oxLDL Uptake Research Articles

Abstract 19002: LRP6 Mutation (LRP6 R611C ) Leads to PDGF Dependent Vascular Smooth Muscle Cell Hyperproliferation and Atherosclerosis

A loss of function mutation in the Wnt co-receptor Low Density Lipoprotein Receptor Related Protein 6, (LRP6 R611C ) causes early atherosclerosis and metabolic syndrome in humans. We have generated LRP6 R611C mice with homologous recombination. LRP6 R611C mice demonstrate enhanced vascular smooth muscle cell (VSMC) proliferation, and exaggerated neointima formation upon carotid injury mice and on LDLR knockout background develop accelerated atherosclerosis compared to their respective controls. The aorta of LRP6 R611C mice exhibits a highly proliferative media marked by an increases in PDGFRβ and ELK1 expression along with decreased myocardin expression. These findings correlate with reduced α-SMA and increased vimentin in the aortic media, indicating proliferative phenotype of VSMCs. Furthermore, in LRP6 R611C mice the aortic adventitia is loaded with PDGFRβ positive cells along with abundance of Sca1 positive progenitor cells in the aortic intimal and adventitial layers. Ex vivo, cultured LRP6 R611C VSMCs exhibit increased expression of PDGF-BB and its transcriptional regulator SP1. These findings suggest that LRP6 R611C promotes maintenance of progenitor cell in the aorta and enhances VSMC proliferation through activated PDGF signaling. Interestingly, the levels of PDGF-BB were also significantly increased in the macrophages as well as plasma of the LRP6 R611C mice. LRP6 R611C macrophages exhibited increased uptake of ox-LDL and augmented expression of vimentin, a regulator of intracellular LDL transport . Thus, LRP6 R611C mutation indicates a single gene defect in Wnt pathway leading to a major aberration in PDGF dependent signaling thereby predisposing to atherosclerosis.

A role for PKC in foam cell formation?

With great interest we have read the recently published article by Lin et al . 1 where PKCδ is described to regulate uptake of oxLDL and the resultant foam cell formation, thereby suggesting a potential role of this protein kinase in atherogenesis. In their study Lin et al . used two cell types, THP-1 monocytic cell line and primary human macrophages, as models for oxLDL uptake and in vitro foam cell formation. Employing RNA interference they decreased expression of PKCδ by an …

LOX-1 in atherosclerosis: biological functions and pharmacological modifiers

Lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1, also known as OLR-1), is a class E scavenger receptor that mediates the uptake of oxLDL by vascular cells. LOX-1 is involved in endothelial dysfunction, monocyte adhesion, the proliferation, migration, and apoptosis of smooth muscle cells, foam cell formation, platelet activation, as well as plaque instability; all of these events are critical in the pathogenesis of atherosclerosis. These LOX-1-dependent biological processes contribute to plaque instability and the ultimate clinical sequelae of plaque rupture and life-threatening tissue ischemia. Administration of anti-LOX-1 antibodies inhibits atherosclerosis by decreasing these cellular events. Over the past decade, multiple drugs including naturally occurring antioxidants, statins, antiinflammatory agents, antihypertensive and antihyperglycemic drugs have been demonstrated to inhibit vascular LOX-1 expression and activity. Therefore, LOX-1 represents an attractive therapeutic target for the treatment of human atherosclerotic diseases. This review aims to integrate the current understanding of LOX-1 signaling, regulation of LOX-1 by vasculoprotective drugs, and the importance of LOX-1 in the pathogenesis of atherosclerosis.

Resveratrol mediates anti-atherogenic effects on cholesterol flux in human macrophages and endothelium via PPARγ and adenosine

