oxLDL Uptake Research Articles

AMP-activated protein kinase attenuates oxLDL uptake in macrophages through PP2A/NF-κB/LOX-1 pathway

The differentiation of macrophages into lipid-laden foam cells is a hallmark in early-stage atherosclerosis. The developmental role of adenosine monophosphate-activated protein kinase (AMPK) in a transformation of foam cells, especially in macrophage cholesterol uptake that remains undetermined. Here we demonstrate that AMPK activation in response to IMM-H007 or AICAR resulted in a decrease in macrophage cholesterol uptake and thus inhibited foam cell formation in macrophages mediated by oxidized low-density lipoprotein (oxLDL). This functional change was caused by a downregulation of mRNA and protein expression of LOX-1 but not other scavenger receptors, including scavenger receptor-A (SR-A), CD36 and scavenger receptor-BI (SR-BI). The expression of LOX-1 was regulated by AMPK activation induced decreased phosphorylation of nuclear transcription factor NF-κB, since siRNA interference or dominant negative AMPK overexpression significantly promotes Ser536 dephosphorylation of NF-κB p65 and thus increases LOX-1 expression. Moreover, pharmacological AMPK activation was shown to promote protein phosphatase 2A (PP2A) activity and the specific PP2A inhibitor, okadaic acid, could prevent the effects of IMM-H007 or AICAR on NF-κB and LOX-1. In vivo, pharmacological AMPK activation reduced the lesion size of atherosclerosis and the expression of LOX-1 in aortas in apolipoprotein E-deficient mice. Our current findings suggest a novel mechanism of LOX-1 regulation by AMPK to attenuate macrophage oxLDL uptake and atherosclerosis.

CD36 Mediated Fatty Acid-Induced Podocyte Apoptosis via Oxidative Stress

BackgroundHyperlipidemia-induced apoptosis mediated by fatty acid translocase CD36 is associated with increased uptake of ox-LDL or fatty acid in macrophages, hepatocytes and proximal tubular epithelial cells, leading to atherosclerosis, liver damage and fibrosis in obese patients, and diabetic nephropathy (DN), respectively. However, the specific role of CD36 in podocyte apoptosis in DN with hyperlipidemia remains poorly investigated.MethodsThe expression of CD36 was measured in paraffin-embedded kidney tissue samples (Ctr = 18, DN = 20) by immunohistochemistry and immunofluorescence staining. We cultured conditionally immortalized mouse podocytes (MPC5) and treated cells with palmitic acid, and measured CD36 expression by real-time PCR, Western blot analysis and immunofluorescence; lipid uptake by Oil red O staining and BODIPY staining; apoptosis by flow cytometry assay, TUNEL assay and Western blot analysis; and ROS production by DCFH-DA fluorescence staining. All statistical analyses were performed using SPSS 21.0 statistical software.ResultsCD36 expression was increased in kidney tissue from DN patients with hyperlipidemia. Palmitic acid upregulated CD36 expression and promoted its translocation from cytoplasm to plasma membrane in podocytes. Furthermore, palmitic acid increased lipid uptake, ROS production and apoptosis in podocytes, Sulfo-N-succinimidyloleate (SSO), the specific inhibitor of the fatty acid binding site on CD36, decreased palmitic acid-induced fatty acid accumulation, ROS production, and apoptosis in podocytes. Antioxidant 4-hydroxy-2,2,6,6- tetramethylpiperidine -1-oxyl (tempol) inhibited the overproduction of ROS and apoptosis in podocytes induced by palmitic acid.ConclusionsCD36 mediated fatty acid-induced podocyte apoptosis via oxidative stress might participate in the process of DN.

