An apolipoprotein E-based therapeutic improves outcome and reduces Alzheimer's disease pathology following closed head injury: evidence of pharmacogenomic interaction

Apolipoprotein E (apoE) modifies glial activation and the CNS inflammatory response in an isoform-specific manner. Peptides derived from the receptor-binding region of apoE have been demonstrated to maintain the functional activity of the intact protein, and to improve histological and functional deficits after closed head injury. In the current study, APOE2, APOE3, and APOE4 targeted replacement (TR) mice expressing the human apoE protein isoforms (apoE2, apoE3 and apoE4) were used in a clinically relevant model of closed head injury to assess the interaction between the humanized apoE background and the therapeutic apoE mimetic peptide, apoE(133-149). Treatment with the apoE-mimetic peptide reduced microglial activation and early inflammatory events in all of the targeted replacement animals and was associated with histological and functional improvement in the APOE2TR and APOE3TR animals. Similarly, brain beta amyloid protein (Abeta)(1-42) levels were increased as a function of head injury in all of the targeted replacement mice, while treatment with apoE peptide suppressed Abeta(1-42) levels in the APOE2TR and APOE3TR animals. These results suggest a pharmacogenomic interaction between the therapeutic effects of the apoE mimetic peptide and the human apoE protein isoforms. Furthermore, they suggest that administration of apoE-mimetic peptides may serve as a novel therapeutic strategy for the treatment of acute and chronic neurological disease.     

Apolipoprotein E inhibits neointimal hyperplasia after arterial injury in mice

The potential cytostatic function of apolipoprotein (apo) E in vivo was explored by measuring neointimal hyperplasia in response to vascular injury in apoE-deficient and apoE-overexpressing transgenic mice. Results showed a significant increase in medial thickness, medial area, and neointimal formation after vascular injury in both apoE knockout and wild-type C57BL/6 mice. Immunochemical analysis with smooth muscle alpha-actin-specific antibodies revealed that the neointima contained proliferating smooth muscle cells. Neointimal area was 3.4-fold greater, and the intima/medial ratio as well as stenotic luminal area was more pronounced in apoE(-/-) mice than those observed in control mice (P < 0.05). The human apoE3 transgenic mice in FVB/N genetic background were then used to verify a direct effect of apoE in protection against neointimal hyperplasia in response to mechanically induced vascular injury. Results showed that neointimal area was reduced threefold to fourfold in mice overexpressing the human apoE3 transgene (P < 0.05). Importantly, suppression of neointimal formation in the apoE transgenic mice also abolished the luminal stenosis observed in their nontransgenic FVB/N counterparts. These results documented a direct role of apoE in modulating vascular response to injury, suggesting that increasing apoE level may be beneficial in protection against restenosis after vascular surgery.     

A Role for apoE in Regulating the Levels of α-1-Antichymotrypsin in the Aging Mouse Brain and in Alzheimer’s Disease

This study was designed to explore the possible functional relationships between apolipoprotein E (apoE) and the protease inhibitor alpha-1-antichymotrypsin in the aging mouse brain and in Alzheimer's disease. For this purpose, levels of EB22/5 (the mouse homologue to human alpha-1-antichymotrypsin) mRNA expression was studied in apoE-deficient mice. These mice showed an age-dependent increase of EB22/5 mRNA expression in the brain. Furthermore, overexpression of allele 3 of human APOE gene in transgenic mice (in an apoE-deficient background) resulted in normalization of levels of EB22/5 mRNA expression compatible with levels found in control mice. In contrast, overexpression of human APOE4 allele or down-regulation of the apoE receptor low density lipoprotein receptor-related protein by deletion of the receptor-associated protein was associated with elevated levels of EB22/5 similar to apoE-deficient mice. Consistent with the findings in murine models, human alpha-1-antichymotrypsin protein was increased in brain homogenates from patients with Alzheimer's disease, and levels of this serpin were the highest in patients with the APOE4 allele. In summary, the present study showed evidence supporting a role for apoE in regulating alpha-1-antichymotrypsin expression. This is relevant to Alzheimer's disease because these two molecules appear to be closely associated with the pathogenesis of this disorder.     