Resveratrol is a bioactive molecule used in dietary supplements and herbal medicines and consumed worldwide. Known cardioprotective and anti-inflammatory properties of resveratrol have spurred investigation of the mechanisms involved. The present study explored potential atheroprotective actions of resveratrol on cholesterol metabolism in cells of the arterial wall, including human macrophages and arterial endothelium. Using QRT-PCR and Western blotting techniques, we measured expression of the proteins involved in reverse cholesterol transport (ABCA1, ABCG1 and SR-B1) and the scavenger receptors responsible for uptake of modified cholesterol (CD36, SR-A1 and LOX-1). We analyzed the effect of resveratrol on apoA-1-and HDL-mediated cholesterol efflux in human THP-1 macrophages. The effect of resveratrol on oxLDL internalization and foam cell formation were evaluated using confocal and light microscopy. Our data indicate that resveratrol regulates expression of major proteins involved in cholesterol transport, promotes apoA-1 and HDL-mediated efflux, downregulates oxLDL uptake and diminishes foam cell formation. Mechanistically, resveratrol effects were dependent upon PPAR-γ and adenosine 2A receptor pathways. For the first time we demonstrate that resveratrol regulates expression of the cholesterol metabolizing enzyme cytochrome P450 27-hydroxylase, providing efficient cholesterol elimination via formation of oxysterols. This study establishes that resveratrol attenuates lipid accumulation in cultured human macrophages via effects on cholesterol transport. Further in vivo studies are needed to determine whether resveratrol may be an additional resource available to reduce lipid deposition and atherosclerosis in humans.

Ursolic Acid Attenuates HMGB1-induced LOX-1 Expression in Vascular Endothelial Cells in vitro and Inhibits Atherogenesis in Hypercholesterolemic Mice in vivo

Ursolic acid (UA), a triterpenoid compound found in plants, is used in both the human diet and in medicinal herbs and possesses a wide range of benefits, including antioxidative and anti-inflammatory effects. Additionally, UA may inhibit lipid absorption in pancreatic cells and enhance lipolysis in adipocytes. Oxidized LDL (oxLDL) acts as a major mediator of endothelium dysfunction, which mediates atherogenesis. Until now, we have not known what role UA plays in the absorption of oxidized LDL in vascular endothelial cells. Regardless of whether UA affects oxLDL uptake mediated by specific oxLDL receptors (such as lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), scavenger receptor expressed by endothelial cells (SREC), and scavenger receptor B1 (SR-B1)), it is unclear if UA acts on endothelial cells. However, high-mobility group box 1 (HMGB1) is known to accumulate in atherosclerotic lesions and mediates vascular inflammation, although the mechanisms are not understood. Therefore, in this study, human coronary artery endothelial cells (HCAECs) were used in vitro and hypercholesterolemic mice were used in vivo to investigate the effects and mechanisms of HMGB1 and UA on oxLDL uptake. The results demonstrated that HMGB1 enhances oxLDL uptake through induction of LOX-1 in HCAECs and hypercholesterolemic mice. In vitro data showed that exposing HMGB1- stimulated HCAECs to UA decreased the LOX-1-mediated absorption of oxLDL through a cyclooxygenase (COX)-2- related nitric oxide (NO) signaling pathway. Similarly, UA administration decreased LOX-1, but not SREC and SR-B1 expression, in HMGB1-treated hypercholesterolemic mice. These findings suggest that UA may be a potential therapeutic agent for hypercholesterolemia-induced atherosclerosis.

Modification of SR-PSOX functions by multi-point mutations of basic amino acid residues

SR-PSOX can function as a scavenger receptor, a chemokine and an adhesion molecule, and it could be an interesting player in the formation of atherosclerotic lesions. Our previous studies demonstrated that basic amino acid residues in the chemokine domain of SR-PSOX are critical for its functions. In this study the combinations of the key basic amino acids in the chemokine domain of SR-PSOX have been identified. Five combinations of basic amino acid residues that may form conformational motif for SR-PSOX functions were selected for multi-point mutants. The double mutants of K61AR62A, R76AK79A, R82AH85A, and treble mutants of R76AR78AK79A, R78AR82AH85A were successfully constructed by replacing the combinations of two or three basic amino acid residues with alanine. After successful expression of these mutants on the cells, the functional studies showed that the cells expressing R76AK79A and R82AH85A mutants significantly increased the activity of oxLDL uptake compared with that of wild-type SR-PSOX. Meanwhile, the cells expressing R76AK79A mutant also dramatically enhanced the phagocytotic activity of SR-PSOX. However, the cells expressing the construct of combination of R78A mutation in R76AK79A or R82AH85A could abolish these effects. More interestingly, the adhesive activities were remarkably down regulated in the cells expressing the multi-point mutants respectively. This study revealed that some conformational motifs of basic amino acid residues, especially R76 with K79 in SR-PSOX, may form a common functional motif for its critical functions. R78 in SR-PSOX has the potential action to stabilize the function of oxLDL uptake and bacterial phagocytosis. The results obtained may provide new insight for the development of drug target of atherosclerosis.

