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Introduction: Oxysterol binding protein Related Proteins (ORPs) mediate intracellular lipid transport and homeostatic regulation. ORP8 downregulates ABCA1 expression in macrophages and cellular cholesterol efflux to apolipoprotein A-I. In line, ORP8 knockout mice display increased amounts of HDL cholesterol in blood. However, the role of macrophage ORP8 in atherosclerotic lesion development is unknown., Methods and Results: LDL receptor knockout (KO) mice were transplanted with bone marrow (BM) from ORP8 KO mice and C57Bl/6 wild type mice. Subsequently, the animals were challenged with a high fat/high cholesterol Western-type diet to induce atherosclerosis. After 9 weeks of Western-Type diet feeding, serum levels of VLDL cholesterol were increased by 50% in ORP8 KO BM recipients compared to the wild-type recipients. However, no differences were observed in HDL cholesterol. Despite the increase in VLDL cholesterol, lesions in mice transplanted with ORP8 KO bone marrow were 20% smaller compared to WT transplanted controls. In addition, ORP8 KO transplanted mice displayed a modest increase in the percentage of macrophages in the lesion as compared to the wild-type transplanted group. ORP8 deficient macrophages displayed decreased production of pro-inflammatory factors IL-6 and TNFα, decreased expression of differentiation markers and showed a reduced capacity to form foam cells in the peritoneal cavity., Conclusions: Deletion of ORP8 in bone marrow-derived cells, including macrophages, reduces lesion progression after 9 weeks of WTD challenge, despite increased amounts of circulating pro-atherogenic VLDL. Reduced macrophage foam cell formation and lower macrophage inflammatory potential are plausible mechanisms contributing to the observed reduction in atherosclerosis.

Patients suffering from cardiovascular disease have well-established atherosclerotic lesions, rendering lesion regression of therapeutic interest. The OX40 (TNFRSF4)-OX40 ligand (OX40L; TNFSF4) pathway is important for the proliferation and survival of T cells, stimulates B cells, and is associated with cardiovascular disease. We hypothesized that interference with the OX40-OX40L pathway, in combination with decreases in cholesterol, may induce regression of atherosclerosis. LDLr(-/-) mice were fed a Western-type diet for 10 wk, after which they received chow diet and were treated with anti-OX40L or PBS for 10 wk. A significant regression of lesions was observed in the aorta and aortic arch of anti-OX40L-treated mice compared with control mice. Interference of the OX40-OX40L pathway reduced Th2 responses, as shown by decreases in GATA-3 and IL-4 levels. Also, IgE levels were decreased, as demonstrated by reduced mast cell presence and activation. Notably, IL-5 production by T and B1 cells was increased, thus enhancing atheroprotective oxidized low-density lipoprotein-specific IgM production. The increase in IL-5 production and IgM was mediated by IL-33 production by APCs upon OX40L blockade. We conclude that interruption of the OX40-OX40L signaling pathway, combined with decreases in dietary cholesterol, induces the regression of atherosclerosis through induction of IL-5-producing T cells and oxidized low-density lipoprotein-specific IgM and reductions in Th2 and mast cells.

Aims: Altered sphingosine 1-phosphate (S1P) homeostasis and signaling is implicated in various inflammatory diseases including atherosclerosis. As S1P levels are tightly controlled by S1P lyase, we investigated the impact of hematopoietic S1P lyase (Sgpl1(-/-)) deficiency on leukocyte subsets relevant to atherosclerosis., Methods and Results: LDL receptor deficient mice that were transplanted with Sgpl1(-/-) bone marrow showed disrupted S1P gradients translating into lymphopenia and abrogated lymphocyte mitogenic and cytokine response as compared to controls. Remarkably however, Sgpl1(-/-) chimeras displayed mild monocytosis, due to impeded stromal retention and myelopoiesis, and plasma cytokine and macrophage expression patterns, that were largely compatible with classical macrophage activation. Collectively these two phenotypic features of Sgpl1 deficiency culminated in diminished atherogenic response., Conclusions: Here we not only firmly establish the critical role of hematopoietic S1P lyase in controlling S1P levels and T cell trafficking in blood and lymphoid tissue, but also identify leukocyte Sgpl1 as critical factor in monocyte macrophage differentiation and function. Its, partly counterbalancing, pro- and anti-inflammatory activity spectrum imply that intervention in S1P lyase function in inflammatory disorders such as atherosclerosis should be considered with caution.

Leukocyte chemotaxis is deemed instrumental in initiation and progression of atherosclerosis. It is mediated by G-protein-coupled receptors (e.g., CCR2 and CCR5), the activity of which is controlled by G-protein-coupled receptor kinases (GRKs). In this study, we analyzed the effect of hematopoietic deficiency of a potent regulator kinase of chemotaxis (GRK2) on atherogenesis. LDL receptor-deficient (LDLr(-/-)) mice with heterozygous hematopoietic GRK2 deficiency, generated by bone marrow transplantation (n=15), displayed a dramatic attenuation of plaque development, with 79% reduction in necrotic core and increased macrophage content. Circulating monocytes decreased and granulocytes increased in GRK2(+/-) chimeras, which could be attributed to diminished granulocyte colony-forming units in bone marrow. Collectively, these data pointed to myeloid cells as major mediators of the impaired atherogenic response in GRK2(+/-) chimeras. LDLr(-/-) mice with macrophage/granulocyte-specific GRK2 deficiency (LysM-Cre GRK2(flox/flox); n=8) failed to mimic the aforementioned phenotype, acquitting these cells as major responsible subsets for GRK2 deficiency-associated atheroprotection. To conclude, even partial hematopoietic GRK2 deficiency prevents atherosclerotic lesion progression beyond the fatty streak stage, identifying hematopoietic GRK2 as a potential target for intervention in atherosclerosis.

Objective: The ABCA2 transporter shares high structural homology to ABCA1, which is crucial for the removal of excess cholesterol from macrophages and, by extension, in atherosclerosis. It has been suggested that ABCA2 sequesters cholesterol inside the lysosomes, however, little is known of the macrophage-specific role of ABCA2 in regulating lipid homeostasis in vivo and in modulating susceptibility to atherosclerosis., Methods: Chimeras with dysfunctional macrophage ABCA2 were generated by transplantation of bone marrow from ABCA2 knockout (KO) mice into irradiated LDL receptor (LDLr) KO mice., Results: Interestingly, lack of ABCA2 in macrophages resulted in a diminished lesion size in the aortic root (-24.5%) and descending thoracic aorta (-36.6%) associated with a 3-fold increase in apoptotic cells, as measured by both caspase 3 and TUNEL. Upon oxidized LDL exposure, macrophages from wildtype (WT) transplanted animals developed filipin-positive droplets in lysosomal-like compartments, corresponding to free cholesterol (FC) accumulation. In contrast, ABCA2-deficient macrophages displayed an abnormal diffuse distribution of FC over peripheral regions. The accumulation of neutral sterols in lipid droplets was increased in ABCA2-deficient macrophages, but primarily in cytoplasmic clusters and not in lysosomes. Importantly, apoptosis of oxLDL loaded macrophages lacking ABCA2 was increased 2.7-fold, probably as a consequence of the broad cellular distribution of FC., Conclusions: Lack of functional ABCA2 generates abnormalities in intracellular lipid distribution/trafficking in macrophages consistent with its lysosomal sequestering role, leading to an increased susceptibility to apoptosis in response to oxidized lipids and reduced atherosclerotic lesion development., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

