Oxidized Low-density Lipoprotein Treatment Research Articles

Abstract P3189: Vcam-1-mediated Mitochondrial Biogenesis In Macrophages Exacerbates Inflammation And Atherosclerosis

Atherosclerotic cardiovascular disease is a leading cause of adult mortality worldwide. Macrophages play a key role in atherogenesis by sustaining the local inflammatory response, facilitating accumulation of immune cells in atheromas that contribute to plaque development. Mitochondrial metabolism plays a key role in governing inflammatory gene expression in macrophages. Yet, the role of mitochondrial biogenesis and mitochondrial DNA synthesis in regulating macrophage inflammation is not known. Vascular cell adhesion protein 1 (VCAM-1) expressed by endothelial cells mediates monocyte adhesion and extravasation in developing atherosclerotic plaques. We observed increased VCAM-1 expression in human and murine plaque macrophages. However, the function of this cell adhesion molecule expressed by macrophages is not known. Here, we evaluated if and how myeloid-VCAM-1 drive mitochondrial biogenesis, inflammation, and atherosclerosis pathology. Increased VCAM-1 expression in plaque macrophages correlated with oxidative DNA damage and mitochondrial volume. To understand the role of myeloid-Vcam1 in atherogenesis, we generated Apoe -/- LyzM cre/+ VCAM-1 fl/fl mice. Consistently, Apoe -/- mice lacking Vcam1 in macrophages exhibited reduced atherosclerosis severity and inflammation. Vcam1-deficient macrophages exhibited decreased inflammation, oxidative phosphorylation, mitochondrial biogenesis, and mitochondrial DNA synthesis genes, including Cmpk2. Cmpk2 deletion in macrophages after oxidized LDL treatment reduced inflammatory mediators that aggravate atherosclerosis. RNA sequencing analysis of Vcam-1 -deficient plaque macrophages and analysis of macrophages lacking Cmpk2 identified Fcor and Lyz1 as the target genes of Vcam1 and Cmpk2 . Interestingly, atherosclerotic plaque macrophages deficient of Sting , which mediates inflammatory signaling in response to oxidized mitochondrial DNA, had increased levels of Fcor and Lyz1 . Our data suggest that VCAM-1 in macrophages signals via CMPK2 and POLG to promote mitochondrial biogenesis, oxidation, and fragmentation, mediating atherogenesis.

CIRC_0091822 CONTRIBUTES TO THE PROLIFERATION, INVASION, AND MIGRATION OF VASCULAR SMOOTH MUSCLE CELLS UNDER OXIDIZED LOW-DENSITY LIPOPROTEIN TREATMENT.

Background: Circular RNAs (circRNAs) have been shown to mediate atherosclerosis (AS) process by regulating vascular smooth muscle cells (VSMCs) function. However, whether circ_0091822 mediates VSMCs function to regulate AS process is unclear. Methods: Oxidized low-density lipoprotein (ox-LDL) was used to treat VSMCs for constructing AS cell models. Vascular smooth muscle cells proliferation, invasion, and migration were examined by cell counting kit 8 assay, EdU assay, transwell assay, and wound healing assay. Protein expression was tested by western blot analysis. The expression of circ_0091822, microRNA (miR)-339-5p, and blocking of proliferation 1 (BOP1) was determined using quantitative real-time PCR. RNA interaction was examined using dual-luciferase reporter assay and RIP assay. Results: Ox-LDL treatment enhanced VSMCs proliferation, invasion, and migration. Circ_0091822 was overexpressed in the serum of AS patients and ox-LDL-induced VSMCs. Circ_0091822 knockdown inhibited ox-LDL-induced VSMCs proliferation, invasion, and migration. Circ_0091822 sponged miR-339-5p, and miR-339-5p inhibitor reversed the function of circ_0091822 knockdown. MiR-339-5p targeted BOP1, and BOP1 also reversed the repressing effect of miR-339-5p on ox-LDL-induced VSMCs functions. Circ_0091822/miR-339-5p/BOP1 axis promoted the activity of Wnt/β-catenin pathway. Conclusions: Circ_0091822 might be a therapeutic target for AS, which facilitated ox-LDL-induced VSMCs proliferation, invasion, and migration through modulating miR-339-5p/BOP1/Wnt/β-catenin pathway.

