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• W2065372271 abstract "Apolipoprotein E is a multifunctional protein synthesized by hepatocytes and macrophages. Plasma apoE is largely liver-derived and known to regulate lipoprotein metabolism. Macrophage-derived apoE has been shown to reduce the progression of atherosclerosis in mice. We tested the hypothesis that liver-derived apoE could directly induce regression of pre-existing advanced atherosclerotic lesions without reducing plasma cholesterol levels. Aged low density lipoprotein (LDL) receptor-deficient (LDLR−/−) mice were fed a western-type diet for 14 weeks to induce advanced atherosclerotic lesions. One group of mice was sacrificed for evaluation of atherosclerosis at base line, and two other groups were injected with a second generation adenoviruses encoding human apoE3 or a control empty virus. Hepatic apoE gene transfer increased plasma apoE levels by 4-fold at 1 week, and apoE levels remained at least 2-fold higher than controls at 6 weeks. There were no significant changes in plasma total cholesterol levels or lipoprotein composition induced by expression of apoE. The liver-derived human apoE gained access to and was retained in arterial wall. Compared with base-line mice, the control group demonstrated progression of atherosclerosis; in contrast, hepatic apoE expression induced highly significant regression of advanced atherosclerotic lesions. Regression of lesions was accompanied by the loss of macrophage-derived foam cells and a trend toward increase in extracellular matrix of lesions. As an index of in vivooxidant stress, we quantitated the isoprostane iPF2α-VI and found that expression of apoE markedly reduced urinary, LDL-associated, and arterial wall iPF2α-VI levels. In summary, these results demonstrate that liver-derived apoE directly induced regression of advanced atherosclerosis and has anti-oxidant properties in vivo that may contribute to its anti-atherogenic effects. Apolipoprotein E is a multifunctional protein synthesized by hepatocytes and macrophages. Plasma apoE is largely liver-derived and known to regulate lipoprotein metabolism. Macrophage-derived apoE has been shown to reduce the progression of atherosclerosis in mice. We tested the hypothesis that liver-derived apoE could directly induce regression of pre-existing advanced atherosclerotic lesions without reducing plasma cholesterol levels. Aged low density lipoprotein (LDL) receptor-deficient (LDLR−/−) mice were fed a western-type diet for 14 weeks to induce advanced atherosclerotic lesions. One group of mice was sacrificed for evaluation of atherosclerosis at base line, and two other groups were injected with a second generation adenoviruses encoding human apoE3 or a control empty virus. Hepatic apoE gene transfer increased plasma apoE levels by 4-fold at 1 week, and apoE levels remained at least 2-fold higher than controls at 6 weeks. There were no significant changes in plasma total cholesterol levels or lipoprotein composition induced by expression of apoE. The liver-derived human apoE gained access to and was retained in arterial wall. Compared with base-line mice, the control group demonstrated progression of atherosclerosis; in contrast, hepatic apoE expression induced highly significant regression of advanced atherosclerotic lesions. Regression of lesions was accompanied by the loss of macrophage-derived foam cells and a trend toward increase in extracellular matrix of lesions. As an index of in vivooxidant stress, we quantitated the isoprostane iPF2α-VI and found that expression of apoE markedly reduced urinary, LDL-associated, and arterial wall iPF2α-VI levels. In summary, these results demonstrate that liver-derived apoE directly induced regression of advanced atherosclerosis and has anti-oxidant properties in vivo that may contribute to its anti-atherogenic effects. low density lipoprotein LDL receptor fast protein liquid chromatography very low density lipoprotein phosphate-buffered saline high density lipoprotein Apolipoprotein E (apoE) is a multifunctional protein produced largely by the liver and mononuclear phagocytes (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3395) Google Scholar). Although its physiologic role is uncertain, macrophage-derived apoE can inhibit progression of atherogenesis. Expression of macrophage apoE in apoE-deficient mice using bone marrow transplantation reduced plasma cholesterol levels and slowed progression of atherosclerosis (2Linton M.F. Atkinson J.B. Fazio S. Science. 1995; 267: 1034-1037Crossref PubMed Scopus (411) Google Scholar, 3Boisvert W. Spangenberg J. Curtiss L. J. Clin. Invest. 1995; 96: 1118-1124Crossref PubMed Scopus (185) Google Scholar). Tissue-specific transgenic expression reduced atherosclerosis independent of plasma cholesterol levels (4Bellosta S. Mahley R.W. Sanan D.A. Murata J. Newland D.L. Taylor J.M. Pitas R.E. J. Clin. Invest. 