E-deficient ApoE Research Articles

Dietary supplementation with Cyperus esculentus L (tiger nut) tubers attenuated atherosclerotic lesion in apolipoprotein E knockout mouse associated with inhibition of inflammatory cell responses

Atherosclerosis is a pathological process, where recruitment of mononuclear cells results in the growth of fibro-fatty plaques. Apolipoprotein E (ApoE) is a legend for the uptake of lipoproteins, and deficiency of ApoE leads to the accumulation of low-density lipoprotein. Utilizing ApoE-/- mouse, which spontaneously develop atherosclerosis on low-fat chow diets, the present study showed that feeding these mice on a diet supplemented with the whole tubers of Cyperus esculentus L (tiger nut) resulted in attenuation of the development of atherosclerotic lesions. The anti-atherosclerotic effect was associated with a decrease in the number of monocytes, macrophages and dendritic cells in blood, and the expression of IL-2Rα and LFA-1 by these cells. Further, in vitro proliferation of blood and spleen cells from tiger nut-fed ApoE-/- mice showed lower proliferation in responses to ConA and LPS, a T and B cell mitogen, respectively. Further, in vitro treatment of blood and spleen cells with water or ethanol extracts of tiger nut markedly increased their proliferation in response to ConA. Collectively, these data indicate that ingredients of tiger nut tubers exhibit anti-inflammatory properties upon inflammation, and immunostimulatory effects in immunocompetent hosts.

Role of ADAMTS-1 in Atherosclerosis

We investigated the potential role of ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motif type I) in atherogenesis. ADAMTS-1 is expressed at the highest levels in the aorta when compared with other human tissues examined. Immunolocalization studies in human aorta and coronary artery indicate that ADAMTS-1 expression is mainly seen at low levels in the medial layer, but upregulated in the intima when plaque is present. We found that ADAMTS-1 mRNA levels are significantly higher in proliferating/migrating cultured primary aortic vascular smooth muscle cells (VSMCs) compared with resting/confluent cells. Using the mouse carotid artery flow cessation model, we show that there are differences in vessel remodeling in ADAMTS-1 transgenic/apoE-deficient mice compared with apoE deficiency alone, particularly a significant increase in intimal hyperplasia. We show that ADAMTS-1 can cleave the large versican containing proteoglycan population purified from cultured human aortic VSMCs. Finally, using versican peptide substrates, we show data suggesting that ADAMTS-1 cleaves versican at multiple sites. We hypothesize that ADAMTS-1 may promote atherogenesis by cleaving extracellular matrix proteins such as versican and promoting VSMC migration.

Apolipoprotein E Modulates Clearance of Apoptotic Bodies In Vitro and In Vivo, Resulting in a Systemic Proinflammatory State in Apolipoprotein E-Deficient Mice

Apolipoprotein E (apoE) is a 34-kDa glycoprotein involved in lipoprotein transport through interaction with the low-density lipoprotein receptor and related receptors. Recently, it has become clear that apoE binding to its receptors plays a role both in development and in control of the immune system. In this study, we show that apoE modulates the rate of uptake of apoptotic cells by macrophages. In vitro, apoE-deficient macrophages ingest less apoptotic thymocytes (but not latex beads) than wild-type macrophages, and this defect can be corrected by addition of exogenous apoE protein. In vivo, the number of dying macrophages is increased in a range of tissues, including lung and brain. Possibly in response to the larger numbers of persistent apoptotic bodies, the number of live macrophages in these tissues are also increased compared with those of wild-type control mice. In addition to the significant changes in macrophage population dynamics we observed, levels of the proinflammatory cytokine TNF-alpha and the positive acute phase reactant fibrinogen are also elevated in the livers from apoE-deficient mice. In contrast, neither deletion of the gene encoding the LDL receptor nor cholesterol feeding of wild-type mice affected either the number of apoptotic bodies or the number of live macrophages. We conclude that apoE deficiency results in impaired clearance of apoptotic cell remnants and a functionally relevant systemic proinflammatory condition in mice, independent of its role in lipoprotein metabolism. Any similar reduction of apoE activity in humans may contribute to the pathogenesis of a wide range of chronic diseases including atherosclerosis, dementia, and osteoporosis.