Apolipoprotein E mediates sulfatide depletion in animal models of Alzheimer's disease

Herein, we tested a recently proposed working model of apolipoprotein E (apoE)-mediated sulfatide metabolism/trafficking/homeostasis with two well-characterized amyloid precursor protein (APP) transgenic (Tg) animal models of Alzheimer's disease (AD) (i.e., APP(V717F) and APPsw) on a wild-type murine apoE background or after being bred onto an Apoe(-/-) background. As anticipated, lipidomics analysis demonstrated that the sulfatide levels in brain tissues were reduced beginning at approximately 6 months of age in APP(V717F) Tg, Apoe(+/+) mice and at 9 months of age in APPsw Tg, Apoe(+/+) mice relative to their respective non-APP Tg littermates. This reduction increased in both APP Tg mice as they aged. In contrast, sulfatide depletion did not occur in APP Tg, Apoe(-/-) animals relative to the Apoe(-/-) littermates. The lack of sulfatide depletion in APP Tg, Apoe(-/-) mice strongly supports the role of apoE in the deficient sulfatide content in APP Tg, Apoe(+/+) mice. Collectively, through different animal models of AD, this study provides evidence for an identified biochemical mechanism that may be responsible for the sulfatide depletion at the earliest stages of AD.     

Apolipoprotein E isoform-dependent effects on anxiety and cognition in female TR mice

Compared with apoE3, apoE4 is associated with increased risk to develop age-related cognitive decline, particularly in women. In this study, young, middle-aged, and old female mice expressing human apoE under control of the mouse apoE promoter were behaviorally analyzed. Cognitive performance in the water maze decreased with age in all mice. Compared with apoE2 and apoE3 mice, apoE4 mice showed better cognitive performance and higher measures of anxiety than apoE2 and apoE3 mice. Measures of anxiety correlated with cognitive performance in the water maze and passive avoidance tests and might have contributed to the enhanced cognitive performance of the apoE4 mice. ApoE4 mice showed better water maze learning and higher cortical apoE levels than mice expressing apoE4 in astrocytes under control of the GFAP promoter. This was not seen in apoE3 mice. There were no line differences in either genotype in spatial memory retention in the probe trial following the last day of hidden platform training. Thus, the promoter used to express apoE4 critically modulates its effects on brain function.     

ApoE isoform-specific regulation of regeneration in the peripheral nervous system

Apolipoprotein E (apoE) is a 34 kDa glycoprotein with three distinct isoforms in the human population (apoE2, apoE3 and apoE4) known to play a major role in differentially influencing risk to, as well as outcome from, disease and injury in the central nervous system. In general, the apoE4 allele is associated with poorer outcomes after disease or injury, whereas apoE3 is associated with better responses. The extent to which different apoE isoforms influence degenerative and regenerative events in the peripheral nervous system (PNS) is still to be established, and the mechanisms through which apoE exerts its isoform-specific effects remain unclear. Here, we have investigated isoform-specific effects of human apoE on the mouse PNS. Experiments in mice ubiquitously expressing human apoE3 or human apoE4 on a null mouse apoE background revealed that apoE4 expression significantly disrupted peripheral nerve regeneration and subsequent neuromuscular junction re-innervation following nerve injury compared with apoE3, with no observable effects on normal development, maturation or Wallerian degeneration. Proteomic isobaric tag for relative and absolute quantitation (iTRAQ) screens comparing healthy and regenerating peripheral nerves from mice expressing apoE3 or apoE4 revealed significant differences in networks of proteins regulating cellular outgrowth and regeneration (myosin/actin proteins), as well as differences in expression levels of proteins involved in regulating the blood-nerve barrier (including orosomucoid 1). Taken together, these findings have identified isoform-specific roles for apoE in determining the protein composition of peripheral nerve as well as regulating nerve regeneration pathways in vivo.     