MicroRNA hsa-let-7g targets lectin-like oxidized low-density lipoprotein receptor-1 expression and inhibits apoptosis in human smooth muscle cells

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been identified as a major receptor for oxidatively modified low density lipoprotein (ox-LDL) in endothelial cells and smooth muscle cells (SMCs). MicroRNAs are small non-coding RNAs that regulate gene expression. Very few studies have reported regulation of LOX-1 expression by microRNAs in SMCs. The present study demonstrates that the microRNA hsa-let-7g can inhibit LOX-1 expression in a time- and dose-dependent fashion, and subsequently inhibit ox-LDL uptake in and proliferation of human aortic SMCs. We also show that hsa-let-7g can reduce SMC apoptosis by down-regulation of cytochrome c and Smac/Diablo and upregulation of Bcl-xL and Bcl-2 expression. Furthermore, we show that hsa-let-7g reduces ox-LDL-induced increase in the expression of NADPH oxidase (p22(phox) and p47(phox) subunits) and subsequent intracellular reactive oxygen species generation, phosphorylation of p44/42 mitogen-activated protein kinase and nuclear factor-kappaB p65 expression. These observations suggest that hsa-let-7g is a critical regulator of SMC apoptosis, and may be suitable for therapeutic intervention in disease states characterized by LOX-1 over-expression.

Degradation of heparan sulfate proteoglycans enhances oxidized-LDL-mediated autophagy and apoptosis in human endothelial cells

BackgroundCell surface heparan sulfate proteoglycans (HSPG) play an important role in atherogenesis. We hypothesized that degradation of HSPG may increase the binding of atherogenic oxidized low density lipoprotein (ox-LDL) to endothelial cells, and result in extensive HSPG degradation as well as autophagy and apoptosis. MethodsPrimary human umbilical vein endothelial cells (HUVECs) were used to study the expression of lectin-like ox-LDL receptor-1 (LOX-1), HSPG, autophagy and apoptosis in response to ox-LDL and heparinase III (Hep III). ResultsAs expected, ox-LDL treatment resulted in LOX-1 expression, ox-LDL uptake and reactive oxygen species (ROS) generation. Ox-LDL treatment also resulted in a modest degradation of HSPG and increase in autophagy (expression of LC3, beclin-1 and Atg5) and apoptosis (enhanced expression of caspases and Bax, and reduced expression of Bcl-2 and Bcl-xL). The effects of ox-LDL were blocked by pretreatment of cells with LOX-1 antibody or apocynin, an NADPH oxidase inhibitor. Hep III alone caused HSPG degradation and slightly, but significantly, increased ROS generation, and induced autophagy and caspase expression. However, autophagy and apoptosis induced by Hep III were not affected by apocynin or LOX-1 antibody. Importantly, Hep III pretreatment of cells significantly enhanced ox-LDL-induced HSPG degradation, LOX-1 expression, ox-LDL uptake and ROS generation as well as autophagy and apoptosis. ConclusionThese data demonstrate that Hep III enhances the pro-atherosclerotic characteristics in HUVECs induced by ox-LDL.

Insulin promotes macrophage foam cell formation: potential implications in diabetes-related atherosclerosis

The prevalence of atherosclerotic cardiovascular disease is higher in patients with type 2 diabetes, a disorder characterized by hyperinsulinemia and insulin resistance. The role of hyperinsulinemia as an independent participant in the atherogenic process has been controversial. In the current study, we tested the effect of insulin and the insulin sensitizer, adiponectin, on human macrophage foam cell formation. We found that both insulin and adiponectin increased the expression of the type 2 scavenger receptor CD36 by approximately twofold and decreased the expression of the ATP-binding cassette transporter ABCA1 by >80%. In both cases regulation was post-transcriptional. As a consequence of these changes, we found that oxidized LDL (oxLDL) uptake was increased by 80% and cholesterol efflux to apolipoprotein A1 (apoA1) was decreased by ∼25%. This led to two- to threefold more cholesterol accumulation over a 16-h period. As reported previously in studies of murine systems, scavenger receptor-A (SR-A) expression on human macrophages was downregulated by insulin and adiponectin. Insulin and adiponectin did not affect oxLDL-induced secretion of monocyte attractant protein-1 (MCP-1) and interleukin-6 (IL-6). These studies suggest that hyperinsulinemia could promote macrophage foam cell formation and thus may contribute to atherosclerosis in patients with type 2 diabetes.