1995; 96: 2170-2179Crossref PubMed Scopus (251) Google Scholar). Transplantation of apoE-deficient bone marrow in wild-type mice increased atherosclerosis (5Fazio S. Babaev V. Murray A. Hasty A. Carter K. Gleaves L. Atkinson J. Linton M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4647-4652Crossref PubMed Scopus (249) Google Scholar), consistent with a role of macrophage-derived apoE in inhibiting progression of atherosclerosis. Liver-derived apoE is the major source of plasma apoE and has an important physiologic role in regulating lipoprotein metabolism (1Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3395) Google Scholar). However, the potential role of liver-derived apoE in atherogenesis independent of its role in lipoprotein metabolism is uncertain. Expression of apoE in the liver of apoE-deficient mice using an adenoviral vector slowed the progression of atherosclerosis (6Kashyap V.S. Santamarina-Fojo S. Brown D.R. Parrott C.L. Applebaum-Bowden D. Meyn S. Talley G. Paigen B. Maeda N. Brewer Jr., H.B. J. Clin. Invest. 1995; 96: 1612-1620Crossref PubMed Scopus (123) Google Scholar). We demonstrated that adenoviral-mediated hepatic expression of apoE in apoE-deficient mice resulted in marked regression of both early and advanced atherosclerotic lesions (7Tsukamoto K. Tangirala R. Chun S.H. Pure' E. Rader D.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2162-2170Crossref PubMed Scopus (97) Google Scholar), an observation confirmed subsequently by another group (8Desurmont C. Caillaud J.M. Emmanuel F. Benoit P. Fruchart J.C. Castro G. Branellec D. Heard J.M. Duverger N. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 435-442Crossref PubMed Scopus (58) Google Scholar). However, in all of these experiments, hepatic expression of apoE in apoE-deficient mice markedly reduced plasma cholesterol levels, making it impossible to assess the direct effect of liver-derived apoE on atherosclerosis. A previous study showed that repeated injection of purified apoE to Watanabe Heritable Hyperlipidemic rabbits lacking functional LDL receptors resulted in reduced progression of atherosclerosis without lowering plasma cholesterol levels (9Yamada N. Inoue I. Kawamura M. Harada K. Watanabe Y. Shimano H. Gotoda T. Shimada M. Kohzaki K. Tsukada T. Shiomi M. Yazaki Y. J. Clin. Invest. 1992; 89: 706-711Crossref PubMed Scopus (74) Google Scholar). We subsequently demonstrated that hepatic expression of apoE in LDL1 receptor-deficient mice fed a western-type diet did not reduce plasma cholesterol levels but nevertheless slowed the progression of early fatty streak atherosclerotic lesions (10Tsukamoto K. Tangirala R. Chun S. Usher D. Pure' E. Rader D.J. Mol. Ther. 2000; 1: 189-194Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Expression of apoE by the adrenals at plasma levels below the threshold for reducing plasma cholesterol levels reduced atherosclerosis in apoE-deficient mice (11Thorngate F.E. Rudel L.L. Walzem R.L. Williams D.L. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1939-1945Crossref PubMed Scopus (109) Google Scholar). However, regression of atherosclerosis by apoE derived from any source in the absence of cholesterol reduction has never been demonstrated. In the current study, we extended previous observations by testing the hypothesis that apoE expression would induce regression of pre-existing advanced atherosclerotic lesions in the absence of significant reduction in plasma cholesterol. The mechanisms by which apoE directly inhibits atherosclerosis are poorly understood. One of the key factors that plays a causative role in development and progression of atherosclerosis is oxidative stress (12Witztum J.L. Lancet. 1994; 344: 793-795Abstract PubMed Scopus (1261) Google Scholar). Oxidative stress in the artery wall contributes to the inflammatory nature of atherosclerotic lesions (12Witztum J.L. Lancet. 1994; 344: 793-795Abstract PubMed Scopus (1261) Google Scholar, 13Berliner J.A. Navab M. Fogelman A.M. Frank J.S. Demer L.L. Edwards P.A. Watson A.D. Lusis A.J. Circulation. 1995; 91: 2488-2496Crossref PubMed Scopus (1593) Google Scholar) and could potentially contribute to plaque rupture (14Rajagopalan S. Meng X.P. Ramasamy S. Harrison D.G. Galis Z.S. J. Clin. Invest. 1996; 98: 2572-2579Crossref PubMed Scopus (1017) Google Scholar). Products of lipoprotein oxidation are present in vessel wall and have proinflammatory effects that could promote atherogenesis (15Palinski W. Ord V.A. Plump A.S. Breslow J.L. Steinberg D. Witztum J.L. Arterioscler. Thromb. Vasc. Biol. 1994; 14: 605-616Crossref Scopus (473) Google Scholar). ApoE-deficient mice exhibit evidence of markedly increased lipoprotein oxidation as assessed by autoantibodies to oxidized lipoproteins (15Palinski W. Ord V.A. Plump A.S. Breslow J.L. Steinberg D. Witztum J.L. Arterioscler. Thromb. Vasc. Biol. 1994; 14: 605-616Crossref Scopus (473) Google Scholar). ApoE has been shown in vitro to have antioxidant (16Miyata M. Smith J.D. Nat. Genet. 1996; 14: 55-61Crossref PubMed Scopus (802) Google Scholar) and anti-inflammatory (17Kelly M.E. Clay M.A. Mistry M.J. Hsieh-Li H.M. Harmony J.A. Cell. Immunnol. 