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M F Linton

Publications and source records attributed to M F Linton.

At least 37 records · Page 2Linked to original sources
PubMed1999

Increased cerebral cortical lipid peroxidation and abnormal phospholipids in aged homozygous apoE-deficient C57BL/6J mice.

Aged homozygous apolipoprotein E gene-deficient (apoE -/-) mice have been proposed as an experimental model for the role of human apoE isoforms in Alzheimer's disease (AD). However, results from different laboratories have been in conflict regarding the presence or absence of neurodegeneration in these mice. Moreover, despite apoE being the major lipid trafficking molecule in the central nervous system, there has been no investigation of brain lipid levels in apoE -/- mice. Here we have examined male and female apoE -/- and control mice aged 10 to 12 months, testing the hypothesis that lack of apoE leads to some of the neuropathological changes seen in AD. Our results failed to demonstrate significant neurodegeneration, histopathological changes, or reduction in cerebral cortical synaptophysin in apoE -/- mice. However, we did observe a significant reduction in cerebral cortical phospholipids and their constituent fatty acids, as well as elevated lipid peroxidation products, in apoE -/- mice compared to apoE +/+ mice with the same genetic background. Our results suggest that the brains of aged apoE -/- mice display some of the lipid abnormalities associated with AD; however, these changes alone, at the magnitudes achieved in the apoE -/- mice, do not directly lead to the major neurodegenerative changes of AD.

Aging↗
PubMed1999

Macrophages, lipoprotein metabolism, and atherosclerosis: insights from murine bone marrow transplantation studies.

The macrophage expresses a variety of genes that may contribute to lipoprotein metabolism and atherosclerotic lesion formation. Bone marrow transplantation experiments using gene-targeted mice, both as donors and recipients, provide a useful approach to examine the contribution of macrophage gene expression to lipoprotein metabolism and atherogenesis in vivo. This article describes recent insights into the role of macrophage expression of apolipoprotein E and the LDL receptor gained from bone marrow transplantation studies in the mouse.

Animals↗
PubMed1999

Disruption of the 12/15-lipoxygenase gene diminishes atherosclerosis in apo E-deficient mice.

Atherosclerosis may be viewed as an inflammatory disease process that includes early oxidative modification of LDLs, leading to foam cell formation. This "oxidation hypothesis" has gained general acceptance in recent years, and evidence for the role of lipoxygenases in initiation of, or participation in, the oxidative process is accumulating. However, the relative contribution of macrophage-expressed lipoxygenases to atherogenesis in vivo remains unknown. Here, we provide in vivo evidence for the role of 12/15-lipoxygenase in atherogenesis and demonstrate diminished plasma IgG autoantibodies to oxidized LDL epitopes in 12/15-lipoxygenase knockout mice crossbred with atherosclerosis-prone apo E-deficient mice (apo E-/-/L-12LO-/-). In chow-fed 15-week-old apo E-/-/L-12LO-/- mice, the extent of lesions in whole-aorta en face preparations (198 +/- 60 microm2) was strongly reduced (P < 0.001, n = 12) when compared with 12/15-lipoxygenase-expressing controls (apo E-/-/L-12LO+/+), which showed areas of lipid deposition (15,700 +/- 2,688 microm2) in the lesser curvature of the aortic arch, branch points, and in the abdominal aorta. These results were observed despite cholesterol, triglyceride, and lipoprotein levels that were similar to those in apo E-deficient mice. Evidence for reduced lesion development was observed even at 1 year of age in apo E-/-/L-12LO-/- mice. The combined data indicate a role for 12/15-lipoxygenase in the pathogenesis of atherosclerosis and suggest that inhibition of this enzyme may decrease disease progression.

Animals↗
PubMed1999

Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in vivo.