MicroRNAs modulate processes associated with cell cycle control and differentiation. Here we explored the potential of microRNAs in the modulation of hepatic lipid metabolism and the development of nonalcoholic fatty liver disease. MicroRNA profiles of hepatocytes from low-density lipoprotein (LDL) receptor knockout mice fed a chow diet or a hypertriglyceridemia/fatty liver-inducing Western-type diet (WTD) were determined using quantitative real-time polymerase chain reaction. Ninety-seven of 103 microRNAs measured were expressed by hepatocytes and low variability between hepatocyte pools was observed. Feeding WTD coincided with a marked fivefold decrease in the relative expression level of miR-216 (P<.05) and miR-302a (P<.01). Interestingly, an increased hepatic miR-216 expression was detected in response to fasting. MicroRNA/biological function linkage analysis suggested that the change in hepatocyte microRNA profiles in response to high dietary lipid levels is associated with changes in cell cycle control and proliferation. In accordance with a diminished miR-302a expression on the WTD, hepatocyte mRNA expression levels of miR-302a target genes ABCA1 and in particular ELOVL6 were increased in response to WTD (twofold to ninefold). This suggests a role for miR-302a in hepatic cholesterol, fatty acid and glucose metabolism. In conclusion, we have shown that fatty liver development in LDL receptor knockout mice is associated with a significant change in the hepatocyte microRNA profile, i.e., a fivefold decrease in miR-216 and miR-302a expression. Based upon our comparative gene and microRNA expression studies it is anticipated that miR-302a may prove to be a valuable therapeutic target in the regulation of hepatic fatty acid utilization and insulin resistance., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: As ABCG1 plays a role in cholesterol efflux, macrophage ABCG1 expression has been suggested to protect against atherosclerosis. However, we and others observed varying effects of ABCG1 deficiency on atherosclerotic lesion size. The objective of this study was to define the effect of ABCG1 deficiency during atherosclerotic lesion progression in LDL receptor knockout (LDLr(-/-)) mice., Methods and Results: ABCG1(-/-)/LDLr(-/-) and ABCG1(+/+)/LDLr(-/-) littermates were fed a Western-type diet for 10 and 12 weeks in order to study the effect of ABCG1 deficiency in the exponential phase of atherosclerotic lesion formation. At 10 weeks of diet feeding, a significant 1.5-fold increase in early atherosclerotic lesion size (130±12×10(3) μm(2)) was observed in ABCG1(-/-)/LDLr(-/-) mice compared to ABCG1(+/+)/LDLr(-/-) mice (88±11×10(3) μm(2); p<0.05). Interestingly, in more advanced lesions, induced by 12 weeks of WTD feeding, ABCG1(-/-)/LDLr(-/-) mice showed a significant 1.7-fold decrease in atherosclerotic lesion size (160±20×10(3) μm(2) vs 273±19×10(3) μm(2) in control mice; p<0.01), indicating that in the ABCG1(-/-)/LDLr(-/-) mice progression of lesion formation is retarded as compared to ABCG1(+/+)/LDLr(-/-) mice. In addition, correlation analysis performed on 7 independent published studies and the current study confirmed that ABCG1 is atheroprotective in early lesions, while the development of advanced lesions is stimulated., Conclusions: It appears that the effect of ABCG1 deficiency on lesion development in LDLr(-/-) mice depends on the stage of atherogenesis, whereby the absence of ABCG1 leads to increased lesions at sizes<167×10(3) μm(2) while in more advanced stages of atherosclerosis enhanced apoptosis and/or compensatory mechanisms lead to retarded lesion progression., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Aim: ATP-binding cassette transporter A1 (ABCA1) is an important mediator of macrophage cholesterol efflux. It mediates the efflux of cellular cholesterol to lipid-poor apolipoprotein A-I. LDL receptor (LDLr) knockout (KO) mice deficient for leukocyte ABCA1 (ABCA1 KO→LDLr KO) show increased atherosclerosis and splenic lipid accumulation despite largely attenuated serum cholesterol levels. In the present study, we aimed to explore the importance of the spleen for the atheroprotective effects of leukocyte ABCA1., Methods: LDLr KO mice were transplanted with bone marrow from ABCA1 KO mice or wild-type (WT) controls. After 8 weeks recovery, mice were either splenectomized (SP-x) or underwent a sham operation, and were subsequently challenged with a Western-type diet (WTD)., Results: In agreement with previous studies, the atherosclerotic lesion area in ABCA1 KO→LDLr KO sham animals (655 ± 82 × 10(3) µm(2)) was 1.4-fold (p = 0.03) larger compared to sham WT→LDLr KO mice (459 ± 33 × 10(3) µm(2)) after 8 weeks WTD feeding, despite 1.7-fold (p<0.001) lower serum cholesterol levels. Interestingly, deletion of ABCA1 in leukocytes led to 1.6-fold higher neutrophil content in the spleen in absence of differences in circulating neutrophils. Levels of KC, an important chemoattractant for neutrophils, in serum, however, were increased 2.9-fold (p = 0.07) in ABCA1 KO→LDLr KO mice. SP-x induced blood neutrophilia as compared to WT→LDLr KO mice (1.9-fold; p<0.05), but did not evoke differences in serum cholesterol and anti-oxLDL antibody levels. Atherosclerotic lesion development, however, was 1.3-fold induced both in the presence and absence of leukocyte ABCA1 (WT: 614 ± 106 × 10(3) µm(2), ABCA1 KO: 786 ± 44 × 10(3) µm(2)). Two-way ANOVA revealed independent effects on atherosclerosis for both leukocyte ABCA1 deficiency and SP-x (p<0.05)., Conclusions: The observed splenic alterations induced by leukocyte ABCA1 deficiency do not play a significant role in the anti-atherogenic effects of leukocyte ABCA1 on lesion development.

Reducing the concentration of circulating lipids leads to decreased cardiovascular morbidity and mortality, but the dynamic remodeling that established atherosclerotic lesions undergo upon lipid lowering is poorly understood. Early or advanced lesions in the aortic root were induced by feeding LDL receptor knockout mice a high-fat, high-cholesterol Western-type diet for 5 or 9 weeks, respectively. In the first week after switching to a chow diet, plasma total cholesterol levels dropped 70%, but both early and advanced lesions increased in size. Early lesions grew because of an increase in smooth muscle cells; advanced lesions had an enlargement of absolute macrophage area. From 1 to 3 weeks after the diet switch, plasma total cholesterol levels were completely normalized, but the size of early lesions remained stable; however, advanced lesions became smaller due to a reduction of the absolute macrophage area. From 3 to 6 weeks, both early and advanced lesions progressed further, as a result of expansion of the absolute collagen and necrotic core area. In contrast, early lesions became proinflammatory, as evidenced by the increased infiltration of neutrophils and increased oxidative stress, probably caused by the activation of mast cells in the adventitia. Thus, the severity of atherosclerotic lesions affects their dynamic response to lipid lowering, indicating the importance of establishing stage-specific therapeutic protocols for the treatment of atherosclerosis., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Interleukin 15 (IL-15) is a pro-inflammatory cytokine involved in inflammatory diseases and IL-15 is expressed in atherosclerotic plaques., Methods: To establish the role of IL-15 in atherosclerosis we studied the effect of IL-15 on atherosclerosis associated cells in vitro and in vivo by neutralizing IL-15 using a DNA vaccination strategy., Results: Upon feeding a Western type diet LDLr(-/-) mice do express higher levels of IL-15 within the spleen and the number of IL-15 expressing cells among blood leukocytes and spleen cells is increased. Addition of IL-15 to macrophages induces the expression TNF-α and CCL-2. After the mice were vaccinated against IL-15, we observe a reduction in plaque size of 75% plaque. Unexpectedly, the relative number of macrophages within the plaque was 2-fold higher in IL-15 vaccinated mice than in control mice. Vaccination against IL-15 leads to an increased cytotoxicity against IL-15 overexpressing target cells, resulting in a reduction in IL-15 expressing cells and macrophages in blood and spleen and a decreased CD4/CD8 ratio., Conclusion: Hypercholesterolemia leads to upregulation of IL-15 within spleen and blood. DNA vaccination against IL-15 does markedly reduces atherosclerotic lesion size, but does not promote lesion stability., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Aim: ABCA1 protects against atherosclerosis by facilitating cholesterol efflux from macrophage foam cells in the arterial wall to extracellular apolipoprotein (apo) A-I. In contrast to apoA-I, apoE is secreted by macrophages and can, like apoA-I, induce ABCA1-mediated cholesterol efflux. Yet, the combined effect of macrophage ABCA1 and apoE on lesion development is unexplored., Methods and Results: LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/apoE double KO (dKO) mice, their respective single KO's, and wild-type (WT) controls and were challenged with a high-fat/high-cholesterol diet for 9 weeks. In vitro cholesterol efflux experiments showed no differences between ABCA1 KO and dKO macrophages. The serum non-HDL/HDL ratio in dKO transplanted mice was 1.7-fold and 2.4-fold (p<0.01) increased compared to WT and ABCA1 KO transplanted mice, respectively. The atherosclerotic lesion area in dKO transplanted animals (650±94×10(3) µm(2)), however, was 1.9-fold (p<0.01) and 1.6-fold (p<0.01) increased compared to single knockouts (ABCA1 KO: 341±20×10(3) µm(2); apoE KO: 402±78×10(3) µm(2), respectively) and 3.1-fold increased (p<0.001) compared to WT (211±20×10(3) µm(2)). When normalized for serum cholesterol exposure, macrophage ABCA1 and apoE independently protected against atherosclerotic lesion development (p<0.001). Moreover, hepatic expression levels of TNFα and IL-6 were highly induced in dKO transplanted animals (3.0-fold; p<0.05, and 4.3-fold; p<0.001, respectively). In agreement, serum IL-6 levels were also enhanced in ABCA1 KO transplanted mice (p<0.05) and even further enhanced in dKO transplanted animals (3.1-fold as compared to ABCA1 KO transplanted animals; p<0.05)., Conclusions: Combined deletion of macrophage ABCA1 and apoE results in a defect in cholesterol efflux and, compared to ABCA1 KO transplanted mice, elevated serum total cholesterol levels. Importantly, these mice also suffer from enhanced systemic and hepatic inflammation, together resulting in the observed augmented atherosclerotic lesion development.