1994; 159: 124-139Crossref PubMed Scopus (110) Google Scholar) properties. In this study, we investigated effects of apoE expression on in vivo oxidative stress by quantitating isoprostanes in multiple tissues. Isoprostanes are stable prostaglandin isomers generated during free radical-mediated lipid peroxidation and have been shown to be reliable markers of lipid peroxidation and oxidative stress generation in vivo (18Patrono C. FitzGerald G.A. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2309-2315Crossref PubMed Scopus (411) Google Scholar). We previously reported that urine, plasma, and arterial iPF2α-VI levels are markedly increased in apoE-deficient mice and are suppressed by the administration of vitamin E accompanied by reduction in atherosclerosis (19Praticó D. Tangirala R.K. Rader D.J. Rokach J. FitzGerald G.A. Nat. Med. 1998; 4: 1189-1192Crossref PubMed Scopus (465) Google Scholar). Therefore, we also tested the hypothesis that hepatic expression of apoE would reduce oxidant stress in LDLR-deficient mice as assessed by quantitation of isoprostanes in urine, plasma, and aorta. Recombinant second generation adenovirus encoding the human apoE3 and the control empty adenovirus (Adnull) were constructed as described previously (7Tsukamoto K. Tangirala R. Chun S.H. Pure' E. Rader D.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2162-2170Crossref PubMed Scopus (97) Google Scholar, 20Tsukamoto K. Smith P. Glick J.M. Rader D. J. Clin. Invest. 1997; 100: 107-114Crossref PubMed Scopus (87) Google Scholar). A total of 27 LDLR−/− mice (back-crossed 10 times to C57BL/6 mice) obtained from Jackson Laboratory were fed a western-type diet (normal chow supplemented with 0.15% cholesterol and 20% butter fat) for a total of 14 weeks prior to base-line analysis or administration of vector. Blood was drawn 1 week prior to vector administration, plasma cholesterol was quantified, and mice were divided into three groups with mean cholesterol levels that were not significantly different. One day prior to vector administration, one group of mice (n = 9) were killed for analysis of atherosclerosis (base-line group). Remaining mice were injected intravenously with either AdhapoE3 (n = 9) or control Adnull virus (n = 9) at a dose of 1.0 × 1011particles (∼6.0 × 109 plaque-forming uints/g of body weight). The western-type diet was continued after vector administration. Blood samples from mice were obtained by retro-orbital plexus prior to injection and 7, 14, 21, 28, and 42 days after injection. Blood samples were collected into tubes containing 2 nm EDTA, 0.2% NaN3, 0.77% gentamycin and were centrifuged to obtained plasma, which was stored at −20 °C for lipid analyses and at 4 °C for FPLC. Urine samples were collected by placing mice in metabolic cages (Nalgene, Rochester, NY) for a 24-h urine collection at various time points. The samples were stored at −80 °C. Mice were killed 6 weeks after vector administration for analysis of atherosclerosis. Plasma cholesterol and triglyceride levels were measured enzymatically on a Cobas Fara II (Roche Diagnostic Systems Inc.) using Sigma reagents. Plasma apoE levels were measured using an immunoturbidometric assay (Sigma). Plasma samples (200 μl pooled from nine mice each of Adnulll and AdhapoE3 group) were analyzed by FPLC gel filtration (Amersham Pharmacia Biotech, Uppsala, Sweden) on two Superose 6 columns as described previously (20Tsukamoto K. Smith P. Glick J.M. Rader D. J. Clin. Invest. 1997; 100: 107-114Crossref PubMed Scopus (87) Google Scholar). The cholesterol concentrations in the FPLC fractions were determined using an enzymatic assay (Wako Pure Chemical Industries Ltd., Osaka, Japan). Lipoproteins were isolated from plasma samples by sequential ultracentrifugation. The plasma samples obtained at various time points during the experiment were pooled from nine mice each (250 μl) of Adnull and AdhapoE3 group. The samples were subjected to ultracentrifugation in 1-ml polycarbonate tubes at 90,000 rpm in a Beckman TLA 100.2 rotor for 3 h at 10 °C (TL-100 centrifuge, Beckman Instruments Inc.). The VLDL (d<1.006), LDL (1.006 < d < 1.063) and HDL (1.063 < density < 1.21) were isolated by tube slicing in a volume of 250 μl. The lipoprotein fractions were analyzed for the lipid and protein content. Western blotting analysis for the detection of human apoE in aortas was performed using extracts prepared from the lower abdominal abdominal portion of the aortas from base-line, Adnull, and AdhapoE3 mice. The aortas were minced on ice and homogenized in 250 μl of PBS containing a mixture of protease inhibitors (complete mini™, Roche Molecular Biochemicals). Aliquots of the aortic extracts (10 and 15 μg of protein) were resuspended in 40 μl of Laemli buffer (Bio-Rad) and heated at 95 °C for 5 min. The samples were subjected to 10–20% linear gradient SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Hybond™ ECL, Amersham Pharmacia Biotech). The presence of human apoE was detected using anti-human apoE3 monoclonal antibody as primary and horseradish peroxidase-labeled donkey anti-mouse IgG as the secondary antibody. The extent of atherosclerosis in the aorta and in aortic cross-sections was analyzed as described previously (7Tsukamoto K. Tangirala R. Chun S.H. Pure' E. Rader D.