Apolipoprotein B stimulates formation of monocyte-macrophage surface-connected compartments and mediates uptake of low density lipoprotein-derived liposomes into these compartments

Much of the cholesterol that accumulates in atherosclerotic plaques is found within monocyte-macrophages transforming these cells into "foam cells." Native low density lipoprotein (LDL) does not cause foam cell formation. Treatment of LDL with cholesterol esterase converts LDL into cholesterol-rich liposomes having >90% cholesterol in unesterified form. Similar cholesterol-rich liposomes are found in early developing atherosclerotic plaques surrounding foam cells. We now show that cholesterol-rich liposomes produced from cholesterol esterase-treated LDL can cause human monocyte-macrophage foam cell formation inducing a 3-5-fold increase in macrophage cholesterol content of which >60% is esterified. Although cytochalasin D inhibited LDL liposome-induced macrophage cholesteryl ester accumulation, LDL liposomes did not enter macrophages by phagocytosis. Rather, the LDL liposomes induced and entered surface-connected compartments within the macrophages, a unique endocytic pathway in these cells that we call patocytosis. LDL liposome apoB rather than LDL liposome lipid mediated LDL liposome uptake by macrophages. This was shown by the findings that: 1) protease treatment of the LDL liposomes prevented macrophage cholesterol accumulation; 2) liposomes prepared from LDL lipid extracts did not cause macrophage cholesterol accumulation; and 3) purified apoB induced and accumulated within macrophage surface-connected compartments. Although apoB mediated the macrophage uptake of LDL liposomes, this uptake did not occur through LDL, LDL receptor-related protein, or scavenger receptors. Also, LDL liposome uptake was not sensitive to treatment of macrophages with trypsin or heparinase. Cholesterol esterase-mediated transformation of LDL into cholesterol-rich liposomes is an LDL modification that: 1) stimulates uptake of LDL cholesterol by apoB-dependent endocytosis into surface-connected compartments, and 2) causes human monocyte-macrophage foam cell formation.     

Sphingomyelinase enhances low density lipoprotein uptake and ability to induce cholesteryl ester accumulation in macrophages.

Cholesteryl ester-loaded macrophages, or foam cells, are a prominent feature of atherosclerotic lesions. Low density lipoprotein (LDL) receptor-mediated endocytosis of native LDL is a relatively poor inducer of macrophage cholesteryl ester accumulation. However, the data herein show that in the presence of a very small amount of sphingomyelinase, LDL receptor-mediated endocytosis of 125I-LDL was enhanced and led to a 2-6-fold increase in 125I-LDL degradation and up to a 10-fold increase in cholesteryl ester accumulation in macrophages. The enhanced lipoprotein uptake and cholesterol esterification was seen after only approximately 12% hydrolysis of LDL phospholipids, was specific for sphingomyelin hydrolysis, and appeared to be related to the formation of fused or aggregated spherical particles up to 100 nm in diameter. Sphingomyelinase-treated LDL was bound by the macrophage LDL receptor. However, when unlabeled acetyl-LDL, a scavenger receptor ligand, was present during or after sphingomyelinase treatment of 125I-LDL, 125I-LDL binding and degradation were enhanced further through the formation of LDL-acetyl-LDL mixed aggregates. Experiments with cytochalasin D suggested that endocytosis, not phagocytosis, was involved in internalization of sphingomyelinase-treated LDL. Nonetheless, the sphingomyelinase effect on LDL uptake was macrophage-specific. These data illustrate that LDL receptor-mediated endocytosis of fused LDL particles can lead to foam cell formation in cultured macrophages. Furthermore, since both LDL and sphingomyelinase are present in atherosclerotic lesions and since some lesion LDL probably is fused or aggregated, there is a possibility that sphingomyelinase-treated LDL is a physiologically important atherogenic lipoprotein.     

