apoE Expression In Macrophages Research Articles

ApoE expression in macrophages communicates immunometabolic signaling that controls hyperlipidemia-driven hematopoiesis & inflammation via extracellular vesicles.

While apolipoprotein E (apoE) expression by myeloid cells is recognized to control inflammation, whether such benefits can be communicated via extracellular vesicles is not known. Through the study of extracellular vesicles produced by macrophages derived from the bone marrow of Wildtype (WT-BMDM-EV) and ApoE deficient (EKO-BMDM-EV) mice, we uncovered a critical role for apoE expression in regulating their cell signaling properties. WT-BMDM-EV communicated anti-inflammatory properties to recipient myeloid cells by increasing cellular levels of apoE and miR-146a-5p, that reduced NF-κB signalling. They also downregulated cellular levels of miR-142a-3p, resulting in increased levels of its target carnitine palmitoyl transferase 1A (CPT1A) which improved fatty acid oxidation (FAO) and oxidative phosphorylation (OxPHOS) in recipient cells. Such favorable metabolic polarization enhanced cell-surface MerTK levels and the phagocytic uptake of apoptotic cells. In contrast, EKO-BMDM-EV exerted opposite effects by reducing cellular levels of apoE and miR-146a-5p, which increased NF-κB-driven GLUT1-mediated glucose uptake, aerobic glycolysis, and oxidative stress. Furthermore, EKO-BMDM-EV increased cellular miR-142a-3p levels, which reduced CPT1A levels and impaired FAO and OxPHOS in recipient myeloid cells. When cultured with naïve CD4+ T lymphocytes, EKO-BMDM-EV drove their activation and proliferation, and fostered their transition to a Th1 phenotype. While infusions of WT-BMDM-EV into hyperlipidemic mice resolved inflammation, infusions of EKO-BMDM-EV increased hematopoiesis and drove inflammatory responses in myeloid cells and T lymphocytes. ApoE-dependent immunometabolic signaling by macrophage extracellular vesicles was dependent on transcriptional axes controlled by miR-146a-5p and miR-142a-3p that could be reproduced by infusing miR-146a mimics & miR-142a antagonists into hyperlipidemic apoE-deficient mice. Together, our findings unveil a novel property for apoE expression in macrophages that modulates the immunometabolic regulatory properties of their secreted extracellular vesicles.

The Opposite Effect of c-Jun Transcription Factor on Apolipoprotein E Gene Regulation in Hepatocytes and Macrophages.

Apolipoprotein E (apoE) is mainly secreted by hepatocytes and incorporated into most plasma lipoproteins. Macrophages, which accumulate cholesterol and are critical for the development of the atherosclerotic plaque, are also an important, albeit smaller, apoE source. Distal regulatory elements control cell-specific activity of the apoE promoter: multienhancers (ME.1/2) in macrophages and hepatic control regions (HCR-1/2) in hepatocytes. A member of AP-1 cell growth regulator, c-Jun regulates the transcription of various apolipoproteins and proinflammatory molecules implicated in atherosclerosis. We aimed to investigate the effect of c-Jun on apoE expression in macrophages versus hepatocytes and to reveal the underlying molecular mechanisms. Herein we show that c-Jun had an opposite, cell-specific effect on apoE expression: downregulation in macrophages but upregulation in hepatocytes. Transient transfections using ME.2 deletion mutants and DNA pull-down (DNAP) assays showed that the inhibitory effect of c-Jun on the apoE promoter in macrophages was mediated by a functional c-Jun binding site located at 301/311 on ME.2. In hepatocytes, c-Jun overexpression strongly increased apoE expression, and this effect was due to c-Jun binding at the canonical site located at −94/−84 on the apoE proximal promoter, identified by transient transfections using apoE deletion mutants, DNAP, and chromatin immunoprecipitation assays. Overall, the dual effect of c-Jun on apoE gene expression led to decreased cholesterol efflux in macrophages resident in the atherosclerotic plaque synergized with an increased level of systemic apoE secreted by the liver to exacerbate atherogenesis.