Expression of lipoprotein lipase (LPL) by the macrophage has been proposed to promote foam cell formation and atherosclerosis, primarily on the basis of in vitro studies. LPL-deficient mice might provide a model for testing the role of LPL secretion by the macrophage in an in vivo system. Unfortunately, homozygous deficiency of LPL in the mouse is lethal shortly after birth. Because the fetal liver is the major site of hematopoiesis in the developing fetus, transplantation of C57BL/6 mice with LPL-/- fetal liver cells (FLCs) was used to investigate the physiologic role of macrophage LPL expression in vivo. Thirty-four female C57BL/6 mice were lethally irradiated and reconstituted with FLCs from day 14 LPL+/+, LPL+/-, and LPL-/- donors. No significant differences were detected in plasma levels of post-heparin LPL activity or in serum cholesterol or triglyceride levels between the 3 groups on either a chow diet or an atherogenic diet. After 19 weeks on the atherogenic diet, aortae were collected for quantitative analysis of the extent of aortic atherosclerosis. LPL expression was detected by immunocytochemistry and in situ hybridization in macrophages of aortic atherosclerotic lesions of LPL+/+-->C57BL/6 and LPL+/--->C57BL/6 mice, but not in LPL-/--->C57BL/6 mice, whereas myocardial cells expressed LPL in all groups. The mean aortic lesion area was reduced by 55% in LPL-/--->C57BL/6 mice compared with LPL+/+-->C57BL/6 mice and by 45% compared with LPL+/--->C57BL/6 mice, respectively. These data demonstrate in vivo that LPL expression by macrophages in the artery wall promotes foam cell formation and atherosclerosis. off

Animals↗
PubMed1999

Determination of the lower threshold of apolipoprotein E resulting in remnant lipoprotein clearance.

Apolipoprotein E (apoE) is the ligand for receptor-mediated clearance of remnant lipoproteins. ApoE at concentrations only 10% of normal, achieved through transplantation of wild-type marrow into apoE(-/-) mice, is sufficient for the maintenance of normal serum lipid and lipoprotein levels. The goal of the present study was to identify the minimal concentration of serum apoE still affecting cholesterol levels, and to determine whether any effects on remnant clearance below this level of apoE were detectable. ApoE(+/+) marrow was mixed with apoE(-/-) marrow in proportions of 1, 5, 10, and 25% to make chimeric mice with serum levels of apoE ranging from 0.005 to 0.46 mg/dl. Analysis of serum cholesterol and apoE levels demonstrated a positive correlation between apoE levels and cholesterol reduction (r = 0.83), with levels of 0.04 mg/dl representing the functional threshold level. There were no differences in lipoprotein profiles and clearance between apoE(-/-) mice and mice with serum apoE of less than 0.04 mg/dl, as assessed by FPLC, non-denaturing gel electrophoresis, and turnover studies. However, electron microscopy of negative stains showed fewer lipoprotein particles with a diameter of <30 nm in the serum of these mice compared to apoE(-/-) mice. These data demonstrate that the threshold of serum apoE resulting in cholesterol reduction is 0. 04 mg/dl, and indicate that apoE below this level affects lipoprotein size distribution possibly by accelerating the clearance of smaller remnants.

Animals↗
PubMed1998

Hepatic apo E expression is required for remnant lipoprotein clearance in the absence of the low density lipoprotein receptor.

According to the secretion-capture model of remnant lipoprotein clearance, apo E secreted by hepatocytes into the space of Disse serves to enrich the remnants with a ligand for receptor-mediated lipoprotein endocytosis. Current evidence supports a two-receptor model of lipoprotein removal, in which apo E-containing remnants bind either the low density lipoprotein receptor (LDLR) or the LDLR-related protein (LRP). Recently, we demonstrated that reconstitution of apo E(-/-) mice with apo E(+/+) marrow results in normalization of plasma lipoprotein levels, indicating that hepatic expression of apo E is not required for remnant clearance and calling into question the relevance of the secretion-capture mechanism. To dissect the relative contributions of LDLR and LRP to the cellular catabolism of remnant lipoproteins by the hepatocyte, bone marrow transplantation (BMT) was used to reconstitute macrophage expression of apo E in mice that were null for expression of both apo E and the LDLR. Reconstitution of macrophage apo E in apo E(-/-)/LDLR(-/-) mice had no effect on serum lipid and lipoprotein concentrations, although it produced plasma apo E levels up to 16-fold higher than in C57BL/6 controls. Immunocytochemistry of hepatic sections revealed abundant staining for apo E in the space of Disse, but no evidence of receptor-mediated endocytosis of remnant lipoproteins. Transient expression of human LDLR in the livers of apo E(+/+)--> apo E(-/-)/LDLR(-/-) mice by adenoviral gene transfer resulted in normalization of serum lipid levels and in the clearance of apo E-containing lipoproteins from the space of Disse. We conclude that whereas the LDLR efficiently clears remnant lipoproteins irrespective of the site of origin of apo E, endocytosis by the chylomicron remnant receptor (LRP) is absolutely dependent on hepatic expression of apo E. These data demonstrate in vivo the physiologic relevance of the apo E secretion-capture mechanism in the liver.