Carbohydrate composition of amphiphilic macromolecules influences physicochemical properties and binding to atherogenic scavenger receptor A

Amphiphilic macromolecules (AMs) based on carbohydrate domains functionalized with poly(ethylene glycol) can inhibit the uptake of oxidized low density lipoprotein (oxLDL) mediated by scavenger receptor A (SR-A) and counteract foam cell formation, the characteristic “atherosclerotic” phenotype. A series of AMs was prepared by altering the carbohydrate chemistry to evaluate the influence of backbone architecture on the physicochemical and biological properties. Upon evaluating the degree of polymer-based inhibition of oxLDL uptake in human embryonic kidney cells expressing SR-A, two AMs (2a and 2c) were found to have the most efficacy. Molecular modeling and docking studies show that these same AMs have the most favorable binding energies and most close interactions with the molecular model of the SR-A collagen-like domain. Thus, minor changes in the AMs’ architecture can significantly affect the physicochemical properties and inhibition of oxLDL uptake. These insights can be critical for designing optimal AM-based therapeutics for the management of cardiovascular disease.

Oxidized low-density lipoprotein induces secretion of interleukin-1β by macrophages via reactive oxygen species-dependent NLRP3 inflammasome activation

Oxidized low-density lipoprotein (ox-LDL) is a critical mediator of atherogenesis. Macrophage uptake of ox-LDL and their subsequent development into foam cells is the principal event in atherosclerosis. Interleukin-1β (IL-1β), a prototypic multifunctional cytokine involved in inflammation, has an important effect on the pathogenesis and progression of atherosclerosis. Here we show that the phagocytosis of ox-LDL can induce human macrophages to secrete IL-1β by activating the NLRP3 inflammasome, and we further show that the activation of the NLRP3 inflammasome is dependent on the generation of reactive oxygen species and is related to the cathepsin B pathway. Furthermore, ox-LDL can upregulate the expression of the pro-IL-1β protein, thus priming IL-1β secretion. Therefore, our results suggest that the role of ox-LDL in atherosclerosis-related inflammation may involve the activation of the NLRP3 inflammasome.

Berberine-induced inhibition of adipocyte enhancer-binding protein 1 attenuates oxidized low-density lipoprotein accumulation and foam cell formation in phorbol 12-myristate 13-acetate-induced macrophages

The phagocytosis of oxidized low-density lipoprotein (oxLDL) by monocyte-derived macrophages and the subsequent differentiation of macrophages into foam cells are the key steps in atherogenesis. Scavenger receptors, such as CD36 and lectin-like low-density lipoprotein receptor 1 (LOX-1), are responsible for the uptake of oxLDL. Adipocyte enhancer-binding protein 1 (AEBP1) regulates many key genes associated with intracellular cholesterol efflux. The present study investigated the function of berberine, a compound isolated from Rhizoma coptidis, on foam cell formation, and explored the possible underlying mechanism. We found that berberine inhibited the oxLDL uptake of macrophages and reduced foam cell formation in a dose-dependent manner. Moreover, AEBP1 expression in macrophages increased and decreased after oxLDL and berberine treatments in a dose-dependent manner, respectively. Berberine reduced the expression of scavenger receptors CD36 and LOX-1, but did not affect the expression of CD68 in oxLDL-stimulated macrophages. Overall, berberine reduced foam cell formation by a dual mechanism, which decreased oxLDL internalization via the suppression of CD36 and LOX-1, and increased cholesterol efflux by inhibiting AEBP1 expression in macrophages.