Abstract 382: Lipin-1 Links Pro-inflammatory Responses and Foam Cell Formation by Oxidized-Low Density Lipoprotein-Elicited Macrophages

Atherosclerosis is a chronic inflammatory disease of large and medium-sized arteries and one of the underlying causes of cardiovascular disease (CVD). Macrophages participate decisively in the development and promotion of atherosclerosis. Macrophages infiltrate the arterial intima to ingest modified low density lipoproteins (e.g. oxLDLs) via scavenger receptors. The scavenging of oxLDLs results in foam cell formation due to enhanced lipid droplet biogenesis. These foam cells eventually release pro-inflammatory cytokines that promote atherosclerosis. However, it is currently unknown whether there is a link between lipid droplet biogenesis and pro-inflammatory cytokine production in macrophages that scavenge oxLDL. Lipin-1, a phosphatidate phosphohydrolase enzyme, partially contributes to macrophage pro-inflammatory cytokine production following stimulation with bacteria. Lipin-1 is also required for lipid droplet biogenesis in macrophages. Finally, we observed lipin-1 protein within macrophages from human atherosclerotic plaques. Thus, we hypothesized that lipid droplet biogenesis, via lipin-1 activity, directly contributes to foam cell pro-inflammatory cytokine production. To test this hypothesis we compared lipid droplet biogenesis and pro-inflammatory cytokine responses of oxLDL-stimulated wild type and lipin-1-depleted macrophages. Depletion of lipin-1 inhibited oxLDL-induced foam cell generation by reducing lipid droplet number, area, and staining intensity. There were no differences in scavenger receptor expression or uptake of oxLDL between wild type and lipin-1-depleted cells. In addition, depletion of lipin-1 also ablated oxLDL-elicited production of the pro-atherogenic cytokines tumor necrosis factor-α and interleukin-6. These findings demonstrate a critical role for lipin-1 in the regulation of macrophage inflammatory responses to oxLDL. Furthermore, these data begin to link foam cell formation, via lipid droplet biogenesis, and pro-inflammatory cytokine production within oxLDL stimulated macrophages. Thus, our studies suggest that lipid droplet biogenesis may be an ideal therapeutic target to inhibit inflammation associated with atherosclerosis to treat CVD.

Abstract 146: Administration of Interleukin-19 Halts Progression of Pre-formed Plaque, Polarizes Macrophage, and Increases Macrophage Lipid Uptake.

Increased uptake of oxLDL by macrophage is proposed to be an atheroprotective mechanism to reduce plaque formation. It has been proposed that macrophage phenotype may be causative or at least associated with plaque severity. Several macrophage phenotypes have been classified, and M2 macrophage are considered to be anti-inflammatory and reparative. Interleukin-19 is a purported anti-inflammatory, Th2 interleukin, and we previously found that addition of rIL-19 could significantly reduce atherosclerosis in susceptible mice. The purpose of this study was to test the hypothesis that administration of exogenous IL-19 could attenuate progression of pre-formed atherosclerotic plaque, and to identify potential molecular mechanisms. LDLR-/- mice were fed high-fat diet for 12 weeks, then administered rIL-19 (10ng/g/day) or PBS for an additional 8 weeks while still consuming HFD. En face analysis demonstrated that IL-19 could halt, but not reverse existing plaque. M2 macrophage marker expression was significantly increased in aorta and spleen from IL-19 treated mice, in IL-19-treated bone marrow derived macrophage (BMDM) and primary human macrophage. IL-19 significantly decreased expression of inflammatory cytokines TNFa, IL-12p40, MCP-1 and IL-1b mRNA in BMDM. Addition of IL-19 to BMDM significantly increased oxLDL uptake as well as expression of lipid uptake receptors CD36, SRA-1, and SRB-1 in primary human macrophage. IL-19 increased expression and activation of PPARg. Knock down of PPARg significantly decreased IL-19 mediated expression of both lipid uptake receptors and uptake of oxLDL. Collectively, these data show that IL-19 can halt progression of established plaque by at least two mechanisms; M2 macrophage polarization, and increasing expression of lipid scavenger receptors and cholesterol uptake in a PPARg-dependent mechanism, suggesting therapeutic potential for this interleukin.