Animals↗
PubMed1997

Reading-frame restoration by transcriptional slippage at long stretches of adenine residues in mammalian cells.

We previously characterized a mutant apoB allele (the apoB86 allele) that produces both a truncated apoB (apoB86) and a full-length apoB100. The mutant allele contained a deletion of a single cytosine in exon 26, creating a stretch of eight consecutive adenines in the -1 reading frame. The altered reading-frame allele was restored, with approximately 10% efficiency, by the transcriptional insertion of an extra adenine into the stretch of eight consecutive adenines, thereby accounting for the synthesis of the full-length apoB100. Here, we demonstrate that this reading-frame restoration does not occur when the long stretch of adenines is interrupted by a cytosine. To assess whether reading-frame restoration is unique to a single site in the apoB gene, the same mutation (eight consecutive adenines in the -1 reading frame) was inserted into another site within the apoB gene. Reading-frame restoration occurred at the second site and was abrogated when the stretch of adenines was interrupted by another base. Of note, a computerized analysis of human cDNA sequences revealed that long stretches of adenines in protein-coding sequences occur at a lower than predicted frequency, suggesting that evolution has selected against these sequences.

Adenine↗
PubMed1997

Increased atherosclerosis in mice reconstituted with apolipoprotein E null macrophages.

Macrophage-derived foam cells express apolipoprotein E (apoE) abundantly in atherosclerotic lesions. To examine the physiologic role of apoE secretion by the macrophage in atherogenesis, bone marrow transplantation was used to reconstitute C57BL/6 mice with macrophages that were either null or wild type for the apoE gene. After 13 weeks on an atherogenic diet, C57BL/6 mice reconstituted with apoE null marrow developed 10-fold more atherosclerosis than controls in the absence of significant differences in serum cholesterol levels or lipoprotein profiles. ApoE expression was absent in the macrophage-derived foam cells of C57BL/6 mice reconstituted with apoE null marrow. Thus, lack of apoE expression by the macrophage promotes foam cell formation. These data support a protective role for apoE expression by the macrophage in early atherogenesis.

Animals↗
PubMed1997

Leukocyte low density lipoprotein receptor (LDL-R) does not contribute to LDL clearance in vivo: bone marrow transplantation studies in the mouse.

The targeted disruption of the low density lipoprotein (LDL) receptor gene in mice results in accumulation of plasma LDL cholesterol and in predisposition to diet-induced aortic atherosclerosis. Although the liver is the central organ for receptor mediated clearance of LDL, the in vivo role of other organs and tissues in LDL catabolism has not been directly studied. Since bone marrow-derived cells such as blood leukocytes and tissue macrophages express LDL receptors and contribute a large mass to the body, we designed bone marrow transplantation (BMT) experiments to reconstitute LDL receptor null mice [LDL-R(-/-)] with marrow obtained from LDL-R wild-type mice [LDL-R(+/+)] and evaluate the effects on parameters of plasma lipid metabolism. Although reconstitution of the transplanted mice with donor bone marrow cells was complete, no differences in plasma lipid levels and lipoprotein distribution were found between groups, irrespective of the diet used, and turnover studies using 125I-labeled LDL showed that LDL receptor expression by leukocytes and macrophages does not significantly contribute to plasma LDL clearance. The complementary experiment of transplanting LDL-R(-/-) marrow into C57BL/6 recipients [LDL-R(-/-)-->LDL(+/+)], performed to evaluate the role of leukocyte LDL-R in normocholesterolemic condition, also produced no effects on plasma lipid parameters. LDL binding studies using macrophages isolated from transplanted mice showed a lack of LDL-R expression. Thus, despite their large number and wide distribution, bone marrow-derived cells do not significantly influence receptor-mediated clearance of plasma LDL.