Objective: The consequences of macrophage triglyceride (TG) accumulation on atherosclerosis have not been studied in detail so far. Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for the initial step in TG hydrolysis. Because ATGL knockout (KO) mice exhibit massive TG accumulation in macrophages, we used ATGL KO mice to study the effects of macrophage TG accumulation on atherogenesis., Methods and Results: Low-density lipoprotein receptor (LDLr) KO mice were transplanted with bone marrow from ATGL KO (ATGL KO→LDLr KO) or wild-type (WT→LDLr KO) mice and challenged with a Western-type diet for 9 weeks. Despite TG accumulation in ATGL KO macrophages, atherosclerosis in ATGL KO→LDLr KO mice was 43% reduced associated with decreased plasma monocyte chemoattractant protein-1 (MCP-1) and macrophage interleukin-6 concentrations. This coincided with a reduced amount of macrophages, possibly because of a 39% increase in intraplaque apoptosis and a decreased migratory capacity of ATGL KO macrophages. The reduced number of white blood cells might be due to a 36% decreased Lin(-)Sca-1(+)cKit(+) hematopoietic stem cell population., Conclusions: We conclude that the attenuation of atherogenesis in ATGL KO→LDLr KO mice is due to decreased infiltration of less inflammatory macrophages into the arterial wall and increased macrophage apoptosis.

We have previously identified the E3 ubiquitin ligase-inducible degrader of the low density lipoprotein receptor (LDLR) (Idol) as a post-translational modulator of LDLR levels. Idol is a direct target for regulation by liver X receptors (LXRs), and its expression is responsive to cellular sterol status independent of the sterol-response element-binding proteins. Here we demonstrate that Idol also targets two closely related LDLR family members, VLDLR and ApoE receptor 2 (ApoER2), proteins implicated in both neuronal development and lipid metabolism. Idol triggers ubiquitination of the VLDLR and ApoER2 on their cytoplasmic tails, leading to their degradation. We further show that the level of endogenous VLDLR is sensitive to cellular sterol content, Idol expression, and activation of the LXR pathway. Pharmacological activation of the LXR pathway in mice leads to increased Idol expression and to decreased Vldlr levels in vivo. Finally, we establish an unexpected functional link between LXR and Reelin signaling. We demonstrate that LXR activation results in decreased Reelin binding to VLDLR and reduced Dab1 phosphorylation. The identification of VLDLR and ApoER2 as Idol targets suggests potential roles for this LXR-inducible E3 ligase in the central nervous system in addition to lipid metabolism.

Aims: Modification of lipoproteins plays an important role in the development of atherosclerosis. Oxidatively modified low-density lipoprotein (oxLDL) has a number of pro-inflammatory effects, whereas immunization with various forms of oxLDL is able to reduce atherosclerosis. The uptake of modified LDL by dendritic cells (DCs) and the presentation of epitopes thereof may form an important step in the immunomodulatory effects of LDL. In this study, we transferred oxLDL-pulsed mature DCs (mDCs) to LDL receptor-null (LDLr(-/-)) mice and examined the effects on atherosclerosis., Methods and Results: Bone marrow-derived DCs were cultured for 10 days in the presence of granulocyte-macrophage colony-stimulating factor. Immature DCs were matured by lipopolysaccharide and pulsed with copper-oxidized LDL. These mDCs were transferred three times to LDLr(-/-) mice before the induction of atherosclerosis by Western-type diet feeding. The transfer of oxLDL-pulsed mDCs resulted in an 87% reduction in carotid artery lesion size (P < 0.001) with a concurrent increase in plaque stability, whereas treatment using mDCs pulsed with the atherosclerosis-irrelevant antigen, ovalbumin, did not influence lesion size or stability. Furthermore, the vaccination procedure resulted in the induction of oxLDL-specific T cells with a reduced Th1 profile and an increase in oxLDL-specific IgG levels, which contributed to a reduction in foam cell formation., Conclusion: These data indicate that vaccination with oxLDL-pulsed mDCs provides a novel and powerful strategy for the immunomodulation of atherosclerosis.

Atherosclerosis is an inflammatory disease characterized by the influx of macrophages and T cells and IL-17 may connect innate and adaptive immune responses involved in atherogenesis. We investigated the role of IL-17 receptor signaling in atherosclerosis and transplanted LDLr deficient recipient mice with IL-17R deficient bone marrow. Induction of atherosclerosis by Western-type diet induced a 46% reduction in lesion size in the aortic root and the plaque composition revealed no significant changes in collagen content and neutrophil counts, but a reduction in mast cell number and an increase in macrophage number. In addition, we observed a decrease in anti-oxLDL antibodies of the IgG class upon IL-17R BMT, while introduction of IL-17R deficient bone marrow resulted in a reduced IL-6 production and an increased IL-10 production. In conclusion, signaling via the IL-17 receptor in bone marrow derived cells enhances the process of atherosclerosis.

Aims: Cathepsin K (CatK), an established drug target for osteoporosis, has been reported to be upregulated in atherosclerotic lesions. Due to its proteolytic activity, CatK may influence the atherosclerotic lesion composition and stability. In this study, we investigated the potential role of leucocyte CatK in atherosclerotic plaque remodelling., Methods and Results: To assess the biological role of leucocyte CatK, we used the technique of bone marrow transplantation to selectively disrupt CatK in the haematopoietic system. Total bone marrow progenitor cells from CatK(+/+), CatK(+/-), and CatK(-/-) mice were transplanted into X-ray irradiated low-density lipoprotein receptor knockout (LDLr(-/-)) mice. The selective silencing of leucocyte CatK resulted in phenotypic changes in bone formation with an increased total bone mineral density in the CatK(-/-) chimeras and an effect of gene dosage. The absence of leucocyte CatK resulted in dramatically decreased collagen and increased macrophage content of the atherosclerotic lesions while lesion size was not affected. The atherosclerotic lesions also demonstrated less elastic lamina fragmentation and a significant increase in the apoptotic and necrotic area in plaques of mice transplanted with CatK(-/-) bone marrow., Conclusion: Leucocyte CatK is an important determinant of atherosclerotic plaque composition, vulnerability, and bone remodelling, rendering CatK an attractive target for pharmaceutical modulation in atherosclerosis and osteoporosis.

To investigate the potential for pregnane X receptor (PXR) ligands as antiatherosclerotic drugs, we have determined the effect of PXR activation on lipid metabolism in an established atherosclerotic mouse model. LDL receptor knockout mice were treated with the PXR agonist PCN. PCN induced a striking 66% decrease in plasma LDL-cholesterol levels. PCN did not affect the cholesterol levels of high-density lipoprotein (HDL) or very-low-density lipoprotein (VLDL). VLDL-triglyceride levels were 2.2-fold increased by PCN, resulting in the presence of triglyceride-rich VLDL particles. This coincided with a 60% decreased hepatic lipase (HL)-mediated plasma lipolysis rate, which could be attributed to a decrease in the hepatic mRNA expression level of both HL (-31%) and its cofactor apolipoprotein A4 (-62%). In the liver, PCN induced a significant increase in the level of triglycerides (+65%) and phospholipids (+72%), a hallmark of hepatic steatosis, leading to a marked increase in Oil red O neutral lipid staining. A similar effect was noticed in ApoE knockout mice. Our studies show that activation of the nuclear receptor PXR by PCN leads to an inhibition of the plasma HL-mediated lipolysis rate, which is associated with a decrease in plasma LDL-cholesterol levels and induction of hepatic steatosis in LDL receptor knockout mice.

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp(-)ldlr(-/-)vldlr(-/-) mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp(-)ldlr(-/-)vldlr(-/-) mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.

Aims: Murine CD99 was recently found to be expressed on leukocytes and endothelial cells, where it is concentrated at inter-endothelial contacts. Blockade of CD99 by specific antibodies inhibits leukocyte extravasation to inflamed sites in vivo. The aim of the present study is to show the role of CD99 in atherosclerosis using a CD99 vaccination protocol to block the function of CD99 during atherosclerosis., Methods and Results: We constructed a DNA vaccine against CD99 by cloning the extracellular domain of murine CD99 into pcDNA3. Vaccination was performed by oral administration of attenuated Salmonella typhimurium transformed with pcDNA3-CD99. This vaccination results in a CD99-specific, CD8-mediated cytotoxic response and subsequent reduction of CD99-expressing cells. We showed that CD99 is expressed on vascular endothelium overlying atherosclerotic plaques and found that CD99 expression is upregulated during western-type diet feeding. CD99 vaccination induced the formation of CD8-positive T cells that were cytotoxic against cells transfected with pcDNA3-CD99. Activation of CD8(+) T cells was demonstrated by a 30% increase in CD8(+)CD69(+) double-positive T cells in spleen and mediastinal lymph nodes. Furthermore, lymphocytes isolated from CD99-vaccinated mice specifically lysed CD99-expressing cells. More importantly, vaccination against CD99 attenuated atherosclerotic lesion formation in the aortic valve leaflets by 38% and in the carotid artery by 69% compared with mice that were vaccinated with a control vector. Furthermore, a lower number of cells were found in atherosclerotic lesions, implying that fewer leukocytes were recruited to these sites. These observations were accompanied by a decrease in CD99 expression on leukocytes., Conclusion: We conclude that vaccination against CD99 decreases atherogenesis by the selective removal of CD99-expressing cells, which could reduce leukocyte recruitment into atherosclerotic lesions and attenuate atherogenesis.

