Physiologically regulated transgenic ABCA1 does not reduce amyloid burden or amyloid-beta peptide levels in vivo

ABCA1-deficient mice have low levels of poorly lipidated apolipoprotein E (apoE) and exhibit increased amyloid load. To test whether excess ABCA1 protects from amyloid deposition, we crossed APP/PS1 mice to ABCA1 bacterial artificial chromosome (BAC) transgenic mice. Compared with wild-type animals, the ABCA1 BAC led to a 50% increase in cortical ABCA1 protein and a 15% increase in apoE abundance, demonstrating that this BAC supports modest ABCA1 overexpression in brain. However, this was observed only in animals that do not deposit amyloid. Comparison of ABCA1/APP/PS1 mice with APP/PS1 controls revealed no differences in levels of brain ABCA1 protein, amyloid, Abeta, or apoE, despite clear retention of ABCA1 overexpression in the livers of these animals. To further investigate ABCA1 expression in the amyloid-containing brain, we then compared ABCA1 mRNA and protein levels in young and aged cortex and cerebellum of APP/PS1 and ABCA1/APP/PS1 animals. Compared with APP/PS1 controls, aged ABCA1/APP/PS1 mice exhibited increased ABCA1 mRNA, but not protein, selectively in cortex. Additionally, ABCA1 mRNA levels were not increased before amyloid deposition but were induced only in the presence of extensive Abeta and amyloid levels. These data suggest that an induction of ABCA1 expression may be associated with late-stage Alzheimer's neuropathology.     

Overexpression of apolipoprotein O does not impact on plasma HDL levels or functionality in human apolipoprotein A-I transgenic mice

Apolipoprotein (apo) O is a newly discovered apolipoprotein preferentially contained within HDL; however, currently, no data are available on the (patho)physiological effects of apoO. Therefore, the present study assessed the impact of apoO overexpression on (i) plasma lipids and lipoproteins as well as on (ii) HDL functionality. Human apoO was overexpressed by means of recombinant adenovirus (AdhapoO) in human apoA-I transgenic mice, a humanized mouse model of HDL metabolism. AdhapoO substantially increased apoO in plasma and within HDL. However, plasma triglycerides, phospholipids, total cholesterol and HDL cholesterol did not change. HDL size distribution, lipid composition and the apoA-I and the apoO distribution over the different HDL fractions separated by FPLC remained unaltered. Furthermore, enrichment of HDL with apoO did not impact on HDL functionality assessed in four independent ways, namely (i) stimulation of cholesterol efflux from macrophage foam cells, (ii) protection against LDL oxidation, (iii) anti-inflammatory activity on endothelial cells, and (iv) induction of vasodilation in isolated aortic rings ex vivo as a measure of stimulating vascular NO production. These results demonstrate that although overexpression of apoO results in a substantial enrichment of HDL particles with this novel apolipoprotein, apoO does not impact the plasma lipoprotein profile or HDL functionality.     

SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I-/- mice.

Serum amyloid A (SAA) is an acute phase protein of unknown function that is involved in systemic amyloidosis and may also be involved in atherogenesis. The precise role of SAA in these processes has not been established. SAA circulates in plasma bound to high density lipoprotein-3 (HDL3). The pathway for the production of SAA-containing HDL is not known. To test whether apolipoprotein (apo)A-I-HDL is required in the production of SAA-HDL, we analyzed the lipopolysaccharide (LPS)-induced changes in apoA-I+/+ and apoA-I-/- mice. In apoA-I+/+ mice, after injection of LPS, remodeling of HDL occurred: total cholesterol increased and apoA-I decreased slightly and shifted to lighter density. Dense (density of HDL3) but large (size of HDL2 ) SAA-containing particles were formed. Upon fast phase liquid chromatography fractionation of plasma, >90% of SAA eluted with HDL that was enriched in cholesterol and phospholipid and shifted "leftward" to larger particles. Non-denaturing immunoprecipitation with anti-mouse apoA-I precipitated all of the apoA-I but not all of the SAA, confirming the presence of SAA-HDL devoid of apoA-I. In the apoA-I-/- mice, which normally have very low plasma lipid levels, LPS injection resulted in significantly increased total and HDL cholesterol. Greater than 90% of the SAA was lipid associated and was found on dense but large, spherical HDL particles essentially devoid of other apolipoproteins.We conclude that serum amyloid A (SAA) is able to sequester lipid, forming dense but large HDL particles with or without apoA-I or other apolipoproteins. The capacity to isolate lipoprotein particles containing SAA as the predominant or only apolipoprotein provides an important system to further explore the biological function of SAA.     

