. Arylacetamide deacetylase (AADA) shares homology with hormone-sensitive lipase and therefore could potentially participate in hepatic lipid metabolism, including the regulation of hepatic triacylglycerol levels. We have established McArdle-RH7777 (rat hepatoma) cell lines stably expressing mouse AADA cDNA and performed metabolic labeling as well as lipid mass analyses. Expression of AADA cDNA in McArdle-RH7777 cells significantly reduced intracellular triacylglycerol levels and apolipoprotein B secretion and increased fatty acid oxidation.

Atherosclerosis is a chronic inflammatory disease, but can regress. Despite the importance of atherosclerotic lesion regression, its mechanisms are not well understood. We induced regression of advanced atherosclerosis in Apoe −/− mice and studied potential mechanisms. A single i.v. injection of helper-dependent adenoviral vector expressing human apoE3 (HDAd-gE3) into Apoe −/− mice resulted in a 28±4% reduction of the advanced atherosclerosis (n= 7 vs. baseline n=8, p=0.012) after 41 weeks. Using this system, we analyzed gene expression profiles in the aorta at day 10, when plasma cholesterol was normalized. Pathway and ontology analyses revealed multiple signaling pathways influenced by the treatment; however, after correction for multiple testing we unexpectedly found that interleukin-7 (IL-7) was significantly downregulated. IL-7 is a nonredundant cytokine for B- and T-cell development. Although several studies have suggested that IL-7 is associated with cardiovascular disease, IL-7 has not been implicated as having a direct role in atherogenesis. IL-7 positive areas in the aorta were also reduced in the HDAd-gE3 group by 53% (n=5– 6/group, p<0.01) after 41 weeks of treatment. IL-7 could be a marker for lesion regression or reduced inflammation, or be directly involved in atherosclerosis. To test this hypothesis, we analyzed the effects of IL-7 on expression of adhesion molecules and chemokines in human aortic endothelial cells (HAECs). IL-7 upregulated adhesion molecules and chemokines which include VCAM-1, ICAM-1, E-selectin and monocyte chemotactic protein-1. This upregulation was time- and dose-dependent and promoted monocyte adhesion to HAECs. Moreover, the increase of plasma IL-7 levels in Apoe −/− mice by HDAd-IL-7 enhanced homing of monocyte/macrophage to the lesions by 2-fold (n=6/group, p<0.01) without increasing plasma cholesterol. In contrast, transplantation of bone marrow cells isolated from Il-7 −/− mice into Ldlr −/− mice reduced macrophage positive areas by 26% after 20 weeks compared with those isolated from wild type mice (n=6/group, p<0.05). These results suggest that IL-7 plays a direct role in atherosclerosis by promoting monocyte/macrophage homing to atherosclerotic lesions.

There is much speculation whether extravascular inflammation accelerates atherosclerosis. We tested this hypothesis in apoE−/− mice using three well-characterized models of non-autoimmune chronic inflammation: croton oil-induced skin inflammation, Aspergillus fumigatus antigen-induced allergic lung disease, and A. fumigatus antigen-induced peritonitis. The croton oil model produced recurrent inflammatory skin ulceration, and marked increases in plasma levels of IL-6 and serum amyloid A (SAA). The allergic lung disease model showed strong local inflammation with eosinophilic infiltration and serum IgE induction. The recurrent peritonitis model was accompanied by mild elevation in plasma SAA levels. Aortic atherosclerosis was quantified by computer-assisted morphometry of en face arteries in apoE−/− mice at 34 weeks for the croton oil model, 26 and 42 weeks for the allergic lung disease model, and 26 weeks for the peritonitis model. We found that all three forms of chronic extravascular inflammation had no effect on the rate of atherosclerosis development.

Mouse esterase-x/carboxylesterase 1 (Es-x/Ces1) is a close homolog of triacylglycerol hydrolase/carboxylesterase 3 (TGH/Ces3). Es-x possesses a conserved esterase/lipase active site motif, suggesting that like TGH it could play a role in hepatic triacylglycerol (TG) metabolism. McArdle-RH7777 cells stably transfected with Es-x cDNA accumulated significantly less TG and had increased production of acid-soluble metabolites (an indicator of β-oxidation) during incubations with 0.4mM oleic acid when compared to empty vector or TGH cDNA transfected cells. Reduction of cellular TG persisted in the presence of esterase/lipase inhibitor E600 indicating that Es-x-mediated TG lowering can be largely explained by reduced partitioning of exogenous fatty acids to TG and increased redirection to β-oxidation, rather than by increased TG turnover. Glycerol supplementation increased TG synthesis in both control and Es-x expressing cells to similar extent suggesting that Es-x expression did not reduce flux of metabolic intermediates through the glycerol-3-phosphate pathway. While Es-x expression reduced cellular TG levels, secretion of TG and apolipoprotein B remained unchanged when compared to control cells. Overall, these results suggest that Es-x limits hepatic TG accumulation by promoting β-oxidation.

Atherosclerosis is now recognized as an inflammatory disease involving the vascular wall. Recent results indicate that acute inflammation does not simply passively resolve as previously assumed but is actively terminated by a homeostatic process that is governed by specific lipid-derived mediators initiated by lipoxygenases. Experiments with animals and humans support a proinflammatory role for the 5-lipoxygenase system. In contrast, results from animal experiments show a range of responses with the 12/15-lipoxygenase pathways in atherosclerosis. To date, the only two clinical epidemiology human studies both support an antiatherogenic role for 12/15-lipoxygenase downstream actions. We tested the hypothesis that atherosclerosis results from a failure in the resolution of local inflammation by analyzing apolipoprotein E-deficient mice with 1) global leukocyte 12/15-lipoxygenase deficiency, 2) normal enzyme expression, or 3) macrophage-specific 12/15-lipoxygenase overexpression. Results from these indicate that 12/15-lipoxygenase expression protects mice against atherosclerosis via its role in the local biosynthesis of lipid mediators, including lipoxin A(4), resolvin D1, and protectin D1. These mediators exert potent agonist actions on macrophages and vascular endothelial cells that can control the magnitude of the local inflammatory response. Taken together, these findings suggest that a failure of local endogenous resolution mechanisms may underlie the unremitting inflammation that fuels atherosclerosis.