Sequestration of aggregated low-density lipoproteins by macrophages

PURPOSE OF REVIEW: Evidence suggests that much of the LDL in atherosclerotic plaques is aggregated. Aggregation of LDL could be an important factor that determines how this lipoprotein is metabolized by plaque macrophages and the fate of aggregated LDL cholesterol within plaques. This review discusses a novel endocytic pathway by which macrophages process aggregated LDL. RECENT FINDINGS: Recently, it has been shown that aggregated LDL can be sequestered in macrophage surface-connected compartments and plasma membrane invaginations by a process termed patocytosis. In contrast to rapid degradation of LDL and aggregated LDL taken up by macrophages through pinocytosis and phagocytosis, respectively, aggregated LDL sequestered in macrophages undergoes only limited degradation. Macrophages can disaggregate and release sequestered aggregated LDL by activating plasminogen to plasmin. Plasmin degrades LDL apolipoprotein B sufficiently to disaggregate the aggregated LDL, releasing it from the macrophage surface-connected compartments. In contrast, activating macrophages with phorbol-myristate-acetate stimulates degradation of aggregated LDL and inhibits plasminogen-mediated release of the aggregated lipoprotein from macrophage surface-connected compartments. SUMMARY: Macrophage sequestration of aggregated LDL is a unique endocytic pathway relevant not only to the processing of aggregated LDL in atherosclerotic plaques but also for the processing of other materials, such as hydrophobic particles that trigger this endocytic pathway. Macrophage sequestration of aggregated LDL can result in different fates for the aggregated LDL, depending on the state of macrophage activation and the functioning of the plasminogen-based fibrinolytic system. Patocytosis of aggregated LDL should be considered in addition to phagocytosis as a possible uptake pathway in studies of macrophage processing of aggregated LDL.     

Group V secretory phospholipase A2-modified low density lipoprotein promotes foam cell formation by a SR-A- and CD36-independent process that involves cellular proteoglycans

Accumulating evidence indicates that secretory phospholipase A2 (sPLA2) enzymes promote atherogenic processes. We have previously showed the presence of Group V sPLA2 (GV sPLA2) in human and mouse atherosclerotic lesions, its hydrolysis of low density lipoprotein (LDL) particles, and the ability of GV sPLA2-modified LDL (GV-LDL) to induce macrophage foam cell formation in vitro. The goal of this study was to investigate the mechanisms involved in macrophage uptake of GV-LDL. Peritoneal macrophages from C57BL/6 mice (wild type (WT)), C57BL/6 mice deficient in LDL receptor (LDLR-/-), or SR-A and CD36 (DKO) were treated with control LDL, GV-LDL, oxidized LDL (ox-LDL) or LDL aggregated by vortexing (vx-LDL). As expected, ox-LDL induced significantly more cholesterol ester accumulation in WT and LDLR-/- compared with DKO macrophages. In contrast, there was no difference in the accumulation of GV-LDL or vx-LDL in the three cell types. 125I-ox-LDL exhibited high affinity, saturable binding to WT cells that was significantly reduced in DKO cells. Vx-LDL and GV-LDL showed low affinity, non-saturable binding that was similar for both cell types, and significantly higher compared with control LDL. GV-LDL degradation in WT and DKO cells was similar. Analyses by confocal microscopy indicated a distinct intracellular distribution of Alexa-568-labeled GV-LDL and Alexa-488-labeled ox-LDL. Uptake of GV-LDL (but not ox-LDL or vx-LDL) was significantly reduced in cells preincubated with heparin or NaClO3, suggesting a role for proteoglycans in GV-LDL uptake. Our data point to a physiological modification of LDL that has the potential to promote macrophage foam cell formation independent of scavenger receptors.     

Metabolism of oxidized LDL by macrophages

Oxidation products of lipids and proteins are found in atherosclerotic plaque and in macrophage foam cells. Macrophages avidly endocytose in-vitro oxidized LDL and accumulate sterols. What is the evidence that such a process is involved in in-vivo foam cell formation? The present review surveys current knowledge on the metabolism of oxidized LDL by macrophages, and the types, amounts and location of oxidation products that accumulate in these cells. Comparable studies of lesion lipoproteins and foam cells indicate that limited extracellular lipoprotein oxidation, perhaps followed by more extensive intracellular oxidation subsequent to uptake by macrophages, is a more likely scenario in vivo.     