Differential action of glucocorticoids on apolipoprotein E gene expression in macrophages and hepatocytes.

Apolipoprotein E (apoE) has anti-atherosclerotic properties, being involved in the transport and clearance of cholesterol-rich lipoproteins as well as in cholesterol efflux from cells. We hypothesized that glucocorticoids may exert anti-inflammatory properties by increasing the level of macrophage-derived apoE. Our data showed that glucocorticoids increased apoE expression in macrophages in vitro as well as in vivo. Dexamethasone increased ~6 fold apoE mRNA levels in cultured peritoneal macrophages and RAW 264.7 cells. Administered to C57BL/6J mice, dexamethasone induced a two-fold increase in apoE expression in peritoneal macrophages. By contrast, glucocorticoids did not influence apoE expression in hepatocytes, in vitro and in vivo. Moreover, dexamethasone enhanced apoE promoter transcriptional activity in RAW 264.7 macrophages, but not in HepG2 cells, as tested by transient transfections. Analysis of apoE proximal promoter deletion mutants, complemented by protein-DNA interaction assays demonstrated the functionality of a putative glucocorticoid receptors (GR) binding site predicted by in silico analysis in the -111/-104 region of the human apoE promoter. In hepatocytes, GR can bind to their specific site within apoE promoter but are not able to modulate the gene expression. The modulatory blockade in hepatocytes is a consequence of partial involvement of transcription factors and other signaling molecules activated through MEK1/2 and PLA2/PLC pathways. In conclusion, our study indicates that glucocorticoids (1) differentially target apoE gene expression; (2) induce a significant increase in apoE level specifically in macrophages. The local increase of apoE gene expression in macrophages at the level of the atheromatous plaque may have therapeutic implications in atherosclerosis.

Abstract 155: MicroRNA-146a Suppression of NF-κB-driven Monocyte/Macrophage Activation and Atherosclerosis is Regulated by Cellular ApoE Expression

Apolipoprotein E (apoE) exerts anti-inflammatory properties that protect against atherosclerosis and other inflammatory diseases. However, the mechanisms by which apoE suppresses the activation of leukocytes remain incompletely understood. An assessment of apoE expression levels among major leukocyte subsets in wild-type mice revealed that only macrophages and monocytes express apoE abundantly. An absence of Apoe expression in mouse macrophages and monocytes resulted in enhanced NF-κB signaling and an exaggerated inflammatory response upon stimulation with lipopolysaccharide. This correlated with reduced levels of microRNA-146a, a critical negative regulator of NF-κB signaling. Restoration of apoE expression in Apoe-/- macrophages and monocytes enhanced miR-146a levels, while silencing apoE expression in wild-type mouse monocytes reduced miR-146a levels. Mechanistically, apoE increased the expression of transcription factor PU.1, which raised levels of pri-miR-146 transcripts, demonstrating that apoE exerts a transcriptional control over miR-146a. In vivo, even a small amount of apoE expression in macrophages and monocytes of hypomorphic apoE mice led to increased miR-146a levels, which inhibited macrophage proinflammatory responses, Ly-6Chigh monocytosis and atherosclerosis in the settings of hyperlipidemia. Accordingly, cellular enrichment of miR-146a through the systemic delivery of miR-146a mimics emulsified in lipid microparticles infused into apoE deficient mice attenuated systemic inflammation and atherosclerosis in the absence of plasma lipid reduction. Together, our data demonstrate that apoE suppresses NF-κB-mediated inflammation and atherosclerosis by enhancing miR-146a levels in monocytes and macrophages. Ongoing studies are exploring the impact of apoE on the cellular release of endogenous miR-146a in exosomes and high density lipoproteins from macrophages that can be communicated to cells in the circulation and artery to suppress inflammation and atherosclerosis.

Apolipoprotein E enhances microRNA-146a in monocytes and macrophages to suppress nuclear factor-κB-driven inflammation and atherosclerosis.