Apolipoprotein E delivery by peritoneal implantation of encapsulated recombinant cells improves the hyperlipidaemic profile in apoE-deficient mice

Plasma apolipoprotein E (apoE) is a 34-kDa polymorphic protein which has atheroprotective actions by clearing remnant lipoproteins and sequestering excess cellular cholesterol. Low or dysfunctional apoE is a risk factor for hyperlipidaemia and atherosclerosis, and for restenosis after angioplasty. Here, in short-term studies designed to establish proof-of-principle, we investigate whether encapsulated recombinant Chinese hamster ovary (CHO) cells can secrete wild-type apoE3 protein in vitro and then determine whether peritoneal implantation of the microcapsules into apoE-deficient (apoE −/−) mice reduces their hypercholesterolaemia. Recombinant CHO-E3 cells were encapsulated into either alginate poly- l-lysine or alginate polyethyleneimine/polybrene microspheres. After verifying stability and apoE3 secretion, the beads were then implanted into the peritoneal cavity of apoE −/− mice; levels of plasma apoE3, cholesterol and lipoproteins were monitored for up to 14 days post-implantation. Encapsulated CHO-E3 cells continued to secrete apoE3 protein throughout a 60-day study period in vitro, though levels declined after 14 days. This cell-derived apoE3 was biologically active. When conditioned medium from encapsulated CHO-E3 cells was incubated with cultured cells pre-labelled with [ 3H]-cholesterol, efflux of cholesterol was two to four times greater than with normal medium (at 8 h, for example, 7.4±0.3% vs. 2.4±0.2% of cellular cholesterol; P<0.001). Moreover, when secreted apoE3 was injected intraperitoneally into apoE −/− mice, apoE3 was detected in plasma and the hyperlipidaemia improved. Similarly, when alginate polyethyleneimine/polybrene capsules were implanted into the peritoneum of apoE −/− mice, apoE3 was secreted into plasma and at 7 days total cholesterol was reduced, while atheroprotective high-density lipoprotein (HDL) increased. In a second study, apoE was detectable in plasma of five mice treated with alginate poly- l-lysine beads, 4 and 7 days post-implantation, though not at day 14. Furthermore, their hypercholesterolaemia was reduced, while HDL was clearly elevated in all mice at days 4 and 7 (from 18.4±6.2% of total lipoproteins to 31.1±6.8% at 7 days; P<0.001); however, these had rebounded by day 14, possibly due to the emergence of anti-apoE antibodies. We conclude that microencapsulated apoE-secreting cells have the potential to ameliorate the hyperlipidaemia of apoE deficiency, but that the technology must be improved to become a feasible therapeutic to treat atherosclerosis.

Effects of high cholesterol diet on gliosis in apolipoprotein E knockout mice: Implications for Alzheimer’s disease and stroke

Hypercholesterolemia has been suggested as a risk factor for Alzheimer’s disease (AD). A genetic risk factor for AD is the E4 allele of apolipoprotein E (apoE). ApoE is the major lipoprotein transporter in the brain, and is mainly produced by glial cells. The present study is focussed on analysing the effects of high cholesterol (HC) diet, duration 9 months, on glial activation in the brain, both in wild type (WT) mice and in mice with a null mutation in the apoE gene (knock-out, KO) mice. The activation of astrocytes and microglia was analysed after immunohistochemical labelling of glial fibrillary acidic protein (GFAP), and F4/80, respectively. In addition, the expression of the antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO1) was analysed. There was a marked stimulation of astrocyte and microglial activation as well as induced expression of NQO1 in the hippocampus and cerebral cortex upon HC diet. Furthermore, there was significant astrocyte activation in the apoE KO mice, as compared to the WT mice, on ND. The long time exposure to HC diet combined with apoE deficiency resulted in a synergistic effect on the expression of NQO1 in the brain.

