Low-density Lipoprotein Uptake In Macrophages Research Articles

Nalmefene, an opioid receptor modulator, aggravates atherosclerotic plaque formation in apolipoprotein E knockout mice by enhancing oxidized low-density lipoprotein uptake in macrophages

Nalmefene, an antagonist of mu- and delta-opioid receptors and a partial agonist of kappa-opioid receptors, has shown promise in reducing alcohol consumption among patients with alcohol dependence. Opioid receptors play pivotal roles in various physiological processes, including those related to peripheral inflammatory diseases such as colitis and arthritis, as well as functions in the immune system and phagocytosis. Atherosclerosis, a chronic inflammatory disease, progresses through the phagocytosis and uptake of oxidized low-density lipoprotein (oxLDL) by macrophages in atherosclerotic plaques. Despite this knowledge, it remains unclear whether nalmefene influences the formation of atherosclerotic plaques and increases the risk of serious cardiovascular events. This study aims to elucidate the impact of nalmefene on atherosclerosis in apolipoprotein E knockout (ApoE KO) mice and peritoneal macrophages in vitro.In this experiment, 8-week-old male ApoE KO mice were fed a high-fat diet intraperitoneally administered either vehicle (saline) or nalmefene (1 mg and 3 mg kg−1 day−1) for 21 days. Oil red O-staining and immunohistochemistry with an anti-MOMA2 (monocyte/macrophage) antibody showed that a dose-dependent increase in atherosclerotic plaque formation and augmentation of macrophage-rich plaque formation in ApoE-KO mice. Further investigations focused on the effects of nalmefene on the expression of scavenger receptor CD36 in RAW264.7 cells, conducted through western blotting analysis. Nalmefene demonstrated a significant increase in CD36 protein expression in RAW264.7 cells. To explore the impact on oxidized LDL uptake in peritoneal macrophages, cells were treated with nalmefene (300 μg/mL) for 24 h, followed by the addition of DiI-labeled oxLDL (DiI-oxLDL) for 4 h. Nalmefene significantly enhanced DiI-oxLDL uptake in macrophages. Additionally, treatment with nalmefene (300 μg/mL) for 24 h decreased the mRNA expression of mu-, delta-, and kappa-opioid receptors in RAW264.7 cells.In conclusion, nalmefene may augment oxLDL uptake by macrophages through increased CD36 expression and decreased opioid receptor, thereby contributing to atherosclerotic plaque formation and vulnerability. Consequently, the use of nalmefene may be associated with an elevated risk of cardiovascular events.

Ubiquitinated ligation protein NEDD4L participates in MiR-30a-5p attenuated atherosclerosis by regulating macrophage polarization and lipid metabolism

MiR-30a-5p plays an important role in various cardiovascular diseases, but its effect in atherosclerosis has not been reported. Apolipoprotein E-deficient (Apo E−/−) mice were used to investigate the role of miR-30a-5p in atherosclerosis, and the underlying mechanism was investigated in vivo and in vitro. The fluorescence in situ hybridization test revealed that miR-30a-5p was expressed in Apo E−/− mice lesions. Nevertheless, in RAW264.7 macrophages, the expression of miR-30a-5p was reduced by lipopolysaccharide (LPS) or oxidized low-density lipoprotein. MiR-30a-5p-ago-treated Apo E−/− mice significantly reduced lesion areas in the aorta and aortic root, reduced levels of lipoprotein and pro-inflammatory cytokines, and increased levels of anti-inflammatory cytokines. The ratio of M1/M2 macrophages was decreased in miR-30a-5p-ago-treated Apo E−/− mice and LPS-treated RAW264.7 macrophages by the regulation of Smad-1/2 phosphorylation. MiR-30a-5p reduced lipid uptake in oxidized low-density lipoprotein-treated macrophages by regulating the expression of PPAR-γ, ABCA1, ABCG1, LDLR, and PCSK9. Ubiquitinated ligase NEDD4L was identified as a target of miR-30a-5p. Interestingly, knockdown of NEDD4L decreased the M1/M2 ratio and oxidized low-density lipoprotein uptake in macrophages by inhibiting the ubiquitination of PPAR-γ and phosphorylation of Smad-1/2 and regulating ABCA1, ABCG1, LDLR, and PCSK9. We demonstrated a novel effect and mechanism of miR-30a-5p in atherosclerosis.