The enigmatic role of VCAM‐1 expressed by macrophages in mitochondrial metabolism and atherosclerosis

BackgroundAtherosclerosis is a primary underlying cause of most cardiovascular diseases, including myocardial infarction, heart failure and stroke. It has been shown that vascular cell adhesion protein 1(VCAM‐1) expressed by endothelial cells mediates rolling and adhesion of monocytes to endothelial cells under atherosclerotic conditions. However, the role of VCAM1 expressed by monocyte and macrophage in atherosclerosis pathology has not been explored. Here, we show that VCAM1 in macrophages signals via CMPK2 and STING to modulate mitochondrial biogenesis and promote atherosclerosis.MethodsLyzMcre/+VCAM‐1fl/fl mice were used to characterize the role of macrophage‐specific VCAM1 in the pathogenesis of atherosclerosis, LyzM+/+VCAM‐1fl/flmice were used as controls. Bone marrow cells were transplanted from VCAM‐1‐deficient donor into Ldlr‐/‐fed atherogenic western diet. Additionally, we also generated Apoe‐/‐ LyzMcre/+VCAM‐1fl/fl mice as a second model to understand the function of myeloid VCAM‐1 in atherosclerosis. Flow cytometry was performed to enumerate atheroma immune cell populations and sort plaque macrophages from the aorta. RNA‐seq was performed on isolated aortic macrophages from VCAM‐1 deficient mice (wild‐type controls). Mitochondrial respiration and complex activity were measured by Seahorse Agilent bioanalyzer. Mitochondrial volume and membrane potential were quantified by flow cytometry and immunofluorescence using MitoTracker dyes. Mitophagy was assessed in mtKeima bone marrow derived macrophages (BMDMs) after silencing VCAM1 and treatment with oxLDL. RT‐PCR was performed to quantify expression of inflammatory cytokine, retention factor and mitochondrial biogenesis genes. Gene silencing was performed using siRNA.ResultsAtherosclerotic plaque macrophages expressed high levels of VCAM‐1 in humans and mice. Concomitantly, plaque macrophages had increased mitochondrial volume, mitochondrial DNA synthesis, oxidized mitochondrial DNA and oxidative phosphorylation. We observed that mice lacking Vcam1 in macrophages had reduced atherosclerotic plaque and necrotic core areas. Additionally, Vcam1 silencing in macrophages diminished inflammation, oxidative phosphorylation, mitochondrial biogenesis and the expression of the genes involved in mitochondrial DNA synthesis including Cmpk2. Cmpk2 knock down in macrophages after oxidized LDL treatment reduced inflammatory mediators that aggravate atherosclerosis. RNA sequencing analysis of Vcam‐1‐deficient plaque macrophages and analysis of macrophages lacking Cmpk2 identified Fcor and Lyz1 as the target genes of Vcam1 and Cmpk2. Interestingly, atherosclerotic plaque macrophages deficient of Sting, which mediates inflammatory signaling in response to oxidized mitochondrial DNA, had increased levels of Fcor and Lyz1.ConclusionWe conclude that macrophage‐specific VCAM‐1 augments mitochondrial biogenesis and DNA oxidation via Cmpk2. Oxidized mitochondrial DNA in plaque macrophages increases inflammation via Sting, resulting in exacerbation of atherosclerosis.

Astragaloside IV Reduces OxLDL-Induced BNP Overexpression by Regulating HDAC.