Abnormal in vivo metabolism of apoB-containing lipoproteins in human apoE deficiency

The present study was undertaken to elucidate the metabolic basis for the increased remnants and lipoprotein(a) [Lp(a)] and decreased LDL apolipoprotein B (apoB) levels in human apoE deficiency. A primed constant infusion of (13)C(6)-phenylalanine was administered to a homozygous apoE-deficient subject. apoB-100 and apoB-48 were isolated, and tracer enrichments were determined by gas chromatography-mass spectrometry, then kinetic parameters were calculated by multicompartmental modeling. In the apoE-deficient subject, fractional catabolic rates (FCRs) of apoB-100 in VLDL and intermediate density lipoprotein and apoB-48 in VLDL were 3x, 12x, and 12x slower than those of controls. On the other hand, the LDL apoB-100 FCR was increased by 2.6x. The production rate of VLDL apoB-100 was decreased by 45%. In the Lp(a) kinetic study, two types of Lp(a) were isolated from plasma with apoE deficiency: buoyant and normal Lp(a). (125)I-buoyant Lp(a) was catabolized at a slower rate in the patient. However, (125)I-buoyant Lp(a) was catabolized at twice as fast as (131)I-normal Lp(a) in the control subjects. In summary, apoE deficiency results in: 1) a markedly impaired catabolism of VLDL/chylomicron and their remnants due to lack of direct removal and impaired lipolysis; 2) an increased rate of catabolism of LDL apoB-100, likely due to upregulation of LDL receptor activity; 3) reduced VLDL apoB production; and 4) a delayed catabolism of a portion of Lp(a).

Differential susceptibity of transgenic mice lacking one or both apolipoprotein alleles to folate and vitamin E deprivation.

The E4 allele of apolipoprotein E (ApoE) is associated with neurodegeneration in part due to increased oxidative stress. Transgenic mice lacking ApoE (-/-) represent a model for the consequences of deficiencies in ApoE function. Dietary deficiency in folate and vitamin E has previously been shown to potentiate the impact of ApoE deficiency; ApoE-/- mice deprived of folate and vitamin E for 1 month demonstrated increased oxidative damage in brain tissue and impaired cognitive performance as compared to ApoE+/+ mice. Since individuals homozygous for E4 can demonstrate more increased risk for neurodegeneration and an earlier age of onset than individuals heterozygous for E4, we tested the impact of folate and vitamin E deprivation on ApoE+/- mice. Thiobarbituric acid-reactive substances in brain tissue of ApoE+/- were significantly increased compared to ApoE+/+ mice, but this increase was less than that observed in ApoE-/- mice. By contrast, livers of ApoE+/- and -/- mice displayed an identical increase over that of +/+ mice. ApoE-/- mice, but not +/- or +/+ mice, exhibited impaired cognitive performance in maze trials when deprived of folate and vitamin E. These findings support the notion that homozygous deficiency of ApoE function can be more severe than heterozygous deficiency. They further suggest that the impact of partial deficiency in ApoE function may present a latent risk that may manifest only when compounded by other factors such as dietary deficiency.

Effect of macrophage-derived apolipoprotein E on hyperlipidemia and atherosclerosis of LDLR-deficient mice

LDL receptor-deficient (LDLR −/−) mice fed a Western diet exhibit severe hyperlipidemia and develop significant atherosclerosis. Apolipoprotein E (apoE) is a multifunctional protein synthesized by hepatocytes and macrophages. We sought to determine effect of macrophage apoE deficiency on severe hyperlipidemia and atherosclerosis. Female LDLR −/− mice were lethally irradiated and reconstituted with bone marrow from either apoE −/− or apoE +/+ mice. Four weeks after transplantation, recipient mice were fed a Western diet for 8 weeks. Reconstitution of LDLR −/− mice with apoE −/− bone marrow resulted in a slight reduction in plasma apoE levels and a dramatic reduction in accumulation of apoE and apoB in the aortic wall. Plasma lipid levels were unaffected when mice had mild hyperlipidemia on a chow diet, whereas IDL/LDL cholesterol levels were significantly reduced when mice developed severe hyperlipidemia on the Western diet. The hepatic VLDL production rate of mice on the Western diet was decreased by 46% as determined by injection of Triton WR1339 to block VLDL clearance. Atherosclerotic lesions in the proximal aorta were significantly reduced, partially due to reduction in plasma total cholesterol levels (r=0.56; P<0.0001) . Thus, macrophage apoE-deficiency alleviates severe hyperlipidemia by slowing hepatic VLDL production and consequently reduces atherosclerosis in LDLR −/− mice.