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.

Circulation Research “In This Issue” Anthology

Circulation Research “In This Issue” Anthology

In This Issue

HomeCirculation ResearchVol. 116, No. 5In This Issue Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published27 Feb 2015https://doi.org/10.1161/RES.0000000000000050Circulation Research. 2015;116:763Macrophage sortilin promotes LDL uptake, foam cell formation, and atherosclerosis (p 789)Sortilin protein drives cholesterol uptake in macrophages and promotes atherosclerosis, report Patel et al.Download figureDownload PowerPointA characteristic feature of atherosclerosis is the formation of foam cells that arise from cholesterol-loaded macrophages. But exactly how macrophages take up cholesterol from transporter proteins such as low-density lipoprotein (LDL) is unclear, especially since the deletion of known macrophage lipoprotein receptors in mice does not prevent foam cell formation. Patel and colleagues thus turned their attention to a protein called sortilin. This protein mediates hepatic LDL uptake and, through genome-wide association studies, has been linked with coronary artery disease. The function of sortilin in macrophages, however, was unknown. The team found that atherosclerosis-prone mice lacking sortilin—either in all cells or just macrophages—showed a significant reduction in the number of atherolsclerotic lesions compared to mice with normal levels of the protein. They also discovered that sortilin-lacking macrophages exhibited a reduction in LDL uptake, while macrophages that over-expressed sortilin actually increased their LDL uptake. These results suggest that preventing the sortilin-driven uptake of LDL in macrophages might reduce foam cell formation and thus be a novel strategy for treating, and perhaps even preventing, atherosclerosis in the future.Transcriptional reversion of cardiomyocyte fate during mammalian cardiac regeneration (p 804)O’Meara et al describe the transcriptional signature of mammalian heart regeneration.Download figureDownload PowerPointThe adult mammalian heart has a limited capacity for regeneration. In contrast, neonatal mice, within the first week of life, can completely repair their hearts after injury. During this regeneration, neonatal heart cells lose their sarcomeric structures, adopt a less-differentiated state, and reenter the cell cycle. If this same regenerative process could be recapitulated in the adult hearts, full recovery from myocardial injury might be possible. Hence to understand the mechanism underlying the regenerative capacity of the neonatal heart, O’Meara and colleagues examined changes in the global gene expression profiles of the neonatal heart after injury and during regeneration. They also studied the expression profiles of normal neonatal and adult cardiomyocytes, mouse embryonic stem cells during differentiation into cardiomyocytes, and adult cardiomyocytes induced to de-differentiate. After cross-referencing these data, the team identified a reference panel of genes involved in the differentiation and regeneration of a normal heart. They found that injury to the neonatal heart essentially reversed the differentiation process such that seven days post-injury the genes encoding sarcomeric proteins were reduced, while the expression of cell cycle genes was elevated. The team highlighted IL-13, STAT3 and STAT6 as regulators of cell cycle re-entry, but their results provide a large number of additional candidates that could be examined in future regeneration studies.Effects of DNA damage in smooth muscle cells in atherosclerosis (p 816)DNA damage promotes atherosclerotic plaque instability report Gray et al.Download figureDownload PowerPointDNA damage occurs in the vascular smooth muscle cells (VSMCs) of atherosclerotic plaques and it becomes more pervasive with disease progression. But whether such damage is a cause or consequence of the condition is unclear. It is known that DNA damage reduces cell proliferation—because cells stop cycling to repair the DNA, and this could arguably slow down plaque growth. If, however, the damage is severe, senescence and apoptosis can occur, which induces proinflammatory cytokine production and immune cell recruitment, thus exacerbating plaque growth. To better define the relationship between plaque growth and DNA damage, Gray and colleagues studied the effects of accelerating or inhibiting DNA repair in human VSMC from normal aorta and atherosclerotic plaques. As expected, they found that VSMCs from plaques had significantly higher levels of DNA damage as well as increased activation of DNA repair pathways. By accelerating this repair in a mouse model of atherosclerosis, they found that while plaque size remained the same, there was an increase in the stability of the fibrous caps of the plaques. Inhibiting repair, on the other hand, promoted plaque instability. Because unstable plaques are associated with a greater risk of myocardial infarction and stroke, preventing DNA damage, or promoting DNA repair could be a novel therapeutic strategy for enhancing plaque stability in patients with atherosclerotic disease. Previous Back to top Next FiguresReferencesRelatedDetails February 27, 2015Vol 116, Issue 5 Advertisement Article InformationMetrics © 2015 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000050 Originally publishedFebruary 27, 2015 PDF download Advertisement

Sugar-based amphiphilic polymers for biomedical applications: from nanocarriers to therapeutics.