Effective drug intervention is the most important method to improve the prognosis, improve the quality of life, and prolong the life of patients with heart failure. This study aimed to explore the protective effect of astragaloside IV on myocardial cell injury induced by oxidized low-density lipoprotein (OxLDL) and its regulatory mechanism on the increase of brain natriuretic peptide (BNP) caused by myocardial cell injury. The model of myocardial cell injury, protection, and histone deacetylase (HDAC) inhibition in HL-1 mice was established by OxLDL treatment, astragaloside IV intervention, and UF010 coincubation. The effects of OxLDL and astragaloside IV on apoptosis were detected by flow cytometry. The expression level of BNP mRNA and protein in cells was investigated by real-time fluorescence quantification, western blot, and enzyme-linked immunosorbent assay. HDAC activity in nucleus was calibrated by fluorescence absorption intensity. Enzyme-linked immunosorbent assay (ELISA) was applied to test eNOS level in myocardial cells. OxLDL significantly promoted apoptosis, upregulated BNP mRNA, increased BNP protein level inside and outside cells, and decreased eNOS level. Compared with OxLDL treatment group, apoptosis decreased, BNP mRNA expression level decreased, BNP protein concentration decreased, and eNOS level increased significantly combined with low and high concentration astragaloside IV treatment group. HDAC activity significantly increased in OxLDL treatment group and significantly decreased after combined incubation with low and high concentrations of astragaloside IV. Inhibition of HDAC significantly increased eNOS level and decreased BNP protein level. In conclusion, astragaloside IV can reverse the low level of eNOS caused by OxLDL by regulating HDAC activity to protect myocardial cells from oxide damage, which is manifested by the decrease of BNP concentration.

Siglec-E retards atherosclerosis by inhibiting CD36-mediated foam cell formation

BackgroundThe accumulation of lipid-laden macrophages, foam cells, within sub-endothelial intima is a key feature of early atherosclerosis. Siglec-E, a mouse orthologue of human Siglec-9, is a sialic acid binding lectin predominantly expressed on the surface of myeloid cells to transduce inhibitory signal via recruitment of SH2-domain containing protein tyrosine phosphatase SHP-1/2 upon binding to its sialoglycan ligands. Whether Siglec-E expression on macrophages impacts foam cell formation and atherosclerosis remains to be established.MethodsApoE-deficient (apoE−/−) and apoE/Siglec-E-double deficient (apoE−/−/Siglec-E−/−) mice were placed on high fat diet for 3 months and their lipid profiles and severities of atherosclerosis were assessed. Modified low-density lipoprotein (LDL) uptake and foam cell formation in wild type (WT) and Siglec-E−/−- peritoneal macrophages were examined in vitro. Potential Siglec-E-interacting proteins were identified by proximity labeling in conjunction with proteomic analysis and confirmed by coimmunoprecipitation experiment. Impacts of Siglec-E expression and cell surface sialic acid status on oxidized LDL uptake and signaling involved were examined by biochemical assays.ResultsHere we show that genetic deletion of Siglec-E accelerated atherosclerosis without affecting lipid profile in apoE−/− mice. Siglec-E deficiency promotes foam cell formation by enhancing acetylated and oxidized LDL uptake without affecting cholesterol efflux in macrophages in vitro. By performing proximity labeling and proteomic analysis, we identified scavenger receptor CD36 as a cell surface protein interacting with Siglec-E. Further experiments performed in HEK293T cells transiently overexpressing Siglec-E and CD36 and peritoneal macrophages demonstrated that depletion of cell surface sialic acids by treatment with sialyltransferase inhibitor or sialidase did not affect interaction between Siglec-E and CD36 but retarded Siglec-E-mediated inhibition on oxidized LDL uptake. Subsequent experiments revealed that oxidized LDL induced transient Siglec-E tyrosine phosphorylation and recruitment of SHP-1 phosphatase in macrophages. VAV, a downstream effector implicated in CD36-mediated oxidized LDL uptake, was shown to interact with SHP-1 following oxidized LDL treatment. Moreover, oxidized LDL-induced VAV phosphorylation was substantially lower in WT macrophages comparing to Siglec-E−/− counterparts.ConclusionsThese data support the protective role of Siglec-E in atherosclerosis. Mechanistically, Siglec-E interacts with CD36 to suppress downstream VAV signaling involved in modified LDL uptake.

Inhibitory Effects of Genistein on Vascular Smooth Muscle Cell Proliferation Induced by Ox-LDL: Role of BKCa Channels.