APOE and the regulation of microglial nitric oxide production: a link between genetic risk and oxidative stress

The mechanism linking the APOE4 gene with increased susceptibility for Alzheimer's disease (AD) and poorer outcomes following closed head injury and stroke is unknown. One potential link is activation of the innate immune system in the CNS. Our previously published data demonstrated that apolipoprotein E regulates production of nitric oxide, a critical cytoactive factor released by immune active macrophages. To determine if immune regulation is different in the presence of apolipoprotein E4 compared to apolipoprotein E3, we have measured NO production by peritoneal and CNS macrophages (microglia) cultured from transgenic mice that only express the human apoE4 or apoE3 protein isoform. Significantly more NO was produced in APOE4 mice compared to APOE3 transgenic mice that only express human apoE3 protein. Similarly, monocyte derived macrophages from humans carrying APOE4 gene alleles also produce significantly greater NO than those individuals with APOE3. The mechanism for this isoform-specific difference in NO production is not known and multiple sites in the NO production pathway may be affected. Expression of inducible nitric oxide synthase (iNOS) mRNA and protein are not significantly different between the APOE3 and APOE4 mice, suggesting that induction of iNOS is not a primary cause of the increased NO production in APOE4 animals. One alternative regulatory mechanism that demonstrates isoform specificity is arginine transport, which is greater in microglia from APOE4 transgenic mice compared to microglia from APOE3 mice. Increased transport is consistent with an increased production of NO and may reflect a direct or indirect effect of the APOE genotype on microglial arginine uptake and microglial activation in general. Overall, greater NO production in APOE4 carriers where characteristically high levels of oxidative/nitrosative stress are found in diseases such as AD provides a mechanism that potentially explains the genetic association between APOE4 and human diseases.     

Altered cholesterol metabolism in human apolipoprotein E4 knock-in mice

Thevarepsilon4 allele of apolipoprotein E (apoE) is associated with an increased risk of developing Alzheimer's disease (AD). To accurately determine the isoform-specific effects of human apoE on brain functions under physiological and pathological situations, we created mice expressing human apoE4 isoform in place of mouse apoE by utilizing the gene-targeting technique on the embryonic stem cells (knock-in). The homozygousvarepsilon4 (4/4) mice correctly expressed human apoE4 in the serum and the brain. The human apoE in the brain was found primarily in the astrocytes as was the mouse apoE in the wild-type (+/+) mice. In the 4/4 mice, the serum cholesterol level was 2.5-fold that of the +/+ littermate controls on a regular diet. This marked elevation was accounted for by an accumulation of very low and low density lipo-proteins. In the brains of the 4/4 mice, however, the amounts of total cholesterol and phospholipids were significantly decreased compared with the +/+ littermates. These findings indicate that cholesterol and lipid metabolism is markedly altered in the 4/4 mice. Our human apoE4 knock-in mice will be useful in clarifying the role of apoE in the etiologies of AD and cardiovascular diseases in relation to cholesterol and lipid metabolism.     

Genetically altering Abeta distribution from the brain to the vasculature ameliorates tau pathology

The inheritance of the ε4 allele of the apolipoprotein E ( apoE ) gene is the major genetic risk factor for developing late-onset Alzheimer disease. In transgenic mice overexpressing amyloid precursor protein (APP), replacing the endogenous mouse apoE gene with the human apolipoprotein E4 ( apoE4 ) gene alters the distribution of amyloid-β (Aβ) deposits from the brain parenchyma to the vasculature. However, the effects of this distribution on the onset and progression of tau pathology remain to be established. To address this issue, we used a genetic approach to replace the endogenous apoE gene with the human apoE4 allele in the 3xTg-AD mice. We showed that changing Aβ distribution from the parenchyma to the vasculature drastically reduces the tau pathology. The 3xTg-AD mice expressing the human apoE4 gene were virtually depleted of any somatodendritic tau deposits. These data strongly suggest that the somatodendritic tau accumulation is dependent on the parenchyma Aβ deposits.     