Animals↗
PubMed1997

Secretion from cell culture of HDL and VLDL bearing apoB-33 with a large internal deletion.

Rat hepatoma McA-RH7777 cells synthesize and secrete two populations of apoB-containing lipoproteins: a larger, VLDL-sized population floating in the Sf 40-150 range and a smaller, LDL and HDL-sized population. Three permanently transfected cell lines of McA-RH7777 cells secreted (in addition to the endogenous lipoproteins) lipoproteins containing 1) a carboxyl-terminally truncated human apoB-53 (2377 amino acids in length); 2) a carboxyl-terminally truncated human apoB-31 (1420 amino acids in length); or 3) an internally deleted human apoB protein, apoB-18/95, containing a total of 1490 amino acid residues, equivalent in length to an apoB33. The apoB-18/95 protein contained amino acid residues 1-782 joined to 708 residues near the C-terminus of apoB (residues 36364343). All three of the apoB peptides, apoB53, apoB-31, and apoB-18/95, were present on smaller LDL-HDL-class lipoproteins, with buoyant densities in the HDL density range. The sizes of the HDL class lipoproteins agreed with prior observations that lipoprotein core circumference is directly proportional to apoB size. As HDL containing apoB-18/95 conformed to this rule, contiguous apoB amino acid sequence is not required for the rule to be obeyed. In addition, apoB-18/95, but not apoB-31, was also present on the VLDL-sized lipoproteins even in the absence of serum or oleate supplementation. As the latter two constructs encode equally sized apoB peptides, their particular amino acid sequences rather than just overall length must determine whether they can assemble into a VLDL particle.

Animals↗
PubMed1995

Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation.

Apolipoprotein E (apoE) deficiency causes severe hyperlipidemia and atherosclerosis in humans and in gene-targeted mice. Although the majority of apoE in plasma is of hepatic origin, apoE is synthesized by a variety of cell types, including macrophages. Because macrophages derive from hematopoietic cells, bone marrow transplantation was used to examine the potential of apoE synthesized by bone marrow-derived cells to correct the hyperlipidemia and atherosclerosis caused by apoE deficiency. After transplantation of bone marrow from mice with the normal apoE gene into apoE-deficient mice, apoE was detected in serum and promoted clearance of lipoproteins and normalization of serum cholesterol levels. ApoE-deficient mice given transplants of normal bone marrow showed virtually complete protection from diet-induced atherosclerosis.

Animals↗
PubMed1995

Transgenic mice expressing high levels of human apolipoprotein B develop severe atherosclerotic lesions in response to a high-fat diet.

We previously generated transgenic mice expressing human apolipoprotein (apo-) B and demonstrated that the plasma of chow-fed transgenic animals contained markedly increased amounts of LDL (Linton, M. F., R. V. Farese, Jr., G. Chiesa, D. S. Grass, P. Chin, R. E. Hammer, H. H. Hobbs, and S. G. Young 1992. J. Clin. Invest. 92:3029-3037). In this study, we fed groups of transgenic and nontransgenic mice either a chow diet or a diet high in fat (16%) and cholesterol (1.25%). Lipid and lipoprotein levels were assessed, and after 18 wk of diet, the extent of aortic atherosclerotic lesions in each group of animals was quantified. Compared with the female transgenic mice on the chow diet, female transgenic mice on the high-fat diet had higher plasma levels of cholesterol (312 +/- 17 vs 144 +/- 7 mg/dl; P < 0.0001) and human apo-B (120 +/- 8 vs 84 +/- 3 mg/dl; P < 0.0001). The higher human apo-B levels were due to increased plasma levels of human apo-B48; the human apo-B100 levels did not differ in animals on the two diets. In mice on the high-fat diet, most of the human apo-B48 and apo-B100 was found in LDL-sized particles. Compared with nontransgenic mice on the high-fat diet, the transgenic animals on the high-fat diet had significantly increased levels of total cholesterol (312 +/- 17 vs 230 +/- 19 mg/dl; P < 0.0001) and non-HDL cholesterol (283 +/- 17 vs 193 +/- 19 mg/dl; P < 0.0001). The extent of atherosclerotic lesion development within the ascending aorta was quantified by measuring total lesion area in 60 progressive sections, using computer-assisted image analysis. Neither the chow-fed transgenic mice nor the chow-fed nontransgenic mice had significant atherosclerotic lesions. Nontransgenic animals on the high-fat diet had relatively small atherosclerotic lesions (< 15,000 microns 2/section), almost all of which were confined to the proximal 400 microns of the aorta near the aortic valve. In contrast, transgenic animals on the high-fat diet had extensive atherosclerotic lesions (> 160,000 microns 2/section) that were widely distributed throughout the proximal 1,200 microns of the aorta. Thus, human apo-B expression, in the setting of a diet rich in fats, causes severe atherosclerosis in mice.