Apolipoprotein E (apoE) exerts anti-inflammatory properties that protect against atherosclerosis and other inflammatory diseases. However, mechanisms by which apoE suppresses the cellular activation of leukocytes commonly associated with atherosclerosis remain incompletely understood. To test the hypothesis that apoE suppresses inflammation and atherosclerosis by regulating cellular microRNA levels in these leukocytes. An assessment of apoE expression among such leukocyte subsets in wild-type mice revealed that only macrophages and monocytes express apoE abundantly. An absence of apoE expression in macrophages and monocytes resulted in enhanced nuclear factor-κB signaling and an exaggerated inflammatory response on stimulation with lipopolysaccharide. This correlated with reduced levels of microRNA-146a, a critical negative regulator of nuclear factor-κB signaling. Ectopic apoE expression in Apoe(-/-) macrophages and monocytes raised miR-146a levels, whereas its silencing in wild-type cells had an opposite effect. Mechanistically, apoE increased the expression of transcription factor purine-rich PU-box-binding protein 1, which raised levels of pri-miR-146 transcripts, demonstrating that apoE exerts transcriptional control over miR-146a. In vivo, even a small amount of apoE expression in macrophages and monocytes of hypomorphic apoE mice led to increased miR-146a levels, and inhibited macrophage proinflammatory responses, Ly-6C(high) monocytosis, and atherosclerosis in the settings of hyperlipidemia. Accordingly, cellular enrichment of miR-146a through the systemic delivery of miR-146a mimetics in Apoe(-/-)Ldlr(-/-) and Ldlr(-/-) mice attenuated monocyte/macrophage activation and atherosclerosis in the absence of plasma lipid reduction. Our data demonstrate that cellular apoE expression suppresses nuclear factor-κB-mediated inflammation and atherosclerosis by enhancing miR-146a levels in monocytes and macrophages.

Metformin reduces the endotoxin-induced down-regulation of apolipoprotein E gene expression in macrophages

The atheroprotective role of macrophage-derived apolipoprotein E (apoE) is well known. Our previous reports demonstrated that inflammatory stress down-regulates apoE expression in macrophages, aggravating atherogenesis. Metformin, extensively used as an anti-diabetic drug, has also anti-inflammatory properties, and thus confers vascular protection. In this study, we questioned whether metformin could have an effect on apoE expression in macrophages in normal conditions or under lipopolysaccharide (LPS)-induced stress. The results showed that metformin slightly increases the apoE expression only at high doses (5–10 mM). Low doses of metformin (1–3 mM) significantly reduce the LPS down-regulatory effect on apoE expression in macrophages. Our experiments demonstrated that LPS-induced NF-κB binds to the macrophage-specific distal regulatory element of apoE gene, namely to the multienhancer 2 (ME.2) and its 5′-deletion fragments. The NF-κB binding on ME.2 and apoE promoter has a down-regulatory effect. In addition, data revealed that metformin impairs NF-κB nuclear translocation, and thus, improves the apoE levels in macrophages under inflammatory stress. The positive effect of metformin in the inflammatory states, its clinical safety and low cost, make this drug a potential adjuvant in the therapeutic strategies for atherosclerosis.

Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions

In atherogenesis, macrophage-derived apolipoprotein E (apoE) has an athero-protective role by a mechanism that is not fully understood. We investigated the regulatory mechanisms involved in the modulation of apoE expression in macrophages. The experiments showed that the promoters of all genes of the apoE/apoCI/apoCIV/apoCII gene cluster are enhanced by multienhancer 2 (ME.2), a regulatory region that is located 15.9 kb downstream of the apoE gene. ME.2 interacts with the apoE promoter in a macrophage-specific manner. Transient transfections in RAW 264.7 macrophages showed that the activity of ME.2 was strongly decreased by deletion of either 87 bp from the 5' end or 131 bp from the 3' end. We determined that the minimal fragment of this promoter that can be activated by ME.2 is the proximal -100/+73 region. The analysis of the deletion mutants of ME.2 revealed the importance of the 5' end of ME.2 in apoE promoter transactivation. Chromatin conformational capture assays demonstrated that both ME.2 and ME.1 physically interacted with the apoE promoter in macrophages. Our data showed that phorbol 12-myristate 13-acetate-induced differentiation of macrophages is accompanied by a robust induction of apoE and STAT1 expression. In macrophages (but not in hepatocytes), STAT1 up-regulated apoE gene expression via ME.2. The STAT1 binding site was located in the 174-182 region of ME.2. In conclusion, the specificity of the interactions between the two multienhancers (ME.1 and ME.2) and the apoE promoter indicates that these distal regulatory elements play an important role in the modulation of apoE gene expression in a cell-specific manner.