Isoform-specific effect of apolipoprotein E on endocytosis of beta-amyloid in cultures of neuroblastoma cells

Investigation of the interactions of nerve cells with human apolipoprotein E (apoE), beta-amyloid (Abeta), and their complex, which are known to be included in senile plaques, is necessary to clarify the functional role of apoE in the pathogenesis of Alzheimer's disease. Using flow cytometric analysis, we investigated the isoform-specific effects of apoE on the endocytosis of Abeta in cultured neuroblastoma cells. The level of internalized Abeta within the cells was dependent on the culture time and the kind of apoE isoform present. Both apoE3 and apoE4 enhanced the internalization of Abeta; however, no difference was observed between their effects. The internalized Abeta was hardly catabolized at all in the presence of apoE4, while rapid clearance of Abeta was observed in the presence of apoE3. Unlike apoE3 and apoE4, apoE2 had no effect on Abeta clearance from the media. The isoform-specific effects of apoE on the endocytosis of Abeta may be implicated in the development of Alzheimer's disease, and if so, each isoform of apoE would induce a different incidence of that disease.     

L-3-n-butylphthalide regulates amyloid precursor protein processing by PKC and MAPK pathways in SK-N-SH cells over-expressing wild type human APP695

Amyloid precursor protein (APP) is cleaved by α-secretase, within the amyloid-β (Aβ) sequence, resulting in the release of a secreted fragment (αAPPs) and precluding Aβ production. We investigated the effects of a promising anti-AD new drug, l-3-n-butylphthalide (L-NBP), on APP processing and Aβ generation in neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. L-NBP significantly increased αAPPs release, and reduced Aβ generation. The steady-state full-length APP levels were unaffected by L-NBP. It suggested that L-NBP regulated APP processing towards to the non-amyloidogenic α-secretase pathway. Protein kinase C (PKC) and mitogen activated protein (MAP) kinase might be involved in L-NBP-induced αAPPs secretion. L-NBP significantly increased PKCα and ? activations, lowered PKCγ activation and increased the phosphorylation of p44/p42 MAPK. Furthermore, PKC and MAPK inhibitors partially reduced L-NBP-induced αAPPs secretion. The results suggested alternative pharmacological mechanisms of L-NBP regarding the treatment of Alzheimer's disease (AD).     

Comparison of astrocytic and myocytic metabolic dysregulation in apolipoprotein E deficient and human apolipoprotein E transgenic mice

The accumulation of tubular aggregates in type II skeletal muscle fibres and fibrillo-granular inclusions in hippocampal protoplasmic astrocytes are characteristic lesions of apolipoprotein E deficient mice. Moreover these inclusions reacted immunocytochemically with an antibody specific to fragment 17-24 of the published sequence of Alzheimer's amyloid peptide. In an effort to evaluate the role of apolipoprotein E in the formation of these abnormal structures, we examined the tibialis anterior muscle and the hippocampus of several groups of animals including: (i) apolipoprotein E "knockout" mice which had been whole body irradiated with 1200 rads and bone marrow replenished with apolipoprotein E sufficient marrow; and (ii) three transgenic murine strains that had been genetically engineered to express either human apolipoprotein E2, E3 or E4 protein on an apoE deficient background. The results of this study showed that the presence of murine apolipoprotein E (even in subnormal levels in the serum) in irradiated bone marrow replenished mice and in all three (E2, E3 or E4) human apoE transgenic strains was sufficient to prevent the aggregation of sarcoplasmic tubules in the tibialis anterior type II muscle fibres. Similarly apolipoprotein E "knockout" bone marrow replenished mice and all three transgenic strains expressing the different human apolipoprotein E alleles reduced the number of the astrocytic inclusions in the hippocampus to levels not significantly different to those observed in control C57Bl6J animals.The data obtained in this study indicate that neurological and neuromuscular abnormalities found in apoE deficient mice are reversed when apoE protein is replaced in the circulation, either by bone marrow transplantation of normal apoE sufficient marrow, or by gene therapy with the apoE gene, albeit of human origin and irrespective of the allele used.     