Various therapeutics exhibit unfavorable physicochemical properties or stability issues that reduce their in vivo efficacy. Therefore, carriers able to overcome such challenges and deliver therapeutics to specific in vivo target sites are critically needed. For instance, anticancer drugs are hydrophobic and require carriers to solubilize them in aqueous environments, and gene-based therapies (e.g., siRNA or pDNA) require carriers to protect the anionic genes from enzymatic degradation during systemic circulation. Polymeric micelles, which are self-assemblies of amphiphilic polymers (APs), constitute one delivery vehicle class that has been investigated for many biomedical applications. Having a hydrophobic core and a hydrophilic shell, polymeric micelles have been used as drug carriers. While traditional APs are typically comprised of nondegradable block copolymers, sugar-based amphiphilic polymers (SBAPs) synthesized by us are comprised of branched, sugar-based hydrophobic segments and a hydrophilic poly(ethylene glycol) chain. Similar to many amphiphilic polymers, SBAPs self-assemble into polymeric micelles. These nanoscale micelles have extremely low critical micelle concentrations offering stability against dilution, which occurs with systemic administration. In this Account, we illustrate applications of SBAPs for anticancer drug delivery via physical encapsulation within SBAP micelles and chemical conjugation to form SBAP prodrugs capable of micellization. Additionally, we show that SBAPs are excellent at stabilizing liposomal delivery systems. These SBAP-lipid complexes were developed to deliver hydrophobic anticancer therapeutics, achieving preferential uptake in cancer cells over normal cells. Furthermore, these complexes can be designed to electrostatically complex with gene therapies capable of transfection. Aside from serving as a nanocarrier, SBAPs have also demonstrated unique bioactivity in managing atherosclerosis, a major cause of cardiovascular disease. The atherosclerotic cascade is usually triggered by the unregulated uptake of oxidized low-density lipoprotein, a cholesterol carrier, in macrophages of the blood vessel wall; SBAPs can significantly inhibit oxidized low-density lipoprotein uptake in macrophages and abrogate the atherosclerotic cascade. By modification of various functionalities (e.g., branching, stereochemistry, hydrophobicity, and charge) in the SBAP chemical structure, SBAP bioactivity was optimized, and influential structural components were identified. Despite the potential of SBAPs as atherosclerotic therapies, blood stability of the SBAP micelles was not ideal for in vivo applications, and means to stabilize them were pursued. Using kinetic entrapment via flash nanoprecipitation, SBAPs were formulated into nanoparticles with a hydrophobic solute core and SBAP shell. SBAP nanoparticles exhibited excellent physiological stability and enhanced bioactivity compared with SBAP micelles. Further, this method enables encapsulation of additional hydrophobic drugs (e.g., vitamin E) to yield a stable formulation that releases two bioactives. Both as nanoscale carriers and as polymer therapeutics, SBAPs are promising biomaterials for medical applications.

Impact of ionizing radiation on physicochemical and biological properties of an amphiphilic macromolecule

An amphiphilic macromolecule (AM) was exposed to ionizing radiation (both electron beam and gamma) at doses of 25 kGy and 50 kGy to study the impact of these sterilization methods on the physicochemical properties and bioactivity of the AM. Proton nuclear magnetic resonance and gel permeation chromatography were used to determine the chemical structure and molecular weight, respectively. Size and zeta potential of the micelles formed from AMs in aqueous media were evaluated by dynamic light scattering. Bioactivity of irradiated AMs was evaluated by measuring inhibition of oxidized low-density lipoprotein uptake in macrophages. From these studies, no significant changes in the physicochemical properties or bioactivity were observed after the irradiation, demonstrating that the AMs can withstand typical radiation doses used to sterilize materials.