Macrophage apolipoprotein A-I expression protects against atherosclerosis in ApoE-Deficient mice and up-regulates ABC transporters

The antiatherogenic effect of high-density lipoprotein (HDL) and its major protein component apolipoprotein A-I (apoA-I) has been largely attributed to their key roles in reverse cholesterol transport (RCT) and cellular cholesterol efflux. Substantial evidence shows that overexpression of human apoA-I reduces atherosclerosis in animal models. However, it is uncertain whether this protection is due to an increase in plasma HDL level or to a local effect in the artery wall. To test the hypothesis that expression of human apoA-I in macrophages can promote RCT in the artery wall, we used a retroviral construct expressing human apoA-I cDNA (MFG-HAI) to transduce ApoE−/− bone marrow cells and then transplanted these cells into ApoE−/− mice with preexisting atherosclerosis. ApoE−/− mice reconstituted with MFG-HAI marrow had a significant reduction (30%) in atherosclerotic lesions in the proximal aorta compared to control mice that received marrow expressing MFG parental virus. Peritoneal macrophages isolated from MFG-HAI mice showed a four- to fivefold increase in mRNA expression levels of both ATP-binding cassette (ABC) A1 and ABCG1 compared to controls. Our data demonstrate that gene transfer-mediated expression of human apoA-I in macrophages can compensate in part for apoE deficiency and delay the progression of atherosclerotic lesions by stimulating ABC-dependent cholesterol efflux and RCT.

Circulating progenitor cells regenerate endothelium of vein graft atherosclerosis, which is diminished in ApoE-deficient mice.

Previously we showed that a large number of endothelial cells in vein grafts undergo apoptosis or necrosis during the first few days followed by endothelial regeneration. In the present study, we investigated endothelial cell death and regeneration in vein grafts using transgenic mice carrying LacZ genes driven by an endothelial TIE2 promoter. When a vein fragment from TIE2-LacZ was isografted into the carotid artery of wild-type mice, the number of beta-gal+ cells were reduced at 3 days and disappeared completely by 4 weeks after grafting. Conversely, beta-gal+ cells were observed on the surface of vein segments donated by wild-type mice isografted into TIE2-LacZ mice at 1 week and reached confluence by 4 weeks, suggesting recipient origins of endothelial cells. Interestingly, beta-gal+ cells were evenly distributed on the surface of the whole vein segment grafted into TIE2-LacZ mice, indicating a contribution of circulating progenitor cells. When wild-type veins were grafted into a chimeric mouse carrying TIE2-LacZ genes in bone marrow cells, a proportion of cells displayed a beta-gal+ staining. Furthermore, the number of CD34+ and Flk+ progenitor cells in blood of apoE-deficient mice were significantly lower than those of wild-type controls, which coincided with diminished beta-gal+ endothelial cells on the surface of vein grafts in TIE2-LacZ/apoE-/- mice. Thus, we provide the first evidence that endothelial cells of vein grafts are derived from circulating progenitor cells, of which one-third are derived from bone marrow progenitor cells. Hyperlipidemia due to apoE deficiency results in a lower number of endothelial progenitors in blood and correlated with enhanced atherosclerosis. The full text of this article is available online at http://www.circresaha.org.

Olfactory function in apoE knockout mice

Apolipoprotein E (apoE), a lipid transporting protein, has been shown to play a vital role in nerve repair and remodeling. Since the olfactory system is in a continuous state of remodeling, the present study tested the hypothesis that apoE is required for normal functioning of the olfactory system. Olfactory behavior of wild-type (WT) and apoE-deficient (apoE KO) mice was assessed by using three standard olfactory tests: (1) the buried food pellet (BFP) test; (2) the odor choice (OC) test; and (3) the odor cued taste avoidance (OCTA) test. ApoE KO mice performed poorly in all the three tests as compared to WT mice, although they learned the tasks at a rate comparable to WT mice. ApoE KO mice had a significantly longer latency to find the buried pellet than WT mice. In the OC experiment, apoE KO mice did not differentiate water from an odorant solution. Furthermore, in the OCTA test the apoE KO mice were significantly less successful than WT mice at avoiding water containing an odorant and a bad tastant. These data demonstrate that apoE deficiency in apoE KO mice leads to a deficit in olfactory function, suggesting an important role for apoE in the olfactory system.