The tryptophan metabolite 3-hydroxyanthranilic acid lowers plasma lipids and decreases atherosclerosis in hypercholesterolaemic mice

Cardiovascular disease is the most common cause of death in the world and atherosclerosis, an inflammatory process in the vessel wall, accounts for the majority of these deaths. The tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) has been shown to inhibit inflammation in different experimental autoimmune disease models. However, the effect of 3-HAA in atherosclerosis has never been explored. In this study, we used the atherosclerosis prone Ldlr-/- mice, and cell culture experiments to evaluate the role of 3-HAA in atherosclerosis. Eight weeks treatment with 3-HAA significantly reduced the lesion size in the aorta, and modulated local and systemic inflammatory responses. 3-hydroxyanthranilic acid inhibited the uptake of oxLDL by macrophages, an initiating event in the formation of foam cells, a major cellular component of atherosclerotic lesions. Surprisingly, 3-HAA significantly affected plasma cholesterol and triglyceride levels in Ldlr-/- mice, likely due to modulation of signalling through peroxisome proliferator-activated receptors. 3-Hydroxyanthranilic acid inhibits atherosclerosis by regulating lipid metabolism and inflammation, two major components of this disease.

An essential function for MKP5 in the formation of oxidized low density lipid-induced foam cells

The uptake of oxidized low density lipoprotein (ox-LDL) by macrophages usually leads to the formation of lipid-laden macrophages known as “foam cells,” and this process plays an important role in the development of atherosclerosis. Ox-LDL activates mitogen-activated protein kinase (MAP) kinases and nuclear factor (NF)-κB, and activations of p38 and NF-κB are important for the formation of foam cells. MAP kinase phosphatase (MKP) 5 is a member of the dual specificity phosphatases (DUSPs) family that can selectively dephosphorylate activated MAPKs to regulate innate and adaptive immune responses. However, the role of MKP5 in the formation of foam cells remains unknown. Here, we found that stimulation of ox-LDL induces the expression of MKP5 in macrophages. MKP5 deficiency blocked the uptake of ox-LDL and the formation of foam cells. Further analysis revealed that deletion of MKP5 reduced the ox-LDL-induced activation of NF-κB. Also, MKP5 deficiency markedly inhibited the production of TNF-α, but enhanced the levels of TGF-β1 in ox-LDL-stimulated macrophages. Moreover, inhibition of NF-κB by p65 RNAi significantly reduced foam cell formation in macrophages from WT mice relative to MKP5-deficient mice. Thus, MKP5 has an essential role in the formation of foam cells through activation of NF-κB, and MKP5 represents a novel target for the therapeutic intervention of atherosclerosis.

Co-Stimulation of PAFR and CD36 Is Required for oxLDL-Induced Human Macrophages Activation

The oxidative process of LDL particles generates molecules which are structurally similar to platelet-activating factor (PAF), and some effects of oxidized LDL (oxLDL) have been shown to be dependent on PAF receptor (PAFR) activation. In a previous study, we showed that PAFR is required for upregulation of CD36 and oxLDL uptake. In the present study we analyzed the molecular mechanisms activated by oxLDL in human macrophages and the contribution of PAFR to this response. Human adherent monocytes/macrophages were stimulated with oxLDL. Uptake of oxLDL and CD36 expression were determined by flow cytometry; MAP kinases and Akt phosphorylation by Western blot; IL-8 and MCP-1 concentration by ELISA and mRNA expression by real-time PCR. To investigate the participation of the PI3K/Akt pathway, Gαi-coupled protein or PAFR, macrophages were treated with LY294002, pertussis toxin or with the PAFR antagonists WEB2170 and CV3988, respectively before addition of oxLDL. It was found that the addition of oxLDL to human monocytes/macrophages activates the PI3K/Akt pathway which in turn activates the MAPK (p38 and JNK). Phosphorylation of Akt requires the engagement of PAFR and a Gαi-coupled protein. The upregulation of CD36 protein and the uptake of oxLDL as well as the IL-8 production are dependent on PI3K/Akt pathway activation. The increased CD36 protein expression is dependent on PAFR and Gαi-coupled protein. Transfection studies using HEK 293t cells showed that oxLDL uptake occurs with either PAFR or CD36, but IL-8 production requires the co-transfection of both PAFR and CD36. These findings show that PAFR has a pivotal role in macrophages response to oxLDL and suggest that pharmacological intervention at the level of PAFR activation might be beneficial in atherosclerosis.