Apolipoprotein E is found in astrocytes but not in microglia in the normal mouse brain

Apolipoprotein E (apoE) is a known risk factor for Alzheimer's disease, but neither its roles in the pathogenesis nor its exact physiological functions in the brain is known. In order to study the apoE protein in the brains of normal mouse and transgenic mouse models of neurodegeneration, hamster monoclonal antibodies (MAbs) specific to mouse apoE were generated. N- and C-terminal fragments of mouse apoE protein were produced in E. coli as fusion proteins and used to immunize Armenian hamsters. Specificity of the antibodies was established by immunoblotting against sera and brain homogenates of wild type and apoE-deficient mice. MAb 884F11 was found most suitable for immunohistochemistry on 4% PFA-fixed brain tissues. The strongly positive structure in the normal brain was the astrocytes as identified by simultaneous staining for GFAP with lesser and regionally variable diffuse staining of the neuropil. GFAP-positive cells were variable in their content of apoE. ApoE immunoreactivity in the hippocampus and neocortex did not coincide with the tomato lectin binding, indicating that this apolipoprotein is not detectable in the microglial cells of the normal adult mouse brain.     

Apolipoprotein E4 isoform-specific actions on neuronal cells in culture.

Apolipoprotein E (apoE) allele epsilon4 is a major risk factor for Alzheimer's disease (AD); however, the molecular mechanism underlying the acceleration of the development of AD in patients possessing epsilon4 remains to be determined. To investigate the isoform-specific effects of apoE on neurons, primary neuron cultures were prepared from fetal rat cerebral cortices. Inhibition of de novo cholesterol synthesis by compactin, a 3-hydroxyl-3-methylglutaryl CoA reductase inhibitor, induced neuronal cell death in a dose dependent manner. In the presence of a sublethal dose of compactin, apoE4 with beta-migrating very low density lipoproteins (beta-VLDL) caused apoptotic cell death in neuronal cultures. The same results were obtained with inhibition of de novo cholesterol synthesis by sublethal doses of squalestatin, an inhibitor of squalene synthase. The de novo cholesterol synthesis was suppressed to a higher degree by apoE4 than by apoE3, administered with beta-VLDL in the presence or absence of compactin. Mevalonate and squalene, which are metabolites of the cholesterol synthesis pathway, protected neuronal cells from apoE4-induced cell death. These results may suggest that apoE4 may exhibit neurotoxic action when de novo cholesterol synthesis is suppressed to a certain level, and that apoE4 induces neuronal cell death through the suppression of de novo cholesterol synthesis via an undetermined isoform-specific mechanism.     

Increased levels of intracellular iron in the brains of ApoE-deficient mice with closed head injury.

Previous studies have revealed that apolipoprotein E (apoE)-deficient mice have distinct memory deficits and neurochemical derangements and are oxidatively stressed prior to and following closed head injury. The objective of this study was to evaluate the possibility that the enhanced susceptibility of apoE-deficient mice to closed head injury is related to impairments in their antioxidative iron-chelating mechanisms. ApoE-deficient and control mice were subjected to closed had injury, after which the extent of brain-damage and the level of iron-containing cells were assessed. Examination of the brain-damaged areas in the injured mice revealed that, by Day 3 post injury, animals of both groups were maximally and similarly affected. While the size of the damaged area of the injured control mice diminished significantly by Day 7, however recovery was not observed in injured apoE-deficient mice up to at least 14 days post-injury. Histopathologically, the decrease in the damaged areas in the control mice was interpreted as related to decreased edema. Numbers of iron-containing cells at Days 3 and 7 after injury were greater in the brains of control mice than in the apoE-deficient mice. Whereas the number of iron-containing cells in injured control mice decreased at days 9 and 14-post injury, that of the injured apoE-deficient mice plateaued by Day 9 at a level more than two-fold higher than the maximal level seen for controls. The size of the damaged areas and the number of iron-containing cells were correlated (P < 0.03) for both mouse groups at days 9 and 14 after injury. The data suggest that the increased susceptibility of apoE-deficient mice to closed head injury may be due, at least in part, to impaired iron scavenging and sustained oxidative stress.     