Oxidized low-density lipoprotein accelerates the destabilization of extracellular-superoxide dismutase mRNA during foam cell formation

Extracellular-superoxide dismutase (EC-SOD) is one of the main anti-oxidative enzymes that protect cells against the damaging effects of superoxide. In the present study, we investigated the regulation of EC-SOD expression during the oxidized low density lipoprotein (oxLDL)-induced foam cell formation of THP-1-derived macrophages. The uptake of oxLDL into THP-1-derived macrophages was increased and EC-SOD expression was decreased in a time-dependent manner by oxLDL. Furthermore, EC-SOD suppression by oxLDL was mediated by the binding to scavenger receptors, especially CD36, from the results with siRNA experience. EC-SOD expression is known to be regulated by histone acetylation and binding of the transcription factor Sp1/3 to the EC-SOD promoter region in human cell lines. However, oxLDL did not affect these processes. On the other hand, the stability of EC-SOD mRNA was decreased by oxLDL. Moreover, oxLDL promoted destabilization of ectopically expressed mRNA from EC-SOD or chimeric Cu,Zn-SOD gene with the sequence corresponding to 3′UTR of EC-SOD mRNA, whereas oxLDL had no effect on ectopic mRNA produced from EC-SOD gene lacking the sequence. These results suggested that oxLDL decreased the expression of EC-SOD, which, in turn, accelerated the destabilization of EC-SOD mRNA, leading to weaker protection against oxidative stress and atherosclerosis.

Potential contributions of intimal and plaque hypoxia to atherosclerosis.

Injury of arterial endothelium by abnormal shear stress and other insults induces migration and proliferation of vascular smooth muscle cells (VSMCs), which in turn leads to intimal thickening, hypoxia, and vasa vasorum angiogenesis. The resultant new blood vessels extend from the tunica media into the outer intima, allowing blood-borne oxidized low-density lipoprotein (oxLDL) particles to accumulate in outer intimal tissues by extravasation through local capillaries. In response to oxLDL accumulation, monocytes infiltrate into arterial wall tissues, where they differentiate into macrophages and subsequently evolve into foam cells by uptaking large quantities of oxLDL particles, the latter process being stimulated by hypoxia. Increased oxygen demand due to expanding macrophage and foam cell populations contributes to persistent hypoxia in plaque lesions, whereas hypoxia further promotes plaque growth by stimulating angiogenesis, monocyte infiltration, and oxLDL uptake into macrophages. Molecularly, the accumulation of hypoxia-inducible factor (HIF)-1α and the expression of its target genes mediate many of the hypoxia-induced processes during plaque initiation and growth. It is hoped that further understanding of the underlying mechanisms may lead to novel therapies for effective intervention of atherosclerosis.

Using image-based flow cytometry to measure monocyte oxidized LDL phagocytosis: A potential risk factor for CVD?

Obesity and cardiovascular disease is a worldwide health concern that has been a major focus in research for several decades. Among these diseases, atherosclerosis is one of the leading causes of death and disability nationwide. Circulating monocytes are believed to be primary cells responsible for foam cell formation. The present report describes a novel method for measuring monocyte oxLDL phagocytosis capacity using image-based flow cytometry. Human venous blood monocytes were incubated with different concentrations of oxLDL for different lengths of time to optimize the assay. High (post-meal) and low (pre-meal) responder samples were generated by feeding human subjects a high-fat (~85% of daily fat allowance), high-calorie (~65% of daily calorie needs) meal. This is a relevant model with respect to obesity and risk of developing atherogenesis. After the functional assay, classic (CD14+/CD16−) and pro-inflammatory (CD14+/CD16+) monocytes were assessed for oxLDL uptake, adhesion molecule expression (CD11b and CD18), and scavenger receptor expression (CD36) using an image-based flow cytometer (FlowSight). The present method represents a novel advance in methods available to detect the propensity of circulating monocytes to become intima foam cells. We found the assay to be most effective at separating high from low responder samples when using a fixed oxLDL concentration (120μL/mL) and incubation length (1-h). In a clinical application, this method demonstrated that consuming a single high-fat meal causes an increase in the proportion of monocyte oxLDL phagocytosis and their adhesion capacity, suggesting a higher propensity to become foam cells.