Characterization of low-density lipoprotein uptake by murine macrophages exposed to Chlamydia pneumoniae

Exposure to Chlamydia pneumoniae is correlated with atherosclerosis in a variety of clinical and epidemiological studies, but how the organism may initiate and promote the disease is poorly understood. One pathogenic mechanism could involve modulation of macrophage function by C. pneumoniae. We recently demonstrated that C. pneumoniae induces macrophages to accumulate excess cholesterol and develop into foam cells, the hallmark of early atherosclerotic lesions. To determine if C. pneumoniae-induced foam cell formation involved increased uptake of low-density lipoprotein (LDL), the current study examined macrophage association of a fluorescent carbocyanine (DiI)-labeled LDL following infection. C. pneumoniae enhanced the association of DiI-LDL with macrophages in a dose-dependent manner with respect to both C. pneumoniae and DiI-LDL. Interestingly, increased association was inhibited by native LDL and occurred in the absence of oxidation byproducts and in the presence of antioxidants. However, enhanced DiI-LDL association occurred without the participation of the classical Apo B/E native LDL receptor, since C. pneumoniae increased DiI-LDL association and induced foam cell formation in macrophages isolated from LDL-receptor-deficient mice. Surprisingly, DiI-LDL association was inhibited not only by unlabeled native LDL but also by high-density lipoprotein, very low density lipoprotein, and oxidized LDL. These data indicate that exposure of macrophages to C. pneumoniae increases the uptake of LDL and foam cell formation by an LDL-receptor-independent mechanism.     

Lipoprotein aggregation protects human monocyte-derived macrophages from OxLDL-induced cytotoxicity

Oxidative modifications render low density lipoprotein cytotoxic and enhance its propensity to aggregate and fuse into particles similar to those found in atherosclerotic lesions. We showed previously that aggregation of oxidized LDL (OxLDL) promotes the transformation of human macrophages into lipid-laden foam cells (Asmis, R., and J. Jelk. 2000. Large variations in human foam cell formation in individuals. A fully autologous in vitro assay based on the quantitative analysis of cellular neutral lipids. Atherosclerosis. 148: 243-253). Here, we tested the hypothesis that aggregation of OxLDL enhances its clearance by human macrophages and thus may protect macrophages from OxLDL-induced cytotoxicity. We found that increased aggregation of OxLDL correlated with decreased macrophage injury. Using 3H-labeled and Alexa546-labeled OxLDL, we found that aggregation enhanced OxLDL uptake and increased cholesteryl ester accumulation but did not alter free cholesterol levels in macrophages. Acetylated LDL was a potent competitor of aggregated oxidized LDL (AggOxLDL) uptake, suggesting that scavenger receptor A plays an important role in the clearance of AggOxLDL. Inhibitors of actin polymerization, cytochalasin B, cytochalasin D, and latrunculin A, also prevented AggOxLDL uptake and restored OxLDL-induced cytotoxicity. This suggests that OxLDL-induced macrophage injury does not require OxLDL uptake and may occur on the cell surface. Our data demonstrate that aggregation of cytotoxic OxLDL enhances its clearance by macrophages without damage to the cells, thus allowing macrophages to avoid OxLDL-induced cell injury.     

E-LDL and Ox-LDL differentially regulate ceramide and cholesterol raft microdomains in human Macrophages.

Atherosclerosis is characterized by the generation of lipid-loaded macrophage-derived foam cells. To study the effect of different types of atherogenic lipoproteins, human macrophages were loaded with enzymatically degraded low density lipoprotein (E-LDL) or oxidized LDL (Ox-LDL). Cellular cholesterol content was increased by E-LDL, whereas Ox-LDL increased the ceramide content. Cell surface expression analysis by flow cytometry and confocal microscopy revealed that Ox-LDL increased ceramide and lactosylceramide expression compared to E-LDL loading and induced ceramide rafts, whereas loading with E-LDL induced cholesterol-rich microdomains. Formation of different rafts may have consequences for raft associated signaling in cholesterol homeostasis and apoptosis in human macrophages.     