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2162-2170Crossref PubMed Scopus (97) Google Scholar, 19Praticó D. Tangirala R.K. Rader D.J. Rokach J. FitzGerald G.A. Nat. Med. 1998; 4: 1189-1192Crossref PubMed Scopus (465) Google Scholar, 21Tangirala R.K. Rubin E.M. Palinski W. J. Lipid Res. 1995; 36: 2320-2328Abstract Full Text PDF PubMed Google Scholar, 22Tangirala R.K. Tsukamoto K. Chun S.H. Usher D. Pure' E. Rader D.J. Circulation. 1999; 100: 1816-1822Crossref PubMed Scopus (321) Google Scholar). Mice were killed by carbon dioxide inhalation, and the aorta was immediately perfused in situwith ice-cold PBS for 10 min via the left ventricle. The heart and aorta were removed en bloc, and the heart was severed from the aorta just above the aortic root. The apical half of the heart was cut off in a plane perpendicular to the aortic root. The upper half of the heart containing the aortic root was immediately embedded in Tissue Tek O.C.T. compound and frozen at −80 °C. The remainder of the aorta was cleaned free of adventitial fat and tissue, opened longitudinally, stained with Sudan IV, and pinned out as described (7Tsukamoto K. Tangirala R. Chun S.H. Pure' E. Rader D.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2162-2170Crossref PubMed Scopus (97) Google Scholar, 19Praticó D. Tangirala R.K. Rader D.J. Rokach J. FitzGerald G.A. Nat. Med. 1998; 4: 1189-1192Crossref PubMed Scopus (465) Google Scholar, 21Tangirala R.K. Rubin E.M. Palinski W. J. Lipid Res. 1995; 36: 2320-2328Abstract Full Text PDF PubMed Google Scholar, 22Tangirala R.K. Tsukamoto K. Chun S.H. Usher D. Pure' E. Rader D.J. Circulation. 1999; 100: 1816-1822Crossref PubMed Scopus (321) Google Scholar). The extent of atherosclerosis in aortas was quantified by capturing images of aorta with a Dage-MTI 3CCD three-chip color camera (Dage-MTI Inc., Michigan City, IN) connected to a Leica MZ 12 dissection microscope. The captured 24-bit digitized color images were analyzed, and the lesion areas covering aortas were determined using Image Pro Plus image analysis software (Media Cybernetics, Silver Spring, MD). The acquisition of aortic images and the analysis of lesion areas were both performed in a blinded fashion. Atherosclerosis was also quantified in the aortic root cross-sections from the fresh-frozen OCT-embedded hearts (7Tsukamoto K. Tangirala R. Chun S.H. Pure' E. Rader D.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2162-2170Crossref PubMed Scopus (97) Google Scholar, 22Tangirala R.K. Tsukamoto K. Chun S.H. Usher D. Pure' E. Rader D.J. Circulation. 1999; 100: 1816-1822Crossref PubMed Scopus (321) Google Scholar). Serial 8-μm sections of the aortic root were cut and mounted on slides, then fixed in acetone, rehydrated in PBS containing 0.02% NaN3, and blocked with 1% bovine serum albumin in PBS/NaN3. For detection of macrophages, sections were immunostained with monoclonal rat anti-murine MAC-1 antibody, monoclonal hamster anti-CD18, and monoclonal hamster anti-CD11c. For detection of an extracellular matrix protein, monoclonal hamster laminin antibody was used as a primary antibody, followed by incubation with mouse anti-rat or goat anti-hamster IgG in the presence of 200 μg/ml normal mouse IgG. Antibody reactivity was detected using horseradish peroxidase-conjugated biotin-streptavidin complexes and developed with diaminobenzidine tetrahydrochloride as substrate. Images of immunostained aortic root sections, captured digitally with a video camera connected to a Leica microscope, were analyzed using computerized image analysis (Image Pro Plus, Media Cybernetics, Silver Spring, MD). Total lesion area in aortic root sections was measured by manually tracing entire lesions in eight equally spaced aortic root sections per mouse. Both macrophage and fibrous areas were quantified in sections by determination of area that stained positively for macrophage markers and laminin, respectively. The acquisition of images and analysis of lesions were performed in a blinded fashion. For isoprostane measurement total lipids from urine, LDL, and aortic homogenate samples were extracted with ice-cold Folch solution, chloroform/methanol (2:1, v/v) as described previously (19Praticó D. Tangirala R.K. Rader D.J. Rokach J. FitzGerald G.A. Nat. Med. 1998; 4: 1189-1192Crossref PubMed Scopus (465) Google Scholar). After removing aliquots for phospholipid measurement, the organic phase was dried under nitrogen. The samples were hydrolyzed by the addition of aqueous KOH (15%), and total iPF2α-VI was measured as described previously (19Praticó D. Tangirala R.K. Rader D.J. Rokach J. FitzGerald G.A. Nat. Med. 1998; 4: 1189-1192Crossref PubMed Scopus (465) Google Scholar). Mice that received Adnull had no significant change in plasma apoE levels during the course of the experiment, whereas mice that received AdhapoE3 had significantly increased plasma apoE levels, with the peak expression at day 7 (Fig.1 A). The plasma apoE levels in AdhapoE3-injected mice remained about 2-fold higher than in Adnull mice 6 weeks after administration at the termination of the experiment. Despite the increase in plasma apoE levels, no significant