Native Low-Density Lipoprotein Uptake by Macrophage Colony-Stimulating Factor–Differentiated Human Macrophages Is Mediated by Macropinocytosis and Micropinocytosis

To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor-differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human macrophage colony-stimulating factor-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

Native low-density lipoprotein uptake by Macrophage Colony-Stimulating Factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis (134.12)

Abstract Human macrophage colony-stimulating factor (M-CSF)-differentiated macrophages—the predominant macrophage phenotype in human atherosclerotic plaques—constitutively take up native low-density lipoprotein (LDL) by fluid-phase pinocytosis, but the pinocytotic pathways mediating LDL uptake by these macrophages have not been thoroughly examined. We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages was only partially inhibited (~40%) by these agents. We therefore investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (~40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human M-CSF-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

Inhibitory effects of fucose-related sugar compounds on oxidised low-density lipoprotein uptake in macrophage cell line J774.1

The interaction between oxidised low-density lipoprotein (LDL) and macrophages is known to be important in the development of arteriosclerosis. Macrophages take up oxidised LDL and then become foam cells, which contribute to the thickening of the blood vessel wall. In our previous paper, some flavonoids found in vegetables and fruits were shown to have a protective effect against arteriosclerosis. In this communication, to elucidate the effect of fucose-related compounds on oxidised LDL uptake in macrophages, the inhibitory activity of various fucose-related compounds on the 1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate-acetylated-LDL (DiI-ac-LDL) uptake reaction in mouse macrophage cell line J774.1 was measured. Fucoidan, 4-deoxy-fucose and fucose-galactose-N-acetyl-glucose significantly inhibited uptake of DiI-ac-LDL into macrophages. The IC50 of 4-deoxy-fucose was 108.4 µM, the highest value of the fucose-related compounds used in the experiments. The protective effects of the fucose-related compounds on atherosclerosis can be used as new health foods.

Cilostazol inhibits modified low-density lipoprotein uptake and foam cell formation in mouse peritoneal macrophages

Internalization of modified low-density lipoprotein (LDL) via macrophage scavenger receptors (e.g. scavenger receptor A and CD36) is thought to play a crucial role in the development of atherosclerotic lesions. Cilostazol, an antiplatelet agent with selective phosphodiesterase 3 inhibitory action, has been reported to ameliorate atherosclerosis in mouse models. However, the effect of cilostazol on modified LDL uptake in macrophages is not known. Thus, we investigated the effect of cilostazol on LDL uptake in mouse peritoneal macrophages (MPM). Cilostazol significantly inhibited oxidized and acetylated LDL uptake in MPM, while cyclic AMP (cAMP)-elevating agents, db-cAMP and other phosphodiesterase 3 or 4 inhibitors, did not inhibit the uptake. Cilostazol did not change cytosolic cAMP levels in MPM, and a protein kinase A (PKA) inhibitor did not influence the inhibitory effects of cilostazol. Cilostazol decreased scavenger receptor A but not CD36 expression. Moreover, cilostazol significantly inhibited foam cell formation, which was represented by an increase in esterified cholesterol content. In conclusion, cilostazol significantly inhibits the uptake of modified LDL and foam cell formation in mouse peritoneal macrophages, and the inhibitory effect of cilostazol can be induced in a cAMP- and PKA-independent manner.

Inhibition of Protein Kinase Cβ Prevents Foam Cell Formation by Reducing Scavenger Receptor A Expression in Human Macrophages

Low-density lipoprotein (LDL) uptake by monocyte-derived macrophages is a crucial step in foam cell formation and early atherosclerotic lesion. Increasing evidence supports the theory that activation of protein kinase Cbeta (PKCbeta) is involved in many mechanisms promoting atherosclerosis. Thus, we investigated whether inhibition of PKCbeta prevents foam cell formation. The differentiation of human primary monocytes or the monocytic THP-1 cell line into monocyte-derived macrophages was induced by phorbol 12-myristate 13-acetate (PMA; 0.1 mmol/L), a potent activator of PKC. Incubation of monocyte-derived macrophages with DiI-modified LDL (acetylated LDL and oxidized LDL, 10 mug/mL) led to lipoprotein uptake. Interestingly enough, the nonselective inhibitor of PKCbeta(1) and PKCbeta(2), LY379196 (5x10(-7) to 10(-5) mol/L), blunted LDL uptake in monocyte-derived macrophages as shown by flow cytometry. Specific siRNA-mediated knockdown of PKCbeta exerted a similar effect. Furthermore, PMA alone and in the presence of modified LDL induced scavenger receptor A mRNA and protein expression, which was abolished by LY379196. CGP53353, a selective inhibitor of PKCbeta(2), did not affect LDL uptake, nor did it prevent scavenger receptor A upregulation. Incubation of monocyte-derived macrophages with PMA/LDL increased PKCbeta(1) phosphorylation at the Thr-642 residue, which was blunted by LY379196. However, the expression of CD68, a marker of activated macrophages, was not affected by LY379196. Moreover, LY379196 did not affect lipopolysaccharide-induced CD14 degradation, tumor necrosis factor-alpha release, or superoxide anion production, ruling out any effect of PKCbeta inhibition on innate immunity. Nonspecific inhibition of PKCbeta prevents LDL uptake in macrophages. These findings suggest that PKCbeta inhibitors may represent a novel class of antiatherosclerotic drugs.