Wnt5a Signaling in Atherosclerosis, its Effect on OxLDL Uptake and Foam Cell Differentiation

Atherosclerosis is a chronic inflammatory disease involving plaque buildup in the arteries and can lead to heart attack and stroke. A key step in the pathogenesis of atherosclerosis is oxLDL uptake by macrophages. Our lab has shown that oxLDL up regulated transcription of Wnt5a in macrophages. Wnt5a and Frizzled 5 (Fz5), a Wnt5a receptor, are highly expressed in foam cells in atherosclerosis. Our hypothesis is that Wnt5a stimulates oxLDL uptake and that macrophages show differential expression of Wnt5a receptors.In vitro studies: THP‐1 cell line was cultured and differentiated into macrophages. Cells were treated with oxLDL, nLDL, and rWnt5a in combinations to determine their effects on oxLDL uptake. Expression of receptors involved in oxLDL uptake such as CD68, CD36, and scavenger receptor (SR) were analyzed using immunofluorescence, western blot and PCR. To study Wnt5a signaling ROR2, Fz5, and Wnt5a were evaluated in THP‐1 cells as well as in human atherosclerotic tissue samplesIn THP‐1 cells oxLDL and Wnt5a increased CD68, CD36, SR and Fz5 at both protein and RNA level. IHC results show that Wnt5a is expressed by macrophages, foam cells, smooth muscle cells (SMC), and endothelial cells. But Fz5 is expressed only by some macrophages, and almost negative in SMC. We also found that ROR2 followed a similar expression pattern to Fz5 in macrophages while SMC near the lipid core were positive for ROR2 but negative for Fz5.In conclusion, our results suggest that oxLDL, through Wnt5a signaling, plays a role in foam cell formation and oxLDL uptake. In addition, expression of Wnt5a receptors is variable between cell types in atherosclerosis.

Fatty acid binding to CD36 affects oxLDL uptake

We investigated the hypothesis that interactions of unesterified fatty acid (FA) with the plasma membrane protein CD36 affect uptake of oxLDL. Because the binding of natural FA to CD36 have not been systematically studied, we first characterized FA binding to CD36 by the biophysical method surface plasmon resonance (SPR). After covalently linking the extracellular domain of CD36 to an SPR chip surface, aqueous FA (solubilized in cyclodextrin) was pulsed across the surface, generating association and dissociation binding curves. We studied structurally different long chain FA: a saturated (palmitic acid); unsaturated (oleic acid); trans‐oleic acid; (elaidic); a polyunsaturated [docosahexaenoic acid (DHA)], and an oxidized FA [9S‐hydroxy‐10E,12Z‐octadecadienoic acid (HODE)]. With the exception of HODE, SPR detected FA binding to CD36, and showed that multiple sites are present with rapid association and dissociation kinetics that were similar to HSA. To study the role in oxLDL uptake of these FA, we used a fluorescent oxLDL (Dii‐oxLDL) live‐cell assay with confocal microscopy imaging. CD36‐mediated uptake in serum‐free media was very low but greatly increased with serum. Addition of FA in serum‐free media increased oxLDL binding and uptake to levels found with serum, and affected CD36 plasma membrane (PM) distribution. Binding/uptake of oxLDL exhibited dose dependency of added FA, with the exception of DHA, which binds avidly to CD36 but did not activate uptake. HODE, which binds poorly to CD36, did not affect oxLDL uptake. The high affinity FA binding to CD36 has important implications that binding at some or all sites could affect its conformation, binding of other ligands, and functional properties.