LRP1 [LDL (low-density lipoprotein) receptor-related protein 1]-null CHO cells (Chinese-hamster ovary cells) (13-5-1 cells) exhibited accelerated cell growth and severe tumour progression after they were xenografted into nude mice. Reconstitution of LRP1 expression in these cells, either with the full-length protein or with a minireceptor, reduced growth rate as well as suppressed tumour development. We tested the role of the tyrosine residue in the FXNPXY63 motif within the LRP1 cytoplasmic domain in signal transduction and cell growth inhibition by site-specific mutagenesis. The LRP1 minireceptors harbouring Tyr63 to alanine or Tyr63 to phenylalanine substitution had diametrically opposite effects on cell growth, cell morphology and tumour development in mice. The Y63F-expressing cells showed suppressed cell growth and tumour development, which were associated with decreased beta-catenin and cadherin concentrations in the cells. On the other hand, the Y63A-expressing cells lacked inhibition on cell growth and tumour development, which were associated with hyperactivation of ERKs (extracellular-signal-regulated kinases), FAK (focal adhesion kinase) and cyclin D1 in the cells. The mutant Y63A minireceptor also exhibited reduced capacity in binding to the Dab2 (disabled 2) adaptor protein. In addition, the Y63A mutant showed increased caveolar localization, and cells expressing Y63A had altered caveolae architecture. However, tyrosine to alanine substitution at the other NPXY29 motif had no effect on cell growth or tumorigenesis. These results suggest that the FXNPXY63 motif of LRP1 not only governs cellular localization of the receptor but also exerts multiple functional effects on signalling pathways involved in cell growth regulation.

Management of patients with autosomal dominant familial hypercholesterolemia continues to be a major challenge. Somatic gene therapy may offer an alternative treatment for these patients. We have previously reported that helper-dependent adenovirus (HDAd)-mediated LDL receptor (LDLR) gene transfer reversed hypercholesterolemia for over 108 weeks and markedly retarded development of atherosclerosis in LDLR-deficient mice (Gene Ther 11:1540-1548, 2004). We also reported that HDAd expressing apolipoprotein A-I (apoA-I) was effective in inhibition of progression of advanced atherosclerosis. After 24 weeks treatment, the lesions had a more stable-appearing phenotype with a decrease in subendothelial lipid deposits and a reduction in macrophage-derived cells and adhesion molecule expression (Circulation 107:2726-2732, 2003). ApoA-I and LDLR work via different mechanisms. ApoA-I elevates HDL, which has anti-inflammatory and anti-oxidative effects, and also promotes reverse cholesterol transfer. LDL works mainly by lowering atherogenic non-HDL lipoproteins. We, therefore, compared the efficacy of HDAd-mediated hepatic transfer of ApoA-I only, LDLR only, and ApoA-I + LDLR on atherosclerosis development in LDL-/- mice. LDLR-deficient mice (6-8 weeks of age) were fed a high cholesterol diet for 36 weeks and were separated into 5 groups (baseline, HDAd-0 empty vector, HDAd-AI + HDAd-0, HDAd-LDLR + HDAd-0, HDAd-AI + HDAd-LDLR). The baseline group was sacrificed, and the remaining mice were treated with 2 |[times]| 10E11 VP of HDAd vectors. The plasma cholesterol levels in HDAd-0 (n=6) or HDAd-AI + HDAd-0 (n=7) group were around 500 mg/dl throughout the experiment. Plasma cholesterol levels were reduced to 72 mg/dl 2 weeks after treatment in HDAd-LDLR group and gradually increased to 300 mg/dl over the next 28 weeks (n=7). The mice treated with both genes had a cholesterol level of 182 mg/dl at 2 weeks which gradually increased to 297 mg/dl at 28 weeks (n=8). FPLC analysis revealed that the prominent IDL/LDL fraction is reduced in HDAd-LDLR treated groups. Atherosclerotic lesion areas were measured by quantitative morphometry. Mean lesion area was 5.7 mm2 in the baseline group. It was 12.5 mm2 in the HDAd-0 group, 9.5 mm2 in the HDAd-AI group, 3.2 mm2 in the HDAd-LDLR group, and 3.3 mm2 in the HDAd-AI + HDAd-LDLR group. Compared with baseline and HDAd-0 group, HDAd-AI treatment inhibited the lesion progression by 25%, while HDAd-LDLR alone or combined treatment induced lesion regression by 44%. The combined treatment is very effective. However, our data suggest that LDLR gene transfer accounts for most of the effects in the combined treatment. Moreover, these results indicate that HDAd-mediated LDLR gene therapy is highly effective in inhibition of lesion progression as well as in inducing atherosclerotic lesion regression in a mouse model of familial hypercholesterolemia.

Condensed abstract: We investigated the molecular mechanism of nicotine-accelerated atherosclerosis in hyperlipidemic LDLR-/mouse model. Our findings reveal a novel mechanism of nicotine in atherogenesis. Nicotine directly activates macrophages via the nicotinic acetylcholine receptors, inducing multiple downstream events, subsequently resulting in NFkB-mediated inflammation in the arterial wall and accelerated atherosclerosis.