Endogenously produced glycosaminoglycans affecting the release of lipoprotein lipase from macrophages and the interaction with lipoproteins

Macrophages are intimately involved in the pathogenesis of atherosclerotic diseases. A key feature of this process is their uptake of various lipoproteins and subsequent transformation to foam cells. Since lipoprotein lipase (LPL) is believed to play a role in foam cell formation, we investigated if endogenously produced proteoglycans (PGs) affect the release of this enzyme from macrophages. The human leukaemic cell line THP-1 which differentiates into macrophages by treatment with phorbol ester (phorbol 12-myristate 13-acetate) served as a model. The differentiation of THP-1 macrophages promoted the release of PGs into the cell medium which caused the detachment of LPL activity from the cell surface, and prevented LPL re-uptake and inactivation. These PGs were mainly composed of chondroitin sulfate type and exerted a heparin-like effect on LPL release. LPL is known to increase the cell association of lipoproteins by the well known bridging function. Exogenous bovine LPL at a concentration of 1 microg/ml enhanced low density lipoprotein (LDL)-binding 10-fold. Endogenously produced PGs reduced LPL-mediated binding of LDL. It is proposed that the differentiation-dependent increase in the release of PGs interferes with binding of LPL and reduces lipoprotein-binding to macrophages.     

The uptake and degradation of matrix-bound lipoproteins by macrophages require an intact actin Cytoskeleton, Rho family GTPases, and myosin ATPase activity

A key cellular event in atherogenesis is the interaction of macrophages with lipoproteins in the subendothelium. In vivo, these lipoproteins are bound to matrix and often aggregated, yet most cell-culture experiments explore these events using soluble monomeric lipoproteins. We hypothesized that the internalization and degradation of matrix-retained and aggregated low density lipoprotein (LDL) by macrophages may involve the actin-myosin cytoskeleton in a manner that distinguishes this process from the endocytosis of soluble LDL. To explore these ideas, we plated macrophages on sphingomyelinase-aggregated LDL bound to smooth muscle cell-derived matrix in the presence of lipoprotein lipase. The macrophages internalized and degraded the LDL, which was mediated partially by the LDL receptor-related protein. Cytochalasin D and latrunculin A, which block actin polymerization, markedly inhibited the uptake and degradation of matrix-retained LDL but not soluble LDL. Inhibition of Rho family GTPases by Clostridium difficile toxin B blocked the degradation of matrix-retained and aggregated LDL by >90% without any inhibition of soluble LDL degradation. However, specific inhibition of Rho had no effect, suggesting the importance of Rac1 and Cdc42. Degradation of matrix-retained, but not soluble, LDL was also blocked by inhibitors of tyrosine kinase, phosphatidylinositol 3-kinase, and myosin ATPase. These findings define fundamental cytoskeletal pathways that may be involved in macrophage foam cell formation in vivo but have been missed by the use of previous cell culture models.     

Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF)

Recently, we have shown that macrophage uptake of low density lipoprotein (LDL) and cholesterol accumulation can occur by nonreceptor mediated fluid-phase macropinocytosis when macrophages are differentiated from human monocytes in human serum and the macrophages are activated by stimulation of protein kinase C (Kruth, H. S., Jones, N. L., Huang, W., Zhao, B., Ishii, I., Chang, J., Combs, C. A., Malide, D., and Zhang, W. Y. (2005) J. Biol. Chem. 280, 2352-2360). Differentiation of human monocytes in human serum produces a distinct macrophage phenotype. In this study, we examined the effect on LDL uptake of an alternative macrophage differentiation phenotype. Differentiation of macrophages from human monocytes in fetal bovine serum with macrophage-colony-stimulating factor (M-CSF) produced a macrophage phenotype demonstrating constitutive fluid-phase uptake of native LDL leading to macrophage cholesterol accumulation. Fluid-phase endocytosis of LDL by M-CSF human macrophages showed non-saturable uptake of LDL that did not down-regulate over 48 h. LDL uptake was mediated by continuous actin-dependent macropinocytosis of LDL by these M-CSF-differentiated macrophages. M-CSF is a cytokine present within atherosclerotic lesions. Thus, macropinocytosis of LDL by macrophages differentiated from monocytes under the influence of M-CSF is a plausible mechanism to account for macrophage foam cell formation in atherosclerotic lesions. This mechanism of macrophage foam cell formation does not depend on LDL modification or macrophage receptors.     