changes in the plasma cholesterol levels from base line were observed (Fig.1 B). On day 7 after injection, the mean plasma cholesterol level in mice injected with AdhapoE3 (573 ± 48 mg/dl) were modestly lower than mice injected with Adnull (696 ± 61 mg/dl, p = not significant), but there were no significant differences in cholesterol levels during the 6 weeks of the study. The mean post-injection cholesterol levels were not different between the two groups (AdhapoE3, 594 ± 18 versus Adnull, 663 ± 23 mg/dl,p = NS). Thus, hepatic expression of apoE had no significant effect on plasma cholesterol levels in LDLR−/− mice fed a western-type diet. Plasma triglyceride levels in mice injected with AdhapoE3 were transiently elevated at day 7 compared with control mice (data not shown) and did not differ at the remaining time points. To evaluate the effect of hepatic apoE expression on plasma lipoproteins, pooled plasma samples drawn on day 7 were analyzed by FPLC gel filtration. This analysis showed no significant differences in the plasma lipoprotein profiles between Adnull control and AdhapoE3 mice (Fig. 1 C). These results demonstrate that gene transfer and hepatic expression of apoE3 in LDLR−/− mice did not significantly alter the distribution of cholesterol among lipoprotein fractions. The distribution of human apoE3 was determined in the lipoprotein fractions, and apoE was found to be primarily associated with VLDL and large HDL particles, with a smaller amount of apoE present on intermediate density lipoprotein and LDL fractions (data not shown). To determine whether lipoprotein composition was influenced, we isolated VLDL, LDL, and HDL by sequential ultracentrifugation from pooled plasma samples. Analysis of the lipoprotein cholesterol (free and esterified), phospholipid and protein composition revealed no significant differences in lipoprotein composition between Adnull and AdhapoE3 groups at days 7, 14, 28, or 42. The extent of atherosclerosis was assessed by two independent methods: 1) en face quantitation of atherosclerosis in the aorta from just above the root to the renal arteries and 2) cross-sectional analysis of atherosclerotic lesions in the aortic root. The Sudan IV stained en facepreparations of the LDLR−/− mouse aortas showed extensive atherosclerotic lesions throughout the entire aorta at base line. The aortas from Adnull-injected control mice demonstrated further progression of atherosclerosis. In contrast, the lesions in AdhapoE3 mice were markedly reduced. Quantitation of atherosclerosis using morphometric image analysis of the aortas demonstrated a marked 61% regression of atherosclerosis in mice injected with AdhapoE3 compared with base-line mice (Fig. 2 A). In LDLR−/− mice fed a western-type diet, atherosclerotic lesions develop initially in the proximal portions of the aorta, the aortic root and arch, and with progression of atherosclerosis, extend distally into thoracic and abdominal portions of the aorta. To assess the pattern of apoE-induced regression in different portions of the aorta, the lesion areas in the arch, thoracic, and abdominal portions of the aorta were analyzed. The results show that hepatic apoE expression not only induced marked regression of advanced lesions in the thoracic and abdominal portions of the aorta but also effectively regressed relatively more abundant lesions in the aortic arch (Fig.2 B). Atherosclerosis was also quantified in aortic root cross-sections by manually tracing the entire lesions in five equally spaced sections from each mouse. Consistent with the en face data of the aorta, Adnull-injected mice showed progression of aortic root atherosclerotic lesions, whereas AdhapoE3-injected mice demonstrated significant 34% regression of lesion compared with base-line mice (Fig. 2 C). Thus, by two independent assays, hepatic expression of apoE induced significant regression of advanced atherosclerosis over a 6-week period. To evaluate the effects of liver-derived apoE on the morphology of lesions, we used immunocytochemistry to characterize their composition. Aortic root cross-sections from base-line, Adnull, and AdhapoE3 mice were immunostained for macrophages with antibodies to β2-integrin (Mac-1, CD18, and CD11c) and for extracellular matrix-rich regions with an antibody to laminin. Lesions contained a large amount of non-macrophage foam cell components and laminin, consistent with advanced atherosclerotic lesions. Collagen staining was similar to laminin staining (data not shown). Quantitative computer-assisted image analysis of immunostained aortic root serial cross-sections revealed significant reduction of macrophage-derived foam cells in atheroscleroic lesions of AdhapoE3 mice compared with base-line and Adnull mice (Fig.3 A). In contrast, the distribution of laminin-rich areas showed no change (data not shown), and therefore the ratio of macrophage foam cells to laminin was significantly reduced in mice injected with AdhapoE3 (Fig.3 B). These results show that liver-derived apoE induced regression of pre-existing advanced lesions primarily by reducing the macrophage-derived components of lesions but not the extracellular matrix components. In previous studies, we have shown that liver-derived apoE after gene transfer gained access to the artery wall and was specifically retained in areas of atherosclerotic lesions in apoE−/− mice (7Tsukamoto K. Tangirala R. Chun S.H. Pure' E. Rader D.