Modified Forms Of Low Density Lipoprotein Research Articles

Atherogenic modification of low-density lipoproteins

One of the first manifestations of atherosclerosis is accumulation of extra- and intracellular cholesterol esters in the arterial intima. Formation of foam cells is considered as a trigger in the pathogenesis of atherosclerosis. Low density lipoprotein (LDL) circulating in human blood is the source of lipids accumulated in the arterial walls. This review considered features and role in atherogenesis different modified forms of LDL: oxidized, small dense, electronegative and especially desialylated LDL. Desialylated LDL of human blood plasma is capable to induce lipid accumulation in cultured cells and it is atherogenic. LDL possesses numerous alterations of protein, carbohydrate and lipid moieties and therefore can be termed multiple-modified LDL. Multiple modification of LDL occurs in human blood plasma and represents a cascade of successive changes in the lipoprotein particle: desialylation, loss of lipids, reduction in the particle size, increase of surface electronegative charge, etc. In addition to intracellular lipid accumulation, stimulatory effects of naturally occurring multiple-modified LDL on other processes involved in the development of atherosclerotic lesions, namely cell proliferation and fibrosis, were shown.

The Evolution of the Scavenger Receptor Cysteine-Rich Domain of the Class A Scavenger Receptors.

The class A scavenger receptor (cA-SR) family is a group of five evolutionarily related innate immune receptors. The cA-SRs are known for their promiscuous ligand binding; as they have been shown to bind bacteria, such as Streptococcus pneumoniae and Escherichia coli, as well as different modified forms of low-density lipoprotein. Three of the five family members possess a scavenger receptor cysteine-rich (SRCR) domain while the remaining two receptors lack the domain. Previous work has suggested that the macrophage-associated receptor with collagenous structure (MARCO) shares a recent common ancestor with the non-SRCR-containing receptors; however, the origin of the SRCR domain within the cA-SRs remains unknown. We hypothesize that the SRCR domains of the cA-SRs have a common origin that predates teleost fish. Using the newly available sequence data from sea lamprey and ghost shark genome projects, we have shown that MARCO shares a common ancestor with the SRCR-containing proteins. In addition, we explored the evolutionary relationships within the SRCR domain by reconstructing the ancestral SRCR domains of the cA-SRs. We identified a motif that is highly conserved between the cA-SR SRCR domains and the ancestral SRCR domain that consist of WGTVCDD. We also show that the GRAEVYY motif, a functionally important motif within MARCO, is poorly conserved in the other cA-SRs and in the reconstructed ancestral domain. Further, we identified three sites within MARCO’s SRCR domain, which are under positive selection. Two of these sites lie adjacent to the conserved WGTVCDD motif, and may indicate a potential biological function for these sites. Together, these findings indicate a common origin of the SRCR domain within the cA-SRs; however, different selective pressures between the proteins may have caused MARCOs SRCR domain to evolve to contain different functional motifs when compared to the other SRCR-containing cA-SRs.

Levels of MDA-LDL in Circulating Immune Complexes Predict Myocardial Infarction in the Veterans Affairs Diabetes Trial (VADT) Cohort