Macrophage, But Not Systemic, Apolipoprotein E Is Necessary for Macrophage Reverse Cholesterol Transport In Vivo

To assess the role of apolipoprotein (apo) E in macrophage reverse cholesterol transport (RCT) in vivo. ApoE exerts an antiatherosclerotic activity by regulating lipoprotein metabolism and promoting cell cholesterol efflux. We discriminated between macrophage and systemic apoE contribution using an assay of macrophage RCT in mice. The complete absence of apoE lead to an overall impairment of the process and, similarly, the absence of apoE exclusively in macrophages resulted in the reduction of cholesterol mobilization from macrophages to plasma, liver, and feces. Conversely, expression of apoE in macrophages is sufficient to promote normal RCT even in apoE-deficient mice. The mechanisms accounting for these results were investigated by evaluating the first step of RCT (ie, cholesterol efflux from cells). Macrophages isolated from apoE-deficient mice showed a reduced ability to release cholesterol into the culture medium, whereas the apoB-depleted plasma from apoE-deficient and healthy mice possessed a similar capacity to promote cellular lipid release from cultured macrophages. Our data demonstrate, for the first time to our knowledge, that apoE significantly contributes to macrophage RCT in vivo and that this role is fully attributable to apoE expressed in macrophages.

Inflammatory Signaling Pathways Regulating ApoE Gene Expression in Macrophages

The atheroprotective role of apolipoprotein E (apoE) is well established. During inflammation, expression of apoE in macrophages is reduced leading to enhanced atheromatous plaque development. In the present study, we investigated the signaling pathways involved in the repression of apoE gene expression in response to lipopolysaccharide (LPS) treatment, a condition that mimics the inflammatory stress, in mouse macrophages RAW 264.7. We identified Tpl-2 and MEKK1 as the kinases that are primarily responsible for the down-regulation of apoE promoter activity by LPS. Using a dominant negative form of IkappaB, we established that Tpl-2 and MEKK1 signaling pathways converge to NF-kappaB acting on the apoE core promoter -55/+73. In addition to NF-kappaB activation, LPS also activated c-Jun via its phosphorylation by JNK. The activity of the apoE promoter was repressed by c-Jun, whereas small interference RNA-mediated inhibition of endogenous c-Jun expression reversed the inhibitory effect of Tpl-2 on the apoE promoter. Transfection experiments and DNA binding assays showed that the binding site for c-Jun is in the -55/+73 region of the apoE promoter. Finally, we showed that LPS inhibited apoE gene expression via activation of the Tpl-2/MEK/ERK pathway acting on a different apoE promoter region. In summary, LPS represses apoE gene expression in macrophages via signaling pathways that involve the upstream kinases Tpl-2 and MEKK1, the intermediate mitogen-activated protein kinases ERK and JNK, and the downstream transcription factors AP-1 and NF-kappaB that inhibit the apoE promoter activity via distinct regions.