Objective: The chemokine receptor CXCR3 is implicated in migration of leukocytes to sites of inflammation. Antagonizing CXCR3 may be a strategy to inhibit inflammation-induced leukocyte migration and subsequently reduce atherosclerosis. We used the CXCR3 specific antagonist NBI-74330 to block CXCR3-mediated signaling in peritonitis and diet-induced atherosclerosis., Methods and Results: Antagonizing CXCR3 with NBI-74330 resulted in a significant reduction in CD4+ T cell and macrophage migration to the peritoneal cavity, which was as shown in ex vivo migration studies totally CXCR3 dependent. Atherosclerotic lesion formation in the aortic valve leaflet area and the entire aorta was significantly inhibited in NBI-74330 treated mice. Lymph nodes draining from the aortic arch were significantly smaller in treated mice and were enriched in regulatory T cells and contained fewer activated T cells, whereas the markers for regulatory T cells within the lesion were enhanced after NBI-74330 treatment., Conclusions: This study shows for the first time that treatment with a CXCR3 antagonist results in attenuating atherosclerotic lesion formation by blocking direct migration of CXCR3+ effector cells from the circulation into the atherosclerotic plaque and by beneficially modulating the inflammatory response in the lesion and the lymph nodes draining from the atherosclerotic lesion.

Objective: HSP60-specific T cells contribute to the development of the immune responses in atherosclerosis. This can be dampened by regulatory T cells activated via oral tolerance induction, and we explored the effect of oral tolerance induction to HSP60 and the peptide HSP60 (253 to 268) on atherosclerosis., Methods and Results: HSP60 and HSP60 (253 to 268) were administered orally to LDLr(-/-) mice before induction of atherosclerosis and resulted in a significant 80% reduction in plaque size in the carotid arteries and in a 27% reduction in plaque size at the aortic root. Reduction in plaque size correlated with an increase in CD4(+)CD25(+)Foxp3(+) regulatory T cells in several organs and in an increased expression of Foxp3, CD25, and CTLA-4 in atherosclerotic lesions of HSP60-treated mice. The production of interleukin (IL)-10 and transforming growth factor (TGF)-beta by lymph node cells in response to HSP60 was observed after tolerance induction., Conclusions: Oral tolerance induction to HSP60 and a small HSP60-peptide leads to an increase in the number of CD4(+)CD25(+)Foxp3(+) regulatory T cells, resulting in a decrease in plaque size as a consequence of increased production of IL-10 and TGF-beta. We conclude that these beneficial results of oral tolerance induction to HSP60 and HSP60 (253 to 268) may provide new therapeutic approaches for the treatment of atherosclerosis.

Objective: Several members of the ATP binding cassette (ABC)-transporter super family expressed in macrophages protect against atherosclerosis by promoting macrophage cholesterol and phospholipid efflux. Systemic disruption of ABCB4 in mice results in a virtual absence of phospholipids in bile and a strongly impaired biliary cholesterol secretion, indicating that ABCB4 plays an essential role in cellular lipid efflux. The aim of the current study was to determine the role of bone marrow-derived ABCB4 in atherosclerotic lesion development., Methods: Chimeras were created that specifically lack ABCB4 in bone marrow-derived cells, including macrophages, by performing a bone marrow transplantation on LDL receptor knockout (LDLr-/-) mice. Atherosclerotic lesion development was induced by feeding a high-cholesterol diet (15% fat and 0.25% cholesterol)., Results: Serum cholesterol levels were significantly lower in mice reconstituted with ABCB4 knockout bone marrow as a result of reduced VLDL and LDL cholesterol levels. Despite the lower serum cholesterol levels, ABCB4 deficiency in bone marrow-derived cells resulted in a 1.8-fold (p=0.005) increase in lesion size. In vitro foam cell formation, induced with acetylated LDL (AcLDL) in peritoneal macrophages, was increased in the absence of ABCB4, possibly due to a 2-fold (p<0.05) increased association of AcLDL, while the efflux of cholesterol was unaffected., Conclusion: Bone marrow-derived ABCB4 has an important anti-atherosclerotic function, probably by limiting macrophage foam cell formation.

Abundant amounts of cholesteryl ester transfer protein (CETP) are found in macrophage-derived foam cells in the arterial wall, but its function in atherogenesis is unknown. To investigate the role of macrophage CETP in atherosclerosis, LDL receptor knockout mice were transplanted with bone marrow from CETP transgenic mice, which express the human CETP transgene under control of its natural promoter and major regulatory elements. CETP production by bone marrow-derived cells induced a 1.8-fold (P<0.01) increase in atherosclerotic lesion development. The increase in lesion size coincided with an increase in VLDL/LDL cholesterol and a decrease in HDL cholesterol. The cholesterol redistribution in serum was a direct effect of the substantial serum CETP activity and mass (38+/-3 nmol/mL/h and 4.8+/-0.5 microg/mL, respectively) induced by CETP production by bone marrow-derived cells. Conversely, specific disruption of CETP production by bone marrow-derived cells in CETP transgenic mice resulted in a approximately 2-fold (P<0.0001) reduction in serum CETP activity and mass, demonstrating the quantitative relevance of bone marrow-derived CETP. Finally, we show that in liver Kupffer cells, hepatic macrophages, contribute approximately 50% to the total hepatic CETP expression. In conclusion, bone marrow-derived CETP induces a proatherogenic lipoprotein profile and promotes the development of atherosclerotic lesions in LDL receptor knockout mice. Most importantly, we show for the first time that bone marrow-derived CETP is an important contributor to total serum CETP activity and mass.

Objective: Systemic phospholipid transfer protein (PLTP) deficiency in mice is associated with a decreased susceptibility to atherosclerosis, whereas overexpression of human PLTP in mice increases atherosclerotic lesion development. PLTP is also expressed by macrophage-derived foam cells in human atherosclerotic lesions, but the exact role of macrophage PLTP in atherosclerosis is unknown., Methods and Results: To clarify the role of macrophage PLTP in atherogenesis, PLTP was selectively disrupted in hematopoietic cells, including macrophages, by transplantation of bone marrow from PLTP knockout (PLTP(-/-)) mice into irradiated low-density lipoprotein receptor knockout mice. Selective deficiency of macrophage PLTP (PLTP(-M/-M)) resulted in a 29% (P<0.01 for difference in lesion area) reduction in aortic root lesion area as compared with mice possessing functional macrophage PLTP (384+/-36*10(3) microm2 in the PLTP(-M/-M) group (n=10), as compared with 539+/-35*10(3) microm2 in the PLTP(+M/+M) group (n=14)) after 9 weeks of Western-type diet feeding. The decreased lesion size in the PLTP(-M/-M) group coincided with significantly lower serum total cholesterol, free cholesterol, and triglyceride levels in these mice. Furthermore, plasma PLTP activity in the PLTP(-M/-M) group was 2-fold (P<0.001) lower than that in the PLTP(+M/+M) group., Conclusion: Macrophage PLTP is a significant contributor to plasma PLTP activity and deficiency of PLTP in macrophages leads to lowered atherosclerotic lesion development in low-density lipoprotein receptor knockout mice on Western-type diet.

Objective: Atherosclerosis is a chronic (auto-)inflammatory disease and T cell activation is an important factor in this process. Tnfrsf4 (OX40) and Tnfsf4 (OX40 ligand) are members of the tumor necrosis factor (TNF) and TNF receptor family and OX40/OX40L mediated signaling is important in co-activation of T cells and facilitates B-T cell interaction. In this study we assessed the role of the OX40/OX40L pathway in atherosclerosis and the effect of interruption of the OX40/OX40L pathway on lesion development., Methods and Results: We treated low-density lipoprotein receptor-deficient (LDLr-/-) mice with an anti-OX40L antibody which lead to a 53% decrease in atherosclerotic lesion formation. Treatment resulted in inhibition of Th2 mediated isotype switching by decreasing interleukin (IL)-4 secretion and subsequent low IgG1 serum levels against oxLDL, whereas protective anti-oxLDL specific IgM titers were increased in treated mice compared with control., Conclusions: We conclude that blocking the OX-40/OX40L interaction reduced atherogenesis by inhibition of IL-4 mediated Th2 induced isotype switching and subsequent increased levels of anti-oxLDL IgM.