Activation of the constitutive androstane receptor decreases HDL in wild-type and human apoA-I transgenic mice

The nuclear hormone receptor constitutive androstane receptor (CAR, NR1I3) regulates detoxification of xenobiotics and endogenous molecules, and has been shown to be involved in the metabolism of hepatic bile acids and cholesterol. The goal of this study was to address potential effects of CAR on the metabolism of HDL particles, key components in the reverse transport of cholesterol to the liver. Wild-type (WT) mice, transgenic mice expressing human apolipoprotein A-I (HuAITg), and CAR-deficient (CAR(-/-)) mice were treated with the specific CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). CAR activation decreased HDL cholesterol and plasma apolipoprotein A-I (apoA-I) levels in both WT and HuAITg mice, but not CAR(-/-) mice. Both mouse apoA-I and human apoA-I were decreased by more than 40% after TCPOBOP treatment, and kinetic studies revealed that the production rate of HDL is reduced in TCPOBOP-treated WT mice. In transient transfections, TCPOBOP-activated CAR decreased the activity of the human apoA-I promoter. Although loss of CAR function did not alter HDL levels in normal chow-fed mice, HDL cholesterol, apoA-I concentration, and apoA-I mRNA levels were increased in CAR(-/-) mice relative to WT mice when both were fed a high-fat diet. We conclude that CAR activation in mice induces a pronounced decrease in circulating levels of plasma HDL, at least in part through downregulation of apoA-I gene expression.     

Deficiency in sPLA(2) does not affect HDL levels or atherosclerosis in mice

Secretory non-pancreatic phospholipase A(2) (sPLA(2)) has been implicated in inflammation and has been found in human atherosclerotic lesions. To test the effect of sPLA(2) deficiency on atherosclerosis, C57BL/Ks mice (apoE(+/+) and PLA(2)(++) were bred with C57BL/6 apoE knockout mice which are sPLA(2)(--) due to a spontaneous mutation. Sibling pairs of mice (apoE(--)/sPLA(2)(++) and apoE(--)/sPLA(2)(--)) on high fat Western diets were dissected at 22 weeks. In vitro enzyme assays confirmed higher serum sPLA(2) activity in the sPLA(2)(++) compared to sPLA(2)(--) for both sexes, while sPLA(2)(--) males had slightly higher serum cholesterol and phospholipids. Analysis of lipoprotein profiles by FPLC showed no effect of sPLA(2) genotype on any measured parameters. Atherosclerosis was quantitated by assaying cholesterol in aortic extracts. Male sPLA(2) trended slightly higher than sPLA(2)(++) with no statistical significance. Female sPLA(2)(++) and sPLA(2)(--) mice showed no significant differences in any of the measured parameters. These results suggest that the endogenous mouse sPLA(2) gene does not significantly affect HDL or atherosclerosis in mice.     

Atherogenic, enlarged, and dysfunctional HDL in human PLTP/apoA-I double transgenic mice

In low density lipoprotein receptor (LDLR)-deficient mice, overexpression of human plasma phospholipid transfer protein (PLTP) results in increased atherosclerosis. PLTP strongly decreases HDL levels and might alter the antiatherogenic properties of HDL particles. To study the potential interaction between human PLTP and apolipoprotein A-I (apoA-I), double transgenic animals (hPLTPtg/hApoAItg) were compared with hApoAItg mice. PLTP activity was increased 4.5-fold. Plasma total cholesterol and phospholipid were decreased. Average HDL size (analyzed by gel filtration) increased strongly, hPLTPtg/hApoAItg mice having very large, LDL-sized, HDL particles. Also, after density gradient ultracentrifugation, a substantial part of the apoA-I-containing lipoproteins in hPLTPtg/hApoAItg mice was found in the LDL density range. In cholesterol efflux studies from macrophages, HDL isolated from hPLTPtg/hApoAItg mice was less efficient than HDL isolated from hApoAItg mice. Furthermore, it was found that the largest subfraction of the HDL particles present in hPLTPtg/hApoAItg mice was markedly inferior as a cholesterol acceptor, as no labeled cholesterol was transferred to this fraction. In an LDLR-deficient background, the human PLTP-expressing mouse line showed a 2.2-fold increased atherosclerotic lesion area. These data demonstrate that the action of human PLTP in the presence of human apoA-I results in the formation of a dysfunctional HDL subfraction, which is less efficient in the uptake of cholesterol from cholesterol-laden macrophages.     