Synopsis: In previous studies, authors have demonstrated a relationship between modified low-density lipoprotein (LDL) and cardiovascular disease (CVD); however, few have examined the relationship between the content of modified LDL in circulating immune complexes (ICs) and CVD, even though the majority of modified LDL in plasma circulates as part of immune complexes. Purpose: To determine whether high levels of oxidized LDL (oxLDL), malondialdehyde-LDL (MDA-LDL), and advanced glycation end products-LDL (AGE-LDL) in circulating IC are associated with incident cardiovascular events in the VADT cohort of type 2 diabetes (T2DM). Methods: We performed prospective analyses to determine associations between modified LDL-IC and vascular end points among 907 VADT subjects (mean age 59.8 years; 96.8% male; 40.9% minority). Modified LDL-IC were measured a median of 2 years after entry into the VADT study, and participants were subsequently followed an average of 3.7 years for outcomes. Cox proportional hazard models were used to calculate hazard ratios (HRs) for macrovascular end points in relation to modified LDL-IC quartile. The primary composite end point included documented myocardial infarction (MI); stroke; death from cardiovascular causes; new or worsening congestive heart failure; surgical intervention for cardiac, cerebrovascular, or peripheral vascular disease; inoperable coronary artery disease; and amputation for ischemic gangrene. Results: During follow-up, 16.8%, 4.7% and 6.4% of participants had a primary composite end point, MI or death, respectively. After wed adjusted for age, minority status, treatment arm, whether an individual had an event before the VADT, as well as systolic blood pressure (SBP) level, LDL-C level and statin use at the time of IC measurement, individuals in the highest quartile of MDALDL IC as compared with individuals in the lowest quartile of MDA-LDL IC were at greater risk of MI (hazard ratio [HR] 2.44; 95% confidence interval [95% CI] 1.0325.77) and the composite end point (HR 1.71; 95% CI 1.0422.80) but similar risk of all-cause mortality (HR 1.15; 95% CI 0.5222.52). In contrast, oxLDL-IC and AGE-LDL-IC were not associated with cardiovascular events. Moreover, subsequent analysis found that individuals with high MDA-LDL IC and low oxLDL-LDL IC were at particularly high risk of MI relative to individuals with low MDA-LDL IC and low oxLDL-IC (HR 3.46; 95% CI 1.5227.87). Results were similar when AGELDL IC was substituted for oxLDL-IC (HR 2.56; 95% CI 1.0626.20). Conclusions: Our study indicates that high levels of MDALDL in isolated IC predict future MI and cardiovascular events in patients with T2DM and supports distinct roles of different modified forms of LDL in the occurrence of macrovascular events.

Eicosapentaenoic Acid and Docosahexaenoic Acid Regulate Modified LDL Uptake and Macropinocytosis in Human Macrophages

There is evidence that long chain n-3 PUFA (such as from fish oils) provide atheroprotection through, in part, changes in macrophage function although it has not been fully determined whether these n-3 PUFA target cellular mechanisms that control macrophage foam cell formation. Therefore, we investigated whether the n-3 PUFA, EPA and DHA, modulate modified low-density lipoprotein (LDL) uptake by human macrophages. The uptake of fluorophore labeled acetylated LDL (AcLDL) and/or oxidized LDL (OxLDL) by THP-1 macrophages and primary human monocyte-derived macrophages were measured by flow cytometry following co-incubation with EPA or DHA in vitro. DHA inhibited both AcLDL and OxLDL uptake in human macrophages whilst EPA reduced AcLDL and increased OxLDL uptake. These effects were only partly explained by changes in the mRNA and protein expression of key scavenger receptors, such as CD36 and scavenger receptor-A, in these cells suggesting the involvement of a scavenger receptor-independent mechanism. EPA and DHA inhibited macropinocytosis, as measured by Lucifer Yellow uptake, in human macrophages and attenuated the expression of Syndecan-4, which has been implicated in the uptake of other modified forms of LDL. EPA and DHA reduced modified LDL uptake by human macrophages through a mechanism that is in part scavenger receptor-independent and may involve inhibition of macropinocytosis and Syndecan-4 expression. This suggests that both EPA and DHA are capable of regulating macrophage foam cell formation and adds to the evidence describing an atheroprotective role for n-3 PUFA, implicating them as potential therapeutic agents for the treatment of clinical atherosclerosis.

Inflammatory stress promotes lipid accumulation in the aorta and liver of SR-A/CD36 double knock-out mice