Objective: ABCG1 has recently been identified as a facilitator of cellular cholesterol and phospholipid efflux to high-density lipoprotein (HDL). Its expression in macrophages is induced during cholesterol uptake in macrophages and by liver X receptor (LXR). The role of macrophage ABCG1 in atherosclerotic lesion development is, however, still unknown., Methods and Results: To assess the role of macrophage ABCG1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDLr-/-) mice that are selectively deficient in macrophage ABCG1 by using bone marrow transfer (ABCG1-/- --> LDLr-/-). Peritoneal macrophages isolated from donor ABCG1-/- mice exhibited a 22% (P=0.0007) decrease in cholesterol efflux to HDL. To induce atherosclerosis, transplanted mice were fed a high-cholesterol diet containing 0.25% cholesterol and 15% fat for 6 and 12 weeks. Serum lipid levels and lipoprotein profiles did not differ significantly between ABCG1-/- --> LDLr-/- mice and controls. In lungs of ABCG1-/- --> LDLr-/- mice a striking accumulation of lipids was observed in macrophages localized to the subpleural region. After 6 weeks of high-cholesterol diet feeding the atherosclerotic lesion size was 49+/-12x10(3) microm2 for ABCG1+/+ --> LDLr-/- mice versus 65+/-15x103 microm2 for ABCG1-/- --> LDLr-/- mice and after 12 weeks of high-cholesterol diet feeding 124+/-17x10(3) microm2 for ABCG1+/+ --> LDLr-/- mice versus 168+/-17x10(3) microm2 for ABCG1-/- --> LDLr-/- mice. Atherosclerotic lesion size depended on both time and the macrophage ABCG1 genotype (P=0.038 by 2-way ANOVA, n > or = 8), indicating a moderately 33% to 36% increase in lesion formation in the absence of macrophage ABCG1., Conclusions: Macrophage ABCG1 deficiency does lead to heavy lipid accumulation in macrophages of the lung, and also a moderately significant effect on atherosclerotic lesion development was observed.

Background: ATP-binding cassette transporter A1 (ABCA1) is a key regulator of cellular cholesterol and phospholipid transport. Previously, we have shown that inactivation of macrophage ABCA1 induces atherosclerosis in low-density lipoprotein receptor knockout (LDLr-/-) mice. However, the possibly beneficial effects of specific upregulation of macrophage ABCA1 on atherogenesis are still unknown., Methods and Results: Chimeras that specifically overexpress ABCA1 in macrophages were generated by transplantation of bone marrow from human ABCA1 bacterial artificial chromosome (BAC) transgenic mice into LDLr-/- mice. Peritoneal macrophages isolated from the ABCA1 BAC --> LDLr-/- chimeras exhibited a 60% (P=0.0006) increase in cholesterol efflux to apolipoprotein AI. To induce atherosclerosis, the mice were fed a Western-type diet containing 0.25% cholesterol and 15% fat for 9, 12, and 15 weeks, allowing analysis of effects on initial lesion development as well as advanced lesions. No significant effect of macrophage ABCA1 overexpression was observed on atherosclerotic lesion size after 9 weeks on the Western-type diet (245+/-36x10(3) microm2 in ABCA1 BAC --> LDLr-/- mice versus 210+/-20x10(3) microm2 in controls). However, after 12 weeks, the mean atherosclerotic lesion area in ABCA1 BAC --> LDLr-/- mice remained only 164+/-15x10(3) microm2 (P=0.0008) compared with 513+/-56x10(3) microm2 in controls (3.1-fold lower). Also, after 15 weeks on the diet, lesions in mice transplanted with ABCA1 overexpressing bone marrow were still 1.6-fold smaller (393+/-27x10(3) microm2 compared with 640+/-59x10(3) microm2 in control transplanted mice; P=0.0015)., Conclusions: ABCA1 upregulation in macrophages inhibits the progression of atherosclerotic lesions.

Objective: The role of Chlamydia pneumoniae in atherosclerosis is still debated. In this study a novel mouse model was applied to determine the direct impact of C. pneumoniae on the arterial wall and the development of atherosclerosis., Methods: Direct effects of C. pneumoniae on collar-induced atherosclerosis were studied after local delivery of C. pneumoniae to carotid arteries of LDL receptor-deficient (LDLr-/-) mice., Results: The presence of C. pneumoniae in the vessel wall was quantified by RT-PCR (6.2 x 10(4) copies/artery) and resulted in a 2.0-fold increase in intima/media ratios (p<0.05) and a 1.7-fold increase in stenosis (p<0.05). Immunostaining revealed a 2.98-fold (p<0.01) increased macrophage content and a tendency towards lower numbers of smooth muscle cells and collagen in lesions of infected carotid arteries. Direct delivery of another respiratory pathogen, Mycoplasma pneumoniae, to the carotids did not affect size or composition of the atherosclerotic lesions. Presence of C. pneumoniae in the carotid arteries resulted within 7 days in a marked upregulation of the expression of MCP-1 (p<0.01) and ICAM-1 as determined on mRNA and protein levels. These in vivo data were in line with data obtained with in vitro infections of macrophages and endothelial cells with C. pneumoniae., Conclusions: We conclude that C. pneumoniae in carotid arteries leads to more pronounced atherosclerotic lesions with a more vulnerable morphology and that this model is suitable to monitor direct effects of C. pneumoniae on atherogenesis.

Objectives: The very-low-density lipoprotein receptor (VLDLr) is highly expressed in macrophage-rich areas of atherosclerotic lesions. The exact role of the macrophage VLDLr in atherosclerotic lesion development, however, is presently unclear., Methods and Results: To assess the role of the macrophage VLDLr in atherosclerotic lesion development in vivo, we used the technique of bone marrow transplantation to selectively disrupt or reconstitute the VLDLr in macrophages in VLDLr+/+ and VLDLr-/- mice, respectively. After 10 weeks high-cholesterol diet feeding, the lesion area in control transplanted wild-type mice was 17+/-4 x 10(3)+/-microm(2). Disruption of the macrophage VLDLr by transplanting bone marrow from VLDLr-/- mice to wild-type VLDLr+/+ littermates resulted in a tendency to a slight reduction in lesion size to 12+/-3 x 10 microm. The mean atherosclerotic lesion area, measured in control transplanted VLDLr-/- mice, lacking the VLDLr in all tissues was 12+/-3 x 10(3)microm(2). Interestingly, reconstitution of the macrophage VLDLr in VLDLr-deficient recipients resulted in a 2.7-fold increase (P<0.05) in the mean atherosclerotic lesion area to 32+/-3 x 10(3)microm(2)., Conclusions: The macrophage VLDLr facilitates atherosclerotic lesion development, probably by mediating the accumulation of atherogenic lipoproteins.

Objective: HIV combination therapy using protease inhibitors is associated with elevated plasma levels of atherogenic lipoproteins and increased risk for atherosclerosis. We investigated whether the HIV entry inhibitor TAK-779 affects lipoprotein levels and atherogenesis in low-density lipoprotein receptor-deficient mice. TAK-779 is an antagonist for the chemokine receptors CCR5 and CXCR3, which are expressed on leukocytes, especially T-helper 1 cells, and these receptors may be involved in recruitment of these cells to atherosclerotic plaques., Methods and Results: TAK-779 treatment of low-density lipoprotein receptor-deficient mice did not elevate the levels of atherogenic lipoproteins, whereas it dramatically reduced atherosclerosis in the aortic root and in the carotid arteries. The number of T cells in the plaque was reduced by 95%, concurrently with a 98% reduction in the relative IFN-gamma area. TAK-779-treated animals showed a decreased percentage of CD4+ and CD8+ T cells in peripheral blood and in mediastinal lymph nodes compared with control-treated animals., Conclusions: TAK-779 not only suppresses HIV entry via blockade of CCR5 but also attenuates atherosclerotic lesion formation by blocking the influx of T-helper 1 cells into the plaque. TAK-779 treatment may be especially beneficial for young HIV patients as they face lifelong treatment, and this drug impairs atherogenesis.

The function of scavenger receptor class B, type I (SR-BI) in the liver as a high-density lipoprotein receptor that promotes the selective uptake of cholesteryl esters is well defined. Its role in macrophages, however, is primarily unknown, because it functions in the uptake of (modified) lipoproteins as well as the secretion of cholesterol to high-density lipoproteins. In this study, the biological role of SR-BI on bone marrow-derived cells, including macrophages, in lipid metabolism and atherosclerosis was assessed by selective disruption of SR-BI in bone marrow in two established models of atherosclerosis: low-density lipoprotein (LDL) receptor-deficient mice that develop extensive atherosclerosis on a Western-type diet and wild-type mice that develop fatty streak lesions when fed a high-cholesterol diet containing 0.5% cholate. The presence of SR-BI in bone marrow-derived cells in LDLr-/- mice decreased lesion development after 9 and 12 weeks of Western-type diet feeding, indicating that macrophage SR-BI protects against lesion development. At 6 weeks, no significant effect of SR-BI in bone marrow-derived cells on lesion development was observed. Interestingly, after only 4 weeks of Western-type diet feeding of transplanted LDLr-/- mice and in wild-type mice on a high-cholesterol/cholate diet, the presence of SR-BI in bone marrow-derived cells increased the development of small fatty streak lesions. It thus appears that, depending on the stage of atherosclerotic lesion development, SR-BI in bone marrow-derived cells is either pro-atherogenic or anti-atherogenic, indicating a unique dual role in the pathogenesis of atherosclerosis.