Molecular mechanism of curcumin on the suppression of cholesterol accumulation in macrophage foam cells and atherosclerosis

Curcumin, a potent antioxidant extracted from Curcuma longa, confers protection against atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined the effect of curcumin on lipid accumulation and the underlying molecular mechanisms in macrophages and apolipoprotein E-deficient (apoE⁻/⁻) mice. Treatment with curcumin markedly ameliorated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages, which was due to decreased oxLDL uptake and increased cholesterol efflux. In addition, curcumin decreased the protein expression of scavenger receptor class A (SR-A) but increased that of ATP-binding cassette transporter (ABC) A1 and had no effect on the protein expression of CD36, class B receptor type I (SR-BI), or ATP-binding cassette transporter G1 (ABCG1). The downregulation of SR-A by curcumin was via ubiquitin-proteasome-calpain-mediated proteolysis. Furthermore, the curcumin-induced upregulation of ABCA1 was mainly through calmodulin-liver X receptor α (LXRα)-dependent transcriptional regulation. Curcumin administration modulated the expression of SR-A, ABCA1, ABCG1, and SR-BI in aortas and retarded atherosclerosis in apoE⁻/⁻ mice. Our findings suggest that inhibition of SR-A-mediated oxLDL uptake and promotion of ABCA1-dependent cholesterol efflux are two crucial events in suppression of cholesterol accumulation by curcumin in the transformation of macrophage foam cells.

Abstract 183: Oxidized LDL Cholesterol Induces Endothelial Dysfunction in the Aorta and Lox-1 Expression in Macrophages

Elevated plasma cholesterol is one of the major risk factors in the development of atherosclerotic lesions. Oxidation of native LDL cholesterol (nLDL) by reactive oxygen species leads to the formation of oxidized LDL (oxLDL). An important receptor for the cellular uptake of oxLDL is the lectin-like oxidized low-density lipoprotein receptor-1 (Lox-1). Lox-1 is highly expressed on macrophages, but also present on arterial endothelial and vascular smooth muscle cells. Especially the uptake by macrophages leads to the formation of foam cells in atherosclerotic lesions. Aim of the present study was to analyze the impact of oxLDL on endothelial function in murine aortas and on Lox-1 expression in human macrophages. In addition, we analyzed the effect of a high-fat diet on vascular function in mice with an endothelial Lox-1 overexpression. First, we incubated aortic rings of wild-type mice for 2 h with 100 μg/mL oxLDL and analyzed the endothelial function using a Mulvany myograph. Compared to basal conditions, oxLDL significantly impaired endothelium-dependent vasodilation. Next, we fed mice with an endothelial overexpression of Lox-1 for 20 weeks a high-fat diet and analyzed the endothelial function in the thoracic aorta. Interestingly, these mice had no impaired endothelium-dependent relaxation after high-fat diet feeding. To get further insight into Lox-1 regulation by oxLDL, we analyzed the impact of oxLDL on Lox-1 expression in human macrophages. Monocytic THP-1 cells were differentiated with phorbol myristate acetate into macrophages and stimulated for 24 h with nLDL or oxLDL. We found a significant induction of Lox-1 mRNA expression after oxLDL incubation, whereas nLDL had no effect. Our data suggest an increased oxLDL uptake in oxLDL-treated macrophages by increased Lox-1 receptor expression. In conclusion, our data support an important role of oxLDL as a proatherosclerotic risk factor by its ability to induce endothelial dysfunction and Lox-1 expression in macrophages. Both processes may be involved in the development of atherosclerotic lesions but the physiological significance and functional role of Lox-1 in contributing to the human disease warrants further investigations.