Animals↗
PubMed1994

Expression of human apolipoprotein B90 in transgenic mice. Demonstration that apolipoprotein B90 lacks the structural requirements to form lipoprotein.

Lipoprotein(a) (Lp(a)) is a lipoprotein formed by the disulfide linkage of apolipoprotein(a) (apo(a)) to the apoB100 of a low density lipoprotein particle. Earlier site-directed mutagenesis studies of apo(a) demonstrated that apo(a) cysteine 4057 is required for the disulfide linkage; however, the cysteine residue within apoB100 that is involved in the disulfide bond has not been identified. We previously demonstrated that the apoB100 produced by human apoB transgenic mice binds to apo(a) and forms Lp(a) (Linton, M.F., Farese, R. V., Jr., Chiesa, G., Grass, D. S., Chin, P., Hammer, R. E., Hobbs, H.H., and Young, S.G. (1993) J. Clin. Invest. 92, 3029-3037). To further explore the structural features of human apoB that are required for the formation of Lp(a), we used a transposon-interrupted human apoB gene clone to develop transgenic mice that express high levels of a truncated form of human apoB, apoB90, which contains the amino-terminal 4084 amino acids of apoB. In vitro incubation of apo(a) with the plasma of human apoB90 transgenic mice did not yield Lp(a), as judged by Western blots of SDS-polyacrylamide gels or by a monoclonal antibody-based radioimmunoassay. In contrast, incubation of apo(a) with the plasma of a mouse that expressed an equivalent amount of the full-length apoB100 did yield Lp(a). In addition to these in vitro incubation studies, no Lp(a) could be detected in the plasma of a "double transgenic" mouse expressing both human apoB90 and apo(a). These data indicate that the carboxyl-terminal 10% of apoB100 contains amino acid sequences that are essential for the formation of Lp(a).

Animals↗
PubMed1994

Expression of P1 DNA in mammalian cells and transgenic mice.

Because the P1 bacteriophage packages DNA inserts of 80-100 kb, which are much larger than inserts of bacteriophage lambda or cosmid vectors, P1 DNA can be used to express large genes in cultured cells and transgenic mice. We obtained a P1 bacteriophage clone with a 79.5-kb insert (p158) that spanned the entire human apolipoprotein (apo-) B gene. We used the insert from p158 to express the human apo-B gene in both cultured rat hepatoma cells and transgenic mice. In this article, we review our apo-B expression studies and discuss the techniques that we have used for these expression studies.

Animals↗
PubMed1994

Transgenic mice expressing human apoB100 and apoB48.

Transgenic mice that express human apolipoprotein (apo)B have been developed by microinjecting fertilized mouse oocytes with an 80 kb genomic DNA fragment that encompasses the entire human APOB gene. In the transgenic mice expressing the largest amounts of human apoB, the concentration of human apoB100 in the plasma is nearly as high as the levels observed in normolipidemic humans (50 mg/dl). Virtually all of the human apoB100 in the transgenic plasma is located in the LDL fraction, resulting in substantially increased levels of LDL cholesterol. These human apoB-transgenic mice should be useful animal models for understanding various aspects of lipoprotein metabolism and for further delineating the role of LDL in atherogenesis.

Animals↗