Background Oxidized low-density lipoprotein (Ox-LDL) is a crucial pathogenic factor for vascular diseases, which can induce the proliferation of vascular smooth muscle cells (VSMCs). Genistein is the main component of soybean isoflavone. Genistein has a variety of pharmacological properties in the treatment of vascular diseases and a promising clinical application. Large-conductance calcium-activated potassium (BKCa) channels are the primary type of potassium channels in VSMCs, which regulate various biological functions of VSMCs. However, whether genistein exerts an antiproliferation effect on Ox-LDL-stimulated VSMCs remains unclear. The current study is aimed at elucidating the effect of genistein on the Ox-LDL-stimulated proliferation of VSMCs and its possible molecular mechanism, especially the electrophysiological mechanism related to BKCa channels. Methods Monoculture VSMC was obtained by an acute enzyme-dispersing method. The proliferation of cells was measured by CCK-8, cell cycle, and proliferating cell nuclear antigen (PCNA) expression. The BKCa whole-cell currents were measured by patch-clamp. Results Ox-LDL treatment induced the proliferation of VSMCs, upregulated the BKCa protein expression, and increased the density of BKCa currents, while genistein significantly inhibited these effects caused by Ox-LDL. BKCa channels exerted a regulatory role in the proliferation of VSMCs in response to Ox-LDL. The inhibition of BKCa channels suppressed Ox-LDL-stimulated VSMC proliferation, while the activation of BKCa channels showed the opposite effect. Moreover, genistein suppressed the activity of BKCa, including protein expression and current density in a protein tyrosine kinase- (PTK-) dependent manner. Conclusion This study demonstrated that genistein inhibited the Ox-LDL-mediated proliferation of VSMCs by blocking the cell cycle progression; the possible molecular mechanism may be related to PTK-dependent suppression of BKCa channels. Our results provided novel ideas for the application of genistein in the treatment of vascular diseases and proposed a unique insight into the antiproliferative molecular mechanism of genistein.

Siglec‐E Retards Atherosclerosis by Inhibiting CD36‐Mediated Foam Cell Formation

The accumulation of lipid‐laden macrophages, foam cells, within sub‐endothelial intima is a key feature of early atherosclerosis. Siglec‐E is a member of sialic acid binding lectin predominantly expressed on myeloid cells to transduce inhibitory signal upon interacting with its ligands. Whether Siglec‐E expression on macrophages impacts foam cell formation and atherosclerosis remains to be established. To this end, both apoE‐deficient and apoE/Siglec‐E‐double deficient mice were placed on high fat diet for 3 months and their lipid profiles and severities of atherosclerosis were then assessed. The results showed that Siglec‐E deficiency accelerated atherosclerosis without affecting lipid profile in apoE deficient mice. In vitro experiments demonstrated that Siglec‐E deletion facilitated the uptake of acetylated or oxidized low density lipoprotein (LDL) and augmented foam cell formation in macrophages. By performing proximity labeling and proteomic analysis, we identified CD36 as a cell surface protein interacting with Siglec‐E. Notably, the interaction between Siglec‐E and CD36 was not affected by the sialylation status of CD36. Further experiments demonstrated that oxidized LDL induced transient Siglec‐E phosphorylation and recruitment of SHP‐1 in macrophages. VAV, a downstream effector implicated in CD36‐mediated oxidized LDL uptake, was shown to interact with SHP‐1 following oxidized LDL treatment. Moreover, Siglec‐E deficiency enhanced VAV phosphorylation induced by oxidized LDL. Collectively, these data demonstrate that Siglec‐E attenuates atherosclerosis in apoE‐deficient mice through suppressing CD36‐mediated signaling responsible for modified LDL uptake and foam cell formation in macrophages.Support or Funding InformationThis work was supported by a grant from Ministry of Science and Technology of Taiwan (MOST‐106‐2320‐B‐001‐020‐MY3).