NOS1-derived nitric oxide facilitates macrophage uptake of low-density lipoprotein

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by a scavenger receptor in macrophages initiates necrosis core formation that characterizes atherosclerosis. We report that NOS1-derived nitric oxide (NO) facilitates low-density lipoprotein (LDL) uptake by macrophages independent of the inflammatory response. LDL uptake could be dramatically suppressed by NOS1 specific inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM). Importantly, the notion that NOS1 can mediate uptake of lipoproteins suggests that the foam cell formation is regulated by NOS1-derived NO-mediated mechanism. This is a novel study involving NOS1 as a critical player of foam cell formation and reveals much about the key molecular proteins involved in atherosclerosis. Targeting NOS1 would be a useful strategy in reducing LDL uptake by macrophages and hence dampening the atherosclerosis progression.     

Cloning and expression of an anti-LDL(-) single-chain variable fragment,and its inhibitory effect on experimental atherosclerosis

The in vivo modified forms of low-density lipoprotein (LDL) are important for the formation of foam cells and as mediators of the immuno-inflammatory process involved in the progression of atherosclerosis. Electronegative LDL, LDL(-), is a LDL subfraction with pro-inflammatory properties that is present in human blood. To investigate possible atheroprotective effects, an anti-LDL(-) single-chain variable fragment (scFv) was expressed in the methylotrophic yeast Pichia pastoris and its activity was evaluated in vitro against macrophages and in experimental atherosclerosis in Ldlr^-/- mice. The recombinant 2C7 scFv was produced in a yield of 9.5 mg of protein/L. The specificity and affinity of purified 2C7 scFv against LDL(-) was confirmed by ELISA. To assess the activity of 2C7 scFv on foam cell formation, RAW 264.7 macrophages were exposed to LDL(-) in the presence or absence of 2C7 scFv. The 2C7 scFv inhibited the uptake of LDL(-) by macrophages in a dose-dependent manner, and internalization of LDL(-) by these cells was found to be mediated by the CD36 and CD14 receptor. In addition, compared with untreated cells, lipid accumulation in macrophages was decreased, and the expression of Cd36, Tlr-4 and Cox-2 was downregulated in macrophages treated with 2C7 scFv. Importantly, compared with untreated mice, the treatment of Ldlr^-/- mice with 2C7 scFv decreased the atherosclerotic lesion area at the aortic sinus. In conclusion, our data show that 2C7 scFv inhibits foam cell formation and atherosclerotic plaque development by modulating the expression of genes relevant to atherogenesis. These results encourage further use of this antibody fragment in the development of new therapeutic strategies that neutralize the pro-atherogenic effects of LDL(-).     

The influence of oxidatively modified low density lipoproteins on expression of platelet-derived growth factor by human monocyte-derived macrophages

Platelet-derived growth factor (PDGF) is secreted by several cells that participate in the process of atherogenesis, including arterial wall monocyte-derived macrophages. Macrophages in human and non-human primate lesions have recently been demonstrated to contain PDGF-B chain protein in situ. In developing lesions of atherosclerosis, macrophages take up and metabolize modified lipoproteins, leading to lipid accumulation and foam cell formation. Oxidatively modified low density lipoproteins (LDL) have been implicated in atherogenesis and have been demonstrated in atherosclerotic lesions. The effects of the uptake of various forms of modified LDL on PDGF gene expression, synthesis, and secretion in adherent cultures of human blood monocyte-derived macrophages were examined. LDL oxidized in a cell-free system in the presence of air and copper inhibited the constitutive expression of PDGF-B mRNA and secretion of PDGF in a dose-dependent fashion. Oxidatively modified LDL also attenuated lipopolysaccharide-induced PDGF-B mRNA expression. These changes were unrelated to the mechanism of lipid uptake and the degree of lipid loading and were detectable within 2 h of exposure to oxidized LDL. The degree of inhibition of both basal and lipopolysaccharide-induced PDGF-B-chain expression increased with the extent of LDL oxidation. Monocyte-derived macrophages exposed to acetylated LDL or LDL aggregates accumulated more cholesterol than cells treated with oxidized LDL, but PDGF expression was not consistently altered. Thus, uptake of a product or products of LDL oxidation modulates the expression and secretion of one of the principal macrophage-derived growth factors, PDGF. This modulation may influence chemotaxis and mitogenesis of smooth muscle cells locally in the artery wall during atherogenesis.     

Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A2 mimics cell-mediated oxidative modification

Low density lipoprotein (LDL) can be oxidatively modified by cultured endothelial cells or by cupric ions, resulting in increased macrophage uptake of the lipoprotein. This process could be relevant to the formation of macrophage-derived foam cells in the early atherosclerotic lesion. The mechanism of endothelial cell modification of LDL is unknown. In the present work we show that incubation of LDL with purified soybean lipoxygenase, in the presence of pure phospholipase A2, can mimic endothelial cell-induced oxidative modification. Typically, incubation with lipoxygenase plus phospholipase A2 caused: 1) generation of about 15 nmol of thiobarbituric acid-reactive substances per mg of LDL protein; 2) a 4- to 7-fold increase in the rate of subsequent macrophage degradation of the LDL; 3) a 10-fold decrease in recognition by fibroblasts; 4) a marked increase in electrophoretic mobility in agarose gels; and, 5) disappearance of intact apoprotein B on SDS polyacrylamide gels. Degradation of the enzymatically modified LDL by macrophages was competitively inhibited by endothelial cell-modified LDL and by polyinosinic acid, but only partially suppressed by acetylated LDL. The lipoxygenase plus phospholipase A2-induced modification of LDL is not necessarily identical to endothelial cell modification, but it is a useful model for studying the mechanism of oxidative modification of LDL. This work also represents the first example of oxidative modification of LDL by specific enzymes leading to enhanced recognition by macrophages.     

Platelet factor 4 enhances the binding of oxidized low-density lipoprotein to vascular wall cells

Accumulation of low-density lipoprotein (LDL)-derived cholesterol by macrophages in vessel walls is a pathogenomic feature of atherosclerotic lesions. Platelets contribute to lipid uptake by macrophages through mechanisms that are only partially understood. We have previously shown that platelet factor 4 (PF4) inhibits the binding and degradation of LDL through its receptor, a process that could promote the formation of oxidized LDL (ox-LDL). We have now characterized the effect of PF4 on the binding of ox-LDL to vascular cells and macrophages and on the accumulation of cholesterol esters. PF4 bound to ox-LDL directly and also increased ox-LDL binding to vascular cells and macrophages. PF4 did not stimulate ox-LDL binding to cells that do not synthesize glycosaminoglycans or after enzymatic cleavage of cell surface heparan and chondroitin sulfates. The effect of PF4 on binding ox-LDL was dependent on specific lysine residues in its C terminus. Addition of PF4 also caused an approximately 10-fold increase in the amount of ox-LDL esterified by macrophages. Furthermore, PF4 and ox-LDL co-localize in atherosclerotic lesion, especially in macrophage-derived foam cells. These observations offer a potential mechanism by which platelet activation at sites of vascular injury may promote the accumulation of deleterious lipoproteins and offer a new focus for pharmacological intervention in the development of atherosclerosis.     

Metabolism of modified LDL and foam cell formation in murine macrophage-like RAW 264 cells.

The uptake of modified low density lipoprotein (LDL) by arterial macrophages is a key event in the atherogenesis. We studied 1) the uptake and degradation of modified LDL, 2) LDL recognition by specific receptors, and 3) the foam cell formation with murine macrophage-like RAW 264 cells in vitro. The cells took up and degraded effectively 125I-labeled acetylated LDL (Ac-LDL) and aggregated LDL (Aggr-LDL). Also oxidized LDL (Ox-LDL) was taken up but it was degraded poorly. The degradation of 125I-Ac-LDL was efficiently competed by both unlabeled Ac-LDL and Ox-LDL, whereas the degradation of 125I-Ox-LDL was partially competed by unlabeled Ox-LDL and Aggr-LDL but not at all by unlabeled Ac-LDL. The incubation with increasing concentrations of Ac-LDL, Aggr-LDL or Ox-LDL resulted in marked foam cell formation in the RAW 264 cells. Ox-LDL was cytotoxic at 500 to 1000 microg/ml concentrations. The results show that RAW 264 cells have at least two classes of receptors for modified lipoproteins: one that recognizes both Ox-LDL and Ac-LDL, and is similar to the scavenger receptors, and another that recognizes Ox-LDL but not Ac-LDL. RAW 264 cells are a convenient model cell line for examining the metabolism of modified lipoproteins, not only that of Ac-LDL but also that of Ox-LDL and Aggr-LDL, and cellular accumulation of lipids derived from modified LDL.     