Disturbed Shear Stress Promotes Caveolae‐Mediated oxLDL Uptake Leading to Increased Endothelial Stiffness

IntroductionOxidized low density lipoprotein (oxLDL) is a major risk factor in the development of atherosclerotic plaques, which preferentially form at vascular bifurcations subject to bidirectional recirculating disturbed shear stress (DSS).MethodsA microfluidic channel with a step barrier was designed, tested and fabricated to generate atheroprotective unidirectional laminar shear stress (LSS) and atheroprone DSS onto an EC monolayer for 48 hours. EC stiffness was assessed using atomic force microscopy.ResultsOur results show a statistically significant increase in oxLDL uptake in DSS vs. LSS regions in three EC types: human aortic (55% increase), human microvascular (40% increase), and mouse microvascular (50% increase). OxLDL uptake was reduced in cells lacking Cav1, the main protein in caveolae (n=4, p<0.05). The mechanism of differential oxLDL uptake was further investigated using siRNA treated ECs for CD36 and Lox1, two oxLDL receptors. Our experiments revealed that CD36 (n=4, p<0.05), but not Lox1 (n=4, p<0.05), is required for increased uptake under DSS. Furthermore, the increased uptake under DSS results in a 60% increase in EC stiffness (n=3, p<0.05). This data is further supported by ex vivo stiffness measurements of vessels from WT mice which show a 50% increase in EC stiffness in the aortic arch (DSS) than the LSS region of the descending aorta (DA) (n=5, p<0.05). In Cav1 KO mice, EC stiffness was markedly reduced (n=5, p<0.05) and no differences observed between the DA and arch.ConclusionTaken together, this study suggests that DSS‐induced increase in oxLDL uptake is mediated by caveolae and plays a significant role in increasing localized EC stiffness.

D4F alleviates macrophage-derived foam cell apoptosis by inhibiting CD36 expression and ER stress-CHOP pathway

This study was designed to explore the protective effect of D4F, an apoA-I mimetic peptide, on oxidized LDL (ox-LDL)-induced endoplasmic reticulum (ER) stress-CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) pathway-mediated apoptosis in macrophages. Our results showed that treating apoE knockout mice with D4F decreased the serum ox-LDL level and apoptosis in atherosclerotic lesions with concomitant downregulation of cluster of differentiation 36 (CD36) and inhibition of ER stress. In vitro, D4F inhibited macrophage-derived foam cell formation. Furthermore, like ER stress inhibitor 4-phenylbutyric acid (PBA), D4F inhibited ox-LDL- or tunicamycin (TM, an ER stress inducer)-induced reduction in cell viability and increase in lactate dehydrogenase leakage, caspase-3 activation, and apoptosis. Additionally, like PBA, D4F inhibited ox-LDL- or TM-induced activation of ER stress response as assessed by the reduced nuclear translocation of activating transcription factor 6 and the decreased phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, as well as the downregulation of glucose-regulated protein 78 and CHOP. Moreover, D4F mitigated ox-LDL uptake by macrophages and CD36 upregulation induced by ox-LDL or TM. These data indicate that D4F can alleviate the formation and apoptosis of macrophage-derived foam cells by suppressing CD36-mediated ox-LDL uptake and subsequent activation of the ER stress-CHOP pathway.

Insulin enhances dendritic cell maturation and scavenger receptor-mediated uptake of oxidised low-density lipoprotein