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2162-2170Crossref PubMed Scopus (97) Google Scholar). To demonstrate the retention of liver-derived human apoE in the artery wall in this study, aortic extracts prepared from base-line mice, as well as Adnull and AdhapoE3 mice at the end of the experiment, were analyzed by Western blotting using monoclonal antibody specific for human apoE. The presence of liver-derived human apoE in the artery wall was detected in AdhapoE3-injected mice but not in Adnull and base-line mice (Fig. 4). No human apoE mRNA was detected (data not shown), indicating that the human apoE present in the aorta was not synthesized within the vessel wall. Thus, liver-derived apoE gained access to the artery wall from the plasma compartment and was retained in substantial amounts within the artery wall several weeks after vector injection. To examine whether hepatic apoE expression affected in vivo oxidant stress, we measured iPF2α-VI levels in urine and plasma LDL before and at several time points after vector administration and in aorta at the termination of the experiment. Injection of Adnull had no effect on urinary iPF2α-VI levels; in contrast, hepatic expression of apoE significantly reduced urinary iPF2α-VI levels by 7 days, and they remained at 60% of base-line values at 6 weeks (Fig.5 A). Analysis of the LDL-associated iPF2α-VI levels showed no change in control mice, but a rapid reduction in the AdhapoE3-injected mice, with levels by 14 days only 6.3% of base-line values and remaining in that range through the 6 weeks of the experiment (Fig. 5 B). Finally, aortic iPF2α-VI levels in AdhapoE mice were significantly reduced compared with both base-line and Adnull mice (Fig. 5 C). These results suggest that hepatic apoE expression reduced oxidant stress in vivo. The apoE-mediated inhibition of lipid peroxidation and oxidative stress may contribute to its anti-atherogenic mechanisms in the regression of advanced atherosclerotic lesions. In this study, we demonstrated that hepatic overexpression of human apoE3 for 6 weeks induced regression of pre-existing advanced atherosclerosis in LDLR-deficient mice without reducing plasma cholesterol levels. This substantially extends our previous finding that apoE expression reduced the progression of early fatty streak lesions in LDLR-deficient mice (10Tsukamoto K. Tangirala R. Chun S. Usher D. Pure' E. Rader D.J. Mol. Ther. 2000; 1: 189-194Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and indicates that liver-derived apoE has the potential to induce loss of foam cell mass from pre-existing advanced atherosclerotic lesions. Furthermore, we demonstrated that apoE overexpression in aged LDLR-deficient mice markedly reduced levels of isoprostanes in urine, plasma, and aorta compared with base-line and control treated mice. These results strongly suggest that hepatic apoE expression resulted in anti-oxidant effects in vivo. Whether these effects were directly responsible for the induction of atherosclerosis regression remains to be determined. It is not surprising that hepatic overexpression of human apoE3 on the background of endogenous mouse apoE and in the absence of the LDLR did not significantly reduce plasma cholesterol levels. The LDLR is the major physiologic receptor for apoE in the liver (23Willnow T.E. Herz J. J. Mol. Med. 1995; 73: 213-220Crossref PubMed Scopus (5) Google Scholar), and although there are backup mechanisms for clearance of lipoprotein remnants, such as LDL receptor-related protein and heparan sulfate proteoglycans, they are much less efficient. Furthermore, the majority of plasma cholesterol in western diet-fed LDLR mouse is in LDL, which are generally not cleared from the plasma through an apoE-mediated mechanism. In fact, apoE-containing remnant lipoproteins can compete with LDL for uptake, resulting in delayed catabolism of LDL (24Woollett L.A. Osono Y. Herz J. Dietschy J.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12500-12504Crossref PubMed Scopus (56) Google Scholar). Furthermore, apoE has been shown to inhibit lipolysis of triglyceride-rich lipoproteins (25van Dijk K.W. van Vlijmen B.J.M. van't Hof H.B. van der Zee A. Fojo S.S. Van Berkel T.J. Havekes L.M. Hofker M.H. J. Lipid Res. 1999; 40: 336-344Abstract Full Text Full Text PDF PubMed Google Scholar). Finally, hepatic apoE expression enhances hepatic VLDL triglyceride (26Mahley R.W. Huang Y. Curr. Opin. Lipidol. 