Vav protein guanine nucleotide exchange factor regulates CD36 protein-mediated macrophage foam cell formation via calcium and dynamin-dependent processes

Atherosclerosis, a chronic inflammatory disease, results in part from the accumulation of modified lipoproteins in the arterial wall and formation of lipid-laden macrophages, known as "foam cells." Recently, we reported that CD36, a scavenger receptor, contributes to activation of Vav-family guanine nucleotide exchange factors by oxidatively modified LDL in macrophages. We also discovered that CD36-dependent uptake of oxidized LDL (oxLDL) in vitro and foam cell formation in vitro and in vivo was significantly reduced in macrophages deficient of Vav proteins. The goal of the present study was to identify the mechanisms by which Vav proteins regulate CD36-dependent foam cell formation. We now show that a Vav-dynamin signaling axis plays a critical role in generating calcium signals in mouse macrophages exposed to CD36-specific oxidized phospholipid ligands. Chelation of intracellular Ca(2+) or inhibition of phospholipase C-γ (PLC-γ) inhibited Vav activation (85 and 70%, respectively, compared with vehicle control) and reduced foam cell formation (approximately 75%). Knockdown of expression by siRNA or inhibition of GTPase activity of dynamin 2, a Vav-interacting protein involved in endocytic vesicle fission, significantly blocked oxLDL uptake and inhibited foam cell formation. Immunofluorescence microscopy studies showed that Vav1 and dynamin 2 colocalized with internalized oxLDL in macrophages and that activation and mobilization of dynamin 2 by oxLDL was impaired in vav null cells. These studies identified previously unknown components of the CD36 signaling pathway, demonstrating that Vav proteins regulate oxLDL uptake and foam cell formation via calcium- and dynamin 2-dependent processes and thus represent novel therapeutic targets for atherosclerosis.     

Relative importance of the LDL receptor and scavenger receptor class B in the beta-VLDL-induced uptake and accumulation of cholesteryl esters by peritoneal macrophages

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.     

Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein

Previously, we reported that fluid-phase endocytosis of native LDL by PMA-activated human monocytederived macrophages converted these macrophages into cholesterol-enriched foam cells (Kruth, H. S., Huang, W., Ishii, I., and Zhang, W. Y. (2002) J. Biol. Chem. 277, 34573-34580). Uptake of fluid by cells can occur either by micropinocytosis within vesicles (<0.1 microm diameter) or by macropinocytosis within vacuoles ( approximately 0.5-5.0 microm) named macropinosomes. The current investigation has identified macropinocytosis as the pathway for fluid-phase LDL endocytosis and determined signaling and cytoskeletal components involved in this LDL endocytosis. The phosphatidylinositol 3-kinase inhibitor, LY294002, which inhibits macropinocytosis but does not inhibit micropinocytosis, completely blocked PMA-activated macrophage uptake of fluid and LDL. Also, nystatin and filipin, inhibitors of micropinocytosis from lipid-raft plasma membrane domains, both failed to inhibit PMA-stimulated macrophage cholesterol accumulation. Time-lapse video phase-contrast microscopy and time-lapse digital confocal-fluorescence microscopy with fluorescent DiI-LDL showed that PMA-activated macrophages took up LDL in the fluid phase by macropinocytosis. Macropinocytosis of LDL depended on Rho GTPase signaling, actin, and microtubules. Bafilomycin A1, the vacuolar H+-ATPase inhibitor, inhibited degradation of LDL and caused accumulation of undegraded LDL within macropinosomes and multivesicular body endosomes. LDL in multivesicular body endosomes was concentrated >40-fold over its concentration in the culture medium consistent with macropinosome shrinkage by maturation into multivesicular body endosomes. Macropinocytosis of LDL taken up in the fluid phase without receptor-mediated binding of LDL is a novel endocytic pathway that generates macrophage foam cells. Macropinocytosis in macrophages and possibly other vascular cells is a new pathway to target for modulating foam cell formation in atherosclerosis.