Macrophage scavenger receptors (SRs), particularly type A SR (SR-A) and CD36, have been implicated in binding and internalizing modified forms of low-density lipoprotein (LDL) through a mechanism not regulated by cellular cholesterol content, resulting in cholesterol accumulation and foam cell formation. To test the impact of the absence of these receptors on lipid-mediated injuries in aortas or livers under inflammatory stress, C57BL/6 mice with knocked out SR-A and CD36 (SR DKO) were administered a combination of a high-fat diet and casein injection for 14 weeks. We analyzed the lipid accumulation in the aortas and livers, assessing lipid droplets, circulating inflammatory cytokines, serum lipid levels and gene expression of cholesterol uptake. Lipid accumulation in the aortas and livers of the inflammatory mice were assessed by morphologic and quantitative assay of cholesterol ester in the livers. The SR DKO mice under inflammatory stress showed an increased level of serum amyloidA, but did not exhibit any significantly increased levels of TNF-α and IL-6, while morphological analysis revealed a significant increase in lipid accumulation in the aortas and livers of the inflammatory mice. Although SR DKO mice exhibit substantially reduced inflammatory cytokine factors, loss of the SR pathway does accelerate lipid accumulation through LDL receptor pathways to lipid accumulation. These data suggest that targeted inhibition of SR pathways in vivo may promote lipid-mediated injuries in aortas and livers under inflammatory stress.

Host Scavenger Receptor SR-BI Plays a Dual Role in the Establishment of Malaria Parasite Liver Infection

An obligatory step of malaria parasite infection is Plasmodium sporozoite invasion of host hepatocytes, and host lipoprotein clearance pathways have been linked to Plasmodium liver infection. By using RNA interference to screen lipoprotein-related host factors, we show here that the class B, type I scavenger receptor (SR-BI) is the strongest regulator of Plasmodium infection among these factors. Inhibition of SR-BI function reduced P. berghei infection in Huh7 cells, and overexpression of SR-BI led to increased infection. In vivo silencing of liver SR-BI expression in mice and inhibition of SR-BI activity in human primary hepatocytes reduced infection by P. berghei and by P. falciparum, respectively. Heterozygous SR-BI(+/-) mice displayed reduced P. berghei infection rates correlating with liver SR-BI expression levels. Additional analyses revealed that SR-BI plays a dual role in Plasmodium infection, affecting both sporozoite invasion and intracellular parasite development, and may therefore constitute a good target for malaria prophylaxis.

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.

Desialylated LDL uptake in human and mouse macrophages can be mediated by a lectin receptor

We have compared the uptake of desialylated low density lipoprotein (LDL) with other modified forms of LDL in mouse peritoneal macrophages and PMA-activated human U937 monocytes. Neuraminidase-treated LDL (NT-LDL) caused significant cholesterol ester accumulation in both cell types, although the efficiency relative to loading with acetylated LDL (AcLDL) was markedly different, suggesting a very different complement of receptors in the cells. We therefore determined the effect of PMA-activation on lipoprotein receptor expression in U937 cells and found that while scavenger receptor concentration was elevated after PMA-activation, there was no significant change in the expression of the LDL receptor. Receptor specificity of NT-LDL uptake was examined by competition experiments using the degradation assay. This showed that 125I-labelled NT-LDL uptake in U937 cells could largely be accounted for by the persistent expression of the LDL receptor in these cells. In contrast, in mouse peritoneal macrophages where LDL receptor expression is very low, 125I-labelled NT-LDL degradation was effectively competed by asialofetuin. Surprisingly, 125I-labelled NT-LDL degradation was also effectively competed by AcLDL. Measurement of sialic acid content of AcLDL showed that approximately 14% of the LDL sialic acid, equivalent to 2 to 3 residues per particle, was lost during acetylation of LDL with acetic anhydride. Thus competition between 125I-labelled NT-LDL and AcLDL could be due to lectin receptor binding rather than competition for scavenger receptor binding.

Macrophage scavenger receptors and atherosclerosis

The fatty streak is the earliest visible atherosclerotic lesion. Microscopically, this lesion consists of a subendothelial collection of macrophage foam cells that are characterized by their content of numerous lipid droplets. Investigations into the mechanisms responsible for foam cell formation have led to the identification of macrophage scavenger receptors that are capable of recognizing and internalizing modified forms of low-density lipoprotein (LDL). The recent cloning of a macrophage scavenger receptor that is capable of recognizing acetylated and oxidized LDL has permitted the application of molecular approaches to the study of scavenger receptor expression and the determinants of ligand recognition. These studies suggest the possibility that treatment strategies that inhibit LDL modification or influence macrophage gene expression might be effective in the prevention or treatment of atherosclerosis.