1999; 10: 207-217Crossref PubMed Scopus (325) Google Scholar, 27Tsukamoto K. Maugeais C. Glick J.M. Rader D.J. J. Lipid Res. 2000; 41: 253-259Abstract Full Text Full Text PDF PubMed Google Scholar, 28Huang Y. Liu X.Q. Rall S.C.J. Taylor J.M. von Eckardstein A. Assmann G. Mahley R.W. J. Biol. Chem. 1998; 273: 26388-26393Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and apoB production (29Maugeais C. Tietge U.J.F. Tsukamoto K. Glick J.M. Rader D.J. J. Lipid Res. 2000; 41: 1673-1679Abstract Full Text Full Text PDF PubMed Google Scholar). Therefore any modest effect of apoE in promoting clearance of VLDL in the absence of the LDLR may be offset by its effect in competing with LDL for clearance, inhibiting VLDL lipolysis, and promoting VLDL production. Our data demonstrate that apoE overexpression had a very modest effect in reducing VLDL cholesterol on day 7, but that this effect was no longer seen at subsequent time points. These data are consistent with studies that expressed apoE using bone marrow transplant (30Linton M.F. Hasty A.H. Babaev V.R. Fazio S. J. Clin. Invest. 1998; 101: 1726-1736Crossref PubMed Scopus (86) Google Scholar) or by liver-directed gene transfer (25van Dijk K.W. van Vlijmen B.J.M. van't Hof H.B. van der Zee A. Fojo S.S. Van Berkel T.J. Havekes L.M. Hofker M.H. J. Lipid Res. 1999; 40: 336-344Abstract Full Text Full Text PDF PubMed Google Scholar) in the absence of both apoE and the LDLR and found little effect on plasma cholesterol levels. Transgenic overexpression of apoE in the absence of the LDL receptor in mice demonstrated reduction in VLDL cholesterol and increased VLDL turnover, but no reduction in LDL cholesterol or change in turnover (31Osuga J. Yonemoto M. Yamada N. Shimano H. Yagyu H. Ohashi K. Harada K. Kamei T. Yazaki Y. Ishibashi S. J. Clin. Invest. 1998; 102: 386-394Crossref PubMed Scopus (15) Google Scholar), and transgenic overexpression of apoE in rabbits resulted in a 70% increase in plasma cholesterol due largely to increased LDL cholesterol (32Fan J. Ji S.J. Huang Y. De Silva H. Sanan D. Mahley R.W. Innerarity T.L. Taylor J.M. J. Clin. Invest. 1998; 101: 2151-2164Crossref PubMed Scopus (78) Google Scholar). Therefore, apoE expression has relatively little ability to reduce plasma cholesterol levels in the absence of the LDLR, making it a good animal model for assessing the direct effects of apoE expression on atherosclerosis and other markers without the confounding effects of a reduction in plasma cholesterol. Macrophage-specific transgenic expression of apoE in apoE-deficient mice reduced progression of atherosclerosis even after controlling for changes in plasma cholesterol levels (4Bellosta S. Mahley R.W. Sanan D.A. Murata J. Newland D.L. Taylor J.M. Pitas R.E. J. Clin. Invest. 1995; 96: 2170-2179Crossref PubMed Scopus (251) Google Scholar). Furthermore, when apoE-deficient bone marrow was transplanted into wild-type mice, atherosclerotic lesion formation was increased despite a lack of effect on plasma cholesterol levels (5Fazio S. Babaev V. Murray A. Hasty A. Carter K. Gleaves L. Atkinson J. Linton M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4647-4652Crossref PubMed Scopus (249) Google Scholar). Thus, macrophage-derived apoE appears sufficient to inhibit the progression of atherosclerotic lesions independent of its effects on plasma lipoproteins. In a previous study, we demonstrated that liver-derived apoE gained access to and accumulated within atherosclerotic lesions (7Tsukamoto K. Tangirala R. Chun S.H. Pure' E. Rader D.J. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2162-2170Crossref PubMed Scopus (97) Google Scholar). Therefore, it is likely that in these studies liver-derived apoE gained access to the vessel wall and had direct effects that promoted regression of lesions. Plasma apoE may gain access to the vessel wall in the context of small lipoprotein particles such as γ-LpE (33Huang Y. von Eckardstein A. Wu S. Maeda N. Assmann G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1834-1838Crossref PubMed Scopus (169) Google Scholar, 34Krimbou L. Tremblay M. Davignon J. Cohn J. J. Lipid Res. 1997; 38: 35-48Abstract Full Text PDF PubMed Google Scholar) and preβ-LpE (34Krimbou L. Tremblay M. Davignon J. Cohn J. J. Lipid Res. 1997; 38: 35-48Abstract Full Text PDF PubMed Google Scholar). Once within the intima, apoE could associate with components of the extracellular matrix, such as heparan sulfate proteoglycans (35Ji Z.S. Brecht W.J. Miranda R.D. Hussain M.M. Innerarity T.L. Mahley R.W. J. Biol. Chem. 1993; 268: 10160-10167Abstract Full Text PDF PubMed Google Scholar,36Zhong-Sheng J. Pitas R.E. Mahley R.W. J. Biol. Chem. 1998; 273: 13452-13460Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and laminin (37Huang D.Y. Weisgraber K.H. Strittmatter W.J. Matthew W.D. Exp. Neurol. 1995; 136: 251-257Crossref PubMed Scopus (57) Google Scholar), for which it has affinity. There are a number of potential mechanisms by which hepatic apoE may have induced regression of atherosclerosis. ApoE promotes cellular cholesterol efflux (38Mazzone T. Curr. Opin. Lipidol. 1996; 7: 303-307Crossref PubMed Scopus (111) Google Scholar, 39Hara H. Yokoyama S. J. Biol. Chem. 1991; 266: 3080-3086Abstract Full Text PDF PubMed Google Scholar, 40Zhang W.-Y. Paulette M. Kruth H.S. J. Biol. Chem. 1996; 271: 28641-28646Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 41Smith J. Miyata M. Ginsberg M. Grigaux C. Shmookler E. Plump A. J. Biol. Chem. 1996; 271: 30647-30655Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 42Huang Y. von Eckardstein A. Wu S. Assmann G. J. Clin. Invest. 