In view of its multifaceted anti-inflammatory properties, interleukin-10 (IL-10) has been deemed to be potentially anti-atherogenic. We have evaluated the capacity of adenoviral gene transfer of IL-10 for the modulation of de novo atherosclerotic lesion formation by systemic and by local overexpression. Atherogenesis was initiated in the carotid arteries of low-density lipoprotein receptor deficient mice by perivascular placement of silastic collars. One week after collar placement, mice were injected intravenously with 1 x 109 plaque-forming units (pfu's) of IL-10 (AdV.IL-10) or control adenovirus (AdV.empty). Administration of AdV.IL-10 resulted in extended systemic expression of IL-10 (peak serum level 3.0 +/- 1.1 ng/ml) and a reduction in atherosclerotic lumen stenosis by 62.2% (P<0.02). This finding was accompanied by monocyte deactivation and lowering of serum cholesterol levels (maximum decrease 44%). In a second experiment, collared arteries were transfected locally by transluminal instillation of adenovirus (titer 1.5x1010 pfu/ml). Systemic parameters remained unchanged following local transfection, but the degree of stenosis was, nonetheless, decreased by 44.9% (P<0.05). We conclude that a marked inhibition of atherogenesis can be achieved by systemic overexpression of AdV.IL-10, owing to its metabolic and immunomodulatory effects. Local IL-10 transfer is virtually equipotent, however, and it may represent a valuable addition to the armory of anti-atherosclerotic therapies.

Background: Perivascular collar placement has been used as a means for localized atherosclerosis induction in a variety of experimental animal species. In mice, however, atherosclerosis-like lesions have thus far not been obtained by this method. The aim of this study was the development of a mouse model of rapid, site-controlled atherogenesis., Methods and Results: Silastic collars were placed around the carotid arteries of apolipoprotein E-deficient (apoE-/-) and LDL receptor-deficient (LDLr-/-) mice. The development of collar-induced lesions was found to occur predominantly in the area proximal to the collar and to be dependent on a high-cholesterol diet. Lesions were evident in apoE-/- mice after 3 weeks and in LDLr-/- mice after 6 weeks and were overtly atherosclerotic in appearance. Lumen stenosis reached 85% in apoE-/- mice and 61% in LDLr-/- mice 6 weeks after collar insertion. Expression levels of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were increased both proximal and distal to the collar, whereas endothelial nitric oxide synthase expression was downregulated at the proximal site., Conclusions: We propose that this model of collar-induced acceleration of carotid atherogenesis is of hemodynamic cause. It may serve as a substrate for sequential mechanistic studies concerned with the underlying cause and pathogenesis of atherosclerosis. The rapidity of lesion development will also aid the efficient screening of new potentially antiatherogenic chemical entities and the evaluation of therapies with limited duration of effectiveness, such as adenoviral gene therapy.

Apolipoprotein E (apoE) is a high affinity ligand for several receptor systems in the liver, including the low-density lipoprotein (LDL) receptor, and non-LDL receptor sites, like the LDL receptor-related protein (LRP), the putative remnant receptor and/or proteoglycans. Although the liver is the major source of apoE synthesis, apoE is also produced by a wide variety of other cell types, including macrophages. In the present study, the role of the LDL receptor in the removal of lipoprotein remnants, enriched with macrophage-derived apoE from the circulation, was determined using the technique of bone marrow transplantation (BMT). Reconstitution of macrophage apoE production in apoE-deficient mice resulted in a serum apoE concentration of only 2% of the concentration in wild-type C57Bl/6 mice. This low level of apoE nevertheless reduced VLDL and LDL cholesterol 12-fold (P<0.001) and fourfold (P<0.001), respectively, thereby reducing serum cholesterol levels and the susceptibility to atherosclerosis. In contrast, reconstitution of macrophage apoE synthesis in mice lacking both apoE and the LDL receptor induced only a twofold (P<0.001) reduction in VLDL cholesterol and had no significant effect on atherosclerotic lesion development, although serum apoE levels were 93% of the concentration in normal C57Bl/6 mice. In conclusion, a functional (hepatic) LDL receptor is essential for the efficient removal of macrophage apoE-enriched lipoprotein remnants from the circulation and thus for normalization of serum cholesterol levels and protection against atherosclerotic lesion development in apoE-deficient mice.

Scavenger receptors, which include various classes, play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins, resulting in the massive accumulation of cholesteryl esters. Because macrophage-derived foam cells are considered to be an important feature in early atherogenesis, we investigated the role of scavenger receptor class A (SR-A) overexpression, especially on macrophages in lipoprotein metabolism and atherosclerosis. Bone marrow from human SR-A (MSR1)-overexpressing mice was transplanted into irradiated low density lipoprotein receptor knockout [LDLR(-/-)] mice. The transplantation resulted in an increase in total serum cholesterol (approximately 15 to 25%), especially in the VLDL fraction, when compared with LDLR(-/-) mice that were transplanted with bone marrow of wild-type littermates. Quantification of atherosclerotic lesions in the mice that were fed a "Western-type" diet for 3 months revealed that there were no differences in mean lesion area between LDLR(-/-) mice transplanted with MSR1 overexpressing and wild-type littermate bone marrow, despite increased scavenger receptor activity in vitro. The presence or absence of the LDLR in the transplanted bone marrow did not influence these results.In conclusion, introduction of MSR1-overexpressing bone marrow in LDLR(-/-) mice via bone marrow transplantation resulted in a slight increase in lipoprotein levels, but had no effect on the atherosclerotic lesion area, despite increased scavenger receptor activity in vitro.

Macrophage-derived foam cells play an important role in the initiation and progression of atherosclerosis. To examine the role of the macrophage low density lipoprotein receptor (LDLr) in atherosclerotic lesion formation, bone marrow from LDLr knockout [LDLr(-/-)] mice was transplanted into irradiated wild-type C57Bl/6 [LDLr(+/+)] mice. After 3 months on an atherogenic diet, C57Bl/6 mice, reconstituted with LDLr(-/-) bone marrow, showed a mean lesion area of 34.7 x 10(3)+/-22.4 x 10(3) microm(2) compared with 100. 8 x 10(3)+/-33.0 x 10(3) microm(2) (P<0.001) in control C57Bl/6 mice that were transplanted with LDLr(+/+) bone marrow. There were no significant differences in total serum cholesterol, triglyceride levels, and lipoprotein profiles between the 2 groups. Histochemical analysis of macrophage LDLr expression in the atherosclerotic lesions indicated that C57Bl/6 mice, reconstituted with LDLr(+/+) bone marrow, showed extensive staining of the foam cells in the atherosclerotic lesions, whereas mice reconstituted with LDLr(-/-) bone marrow showed only a few LDLr-positive foam cells. In vitro, peritoneal macrophages isolated from wild-type C57Bl/6 mice were, respectively, 4.7- and 10.7-fold more effective in cell association and degradation of atherogenic (125)I-beta-very low density lipoprotein than were LDLr(-/-) peritoneal macrophages, establishing that the LDLr on macrophages is important for the interaction of macrophages with beta-very low density lipoprotein. It is concluded that the LDLr on macrophages can facilitate the development of atherosclerosis, possibly by mediating the uptake of atherogenic lipoproteins.

We investigated the mechanism of beta-very low density lipoprotein (beta-VLDL)-induced foam cell formation derived from peritoneal macrophages from control mice and low density lipoprotein (LDL) receptor-deficient mice to elucidate the role of the LDL receptor in this process. The LDL receptor appeared to be of major importance for beta-VLDL metabolism. Consequently, the accumulation of cholesteryl esters in LDL receptor(-)(/)- macrophages is 2.5-fold lower than in LDL receptor(+)(/)(+) macrophages. In the absence of the LDL receptor, however, beta-VLDL was still able to induce cholesteryl ester accumulation and subsequently we characterized the properties of this residual beta-VLDL recognition site(s) of LDL receptor(-)(/)- macrophages. Although the LDL receptor-related protein is expressed on LDL receptor(-)(/)- macrophages, the cell association of beta-VLDL is not influenced by the receptor-associated protein, and treatment of the macrophages with heparinase and chondroitinase was also ineffective. In contrast, both oxidized LDL (OxLDL) and anionic liposomes were able to inhibit the cell association of (125)I-labeled beta-VLDL in LDL receptor(-)(/)- macrophages by 65%. These properties suggest a role for scavenger receptor class B (SR-B), and indeed, in the LDL receptor(-)(/)- macrophages the selective uptake of cholesteryl esters from beta-VLDL was 2.2-fold higher than that of apolipoproteins, a process that could be inhibited by OxLDL, high density lipoprotein (HDL), and beta-VLDL. In conclusion, the LDL receptor on peritoneal macrophages is directly involved in the metabolism of beta-VLDL and the subsequent foam cell formation. When the LDL receptor is absent, SR-B appears to mediate the remaining metabolism of cholesteryl esters from beta-VLDL.