Apolipoprotein E isoform-specific regulation of dendritic spine morphology in apolipoprotein E transgenic mice and Alzheimer's disease patients

Dendritic spines are postsynaptic sites of excitatory input in the mammalian nervous system. Apolipoprotein (apo) E participates in the transport of plasma lipids and in the redistribution of lipids among cells. A role for apoE is implicated in regeneration of synaptic circuitry after neural injury. The apoE4 allele is a major risk factor for late-onset familial and sporadic Alzheimer's disease (AD) and is associated with a poor outcome after brain injury. ApoE isoforms are suggested to have differential effects on neuronal repair mechanisms. In vitro studies have demonstrated the neurotrophic properties of apoE3 on neurite outgrowth. We have investigated the influence of apoE genotype on neuronal cell dendritic spine density in mice and in human postmortem tissue. In order to compare the morphology of neurons developing under different apoE conditions, gene gun labeling studies of dendritic spines of dentate gyrus (DG) granule cells of the hippocampus were carried out in wild-type (WT), human apoE3, human apoE4 expressing transgenic mice and apoE knockout (KO) mice; the same dendritic spine parameters were also assessed in human postmortem DG from individuals with and without the apoE4 gene. Quantitative analysis of dendritic spine length, morphology, and number was carried out on these mice at 3 weeks, 1 and 2 years of age. Human apoE3 and WT mice had a higher density of dendritic spines than human E4 and apoE KO mice in the 1 and 2 year age groups (P<0.0001), while at 3 weeks there were no differences between the groups. These age dependent differences in the effects of apoE isoforms on neuronal integrity may relate to the increased risk of dementia in aged individuals with the apoE4 allele. Significantly in human brain, apoE4 dose correlated inversely with dendritic spine density of DG neurons cell in the hippocampus of both AD (P=0.0008) and aged normal controls (P=0.0015). Our findings provide one potential explanation for the increased cognitive decline seen in aged and AD patients expressing apoE4.     

Cryptotanshinone,a compound from Salvia miltiorrhiza modulates amyloid precursor protein metabolism and attenuates β-amyloid deposition through upregulating α-secretase in vivo and in vitro

The amyloid precursor protein (APP) is cleaved enzymatically by non-amyloidogenic and amyloidogenic pathways. α-Secretase cleaves APP within β-amyloid protein (Aβ) sequence, resulting in the release of a secreted fragment of APP (sAPPα) and precluding Aβ generation. Cryptotanshinone (CTS), an active component of the medicinal herb Salvia miltiorrhiza, has been shown to improve learning and memory in several pharmacological models of Alzheimer's disease (AD). However, the effects of CTS on the Aβ plaque pathology and the APP processing in AD are unclear. Here we reported that CTS strongly attenuated amyloid plaque deposition in the brain of APP/PS1 transgenic mice. In addition, CTS significantly improved spatial learning and memory in APP/PS1 mice assessed by the Morris water maze testing. To define the exact molecular mechanisms involved in the beneficial effects of CTS, we investigated the effects of the CTS on APP processing in rat cortical neuronal cells overexpressing Swedish mutant human APP695. CTS was found to decrease Aβ generation in concentration-dependent (0–10 μM) manner. Interestingly, the N-terminal APP cleavage product, sAPPα was markedly increased by CTS. Further study showed that α-secretase activity was increased by CTS. Taken together, our results suggested CTS improved the cognitive ability in AD transgenic mice and promoted APP metabolism toward the non-amyloidogenic products pathway in rat cortical neuronal cells. CTS shows a promising novel way for the therapy of AD.     

AMPA receptor regulation and LTP in the hippocampus of young and aged apolipoprotein E-deficient mice

In the present study, modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors by phosphatidylserine (PS) and synaptic plasticity were investigated in the hippocampus of young (4-month-old) and aged (18-month-old) apolipoprotein E (apoE)-deficient mice. Qualitative as well as quantitative analysis of brain sections in both young and aged apoE-deficient mice did not reveal any substantial changes of AMPA receptor binding in the various hippocampal regions, compared to age-matched controls. Nevertheless, enhancement of AMPA receptor binding elicited by PS treatment was found to be abolished in most hippocampal regions of young apoE-deficient mice, while modulation of AMPA receptors by this phospholipid was not significantly altered in the hippocampal formation of aged apoE-deficient animals. At the electrophysiological level, long-term potentiation (LTP) induced by theta burst stimulation was lower in area CA1 of the hippocampus of young, but not aged, apoE-deficient mice compared to age-matched controls. These results confirm that apoE is important for AMPA receptor regulation and LTP expression in the hippocampal formation. However, the presence of LTP in aged apoE-deficient animals, together with apparent recovery of the PS action on AMPA receptors, suggests that aged apoE-knockout mice possess compensatory mechanisms that reduce biochemical and electrophysiological alterations of glutamatergic neurons.     