Squalene ameliorates atherosclerotic lesions through the reduction of CD36 scavenger receptor expression in macrophages

Anti-atherogenic features of olive oil (OO) have been attributed, in part, to minor compounds, via diverse mechanisms, although its effects on the CD36 receptor have not been examined. We investigated the effects of minor compounds of OO (squalene (SQ), tyrosol (Tyr) and hydroxytyrosol (OH-Tyr)), on the expression of the CD36 receptor, as well as on monocyte/macrophage differentiation and proliferation. U937 monocytic cells and macrophages (obtained with 10 nM phorbol-myristate-acetate) were exposed to Tyr, OH-Tyr or SQ at 0, 10, 75 and 200 μM with/without native or oxidised LDL(oxLDL). Flow cytometry was used to achieve the expression of CD36 in both cell types exposed to oxLDL plus antioxidants, as well as the inhibition of monocyte/macrophage differentiation after oxLDL and apoptosis. SQ caused a dose-dependent reduction of CD36 in the presence of native and moderate LDL in monocytes and macrophages. Phenotype-dependent cytotoxic and antiproliferative effects were found for OH-Tyr (p < 0.05), while SQ affected neither monocytes nor macrophages (p < 0.01). SQ does not prevent monocyte migration and activation into macrophages, but it would inhibit oxLDL uptake by macrophages, by reducing CD36 expression. This study provides new data about the role of the components of OO in the prevention of atherosclerosis.

Macrophage/foam cell is an attribute of inflammation: Mechanisms of formation and functional role

Transformation of macrophages into foam cells is traditionally considered in the context of atherogenesis, because lipid accumulation is believed to be a consequence of uptake of oxidized low density lipoproteins (oxLDL) through scavenger receptors (SR) of macrophages. However, an excessive uptake of oxLDL is recently shown to trigger compensatory mechanisms of cholesterol elimination from macrophages. Maintaining the lipid homeostasis in macrophages is mediated by regulation of a system of lipid sensors, which is reprogrammed under conditions of inflammation leading to formation of foam cell phenotype without involvement of SR. The increase in the inflammatory potential on macrophage polarization into the M1 phenotype is associated with suppression of LXR and PPAR, their target genes, induction of expression of genes responsible for fatty acid and cholesterol metabolism controlled by SREBP1c and SREBP2, proteins associated with lipid inclusions, macropinocytosis activation, secretion of LXR and PPAR endogenous ligands, and development of apoptosis. In this review the role of foam cells in development and resolution of acute inflammation, mechanisms of their formation from macrophages infected by some bacterial and virus pathogens causing chronic inflammation, and the significance of LXR and PPAR as therapeutic targets in chronic infectious and inflammatory diseases are also discussed.

Tanshinone II-A inhibits oxidized LDL-induced LOX-1 expression in macrophages by reducing intracellular superoxide radical generation and NF-κB activation

Lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1), a novel scavenger receptor highly expressed in human and experimental atherosclerotic lesions, is responsible for the uptake of oxLDL in vascular cells. We demonstrated previously that Tanshinone II-A (Tan), a pharmacologically active compound extracted from the rhizome of the Chinese herb Salvia miltiorrhiza Bunge, inhibits atherogenesis in hypercholesterolemic rats, rabbits, and apolipoprotein-E deficient (ApoE⁻/⁻) mice. However, the precise mechanism by which Tan protects against atherogenesis remains to be elucidated. Therefore, we hypothesized that Tan can suppress the uptake of oxLDL by diminishing the expression of LOX-1 via suppression of NF-κB signaling pathway, thereby contributing to reduced macrophage foam cell formation. In cultured murine macrophages, oxLDL induced LOX-1 expression at the mRNA and protein levels, was abrogated by addition of Tan or pyrrolidinedithiocarbamic acid ammonium salt (PDTC), a widely used inhibitor of NF-κB, suggesting the involvement of NF-κB. Tan also reduced LOX-1 expression in atherosclerotic lesions of ApoE⁻/⁻ mice fed a high cholesterol diet. Mechanistically, Tan suppressed the nuclear translocation of NF-κB P65 subunit and phosphorylation of IκB-α induced by oxLDL. Electrophoretic mobility shift assay (EMSA) demonstrated that Tan inhibited the nuclear protein binding to NF-κB consensus sequence. Functionally, we observed that Tan inhibited DiI-oxLDL uptake by macrophages in a fashion similar to that produced by LOX-1 neutralizing antibody. Our current findings reveal a novel mechanism by which Tan protects against atherogenesis and shed new light on the potential therapeutic application of Tan to the treatment and prevention of atherosclerotic cardiovascular diseases.