The inhibitory effects of isoflavonoids and resveratrol on oxdized low-density lipoprotein uptake in macrophage cell line J774.1

The interaction between oxidized low-density lipoprotein (LDL) and macrophages are known to be important in the development of arteriosclerosis. Macrophages take up oxidized LDL, becoming foam cells, which contribute to the thickening of the blood vessel wall. The antioxidant properties of polyphenols found in vegetables and other foods have been shown to have a protective effect against arteriosclerosis. To elucidate the effect of flavonoids from fruits, vegetables and cereals on oxidized LDL uptake in macrophages, the inhibitory activity of various flavonoids on DiI-ac-LDL uptake reaction in mouse macrophage cell line J774.1 was measured. We found significant uptake of DiI-ac-LDL, but not DiI-LDL, into the J774.1 cells. In addition, polyinosin and dextran sulphate inhibited uptake, but apigenin, kaempferol, and naringenin, did not. Isoflavones and resveratrol significantly inhibit uptake of DiI-ac-LDL into macrophages, and have a protective effect against arteriosclerosis.

Retinoids Potentiate Peroxisome Proliferator-Activated Receptor γ Action in Differentiation, Gene Expression, and Lipid Metabolic Processes in Developing Myeloid Cells

Nuclear hormone receptors have been shown to be important transcription factors for regulating lipid metabolism in myeloid cells and were also implicated in differentiation processes of the myeloid lineage and macrophages. Peroxisome proliferator-activated receptor gamma (PPARgamma) seems to be a key component of lipid uptake by inducing the scavenger receptor CD36 that mediates oxidized low-density lipoprotein uptake in macrophages. Retinoic acid receptors, on the other hand, were also shown to play important roles in myeloid cell differentiation. In this study, we present evidence for a cross-talk between these two nuclear receptor pathways in myeloid cells. We show that expression level of PPARgamma increases with the degree of monocyte/macrophage commitment during maturation. Activation of PPARgamma leads to the increased expression of maturation markers (e.g., CD14, CD36). It is interesting that retinoid treatment potentiates PPARgamma's ability to induce transcription of its target genes. Retinoid-increased PPARgamma response is sufficient for enhancing lipid uptake. Our data, taken together, indicate that the expression level of PPARgamma increases during monocyte/macrophage development. PPARgamma activity can be enhanced by retinoids at least in part via increasing PPARgamma expression level. These observations can be exploited to enhance therapeutically beneficial PPAR responses in myeloid cells.

Scavenger Receptors Class A-I/II and CD36 Are the Principal Receptors Responsible for the Uptake of Modified Low Density Lipoprotein Leading to Lipid Loading in Macrophages

Modification of low density lipoprotein (LDL) can result in the avid uptake of these lipoproteins via a family of macrophage transmembrane proteins referred to as scavenger receptors (SRs). The genetic inactivation of either of two SR family members, SR-A or CD36, has been shown previously to reduce oxidized LDL uptake in vitro and atherosclerotic lesions in mice. Several other SRs are reported to bind modified LDL, but their contribution to macrophage lipid accumulation is uncertain. We generated mice lacking both SR-A and CD36 to determine their combined impact on macrophage lipid uptake and to assess the contribution of other SRs to this process. We show that SR-A and CD36 account for 75-90% of degradation of LDL modified by acetylation or oxidation. Cholesteryl ester derived from modified lipoproteins fails to accumulate in macrophages taken from the double null mice, as assessed by histochemistry and gas chromatography-mass spectrometry. These results demonstrate that SR-A and CD36 are responsible for the preponderance of modified LDL uptake in macrophages and that other scavenger receptors do not compensate for their absence.