Reduced atherosclerosis in interleukin-18 deficient apolipoprotein E-knockout mice

Atherosclerosis is an inflammatory disease in which T helper 1 (Th1) immunity has been proposed to play an important role. Nai;ve CD4+ T cells differentiate into interferon-gamma (IFN-gamma) producing Th1 effector cells when stimulated by interleukin-18 (IL-18) and IL-12. We wanted to directly test whether the Th1 pathway is proatherogenic. We bred IL-18(-/-) mice with apolipoprotein E(-/-) (apoE(-/-)) mice and assessed atherosclerosis in the aortic root of the offspring. 24-week-old IL-18 deficient apoE(-/-) mice exhibited substantially reduced lesion size (93,866+/-11273 vs. 144,019+/-9667 microm(2) in IL-18(+/+)xapoE(-/-) mice, P=0.005). Lesion cells in compound knockout mice displayed reduced I-A(b) expression, implying reduced local IFN-gamma stimulation. These mice also had an increased proportion of alpha-SM-actin+ smooth muscle cells, compatible with a more stable lesion phenotype. Immunoglobulin G (IgG) subclass analysis of antibodies to malondialdehyde-modified low density lipoprotein indicated increased Th2 and reduced Th1 helper to B cell antibody production. Surprisingly, serum cholesterol and triglyceride levels were significantly higher in IL-18(-/-)xapoE(-/-) mice in spite of their reduced atherosclerosis. However, no changes in lipoprotein cholesterol patterns were registered. These data show reduced atherosclerosis and Th1 activity in spite of increased serum cholesterol in IL-18 deficient apoE(-/-) mice. They support a proatherogenic role for IL-18.

Apolipoprotein E protects against oxidative stress in mixed neuronal–glial cell cultures by reducing glutamate toxicity

Apolipoprotein E (ApoE) deficiency has been shown to adversely affect outcome after transient cerebral ischemia and head trauma. Since oxidative stress contributes to these injuries, the ability of ApoE to reduce irreversible oxidative damage was studied in primary mixed neuronal–glial cell cultures. Cells (13–16 days in vitro) were exposed to 50 μM hydrogen peroxide (H 2O 2) for 30 min, and toxicity was determined by the release of lactate dehydrogenase (LDH) 24 h after exposure. The presence of recombinant human ApoE2 (100, 300, or 1000 nM) in the culture media partially protected against oxidative injury. This protection was not reversed by pre-treatment with receptor associated protein. The NMDA receptor antagonist, MK-801, also provided partial protection against H 2O 2 toxicity. The degree of protection was similar to that conferred by ApoE treatment. The protective effects of ApoE and MK-801 were not additive; no ApoE protection was observed in cultures treated with MK-801 prior to H 2O 2 exposure. ApoE treatment had no effect on H 2O 2 stimulated glutamate release, but did increase the rate of glutamate uptake via the high affinity glutamate transporter in H 2O 2 treated cultures. Pre-treatment with ApoE also conferred partial protection against glutamate-induced LDH release. Taken together, these findings suggest that ApoE protects mixed neuronal–glial cell cultures against irreversible oxidative injury from H 2O 2 by reducing secondary glutamate excitotoxicity.

Altered adrenal gland cholesterol metabolism in the apoE-deficient mouse

Previous studies suggest the hypothesis that apoE produced by adrenocortical cells modulates cellular cholesterol metabolism to enhance the storage of esterified cholesterol (EC) at the expense of cholesterol delivery to the steroidogenic pathway. In the present study, parameters of adrenal cholesterol metabolism and corticosteroid production were examined in wild type and apoE-deficient (apoe(-/-)) mice. Adrenal gland EC content and the EC/free cholesterol (FC) ratio in mice stressed by adrenocorticotropin (ACTH) treatment or saline injection were reduced in apoe(-/-) compared to apoe(+/+) mice. Relative to apoe(+/+) mice, apoE deficiency also resulted in increased levels of plasma corticosterone in the basal state, in response to acute or long-term ACTH treatment, and after a swim-induced neuroendocrine-directed stress test. Measurements of adrenal gland scavenger receptor class B, type I (SR-BI), LDL receptor, and LDL receptor related protein (LRP) levels and the activities of ACAT or HMG-CoA reductase showed no difference between genotypes. Apoe(-/-) and apoe(+/+) mice showed similar quantitative increases in LDL receptors, SR-BI, adrenal weight gain, and ACAT activities in response to ACTH, and both genotypes had similar basal plasma ACTH concentrations. These results suggest that the effects of apoE deficiency reflect events at the level of the adrenal gland and are specific to changes in cholesterol accumulation and corticosterone production. Further, these findings support the hypothesis that apoE acts to enhance adrenocortical EC accumulation and diminish corticosterone production.