Reduced ABCA1-Mediated Cholesterol Efflux and Accelerated Atherosclerosis in Apolipoprotein E–Deficient Mice Lacking Macrophage-Derived ACAT1

Macrophage acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) and apolipoprotein E (apoE) have been implicated in regulating cellular cholesterol homeostasis and therefore play critical roles in foam cell formation. Deletion of either ACAT1 or apoE results in increased atherosclerosis in hyperlipidemic mice, possibly as a consequence of altered cholesterol processing. We have studied the effect of macrophage ACAT1 deletion on atherogenesis in apoE-deficient (apoE-/-) mice with or without the restoration of macrophage apoE. We used bone marrow transplantation to generate apoE-/- mice with macrophages of 4 genotypes: apoE+/+/ACAT1+/+ (wild type), apoE+/+/ACAT1-/- (ACAT-/-), apoE-/-/ACAT1+/+ (apoE-/-), and apoE-/-/ACAT1-/- (2KO). When macrophage apoE was present, plasma cholesterol levels normalized, and ACAT1 deficiency did not have significant effects on atherogenesis. However, when macrophage apoE was absent, ACAT1 deficiency increased atherosclerosis and apoptosis in the proximal aorta. Cholesterol efflux to apoA-I was significantly reduced (30% to 40%; P<0.001) in ACAT1-/- peritoneal macrophages compared with ACAT1+/+ controls regardless of apoE expression. 2KO macrophages had a 3- to 4-fold increase in ABCA1 message levels but decreased ABCA1 protein levels relative to ACAT1+/+ macrophages. Microarray analyses of ACAT1-/- macrophages showed increases in proinflammatory and procollagen genes and decreases in genes regulating membrane integrity, protein biosynthesis, and apoptosis. Deficiency of macrophage ACAT1 accelerates atherosclerosis in hypercholesterolemic apoE-/- mice but has no effect when the hypercholesterolemia is corrected by macrophage apoE expression. However, ACAT1 deletion impairs ABCA1-mediated cholesterol efflux in macrophages regardless of apoE expression. Changes in membrane stability, susceptibility to apoptosis, and inflammatory response may also be important in this process.

Absence of the protease inhibitor cystatin C in inflammatory cells results in larger plaque area in plaque regression of apoE-deficient mice

Matrix remodelling plays an important role in regulating plaque stability. Cystatin C, an inhibitor of the elastin-degrading cysteine proteases of the cathepsin family, is believed to be one of the key protease inhibitors in this process. The aim of the present study was to investigate the role of leukocyte-specific cystatin C expression under conditions that favour plaque regression. Apolipoprotein E-deficient mice (apoE −/−) were given a Western-type diet 15 weeks prior transplantation with bone marrow from mice lacking cystatin C (cysC −/−) or cystatin C positive (cysC +/+) mice, in both cases apoE +/+ to create conditions favouring plaque regression. Transplantations were verified with PCR and Western analyses. Transplanted mice showed a 70% decrease in lipid content and reduction in plaque area compared to baseline ApoE −/− mice, demonstrating plaque regression due to apoE expression in macrophages. apoE −/− mice transplanted with cysC −/− bone marrow were then compared to mice transplanted with cysC +/+ bone marrow. Mice receiving cysC −/− bone marrow had a 30% larger plaque area, despite absence of significant differences in plasma cholesterol and lipid contents in plaque. Unexpectedly, mice transplanted with cystatin C-deficient bone marrow cells had increased elastin and collagen content in lesions. These observations suggest that leukocyte-specific expression of cystatin C is actively involved in matrix remodelling associated with plaque regression.