The acute phase protein serum amyloid A primes neutrophils

We studied here the effect of the acute phase protein serum amyloid A (SAA) on the oxidative burst of neutrophils. Incubation of neutrophils with SAA increased the rate of oxygen uptake and the production of reactive oxygen species of neutrophils activated with opsonized zymosan (OZ). The increment in the neutrophil oxidative burst was dependent on SAA concentration in the range of 3-33 microg protein ml(-1) and was observed only in the presence of a relatively low amount of OZ (1 x 10(6) particles ml(-1)). SAA did not affect oxygen consumption and reactive oxygen production triggered by other stimuli, such as f-Met-Leu-Phe, phorbol myristate acetate or non-opsonized zymosan. Our finding points to a priming effect of SAA probably associated with mobilization of receptors for opsonized particles and strengthens the role of SAA as an effector of neutrophil functions in inflammation.     

Lipopolysaccharide binding protein and serum amyloid A secretion by human intestinal epithelial cells during the acute phase response.

The acute phase proteins LPS binding protein (LBP) and serum amyloid A (SAA) are produced by the liver and are present in the circulation. Both proteins have been shown to participate in the immune response to endotoxins. The intestinal mucosa forms a large surface that is continuously exposed to these microbial products. By secretion of antimicrobial and immunomodulating agents, the intestinal epithelium contributes to the defense against bacteria and their products. The aim of this study was to explore the influence of the inflammatory mediators TNF-alpha, IL-6, and IL-1beta on the release of LBP and SAA by intestinal epithelial cells (IEC). In addition, the induction of LBP and SAA release by cell lines of intestinal epithelial cells and hepatic cells was compared. The data obtained show that in addition to liver cells, IEC also expressed LBP mRNA and released bioactive LBP and SAA upon stimulation. Regulation of LBP and SAA release by IEC and hepatocytes was typical for class 1 acute phase proteins, although differences in regulation between the cell types were observed. Endotoxin did not induce LBP and SAA release. Glucocorticoids were demonstrated to strongly enhance the cytokine-induced release of LBP and SAA by IEC, corresponding to hepatocytes. The data from this study, which imply that human IEC can produce LBP and SAA, suggest a role for these proteins in the local defense mechanism of the gut to endotoxin. Furthermore, the results demonstrate that tissues other than the liver are involved in the acute phase response.     

Acute-phase high-density lipoprotein in the rat does not contain serum amyloid A protein.

Serum amyloid A protein (SAA) is an acute-phase apolipoprotein of high-density lipoprotein (HDL). Its N-terminal sequence is identical with that of amyloid A protein (AA), the subunit of AA amyloid fibrils. However, rats do not develop AA amyloidosis, and we report here that neither normal nor acute-phase rat HDL contains a protein corresponding to SAA of other species. mRNA coding for a sequence homologous with the C-terminal but not with the N-terminal part of human SAA is synthesized in greatly increased amounts in acute-phase rat liver. These observations indicate that the failure of rats to develop AA amyloid results from the absence of most of the AA-like part of their SAA-like protein, and that the N-terminal portion of SAA probably contains the lipid-binding sequences.     

Genetically augmenting tau levels does not modulate the onset or progression of Abeta pathology in transgenic mice

The two hallmark pathologies of Alzheimer's disease (AD) are amyloid plaques, composed of the small amyloid-beta (Abeta) peptide, and neurofibrillary tangles, comprised aggregates of the microtubule binding protein, tau. The molecular linkage between these two lesions, however, remains unknown. Based on human and mouse studies, it is clear that the development of Abeta pathology can trigger tau pathology, either directly or indirectly. However, it remains to be established if the interaction between Abeta and tau is bidirectional and whether the modulation of tau will influence Abeta pathology. To address this question, we used the 3xTg-AD mouse model, which is characterized by the age-dependent buildup of both plaques and tangles. Here we show that genetically augmenting tau levels and hyperphosphorylation in the 3xTg-AD mice has no effect on the onset and progression of Abeta pathology. These data suggest that the link between Abeta and tau is predominantly if not exclusively unidirectional, which is consistent with the Abeta cascade hypothesis and may explain why tauopathy-only disorders are devoid of any Abeta pathology.     