ObjectivesThe prevalence of atherosclerotic cardiovascular disease is increased in patients with type 2 diabetes. The role of hyperinsulinaemia as an independent participant in the atherogenic process is controversial. Therefore, we examined whether insulin regulates the expression of scavenger receptors responsible for oxidised low-density lipoprotein (oxLDL) uptake in dendritic cells (DCs). In addition, we investigated the impact of insulin on DC maturation with regard to changes in phenotype and cytokine secretion. MethodsImmature DCs were cultured with different concentrations of insulin (1nmol/L, 10nmol/L, 50nmol/L, and 100nmol/L) in the absence or presence of LY294002 or PD98059 for 24h. The expression of the scavenger receptors SR-A and CD36 was determined by real-time PCR and Western blot analysis. Furthermore, DCs were incubated with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labelled oxLDL. The DiI-oxLDL-incorporated fraction was investigated by flow cytometry. Finally, flow cytometry was used to investigate immunophenotypic protein expression (CD83, CD86, and CD11a). Supernatant cytokine measurements were used as indicators of immune function. ResultsThe incubation of DCs with insulin enhanced SR-A and CD36 gene and protein expression in a dose-dependent manner. This effect was partially abolished by PD98059, which is an extracellular signal-regulated kinase (ERK) inhibitor. However, LY294002 did not inhibit the effect of insulin on scavenger receptor expression. A high concentration of insulin increased the oxLDL-uptake capacity of DCs. Inhibition of the scavenger receptors SR-A and CD36 significantly reduced oxLDL uptake. Furthermore, a high concentration of insulin induced DC maturation. The pro-atherosclerotic chemokines IL-6 and IL-12 were induced by a high concentration of insulin, whereas the release of anti-atherosclerotic IL-10 was reduced. ConclusionThis study suggests that hyperinsulinaemia can promote DC activation and up-regulate the expression of the scavenger receptors SR-A and CD36, which can increase the oxLDL-uptake capacity of DCs. The results of the present study indicate that one of the mechanisms by which insulin promotes atherogenesis is mediated by its effects on DCs.

Tartaric acid-based amphiphilic macromolecules with ether linkages exhibit enhanced repression of oxidized low density lipoprotein uptake

Cardiovascular disease initiates with the atherogenic cascade of scavenger receptor- (SR-) mediated oxidized low-density lipoprotein (oxLDL) uptake. Resulting foam cell formation leads to lipid-rich lesions within arteries. We designed amphiphilic macromolecules (AMs) to inhibit these processes by competitively blocking oxLDL uptake via SRs, potentially arresting atherosclerotic development. In this study, we investigated the impact of replacing ester linkages with ether linkages in the AM hydrophobic domain. We hypothesized that ether linkages would impart flexibility for orientation to improve binding to SR binding pockets, enhancing anti-atherogenic activity. A series of tartaric acid-based AMs with varying hydrophobic chain lengths and conjugation chemistries were synthesized, characterized, and evaluated for bioactivity. 3-D conformations of AMs in aqueous conditions may have significant effects on anti-atherogenic potency and were simulated by molecular modeling. Notably, ether-linked AMs exhibited significantly higher levels of inhibition of oxLDL uptake than their corresponding ester analogues, indicating a dominant effect of linkage flexibility on pharmacological activity. The degradation stability was also enhanced for ether-linked AMs. These studies further suggested that alkyl chain length (i.e., relative hydrophobicity), conformation (i.e., orientation), and chemical stability play a critical role in modulating oxLDL uptake, and guide the design of innovative cardiovascular therapies.

CD36 Binds Oxidized Low Density Lipoprotein (LDL) in a Mechanism Dependent upon Fatty Acid Binding

The association of unesterified fatty acid (FA) with the scavenger receptor CD36 has been actively researched, with focuses on FA and oxidized low density lipoprotein (oxLDL) uptake. CD36 has been shown to bind FA, but this interaction has been poorly characterized to date. To gain new insights into the physiological relevance of binding of FA to CD36, we characterized FA binding to the ectodomain of CD36 by the biophysical method surface plasmon resonance. Five structurally distinct FAs (saturated, monounsaturated (cis and trans), polyunsaturated, and oxidized) were pulsed across surface plasmon resonance channels, generating association and dissociation binding curves. Except for the oxidized FA HODE, all FAs bound to CD36, with rapid association and dissociation kinetics similar to HSA. Next, to elucidate the role that each FA might play in CD36-mediated oxLDL uptake, we used a fluorescent oxLDL (Dii-oxLDL) live cell assay with confocal microscopy imaging. CD36-mediated uptake in serum-free medium was very low but greatly increased when serum was present. The addition of exogenous FA in serum-free medium increased oxLDL binding and uptake to levels found with serum and affected CD36 plasma membrane distribution. Binding/uptake of oxLDL was dependent upon the FA dose, except for docosahexaenoic acid, which exhibited binding to CD36 but did not activate the uptake of oxLDL. HODE also did not affect oxLDL uptake. High affinity FA binding to CD36 and the effects of each FA on oxLDL uptake have important implications for protein conformation, binding of other ligands, functional properties of CD36, and high plasma FA levels in obesity and type 2 diabetes.