Macrophage uptake of low-density lipoprotein bound to aggregated C-reactive protein: possible mechanism of foam-cell formation in atherosclerotic lesions

Foam cells found in atherosclerotic lesions are believed to derive from macrophages that take up aggregated low-density lipoprotein (LDL) particles bound to the extracellular matrix of arterial walls. C-reactive protein (CRP) is an acute-phase protein found in atherosclerotic lesions, which when immobilized on a solid phase, can bind and cluster LDL particles in a calcium-dependent manner. In the present study, we examined whether CRP-bound aggregated LDL could be taken up by macrophages in culture. CRP molecules were aggregated in the presence of calcium and immobilized on the surface of polystyrene microtitre wells. Human LDL added to the wells bound to and aggregated on the immobilized CRP, also in a calcium-dependent manner. On incubation with macrophages, the immobilized CRP-bound LDL aggregates were readily taken up by the cells, as demonstrated by immunofluorescence microscopy, by the cellular accumulation of cholesterol and by the overexpression of adipophilin. Immunofluorescence microscopy and flow-cytometry analysis established that the uptake of the LDL-CRP complex was not mediated by the CRP receptor CD32. These observations with immobilized CRP and LDL, approximating the conditions that exist in the extracellular matrix of the arterial wall, thus suggest that CRP may contribute to the formation of foam cells in atherosclerotic lesions by causing the aggregation of LDL molecules that are then taken up by macrophages through a CD32-independent pathway.

Microglia in Alzheimer's Disease and Transgenic Models: How Close the Fit?

Research on microglia has grown nearly exponentially over the last two decades, 1 fueled in part by the recognition that inflammation plays a role in the pathogenesis of Alzheimer’s disease (AD). 2 Whereas their existence as a distinct central nervous system (CNS) cell type was once debated, it is now widely accepted that microglia are monocyte-derived cells that enter the CNS at early stages of development as highly mobile, ameboid cells that are distributed widely throughout the brain and spinal cord, developing a highly ramified phenotype that eventually forms a uniform reticular array in both gray and white matter. Being related to other cells of the mononuclear phagocytic system they are considered to be poised for activation, each cell policing a discrete and nonoverlapping domain and acting as a sentinel to disruption of the normal homeostasis of the neural tissue. 3 Microglia respond rapidly to subtle alterations not associated with any apparent tissue damage such as spreading electrical depression 4 as well as more severe insults such as anoxic-ischemic injury and neuronal loss associated with a host of degenerative disorders with a sequence of antigenic and morphological changes. The latter form is less ramified, with more abundant cytoplasm in soma and cell processes. It has cell markers, such as class II major histocompatibility antigen, that are barely detectable in the ramified form and is referred to as activated. The full repertoire of ramified microglia is not known, but they appear to be long-lived cells with a limited potential to divide, which contrasts with perivascular macrophages. 5 The latter have a more dynamic life cycle and enter and leave the CNS compartment more readily throughout the life of the individual. The perivascular macrophage, which constitutively express class II major histocompatibility antigen (HLA-DR) participates in the immune response and is fully capable of antigen presentation. 6 The phagocytic potential of the perivascular macrophage is not disputed, but whether ramified microglia are phagocytic cells is debated. Progressive transformation of ramified microglia to macrophages has been shown in a limited number of demyelinating conditions, where it appears that at least some microglia may differentiate into brain macrophages. 3,7

Radioiodination of low density lipoprotein initiates lipid peroxidation: protection by use of antioxidants