Acute regression of advanced and retardation of early aortic atheroma in immunocompetent apolipoprotein-E (apoE) deficient mice by administration of a second generation [E1(-), E3(-), polymerase(-)] adenovirus vector expressing human apoE.

Apolipoprotein E (apoE) is a 34 kDa glycoprotein with multiple actions that help protect against the development of atherosclerosis. Here, we have assessed the atheroprotective potential of an [E1(-), E3(-), polymerase(-)] adenovirus vector expressing human apoE, comparing intramuscular and intravenous (liver-directed) injections in hypercholesterolaemic apoE-deficient mice (apoE(-/-)). Intramuscular injections resulted in low expression of apoE and afforded no protection against atherogenesis. In contrast, 3 and 7 days after intravenous injections into young (6-8-week-old) apoE(-/-) mice, plasma levels of apoE were elevated and were accompanied by reductions in plasma cholesterol and normalization of lipoprotein profiles. Thereafter, plasma apoE was still detectable up to day 70, but gradually declined, although no humoral immune response was evoked, and there was a return to dyslipoproteinaemia. High levels of the vector genome were still present in livers of treated animals at 70 days, implying that decrease in apoE expression was due to cellular shutdown of the cytomegalovirus promoter. Importantly, liver-directed apoE gene transfer to these young mice retarded progression of atherosclerosis by 38% (treated, 8.21 +/- 1.05%; untreated, 13.26 +/- 0.98%, P < 0.05), during the 70 day study period. Moreover, when 10-month-old apoE(-/-) mice with advanced atherosclerosis were treated with the adenovirus vector, there was clear regression of aortic lesion area by 1 month [24.3 +/- 1.7% compared to 40.7 +/- 2.6% in baseline controls (P < 0.002)]. We conclude that the stability of the adenovirus vector genome in the livers of intravenously treated animals provides an ideal platform to evaluate liver-specific promoters for sustained transgene expression and control of atherosclerotic lesion pathology.     

Amyloid A protein amyloidosis induced in apolipoprotein-E-deficient mice.

Apolipoprotein E (apoE) is a constituent of lipoproteins other than low-density lipoprotein, and it principally acts in the transport and metabolism of plasma cholesterol and triglyceride. ApoE is a minor constituent of various kinds of amyloidoses and may play a role as a pathological chaperone for fibrillogenesis of amyloid fibril protein with the amyloid P component and proteoglycans. In this study, we examined the role of apoE in amyloidogenesis in vivo in apoE-deficient mutant mice with amyloid A protein (AA) amyloidosis induced by inflammatory stimulation. Amyloid deposition was seen in six of nine C57BL/6J control mice and in six of eight apoE-deficient mutant mice after the intraperitoneal and subcutaneous injections of the mixture of complete Freund's adjuvant and Mycobacterium butyricum. Moreover, amyloid deposition in apoE-deficient mice as well as C57BL/6J control mice started 48 or 72 hours after injection of amyloid-enhancing factor and silver nitrate, although the amount of amyloid deposit in C57BL/6J control mice was slightly larger than that in apoE-deficient mice. These amyloid deposits reacted with anti-mouse AA antibody were seen in the perifollicular area of the spleen. Immunoreactivity of apoE was seen irregularly in the amyloid deposits of C57BL/6J control mice but not in the amyloid deposit of apoE-deficient mice. From these results, we concluded that apoE is not always necessary for amyloid deposition and that the existence of apoE might slightly accelerate AA amyloid deposition in the earliest phase of AA amyloid deposition.