Lipoprotein inflammatory properties and serum amyloid A levels but not cholesterol levels predict lesion area in cholesterol-fed rabbits

Rabbits on a 1% cholesterol diet received injections of vehicle with or without D-4F or L-4F. After 1 month, the percent of aorta with atherosclerotic lesions was 24 +/- 15% (vehicle), 10 +/- 6% (D-4F) (P < 0.01 vs. vehicle), and 13 +/- 9% (L-4F) (P < 0.05 vs. vehicle). Inflammatory indexes for HDL and LDL were determined by measuring monocyte chemotactic activity after adding rabbit lipoproteins to human endothelial cells. HDL-inflammatory index (HII) and LDL-inflammatory index (LII), respectively, were 1.39 +/- 0.24; 1.35 +/- 0.29 (vehicle), 0.67 +/- 0.26; 0.63 +/- 0.38 (D-4F) (P < 0.001 vs. vehicle), and 0.67 +/- 0.2; 0.68 +/- 0.32 (L-4F) (P < 0.01 vs. vehicle). Serum amyloid A (SAA) levels were 95 +/- 39, 8 +/- 22, and 7 +/- 19 mug/ml, respectively, for vehicle, D-4F, and L-4F (P < 0.001 vs. vehicle). There was no correlation between lesion area and total plasma or HDL-cholesterol levels. In contrast, there was a positive correlation with HII, LII, and SAA (P = 0.002, P = 0.0026, P = 0.0079, respectively). HII correlated closely with SAA levels (r = 0.6616; r(2) = 0.4377, P < 0.0001). Thus, HII, LII, and SAA are better predictors of lesion area than are total plasma or HDL-cholesterol levels in cholesterol-fed rabbits.     

Expression of hepatitis B virus X protein does not alter the accumulation of spontaneous mutations in transgenic mice

Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens, although the mechanism for this cofactor role remains unknown. The ability of HBx to inhibit DNA repair in transiently transfected cell lines suggests one possible pathway. In the present study, primary hepatocytes isolated from transgenic mice that possess the HBV X gene under the control of the human alpha-1-antitrypsin regulatory region (ATX mice) were found to be deficient in their ability to conduct unscheduled DNA synthesis in response to UV-induced DNA damage. In order to measure the impact of HBx expression on DNA repair in vivo, double-transgenic mice that express HBx and possess a bacteriophage lambda transgene were sacrificed at 30, 90, and 240 days of age. Mutation frequency was determined for high-molecular-weight liver DNA of ATX and control mice by functional analysis of the lambda transgene. Expression of HBx did not significantly increase the accumulation of spontaneous mutations. These results are consistent with previous studies of HBx transgenic mice in which no effect of HBx on liver histology was apparent. This new animal model provides a powerful system in which to investigate the in vivo cooperation between HBx expression and environmental carcinogens.     

Extrahepatic production of acute phase serum amyloid A

Amyloidosis is a group of diseases characterized by the extracellular deposition of protein that contains non-branching, straight fibrils on electron microscopy (amyloid fibrils) that have a high content of beta-pleated sheet conformation. Various biochemically distinct proteins can undergo transformation into amyloid fibrils. The precursor protein of amyloid protein A (AA) is the acute phase protein serum amyloid A (SAA). The concentration of SAA in plasma increases up to 1000-fold within 24 to 48 h after trauma, inflammation or infection. Individuals with chronically increased SAA levels may develop AA amyloidosis. SAA has been divided into two groups according to the encoding genes and the source of protein production. These two groups are acute phase SAA (A-SAA) and constitutive SAA (C-SAA). Although the liver is the primary site of the synthesis of A-SAA and C-SAA, extrahepatic production of both SAAs has been observed in animal models and cell culture experiments of several mammalian species and chicken. The functions of A-SAA are thought to involve lipid metabolism, lipid transport, chemotaxis and regulation of the inflammatory process. There is growing evidence that extrahepatic A-SAA formation may play a crucial role in amyloidogenesis and enhances amyloid formation at the site of SAA production.     