Heterogeneous expression of apolipoprotein‐E by human macrophages

Apolipoprotein-E (apoE) is expressed at high levels by macrophages. In addition to its role in lipid transport, macrophage-derived apoE plays an important role in immunoregulation. Previous studies have identified macrophage subpopulations that differ substantially in their ability to synthesize specific cytokines and enzymes, however, potential heterogeneous macrophage apoE expression has not been studied. Here we examined apoE expression in human THP-1 macrophages and monocyte-derived macrophages (MDM). Using immunocytochemistry and flow cytometry methods we reveal a striking heterogeneity in macrophage apoE expression in both cell types. In phorbol-ester-differentiated THP-1 macrophages, 5% of the cells over-expressed apoE at levels more than 50-fold higher than the rest of the population. ApoE over-expressing THP-1 macrophages contained condensed/fragmented nuclei and increased levels of activated caspase-3 indicating induction of apoptosis. In MDM, 3-5% of the cells also highly over-expressed apoE, up to 50-fold higher than the rest of the population; however, this was not associated with obvious nuclear alterations. The apoE over-expressing MDM were larger, more granular, and more autofluorescent than the majority of cells and they contained numerous vesicle-like structures that appeared to be coated by apoE. Flow cytometry experiments indicated that the apoE over-expressing subpopulation of MDM were positive for CD14, CD11b/Mac-1 and CD68. These observations suggest that specific macrophage subpopulations may be important for apoE-mediated immunoregulation and clearly indicate that subpopulation heterogeneity should be taken into account when investigating macrophage apoE expression.

Oleic Acid Modulates the Post-translational Glycosylation of Macrophage ApoE to Increase Its Secretion

There has been increasing interest in a potential role for fatty acids in adversely affecting organismal substrate utilization and contributing to the cardiovascular complications in insulin resistance. Fatty acids have already been implicated in regulating the expression of a number of genes in resident cells of the vessel wall. In the current studies, we evaluated a potential role for fatty acids in the regulation of macrophage apoE expression. Incubation in oleic acid increased the synthesis and secretion of apoE by human monocyte-derived macrophages. Part of this stimulation was mediated at a post-translational locus. Oleic acid increased the secretion of apoE from macrophages that constitutively expressed a human apoE3 cDNA. Incubation in palmitic acid decreased apoE secretion from these cells. The effect of oleic acid on apoE secretion could not be accounted for by the known effect of fatty acid on cellular sterol, because incubation in oleic acid did not suppress the degradation of nascent apoE. Incubation in oleic acid for at least 6 h was required to observe an effect on apoE secretion. Oleic acid altered the glycosylation pattern of cellular and secreted apoE, with a loss of the most heavily sialylated isoform. Oleic acid had no effect on the glycosylation of interleukin 6 secreted from macrophages. Elimination of apoE glycosylation, by substitution of threonine 194 with alanine, eliminated oleic acid-mediated stimulation of apoE secretion. These results indicate that oleic acid increases apoE secretion from macrophages at a locus involving post-translational glycosylation.

Apolipoprotein E modulates immune activation by acting on the antigen-presenting cell.

Apolipoprotein E (ApoE) is synthesized by a variety of cells including macrophages. These cells activate T lymphocytes by antigen presentation, while the T-cell cytokine, interferon-gamma, inhibits macrophage ApoE expression. ApoE inhibits T-cell proliferation in culture but its role in immune responses has been unclear. The ApoE-deficient (E0) mouse permits an evaluation of the immunological role of ApoE. We have analysed T-cell responses to an exogenous antigen (ovalbumin) and polyclonal mitogen (concanavalin A) in E0 and ApoE+/+ mice. Macrophages of E0 mice stimulated T-cell activation more effectively as antigen-presenting cells than macrophages from ApoE+/+ mice. Both proliferation and interferon-gamma secretion were enhanced in T cells activated in the context of antigen-presenting cells from E0 mice. Since the macrophage-T-cell interaction depends on interactions between cell surface molecules, we assessed the expression of such molecules after in vivo stimulation with interferon-gamma. This treatment caused an increased expression of the co-stimulatory surface proteins CD40 and CD80, and also of the major histocompatibility complex class II molecules I-Ab on macrophages of E0 mice compared with ApoE+/+. Our data suggest that ApoE inhibits T-cell activation by reducing the density of immune stimulatory proteins on antigen-presenting cells.