Role of pyruvate kinase M2 in oxidized LDL-induced macrophage foam cell formation and inflammation

Pyruvate kinase M2 (PKM2) links metabolic and inflammatory dysfunction in atherosclerotic coronary artery disease; however, its role in oxidized LDL (Ox-LDL)-induced macrophage foam cell formation and inflammation is unknown and therefore was studied. In recombinant mouse granulocyte-macrophage colony-stimulating factor-differentiated murine bone marrow-derived macrophages, early (1-6 h) Ox-LDL treatment induced PKM2 tyrosine 105 phosphorylation and promotes its nuclear localization. PKM2 regulates aerobic glycolysis and inflammation because PKM2 shRNA or Shikonin abrogated Ox-LDL-induced hypoxia-inducible factor-1α target genes lactate dehydrogenase, glucose transporter member 1, interleukin 1β (IL-1β) mRNA expression, lactate, and secretory IL-1β production. PKM2 inhibition significantly increased Ox-LDL-induced ABCA1 and ABCG1 protein expression and NBD-cholesterol efflux to apoA1 and HDL. PKM2 shRNA significantly inhibited Ox-LDL-induced CD36, FASN protein expression, DiI-Ox-LDL binding and uptake, and cellular total cholesterol, free cholesterol, and cholesteryl ester content. Therefore, PKM2 regulates lipid uptake and efflux. DASA-58, a PKM2 activator, downregulated LXR-α, ABCA1, and ABCG1, and augmented FASN and CD36 protein expression. Peritoneal macrophages showed similar results. Ox-LDL induced PKM2- SREBP-1 interaction and FASN expression in a PKM2-dependent manner. Therefore, this study suggests a role for PKM2 in Ox-LDL-induced aerobic glycolysis, inflammation, and macrophage foam cell formation.

Sestrin2 regulates monocyte activation through AMPK-mTOR nexus under high-glucose and dyslipidemic conditions.

The vicious cycle between hyperinsulinemia and insulin resistance results in the progression of atherosclerosis in the vessel wall. The complex interaction between hyperglycemia and lipoprotein abnormalities promotes the development of atherogenesis. In the early phase of atherosclerosis, macrophage-derived foam cells play an important role in vascular remodeling. Mechanistic target of rapamycin (mTOR) signaling pathway has been identified to play an essential role in the initiation, progression, and complication of atherosclerosis. Recently sestrin2, an antioxidant, was shown to modulate TOR activity and thereby regulating glucose and lipid metabolism. But the role of sestrin2 in monocyte activation is still not clearly understood. Hence, this study is focussed on investigating the role of sestrin2 in monocyte activation under hyperglycemic and dyslipidemic conditions. High-glucose and oxidized low-density lipoprotein (LDL) treatments mediated proinflammatory cytokine production (M1) with a concomitant decrease in the anti-inflammatory cytokine (M2) levels in human monocytic THP1 cells. Both glucose and oxidized LDL (OxLDL) in a dose and time-dependent manner increased the mTOR activation with a marked reduction in the levels of pAMPK and sestrin2 expression. Both high-glucose and OxLDL treatment increased foam cell formation and adhesion of THP1 cells to endothelial cells. Experiments employing activator or inhibitor of adenosine monophosphate kinase (AMPK) as well as overexpression or silencing of sestrin2 indicated that high-glucose mediated monocyte polarization and adhesion of monocytes to the endothelial cells were appeared to be programmed via sestrin2-AMPK-mTOR nexus. Our results evidently suggest that sestrin2 plays a major role in regulating monocyte activation via the AMPK-mTOR-pathway under diabetic and dyslipidemic conditions and also AMPK regulates sestrin2 in a feedback mechanism.

Abstract 17333: Myeloid-Specific Deletion of Epsins 1 and 2 Reduces Atherosclerosis by Preventing LRP-1 Downregulation