Expression of human serum albumin in the milk of transgenic mice

We have tested the feasibility of producing large quantities of human serum albumin (HSA) in the milk of transgenic livestock by generating transgenic mice as a model system. The sheep β-lactoglobulin (BLG) 5′-regulatory promoter sequences were used to support expression of BLG or HSA in transgenic mice. Transgenic animals generated from the entire BLG gene including 3, 5.5 or 10.8 kb of 5′-sequences demonstrated that 3 kb of 5′-sequences were sufficient to support high levels of expression of BLG, and that the longer 5′-sequences did not improve upon the levels of expression. As such, the 3 kb 5′-sequences were used to drive expression of HSA in BLG-HSA constructs. HSA was not detectably expressed in eight transgenic lines generated from a BLG-HSA construct containing the HSA cDNA. Two transgenic lines of 26 generated, using five different constructs, with an HSA minigene possessing the first intron expressed HSA in their milk. One of these expressed HSA at high levels (2.5 mg ml⁻¹) and has stably transmitted this ability to its progeny. A high percentage of transgenic mouse lines (four of six) generated from a vector containing an HSA minigene possessing introns 1 and 2 expressed HSA in their milk at levels which ranged from 1 to 35 μg ml⁻¹. In a similar trend, levels of expression of HSA by transfected tissue culture cells from BLG-HSA vectors containing an introduced SV40 enhancer were low with the HSA cDNA, increased with the HSA minigene with intron 1 and increased further with the minigene containing introns 1 and 2. This study demonstrates that high levels of HSA can be expressed in the milk of transgenic animals, that introns of the HSA gene play a role in its expression and that transfected cell lines may be used to quickly evaluate the relative expression efficiencies of various vector constructs intended for future transgenic evaluation.     

A thiocarbamate inhibitor of endothelial lipase raises HDL cholesterol levels in mice

By screening directed libraries of serine hydrolase inhibitors using the cell surface form of endothelial lipase (EL), we identified a series of carbamate-derived (EL) inhibitors. Compound 3 raised plasma HDL-C levels in the mouse, and a correlation was found between HDL-C and plasma compound levels. Spectroscopic and kinetic studies support a covalent mechanism of inhibition. Our findings represent the first report of EL inhibition as an effective means for increasing HDL-C in an in vivo model.     

Fenofibrate reverses the decline in HDL cholesterol in mice overexpressing human phospholipid transfer protein

In humans, fibrates are used to treat dyslipidemia, because these drugs lower plasma triglycerides and raise HDL cholesterol. Treatment with fibrates lowers plasma phospholipid transfer protein (PLTP) activity in humans, but increases PLTP activity in mice, without a consistent effect on HDL-cholesterol concentration. Earlier, we found that PLTP overexpression in transgenic mice results in decreased plasma HDL levels and increased diet-induced atherosclerosis. So it seems that the interplay between fibrates, PLTP and HDL is different in mice and man, which may be important for atherosclerosis development. In the present study, we measured the effects of fibrates on PLTP expression in cultured human hepatocytes and effects of fibrate treatment on human PLTP expression, plasma PLTP activity and HDL levels in human PLTP transgenic mice. Fibrate treatment did not influence PLTP mRNA levels in human hepatocytes. Hepatic human PLTP mRNA levels and PLTP activity were both moderately elevated by fenofibrate treatment in human PLTP transgenic mice. In wild-type mice, however, feeding fenofibrate resulted in a strong induction of PLTP mRNA in the liver and a more than 4-fold increase of plasma PLTP activity. Plasma triglycerides were reduced in all mice by 48% or more by fenofibrate treatment. HDL-cholesterol concentrations were substantially increased by fenofibrate in PLTP overexpressing mice (+72%), but unaffected in wild-type mice. We conclude that fenofibrate treatment reverses the HDL-lowering effect of PLTP overexpression in human PLTP transgenic mice.     

Differential regulation of cholesterol homeostasis in transgenic mice expressing human cholesterol ester transfer protein

We investigated whether expression of cholesterol ester transfer protein (CETP) in mice alters the regulation of cholesterol metabolism. Transgenic mice expressing human CETP (CETP-TG) and nontransgenic littermates (non-TG) were fed either a monounsaturated fatty acid (MUFA) or a saturated fatty acid (SFA)-rich diet in the presence or absence of cholesterol. Mice fed with MUFA diet had higher CETP activity compared with SFA-fed mice. Addition of cholesterol to the MUFA diet decreased CETP activity, whereas addition of cholesterol to the SFA diet had no effect. Cholesterol 7alpha-hydroxylase (Cyp7a) activity was higher in CETP-TG mice compared with non-TG mice when fed a MUFA diet, whereas SFA fed CETP-TG mice showed lower Cyp7a activity as compared with non-TG. Microsomal triglyceride transfer protein (MTTP) activity was higher in CETP-TG mice compared with non-TG mice when fed a MUFA diet. HMG-CoA reductase activity was lower in CETP-TG mice compared with non-TG mice when fed a MUFA or a SFA diet. These data demonstrate that the regulation of Cyp7a, HMG-CoA reductase, and MTTP is altered in CETP-TG mice as compared with non-TG mice and these alterations are further modulated by the quality of dietary fats. These findings highlight the importance of CETP in regulating cholesterol homeostasis.