Oxidized LDL induces phosphorylation of non-muscle myosin IIA heavy chain in macrophages.

Oxidized LDL (oxLDL) performs critical roles in atherosclerosis by inducing macrophage foam cell formation and promoting inflammation. There have been reports showing that oxLDL modulates macrophage cytoskeletal functions for oxLDL uptake and trapping, however, the precise mechanism has not been clearly elucidated. Our study examined the effect of oxLDL on non-muscle myosin heavy chain IIA (MHC-IIA) in macrophages. We demonstrated that oxLDL induces phosphorylation of MHC-IIA (Ser1917) in peritoneal macrophages from wild-type mice and THP-1, a human monocytic cell line, but not in macrophages deficient for CD36, a scavenger receptor for oxLDL. Protein kinase C (PKC) inhibitor-treated macrophages did not undergo the oxLDL-induced MHC-IIA phosphorylation. Our immunoprecipitation revealed that oxLDL increased physical association between PKC and MHC-IIA, supporting the role of PKC in this process. We conclude that oxLDL via CD36 induces PKC-mediated MHC-IIA (Ser1917) phosphorylation and this may affect oxLDL-induced functions of macrophages involved in atherosclerosis. [BMB Reports 2015; 48(1): 48-53]

Nifedipine inhibits ox-LDL-induced lipid accumulation in human blood-derived macrophages

Studies have shown that nifedipine, an anti-hypertensive drug, protects against atherosclerotic progression, but the underlying mechanisms remain elusive. Oxidized low-density lipoprotein (ox-LDL) is critically implicated in macrophage lipid deposition seen in atherosclerosis. In this study, we examined the effects of nifedipine on some ox-LDL-associated changes in human blood-derived macrophages. We isolated monocytes from normal human blood and differentiated them into macrophages. We then treated these human macrophages with ox-LDL and/or nifedipine, and examined lipid accumulation and expression levels of two scavenge receptors CD36 and SR-A as well as a protein kinase PKC-θ. Nifedipine treatment substantially reduced lipid accumulation and the expression of CD36, SR-A, and protein kinase C (PKC)-θ in human macrophages treated with ox-LDL. Silencing of PKC-θ using siRNA also reduced the expression of CD36 and SR-A in these cells. Our results thus suggest that nifedipine may inhibit atherosclerosis by reducing ox-LDL-induced lipid deposition through suppression of the CD36/SR-A-mediated uptake of ox-LDL by macrophages via a PKC-θ-dependent mechanism.

Naturally-ocurring biflavonoids modulate macrophage response in vitro and are atheroprotective in vivo

Naturally-ocurring biflavonoids modulate macrophage response in vitro and are atheroprotective in vivo

High-density lipoprotein increases the uptake of oxidized low density lipoprotein via PPARγ/CD36 pathway in inflammatory adipocytes.

Aim: Previous studies have demonstrated that the dysregulated-secretion of adipokines by adipocytes may contribute to obesity-associated atherosclerosis (As) and high density lipoprotein (HDL) may protect against atherogenesis through multiple pathways. This study was to explore the effect of HDL on the oxLDL uptake in inflammatory adipocytes stimulated by endotoxin lipopolysaccharide (LPS) and the possible mechanism.Methods and Results: 3T3-L1 adipocytes were cultured and induced to differentiation and maturation. Acute inflammation in adipocytes was induced by LPS (100 ng/ml) for 6 hours. The adipocytes were pretreated with HDL in various concentrations (10, 50, 100 μg/ml) for 16 hours or with specific PPARγ antagonist (GW9662, 10 μM) or agonist (Rosiglitazone, 10 μM) for 30 min before administration of LPS. The results showed that LPS significantly increased the release of inflammation-related adipokines, such as monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor 1 (PAI-1), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-8 and IL-6, while decreasing the release of leptin and adiponectin. Meanwhile, LPS reduced the uptake and degradation of 125I-oxLDL, and down-regulated the expression of PPARγ and CD36. Pretreatment with HDL dose-dependently affected the release of IL-8 and IL-6 and the reduced uptake and degradation of oxLDL of adipocytes stimulated by LPS, accompanied with marked upregulation of PPARγ and CD36 expression. Pretreatment with GW9662 markedly inhibited the upregulation of CD36 expression mediated by HDL (100 μg/ml), while the effects of Rosiglitazone were opposite to GW9662.Conclusions: HDL may increase oxLDL uptake of inflammatory adipocytes stimulated by LPS via upregulation of PPARγ/CD36 pathway, which may be a new mechanism of anti-atherosclerosis mediated by HDL.