Apolipoprotein E deficiency promotes increased oxidative stress and compensatory increases in antioxidants in brain tissue.

The epsilon 4 allele of the apolipoprotein E gene (ApoE) is associated with Alzheimer's disease (AD). The extent of oxidative damage in AD brains correlates with the presence of the E4 allele of ApoE, suggesting an association between the ApoE4 genotype and oxygen-mediated damage in AD. We tested this hypothesis by subjecting normal and transgenic mice lacking ApoE to oxidative stress by folate deprivation and/or excess dietary iron. Brain tissue of ApoE-deficient mice displayed increased glutathione and antioxidant levels, consistent with attempts to compensate for the lack of ApoE. Folate deprivation and iron challenge individually increased glutathione and antioxidant levels in both normal and ApoE-deficient brain tissue. However, combined treatment with folate deprivation and dietary iron depleted antioxidant capacity and induced oxidative damage in ApoE-deficient brains despite increased glutathione, indicating an inability to compensate for the lack of ApoE under these conditions. These data support the hypothesis that ApoE deficiency is associated with oxidative damage, and demonstrate a combinatorial influence of genetic predisposition, dietary deficiency, and oxidative stress on oxidative damage relevant to AD.

Alteration of nitric oxide synthase activity in young and aged apolipoprotein E-deficient mice

Impairments in cognitive performance have been observed in aged apolipoprotein E (apoE)-deficient mice, and apoE ε4 allele is a risk factor in Alzheimer’s disease (AD). The absence of apoE correlates with diminished antioxidative capacity in animals, and elevated cerebral oxidative stress has been observed in AD individuals carrying the ε4 alleles. Nitric oxide (NO) is a neurosignaling molecule that has significant roles in cognition. NO has also been implicated in neurodegenerative diseases due to its oxidative properties. The current study examined the possible relationship between apoE and nitric oxide synthase (NOS) by comparing hippocampal and cortical NOS activities in wild-type and apoE-knockout mice. Our results showed that apoE deficiency had no effect on NOS activity in these animals; however, aged animals uniformly exhibited significantly higher NOS activity levels. These findings suggest that increased NOS activity may contribute to cognitive impairments in aged wild-type and apoE-knockout mice due to excess accumulation of oxidative damages in areas involved in learning and memory.

Extracellular proteolysis in the development and progression of atherosclerosis

Clinical complications of atherosclerosis are often triggered by the rupture of unstable plaques, while thinning of the atherosclerotic vessel wall owing to elastin and collagen degradation and media necrosis may result in aneurysm formation and bleeding. Proteolysis, mediated via the plasminogen/ plasmin and/or matrix metalloproteinase (MMP) systems may contribute to neovascularization and rupture of plaques, or to ulceration and rupture of aneurysms. In an in vivo model of atherosclerosis, using mice that had a combined deficiency of apolipoprotein E (ApoE) and urokinase-type plasminogen activator (u-PA) and that were maintained on a cholesterol-rich diet, it was observed that u-PA deficiency protects against aneurysm formation. This was explained by the findings that plasmin, generated from plasminogen by u-PA, activates several macrophage-secreted proMMPs (e.g. proMMP-3, −9, −12 and −13), which in turn cause extracellular matrix degradation. A potential role for MMP-3 (stromelysin-1) was confirmed in a subsequent study using mice with a combined deficiency of ApoE and MMP-3, that were kept on a cholesterol-rich diet. The results suggest that MMP-3 contributes to plaque destabilization, possibly by degrading extracellular matrix components, but also promotes aneurysm formation by degrading the elastic lamina. These effects may be mediated by MMP-3 directly or by activation of other proMMPs or other (proteolytic) systems. A functional role of MMPs is further supported by the finding that deficiency in TIMP-1 (tissue inhibitor of MMPs type 1) reduces atherosclerotic plaque size but enhances aneurysm formation. Taken together, these results suggest that u-PA has an important role in the structural integrity of the atherosclerotic vessel wall, which is likely to involve triggering the activation of MMPs and, furthermore, they suggest that increased u-PA levels are a risk factor for aneurysm formation.