Gene therapy of apolipoprotein E–deficient mice using a novel macrophage-specific retroviral vector

The use of retroviral gene transfer into hematopoietic stem cells for human gene therapy has been hampered by the absence of retroviral vectors that can generate long-lasting, lineage-specific gene expression. We developed self-inactivating retroviral vectors that incorporate gene-regulatory elements from the macrophage-restricted human CD68 gene. Through the transplantation of transduced murine hematopoietic stem cells (HSCs), we show that a vector incorporating a 342-base pair (bp) fragment of 5' flanking sequence from the CD68 gene, in addition to the CD68 first intron, was able to direct macrophage-specific expression of an enhanced green fluorescent protein (EGFP) reporter gene in inflammatory cell exudates and lymphoid organs in vivo. Levels of EGFP expression generated by this vector were greater than those generated by a standard Moloney murine leukemia retroviral vector, and they were stable for at least a year after transplantation of transduced HSCs. To evaluate the ability of this vector to generate therapeutically useful levels of gene expression, we transplanted apolipoprotein E (ApoE)-deficient HSCs transduced with a virus encoding ApoE into ApoE-deficient mice. Macrophages from these mice expressed levels of ApoE that were comparable to those from wild-type mice, and vector-driven expression of ApoE in macrophages was sufficient to reverse both hypercholesterolemia and atherosclerotic lesion development. The future application of this retroviral vector should provide a powerful tool to further elucidate macrophage function and for human gene therapy.

Regulation of Macrophage ApoE Expression and Processing by Extracellular Matrix

Macrophage-derived apoE in the vessel wall has important effects on atherogenesis in vivo, making it important to understand factors that regulate its expression. Vessel wall macrophages are embedded in an extracellular matrix produced largely by arterial smooth muscle cells and endothelial cells. In this series of studies, we evaluated the influence of extracellular matrix on macrophage apoE expression. Subendothelial matrix, fibronectin, or collagen I stimulated macrophage apoE gene expression and apoE synthesis. Adhesion of macrophages to a polylysine substrate had no effect. An increase in apoE synthesis after plating on fibronectin could be observed by 2 h and was inhibited by blocking antibodies to the alpha(5)beta(1) integrin receptor for fibronectin. Fibronectin also regulated the post-translational processing of newly synthesized macrophage apoE by inhibiting its degradation. The increment in apoE resulting from suppressed degradation was retained in the cell-fibronectin monolayer in a pool that was resistant to release by exogenous high density lipoprotein subfraction 3. These observations establish a new pathway for the regulation of macrophage apoE expression in the vessel wall. The composition of the extracellular matrix changes after vessel wall injury and in response to locally produced cytokines and growth factors. The evolving composition of this matrix will, therefore, be important for regulating apoE expression and processing by vessel wall macrophages.

Sterol efflux mediated by endogenous macrophage ApoE expression is independent of ABCA1.

Sterol efflux importantly contributes to preservation of cellular cholesterol homeostasis, and multiple pathways may be involved for mediating such efflux. Recently, an important role has been ascribed to ABCA1 in facilitating lipid efflux from cells, including macrophages, to extracellular lipid-free apolipoproteins. Macrophages are relatively unique among cells because they express apoprotein E (apoE) as a major protein product, and this endogenous expression of apoE increases sterol and phospholipid efflux from macrophages. The studies in this article were designed to test whether the sterol efflux mediated by the endogenous expression of apoE in macrophages was dependent on ABCA1 expression. These studies were facilitated by comparing apoE-expressing J774 cells (J774E(+)) with nonexpressing parental cells (J774E(-)). Sterol efflux was higher from J774E(+) cells compared with J774E(-) cells, but the increment in efflux between these cell types was not increased by induction of ABCA1 expression with cAMP. Induction of ABCA1 with cAMP, however, did increase sterol efflux to exogenously added apoA1 from both cell types. Inhibitors of ABCA1 activity significantly reduced (by 40% to 50%) sterol efflux from both J774E(+) and J774E(-) cells treated with cAMP and apoA1. This inhibitor did not, however, reduce the increment in sterol efflux due to the expression of endogenous apoE. The results of these studies indicate that the increment in sterol efflux mediated by the endogenous expression of apoE in macrophages does not depend on ABCA1 expression or activity.