Quercetin Alleviates High-Fat Diet-Induced Oxidized Low-Density Lipoprotein Accumulation in the Liver: Implication for Autophagy Regulation.

A growing body of evidence has indicated that high-fat diet-induced nonalcoholic fatty liver disease is usually accompanied by oxidized low-density lipoprotein (ox-LDL) deposited in the liver. The current study aimed to investigate the effect of quercetin on high-fat diet-induced ox-LDL accumulation in the liver and to explore the potential underlying mechanisms. The results demonstrate that quercetin supplementation for 24 weeks significantly alleviated high-fat diet-induced liver damage and reduced hepatic cholesterol and ox-LDL level. Quercetin notably inhibited both mRNA and protein expression of CD36 (reduced by 53% and 71%, resp.) and MSR1 (reduced by 25% and 45%, resp.), which were upregulated by high-fat diet. The expression of LC3II was upregulated by 2.4 times whereas that of p62 and mTOR was downregulated by 57% and 63% by quercetin treatment. Therefore, the significantly improved autophagy lysosomal degradation capacity for ox-LDL may be implicated in the hepatoprotective effect of quercetin; scavenger receptors mediated ox-LDL uptake might also be involved.

Niacin reverses migratory macrophage foam cell arrest mediated by oxLDL in vitro.

IntroductionNiacin reduces vascular oxidative stress and down regulates inducible nitric oxide synthase, an enzyme mediating proatherosclerotic effects in part by increasing oxidative stress. Here, we evaluate whether Niacin reverses the redox sensitive migratory arrest of macrophages in response to oxidised(ox) LDL uptake.Material and MethodsMigration of RAW264.7 cells, a murine macrophage cell line and bone marrow derived macrophages from wildtype and iNOS knockout mice was quantified using a modified Boyden chamber. Unstimulated cells or cells preincubated with oxLDL or non-oxidised (n)LDL were treated with Nicotinic acid or Nicotinamide. Nitric oxide, peroxynitrite and ROS production were assessed using electron paramagnetic resonance (ESR). Additionally, flow cytometry analysis of apoptosis, fokal adhesion kinase (FAK), phalloidin, CD36, F4/80 macrophage marker and iNOS gene expression (PCR) were assessed.ResultsMigration of Nicotinic acid, Nicotinamide treated cells or unstimulated cells did not differ (P>0.05). oxLDL treatment significantly reduced migration vs. unstimulated cells (p<0.05). In contrast, migratory arrest in response to oxLDL treatment was reversed by co-incubation with Nicotinic acid and Nicotinamide. The oxLDL-induced peroxynitrite formation in RAW264.7 cells was abolished by Niacin and glutathion (GSH) oxidation was significantly reduced. However, nitric oxide (NO)- and reactive oxygen species (ROS) production induced by oxLDL were not affected by Niacin treatment of RAW264.7 cells. In addition, Nicotinic acid and Nicotinamide reduced actin polymerization, a marker for migratory arrest.DiscussionOur data shows that oxLDL induced inhibition of macrophage migration in vitro can be reversed by Niacin. Furthermore, Niacin reduces peroxynitite formation and improves antioxidant GSH.