It is now apparent that low density lipoprotein (LDL) is very susceptible to lipid peroxidation and that the resulting oxidized LDL has altered biological properties. Radiation, particularly of longer duration and lower intensities, initiates lipid peroxidation, yet radioiodination with 125I and 131I is a frequently used method to label LDL for biological studies. To test the possibility that this procedure alters the biological properties of LDL, native LDL was radioiodinated with 125I/131I using ICl to average specific activities of approximately 300 and approximately 100 cpm/ng protein, respectively. Lipid peroxidation was monitored by TBARS and conjugated diene formation. Biological properties were monitored by fibroblast and macrophage uptake of LDL as well as by rate of plasma clearance (FCR) in guinea pigs. 131I-labeled LDL showed enhanced indices of lipid peroxidation compared to 125I-labeled LDL and both were greater than native LDL. The FCR of 131I-labeled LDL was greater than that of 125I-labeled LDL (by 20-40%) and both increased progressively (by > 250%) when measured at 2, 6, and 13 days after iodination. The radioiodinated LDL samples were also more susceptible to pro-oxidant conditions. Thus, after exposure to Cu2+, 131I-labeled LDL showed greatly enhanced lipid peroxidation, decreased uptake by fibroblasts, increased uptake by macrophages and greatly accelerated FCR in guinea pigs. Exposure of LDL to 131I-labeled albumin produced similar changes. Protecting LDL with antioxidants such as BHT and ascorbate immediately after radioiodination generally ameliorated the adverse effects.

Dietary Olive Oil Reduces Low-Density Lipoprotein Uptake by Macrophages and Decreases the Susceptibility of the Lipoprotein to Undergo Lipid Peroxidation

Low-density lipoprotein (LDL) oxidation and macrophage cholesterol accumulation are both involved in atherogenesis. Recently it was shown that feeding rabbits or humans with an oleic-acid-rich diet reduced the susceptibility of their LDL to in vitro oxidation. Since olive oil is highly enriched with oleic acid, we tested the effect of olive oil supplementation (50 g/day) to the diet of 10 healthy male subjects, during a 2-week period, on macrophage uptake of their LDL and on the propensity of their LDL to oxidation (with copper ions). Olive oil supplementation to the diet resulted in LDL enrichment with oleic acid (C18:1) and sitosterol. No effect on plasma cholesterol levels was found, but the LDL cholesteryl ester content was reduced (16%) whereas its unesterified cholesterol was increased (41%). Even after 1 week of the olive oil diet, the LDL susceptibility to in vitro oxidation was significantly reduced (p < 0.01). Macrophage uptake of LDL was studied by analysis of cellular cholesterol content and by analysis of the macrophage cholesterol esterification rates. LDL obtained after 1 and 2 weeks of the olive oil diet demonstrated reduced cellular uptake in comparison with LDL obtained before the supplementation of olive oil, by 50 and 61%, respectively. The LDL resistance to oxidation was shown by a reduction in its peroxide, malondialdehyde and conjugated diene content by 73, 28 and 32%, respectively. LDL incubation with oleic acid for the period of its oxidation with copper ions demonstrated a dose-dependent inhibition of lipoprotein oxidation by up to 72% as opposed to linoleic and arachidonic acids (50 microM) which increased LDL oxidation by 22 and 72%, respectively. Sitosterol, in a similar incubation system, inhibited LDL oxidation by up to 26%. We conclude that olive oil supplementation to the diet modifies LDL lipid composition and enriches the lipoprotein with oleic acid and sitosterol. The antiatherogenic properties of this modified lipoprotein may be related to its resistance to in vitro peroxidation and its reduced uptake by macrophages.

Lipid-protein particles secreted from activated platelets reduce macrophage uptake of low density lipoprotein

Cellular uptake of low density lipoprotein (LDL) was reduced by 30–40% in macrophages that were preincubated with platelet conditioned medium (PCM) obtained from activated platelets. LDL mediated cholesterol accumulation and cholesterol esterification in macrophages were substantially inhibited by macrophages preincubation with PCM. This inhibitory effect was found to be dose dependent, and resulted from a reduction in the number of LDL receptors (decrement of 35% in “apparent V max”). The active component in PCM was present only in medium obtained from activated platelets and was found to be of a molecular weight higher than 25,000 dalton. It comprised of both protein and cholesterol but upon PCM delipidation only the lipid fraction demonstrated the inhibitory effect on macrophage uptake of LDL. Specific uptake of the PCM lipoprotein-like particle via the scavenger receptor on macrophages was found to be essential for the expression of LDL receptor reduced activity. Furthermore, LDL mediated cholesterol esterification was not inhibited by PCM in U937 macrophages, a cell line that lacks the scavenger receptors. It is concluded that activated platelets secrete a lipoprotein-like particle which is recognized by the macrophage scavenger receptor. Subsequent to PCM-macrophage interaction, cellular LDL uptake was reduced. This effect could be attributed to the PCM lipid constituents.