Low-dose expression of a human apolipoprotein E transgene in macrophages restores cholesterol efflux capacity of apolipoprotein E-deficient mouse plasma.

Apolipoprotein E- (apoE) deficient (E-/-) mice develop severe hyperlipidemia and diffuse atherosclerosis. Low-dose expression of a human apoE3 transgene in macrophages of apoE-deficient mice (E-/-hTgE+/0), which results in about 5% of wild-type apoE plasma levels, did not correct hyperlipidemia but significantly reduced the extent of atherosclerotic lesions. To investigate the contribution of apoE to reverse cholesterol transport, we compared plasmas of wild-type (E+/+), E-/-, and E-/-hTgE+/0 mice for the appearance of apoE-containing lipoproteins by electrophoresis and their capacity to take up and esterify 3H-labeled cholesterol from radiolabeled fibroblasts or J774 macrophages. Wild-type plasma displayed lipoproteins containing apoE that were the size of high density lipoprotein and that had either electrophoretic alpha or gamma mobilities. Similar particles were also present in E-/-hTgE+/0 plasma. Depending on incubation time, E-/- plasma released 48-74% less 3H-labeled cholesterol from fibroblasts than E+/+ plasma, whereas cholesterol efflux into E-/-hTgE+/0 plasma was only 11-25% lower than into E+/+ plasma. E-/-hTgE+/0 plasma also released 10% more 3H-labeled cholesterol from radiolabeled J774 macrophages than E-/- plasma. E+/+ and E-/-hTgE+/0 plasma each esterified significantly more cell-derived 3H-labeled cholesterol than E-/- plasma. Moreover, E-/- plasma accumulated much smaller proportions of fibroblast-derived 3H-labeled cholesterol in fractions with electrophoretic gamma and alpha mobility than E+/+ and E-/-hTgE+/0 plasma. Thus, low-dose expression of apoE in macrophages nearly restored the cholesterol efflux capacity of apoE-deficient plasma through the formation of apoE-containing particles, which efficiently take up cell-derived cholesterol, and through the increase of cholesterol esterification activity. Thus, macrophage-derived apoE may protect against atherosclerosis by increasing cholesterol efflux from arterial wall cells.

Macrophage-specific expression of human apolipoprotein E reduces atherosclerosis in hypercholesterolemic apolipoprotein E-null mice.

apoE deficiency causes hyperlipidemia and premature atherosclerosis. To determine if macrophage-specific expression of apoE would decrease the extent of atherosclerosis, we expressed human apoE in macrophages of apoE-null mice (apoE-/-) and assessed the effect on lipid accumulation in cells of the arterial wall. Macrophage-specific expression of human apoE in normal mice was obtained by use of the visna virus LTR. These animals were bred with apoE-/- mice to produce animals hemizygous for expression of human apoE in macrophages in the absence of murine apoE (apoE-/-,hTgE+/0). Low levels of human apoE mRNA were present in liver and spleen and high levels in lung and peritoneal macrophages. Human apoE was secreted by peritoneal macrophages and was detected in Kupffer cells of the liver. Human apoE in the plasma of apoE-/-,hTgE+/0 mice (n = 30) was inversely correlated (P < 0.005) with the plasma cholesterol concentration. After 15 wk on a normal chow diet, atherosclerosis was assessed in apoE-/-,hTgE+/0 animals and in apoE-/-,hTgE0/0 littermates matched for plasma cholesterol level (approximately 450 mg/dl) and lipoprotein profile. There was significantly less atherosclerosis in both the aortic sinus and in the proximal aorta (P < 0.0001) in the animals expressing the human apoE transgene. In apo-E-/-,hTgE+/0 animals, which had detectable atherosclerotic lesions, human apoE was detected in the secretory apparatus of macrophage-derived foam cells in the arterial wall. The data demonstrate that expression of apoE by macrophages is antiatherogenic even in the presence of high levels of atherogenic lipoproteins. The data suggest that apoE prevents atherosclerosis by promoting cholesterol efflux from cells of the arterial wall.