We have used adenovirus-mediated gene transfer in mice to investigate low density lipoprotein receptor (LDLR) and LDLR-related protein (LRP)-independent mechanisms that control the metabolism of chylomicron and very low density lipoprotein (VLDL) remnants in vivo. Overexpression of receptor-associated protein (RAP) in mice that lack both LRP and LDLR (MX1cre(+)LRP(flox/flox)LDLR(-/-)) in their livers elicited a marked hypertriglyceridemia in addition to the pre-existing hypercholesterolemia in these animals, resulting in a shift in the distribution of plasma lipids from LDL-sized lipoproteins to large VLDL-sized particles. This dramatic increase in plasma lipids was not due to a RAP-mediated inhibition of a unknown hepatic high affinity binding site involved in lipoprotein metabolism, because no RAP binding could be detected in livers of MX1cre(+)LRP(flox/flox)LDLR(-/-) mice using both membrane binding studies and ligand blotting experiments. Remarkably, RAP overexpression also resulted in a 7-fold increase (from 13.6 to 95.6 ng/ml) of circulating, but largely inactive, lipoprotein lipase (LPL). In contrast, plasma hepatic lipase levels and activity were unaffected. In vitro studies showed that RAP binds to LPL with high affinity (K(d) = 5 nM) but does not affect its catalytic activity, in vitro or in vivo. Our findings suggest that an extrahepatic RAP-sensitive process that is independent of the LDLR or LRP is involved in metabolism of triglyceride-rich lipoproteins. There, RAP may affect the functional maturation of LPL, thus causing the accumulation of triglyceride-rich lipoproteins in the circulation.

Many tumors express elevated levels of low-density lipoprotein (LDL) receptors. Therefore, native LDL and synthetic LDL-like particles have been proposed as carriers for antineoplastic drugs. We demonstrated earlier that small apolipoprotein E (apoE)-exposing liposomes were specifically recognized by the LDL receptor. In this study, we incorporated a lipophilic derivative of daunorubicin (LAD) into the apoE liposomes. Up to 11 molecules of LAD could be incorporated per particle without significantly changing the size, lipid composition, and ability to bind apoE of the liposomes. The biological fate of the prodrug was largely determined by its carrier (70% of the initially incorporated LAD was still associated to the liposomes after 4 h of circulation in mice). Compared with free daunorubicin, the circulation half-life of the liposome-associated prodrug was substantially prolonged and undesired tissue disposition was reduced. The role of the LDL receptor in the metabolism of LAD-loaded apoE liposomes was demonstrated in rats with up-regulated hepatic LDL receptors. In these rats, the liver uptake of the prodrug and carrier was increased 5-fold. The addition of apoE was essential for LDL receptor-mediated uptake of the drug-carrier complex. In LDL receptor-deficient mice, the circulation time of both the prodrug and the carrier increased approximately 2-fold compared with wild-type mice. We conclude that LAD-loaded apoE liposomes constitute a stable drug-carrier complex that is well suited for LDL receptor-mediated selective drug delivery to tumors.

To investigate the quantitative requirement for apolipoprotein (apo) E in the clearance of lipoproteins via the non-low density lipoprotein (LDL) receptor mediated pathway, human APOE was overexpressed at various levels in the livers of mice deficient for both the endogenous Apoe and Ldlr genes (Apoe -/-. Ldlr -/-) using adenovirus-mediated gene transfer. We found that a low level of APOE expression, that was capable of reducing the hyperlipidemia in Apoe -/- mice, did not result in a reduction of the hyperlipidemia in Apoe -/-. Ldlr -/- mice. Surpisingly, a very high level of APOE expression also did not result in a reduction of hypercholesterolemia in Apoe -/-. Ldlr -/- mice, despite very high levels of circulating apoE (>160 mg/dl). Only a moderately high level of APOE expression resulted in a reduction of serum cholesterol level (from 35.2 +/- 6.7 to 14.6 +/- 2.3 mmol/l) and the disappearance of VLDL from the serum. Moreover, the very high level of APOE expression resulted in a severe hypertriglyceridemia in Apoe -/-. Ldlr -/- mice and not Apoe -/- mice (25.7 +/- 8.9 and 2.2 +/- 1.8 mmol/l, respectively). This hypertriglyceridemia was associated with an APOE-induced increase in the VLDL triglyceride production rate and an inhibition of VLDL-triglyceride lipolysis. We conclude from these data that, for efficient clearance, the non-LDL receptor-mediated pathway requires a higher level of APOE expression as compared to the LDL receptor, but is more sensitive to an APOE-induced increase in VLDL production and inhibition of VLDL-triglyceride lipolysis.-van Dijk, K. W., B. J. M. Van Vlijmen, H. B. van't Hof, A. van der Zee, S. Santamarina-Fojo, T. J. C. van Berkel, L. M. Havekes, and M. H. Hofker. In LDL receptor-deficient mice, catabolism of remnant lipoproteins requires a high level of apoE but is inhibited by excess APOE.

Many tumours express relatively high levels of low-density lipoprotein (LDL) receptors on their membranes. The LDL receptor is, therefore, an attractive target for the selective delivery of antineoplastic drugs to tumour cells. We reported previously on the synthesis of small apolipoprotein E (apoE)-containing liposomes that behave in vivo in a very similar way to native LDL. In this study, we examined the interaction of this liposomal carrier with cultured B16 melanoma cells. Binding of apoE liposomes to the cells is saturable, with a maximum binding of approximately 90000 particles per cell. Cross-competition studies indicated that apoE liposomes are bound by the LDL receptor. Association of apoE liposomes to B16 cells is strictly Ca2+ dependent, which forms additional evidence for a role of the LDL receptor. The affinity of apoE liposomes for the LDL receptor on B16 cells is 15-fold higher than that of LDL (0.77 vs 11.5 nM respectively). ApoE is essential for the LDL receptor recognition because liposomes lacking apoE were, in competition studies, 20- to 50-fold less effective than apoE-containing liposomes. We examined in B16 tumour-bearing mice the tumour-localizing properties of apoE liposomes and the disposition of an incorporated lipophilic derivative of daunorubicin (LAD). Tissue distribution studies showed that LAD-loaded apoE liposomes were taken up and processed by the major LDL receptor-expressing organs (i.e. adrenals, liver and spleen). Of all other tissues, the tumour showed the highest uptake. The distribution patterns of LAD-loaded apoE liposomes and native LDL in the tumour-bearing mice were very similar, which supports the role of the LDL receptor in the disposition of the prodrug-loaded particles. The disposition of LAD followed the pattern of the liposomal carrier. We conclude that apoE liposomes enable LDL receptor-mediated specific delivery of antineoplastic (pro)drugs to tumours, and, therefore, constitute an attractive novel option for anti-tumour chemotherapy.

Purpose: Many tumors express elevated levels of LDL receptors (apoB, E receptors) on their membranes. Selective delivery of anti-neoplastic drugs to tumors by incorporation of these drugs into LDL or LDL-resembling particles should improve the efficacy of tumor therapy and minimize the severe side-effects. Since the apolipoproteins on the particles are essential for the LDL receptor recognition, drugs should preferably be incorporated into the lipid moiety. Most anti-tumor agents are too hydrophilic for incorporation into these carriers., Methods: We synthesized LAD, a lipophilic prodrug of daunorubicin, by coupling the drug via a lysosomally degradable peptide spacer to a cholesteryl oleate analog., Results: The overall yield of the synthesis was 50% with a purity of > 90%. Radioactively ([3H]) labeled LAD was obtained via a slightly modified procedure (yield 40%). The octanol/water partition coefficient of LAD is 30-fold higher than that of daunorubicin. LAD could be incorporated into triglyceride-rich lipid emulsions and small liposomes, which, if provided with apoE, have been demonstrated earlier to be cleared in vivo via the LDL receptor. The liposomes contained approximately 10 molecules of LAD per liposomal particle. Analysis of differently sized LAD-containing emulsions suggests that LAD associates with the surface of lipidic particles. In the presence of human serum, LAD did not dissociate from the emulsion particles, indicating a firm association of LAD with the carrier., Conclusions: The coupling of a cholesterol ester analog to daunorubicin results in a lipophilic prodrug that can be firmly anchored into lipidic carries. LAD-loaded emulsions and liposomes provided with recombinant apoE will be tested in the near future for their ability to deliver LAD to tumor tissue in vivo via the LDL receptor.

The LDL receptor (LDLR) plays an important role in the removal of LDL and its precursors, the intermediate and very low density lipoproteins, from the blood circulation. The receptor is expressed on various cell types. In this study the relative importance of the LDLR on macrophages for lipoprotein metabolism and atherogenesis was assessed. For this purpose, irradiated LDLR-knockout (-/-) mice were transplanted with bone marrow of normal C57BL/6J mice. DNA analysis showed that the transplanted mice were chimeric. The transplantation resulted in a slight decrease of total serum cholesterol when compared with LDLR-/- mice that were transplanted with LDLR-/- bone marrow. This modest decrease, however, did not reach statistical significance at all time points examined. This decrease can be almost completely attributed to a decrease in LDL cholesterol. The specific lowering of LDL cholesterol could clearly be observed at 4 weeks after transplantation, but the decrease was less at 12 weeks after transplantation. Quantification of atherosclerotic lesions of mice fed a 1% cholesterol diet for 6 months revealed that there were no differences in mean lesion area between mice transplanted with wild-type bone marrow or LDLR-/- bone marrow. We anticipate that in LDLR-/- mice transplanted with wild-type bone marrow, the LDLR is downregulated by the relatively high concentrations of circulating cholesterol. In vitro incubations of peritoneal macrophages with 125I-LDL indicated that the LDLR of these cells could be downregulated by 25-hydroxycholesterol. Peritoneal macrophages isolated from LDLR-/- mice transplanted with wild-type bone marrow, in contrast to those transplanted with LDLR-/- bone marrow, were able to degrade 125I-LDL, indicating that the capacity to express functional LDLR was achieved. In conclusion, introduction of the LDLR into LDLR -/- mice via bone marrow transplantation resulted in only a relatively modest decrease of LDL cholesterol that became less pronounced at later time points, possibly due to downregulation of the LDLR. To utilize the LDLR in macrophages for effective cholesterol lowering, either the sterol-regulatory elements have to be "silenced" or a high-expression LDLR construct has to be introduced into macrophages, eg, via transplantation of in vitro transfected hematopoietic stem cells.