A phenotype-sensitizing Apoe-deficient genetic background reveals novel atherosclerosis predisposition loci in the mouse.

Therapeutic intervention for atherosclerosis has predominantly concentrated on regulating cholesterol levels; however, these therapeutics are not efficacious for all patients, suggesting that other factors are involved. This study was initiated to identify mechanisms that regulate atherosclerosis predisposition in mice other than cholesterol level regulation. To do so we performed quantitative trait locus analysis using two inbred strains that each carry the atherosclerosis phenotype-sensitizing Apoe deficiency and that have been shown to have widely disparate predilection to atherosclerotic lesion formation. One highly significant locus on chromosome 10 (LOD = 7.8) accounted for 19% of the variance in lesion area independent of cholesterol. Two additional suggestive loci were identified on chromosomes 14 (LOD = 3.2) and 19 (LOD = 3.2), each accounting for 7-8% of the lesion variance. In all, five statistically significant and suggestive loci affecting lesion size but not lipoprotein levels were identified. Many of these were recapitulated in an independent confirmatory cross. In summary, two independently performed crosses between C57BL/6 and FVB/N Apoe-deficient mice have revealed several previously unreported atherosclerosis susceptibility loci that are distinct from loci linked to lipoprotein levels.

Apolipoprotein E expression and behavioral toxicity of high charge, high energy (HZE) particle radiation.

Sixteen male apoE knockout (KO) mice and sixteen genetically matched wild-type (WT) C57BL mice were used in this experiment. Half of the KO and half of the WT animals were irradiated with 600 MeV/amu iron particles (2 Gy whole body). The effect of irradiation on motor coordination and stamina (Rotarod test), exploratory behavior (open field test), and spatial working and reference memory (Morris water maze) was assessed. Performance was adversely affected by radiation exposure in both KO and WT groups at 30 d after irradiation. By 60 d after radiation, the radiation effect was lost in WT, but still apparent in irradiated KO mice. Radiation reduced open field exploratory activity 14, 28, 56, 84, and 168 d after irradiation of KO mice, but had no effect on WT mice. Radiation adversely affected spatial working memory in the KO mice, but had no discernible effect in the WT mice as assessed 180 d after irradiation. In contrast, irradiated WT mice showed marked impairment of spatial reference memory in comparison to non-irradiated mice, while no effect of radiation was observed in KO mice. These studies show that apoE expression influences the behavioral toxicity of HZE particle radiation and suggest that apoE plays a role in the repair/recovery from radiation injury of the CNS. ApoE deficiency may exacerbate the previously reported effects of HZE particle radiation in accelerating the brain aging process.

Peripheral nerve regeneration in the apolipoprotein-E-deficient mouse.

Apolipoprotein E (apo E) is thought to mediate the reutilization of myelin cholesterol for nerve regeneration. Prior research suggests that apo E is not essential for nerve regeneration following a nerve crush injury. This study was conducted to determine if apo E is essential for nerve regeneration after nerve transection and interposition nerve autograft. Nerve regeneration of transgenic apo E-deficient mice was compared with control mice after a sciatic nerve neurolysis and repair and interposition autograft. Histomorphometric assessment and histology were performed on distal nerve segments to evaluate nerve regeneration. Apo E-deficient mice demonstrated no difference in total fiber number or nerve fiber width when compared with controls; however, the nerve fiber density and percent neural tissue of apo E-deficient mice were significantly less than controls following nerve repair. Apo E deficiency does not affect nerve regeneration. It is likely that the low nerve fiber density and the low percent neural tissue associated with apo E-deficiency result from impairment in the disposal of myelin debris.