1995; 96: 2693-2701Crossref PubMed Scopus (58) Google Scholar) and is a contributor to the ability of plasma to induce cholesterol efflux from cells (43Huang Y. Zhu Y. Raabe M. Wu S. Weisenhutter B. Seedorf U. Maeda N. Assmann G. von Eckardstein A. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2010-2019Crossref PubMed Scopus (28) Google Scholar, 44Zhu Y. Bellosta S. Langer C. Bernini F. Pitas R.E. Mahley R.W. Assmann G. von Eckardstein A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7580-7585Crossref PubMed Scopus (145) Google Scholar). However, apoE has been demonstrated in vitro to have other cellular effects such as inhibition of T-lymphocyte proliferation (17Kelly M.E. Clay M.A. Mistry M.J. Hsieh-Li H.M. Harmony J.A. Cell. Immunnol. 1994; 159: 124-139Crossref PubMed Scopus (110) Google Scholar), inhibition of vascular smooth muscle cell proliferation (45Ishigami M. Swertfeger D.K. Granholm N.A. Hui D.Y. J. Biol. Chem. 1998; 273: 20156-20161Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), and anti-oxidant effects (16Miyata M. Smith J.D. Nat. Genet. 1996; 14: 55-61Crossref PubMed Scopus (802) Google Scholar). However, the in vivo relevance of these observations has been uncertain. In these studies, we used quantitation of iPF2α-VI, a major isoprostane that is not influenced by cyclo-oxygenase activity and therefore felt to be a specific marker for lipid peroxidation and oxidant stress (18Patrono C. FitzGerald G.A. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2309-2315Crossref PubMed Scopus (411) Google Scholar, 46Lawson J.A. Rokach J. FitzGerald G.A. J. Biol. Chem. 1999; 27: 24441-24444Abstract Full Text Full Text PDF Scopus (350) Google Scholar), to test the hypothesis that expression of apoE reduced oxidant stress in vivo. We found a dramatic effect of hepatic apoE expression in reducing urine and LDL-associated and aortic levels of iPF2α-VI. This is the first in vivoevidence that apoE has direct anti-oxidant effects. Combined with our previous study in which vitamin E administration in apoE-deficient mice reduced isoprostane generation accompanied by reduction in atherosclerosis (19Praticó D. Tangirala R.K. Rader D.J. Rokach J. FitzGerald G.A. Nat. Med. 1998; 4: 1189-1192Crossref PubMed Scopus (465) Google Scholar), this finding further supports the concept that isoprostanes may be a surrogate marker for anti-oxidant effects of interventions, which are likely to beneficially influence atherosclerosis. The findings in our studies advance the concept of apoE as an anti-atherogenic molecule in several important ways. First, we expressed apoE in the liver instead of macrophages, demonstrating that apoE is anti-atherogenic regardless of whether it is presented to the vessel wall via macrophages or via the plasma compartment. This suggests that interventions to raise hepatic production and plasma levels of apoE could be anti-atherogenic, even if macrophage production of apoE is not increased. Second, we demonstrated that apoE induced regression of pre-existing advanced atherosclerotic lesions, which has not been previously demonstrated for apoE derived from any source. Inhibition of progression and induction of regression involve very different cellular and molecular processes (47Dansky H.M. Fisher E.A. Circulation. 1999; 100: 1762-1763Crossref PubMed Scopus (18) Google Scholar), and the difference between these two processes is of potential clinical significance. Third, we demonstrated that apoE changed the morphology of advanced lesions, reduced foam cells and increased matrix, in a way that could be interpreted as consistent with “stabilization” of lesions (48Lee R.T. Libby P. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1859-1867Crossref PubMed Scopus (509) Google Scholar). Finally, we showed that hepatic apoE expression markedly reduced isoprostanes, an accepted index of in vivo oxidant stress, providing important evidence that the in vitroanti-oxidant properties of apoE that were described previously are also functional in vivo and suggesting another mechanism by which apoE may be anti-atherogenic. In summary, we demonstrate that liver-directed gene transfer and hepatic expression of apoE in LDLR−/− mice induced significant regression of advanced atherosclerotic lesions without substantial alteration in plasma cholesterol and lipoprotein levels. In addition, apoE expression markedly reduced isoprostane levels in urine, plasma LDL, and aortic tissue. These results demonstrate that liver-derived apoE has direct anti-atherogenic and antioxidant properties in vivo that are independent of cholesterol and lipoprotein modulation. We are indebted to Dawn Marchadier, Robert Hughes, Anna Lillethun, and Linda Morrell for excellent technical assistance and Dr. Jane Glick for helpful discussions." @default.
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• W2065372271 title "Reduction of Isoprostanes and Regression of Advanced Atherosclerosis by Apolipoprotein E" @default.
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