Apolipoprotein E (apoE) is an important determinant for the uptake of triglyceride-rich emulsions and lipoproteins by the liver, and exerts affinity for both the LDL receptor (LDLr) and a distinct liver-specific recognition site. Our current aim was to assess the mechanism underlying the receptor-specificity of apoE-carrying lipoproteins. Triglyceride-rich emulsions were synthesized, with mean sizes of 50, 80, and 150 nm. These fractions efficiently acquired apoE from rat serum, and were processed by LPL in vivo with a similar efficiency. Upon injection of the [5H]cholesteryl oleate-labeled emulsions into rats, the liver association rate was positively correlated with particle size (24 +/- 2%, 54 +/- 1%, and 64 +/- 3% of the injected dose at 20 min after injection, respectively) and the liver uptake was predominantly exerted by parenchymal cells. The role of the LDLr in emulsion clearance was established in wild-type versus LDLr knockout mice. In the absence of the LDLr, an 8-fold increased serum half-life was observed for the small emulsion, concomitant with a 6- and 15-fold decreased uptake by the liver and adrenals at 60 min after injection, respectively. In contrast, the in vivo behavior of the large emulsion was independent of the LDLr. Both the ratio of apoE:C on the emulsions upon serum incubation and the alpha-helical content of apoE were inversely correlated with particle size, indicating that these factors may be involved in the emulsion size-dependent receptor specificity in vivo. It is concluded that the contribution of the LDLr to the apoE-mediated clearance of emulsions by the liver and adrenals strongly increases with decreasing particle size, while large particles initially associate with a distinct liver-specific recognition site. As these emulsions mimic chylomicrons, we anticipate that the apoE-dependent metabolic behavior of chylomicrons (remnants) is largely dependent on their size.

Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91% and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= delta 125-rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-delta 125-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

Rat liver Kupffer cell membranes contain a protein that recognizes specifically oxidized low-density lipoproteins (oxLDL). Visualization after blotting under reducing conditions indicates that the receptor is a monomeric protein, with an estimated molecular mass of 115-120 kDa. N-Glycosidase F and endoglycosidase F treatment resulted in a fall in estimated molecular mass of 24 and 11 kDa respectively, whereas O-glycosidase was ineffective. No effect on the extent of interaction with oxLDL was noticed, suggesting that glycans are not essential for ligand recognition. Using a polyclonal antibody to mouse macrosialin, we visualized macrosialin on blot, and compared this glycoprotein with the oxLDL-binding protein. It appears that the two glycoproteins have a similar molecular mass and are comparably affected by treatment with the different glycosidases. Incubation with trypsin resulted in a reduction in the estimated molecular mass of about 25 kDa for both the oxLDL-binding protein and macrosialin. These results indicate that the oxLDL-binding protein and macrosialin are identical, suggesting a role for macrosialin in modified LDL catabolism.

Selective delivery of cytotoxic anti-neoplastic drugs can diminish the severe side-effects associated with these drugs. Many malignant tumours express high levels of low-density lipoprotein (LDL) receptors on their membranes. Therefore, LDL may be used as a carrier to obtain selective delivery of anti-neoplastic drugs to tumours. The present study was performed to investigate the feasibility of the murine B16 tumour/mouse model for the evaluation of LDL-mediated tumour therapy. LDL binds with high affinity to LDL receptors on cultured B16 cells (Kd, 5.9 +/- 2.3 micrograms ml-1; Bmax 206 +/- 23 ng LDL mg-1 cell protein). After binding and internalisation, LDL was very efficiently degraded: 724 +/- 19 ng LDL mg-1 cell protein h-1. Chloroquine and ammonium chloride completely inhibited the degradation of LDL by the B16 cells, indicating involvement of lysosomes. LDL receptors were down-regulated by 70% after preincubation of B16 cells with 300 micrograms ml-1 LDL, indicating that their expression is regulated by intracellular cholesterol. To evaluate the uptake of LDL by the B16 tumour in vivo, tissue distribution studies were performed in C57/B1 mice inoculated with B16 tumours. For these experiments, LDL was radiolabelled with tyramine cellobiose, a non-degradable label, which is retained in cells after uptake. At 24 h after injection of LDL, the liver, adrenals and the spleen were found to be the major organs involved in LDL uptake, with tissue-serum (T/S) ratios of 0.82 +/- 0.08, 1.17 +/- 0.20 and 0.69 +/- 0.08 respectively. Of all the other tissues, the tumour showed the highest uptake of LDL (T/S ratio of 0.40 +/- 0.07). A large part of the LDL uptake was receptor mediated, as the uptake of methylated LDL was much lower. Although the LDL uptake by the liver, spleen and adrenals is higher than that by the tumour, the LDL receptor-mediated uptake by these organs may be selectively down-regulated by methods that do not affect the expression of LDL receptors on tumour cells. It is concluded that the B16 tumour-bearing mouse constitutes a good model to evaluate the effectiveness of LDL-mediated delivery of cytotoxic (pro)drugs to tumours in vivo.

1. Lactoferrin and aminopeptidase M-modified lactoferrin (APM-lactoferrin; which lacks its 14 N-terminal amino acids) inhibit the liver uptake of lipoprotein remnant. In the present study, the role of proteoglycans in the initial interaction of beta-migrating very-low-density lipoprotein (beta-VLDL), native and APM-lactoferrin with isolated rat parenchymal liver cells was investigated. Treatment of the cells with chondroitinase lowered the Kd of lactoferrin binding (from 10 to 2.4 microM), and the number of sites/cell (from 20 x 10(6) to 7 x 10(6)), while heparinase treatment did not affect the binding. The binding characteristics of APM-lactoferrin and beta-VLDL were not altered by treatment of the cells with chondroitinase or heparinase. It is concluded that proteoglycans are not involved in the initial binding of APM-lactoferrin and beta-VLDL to parenchymal cells, while chondroitin sulphate proteoglycans are mainly responsible for the massive, low-affinity binding of native lactoferrin..2. The binding of lactoferrin, APM-lactoferrin and beta-VLDL to parenchymal liver cells was not influenced by the glutathione S-transferase-receptor-associated protein (GST-RAP) (97.2% +/- 4.0%, 95.5 +/- 3.7% and 98.5% of the control binding), while the binding of alpha 2-macroglobulin was fully blocked at 10 micrograms/ml GST-RAP (1.8 +/- 0.5% of the control binding). Since GST-RAP blocks the binding of all the known ligands to the low-density lipoprotein (LDL)-receptor-related protein (LRP), it is concluded that LRP is not the initial primary recognition site for lactoferrin, APM-lactoferrin and beta-VLDL on parenchymal liver cells. 3. We showed earlier that.APM-lactoferrin, as compared with lactoferrin, is a more effective inhibitor of the liver uptake of lipoprotein remnants (49.4 +/- 4.0% versus 80.8 +/- 4.8% of the control at 500 micrograms/ml respectively). We found in the present study that beta-VLDL is able to inhibit the binding of APM-lactoferrin to parenchymal liver cells significantly (74.9 +/- 3.3% of the control; P < 0.002), while the lactoferrin binding was unaffected. It is concluded that a still unidentified specific recognition site (the putative remnant receptor) is responsible for the initial binding of remnants to parenchymal cells and it is suggested that the partial cross-competition between APM-lactoferrin and beta-VLDL may be of further help in the elucidation of the molecular nature of this recognition site.

The liver plays a decisive role in the regulation of the plasma levels of atherogenic lipoproteins. The primary liver interaction site for chylomicron-remnants and VLDL remnants (beta-VLDL) is still unidentified, while the subsequent cellular uptake is likely to be mediated in concert by the LDL receptor related protein (LRP) and the LDL receptor. The nature of the primary interaction site of remnants (remnant-receptor) might be a liver-specific proteoglycan or a liver-specific protein. Atherogenic modified LDL can be recognized by a family of scavenger-receptors. A newly identified 95-kDa protein forms the most likely candidate for mediating the in vivo uptake of oxidized LDL from the circulation and might thus protect the body against the presence of oxidized LDL in the blood compartment.