Introduction: Atherosclerosis is in part caused by immune and inflammatory cell infiltration into the vascular wall, leading to enhanced inflammation and lipid accumulation in the aortic endothelium. Understanding the molecular mechanisms underlying this disease is critical for the development of new therapies. Our recent studies demonstrate that epsins, a family of ubiquitin-binding endocytic adaptors, are critical regulators of atherogenicity. Given the fundamental contribution lesion macrophages make to fuel atherosclerosis, whether and how myeloid specific epsins promote atherogenesis is an open and significant question. Hypothesis: We hypothesize that myeloid specific epsins regulate lesion macrophage function during atherosclerosis. Methods and Results: We engineered myeloid cell-specific epsins double knockout mice (LysM-DKO)on an ApoE -/- background. On Western diet, these mice exhibited marked decrease in atherosclerotic lesion formation, diminished immune and inflammatory cell content in aortas, and reduced necrotic core content but increased smooth muscle cell content in aortic root sections.Epsins deficiency hindered foam cell formation and suppressed the pro-inflammatory macrophage phenotype but increased efferocytosis and the anti-inflammatory macrophage phenotype in primary macrophages.Mechanistically, we show that epsins loss specifically increased total and surface levels of LRP-1, an efferocytosis receptor with anti-atherosclerotic properties. We further show thatepsin and LRP-1 interact via epsin’s Ubiquitin Interacting Motif (UIM) domain. Oxidized LDL treatment increased LRP-1 ubiquitination and subsequent binding to epsin while mutation of cytoplasmic lysine residues attenuated LRP-1 ubiquitination, suggesting that epsins promote the ubiquitin-dependent internalization and downregulation of LRP-1. Crossing ApoE -/- /LysM-DKO mice onto a LRP-1 heterozygous background restored, in part, atherosclerosis, suggesting that epsin-mediated LRP-1 downregulation in macrophages plays a pivotal role in propelling atherogenesis. Conclusions: Myeloid epsins promote atherogenesis by facilitating pro-inflammatory macrophage recruitment and inhibiting efferocytosis in part by downregulating LRP-1, implicating that targeting epsins in macrophages may serve as a novel therapeutic strategy to treat atherosclerosis. Key words: epsin, macrophage, atherosclerosis, LRP-1, inflammation, efferocytosis

Quercetin attenuates Ox-LDL-induced calcification in vascular smooth muscle cells by regulating ROS-TLR4 signaling pathway

To determine whether quercetin inhibits oxidized low-density lipoprotein (Ox-LDL)-induced osteogenic differentiation and calcification of vascular smooth muscle cells (VSMCs) and understand the underlying mechanism. The calcification of human VSMCs following Ox-LDL treatment was assessed using alizarin red staining and by detecting ALP activity. The mRNA expressions of the bone-related genes including Msx2, BMP2 and Osterix, and the contractile proteins including SMA and SM22a were analyzed using qPCR. The effects of quercetin were investigated on OxLDL-induced VSMC calcification and changes in ALP activity, expressions of Msx2, BMP2, Osterix, SMA and SM22a, ROS levels and SOD activity. The effect of Toll like receptor 4 (TLR4) silencing mediated by siRNA transfection on cell calcification, ALP activity, gene expressions and ROS levels were investigated. Ox-LDL treatment promoted VSMC calcification and up-regulated TLR4 expression. Quercetin treatment significantly attenuated Ox-LDL-induced VSMC calcification, reduced ALP activity, down-regulated the expression levels of Msx2, BMP2 and Osterix, and up-regulated the expressions of vascular smooth muscle contractile proteins SMA and SM22a. In addition, Quercetin treatment markedly increased SOD activity, reduced ROS levels and TLR4 expression in VSMCs. Silencing TLR4 expression using TLR4 siRNA also significantly decreased calcification of the VSMCs. Quercetin inhibits Ox-LDL-induced VSMC calcification in VSMCs possibly by targeting the ROS/TLR4 signaling pathway.

Expression of LOX-1 in human mesangial cells is increased by Ox-LDL and IL-1β treatment.

The present study aimed to determine whether the expression of lectin-like oxidized LDL receptor 1 (LOX-1) could be induced by oxidized low-density lipoprotein (Ox-LDL) and interleukin 1β (IL-1β) in human mesangial cells (HMCs). Oil Red O staining was used to observe the uptake of Ox-LDL by HMCs stimulated with IL-1β, and reverse transcription-quantitative polymerase chain reaction analysis and western blotting were used to examine the expression of LOX-1 in HMC following Ox-LDL and IL-1β treatment. Uptake of Ox-LDL by HMCs was upregulated upon stimulation with IL-1β. Furthermore, Ox-LDL (10–40 µg/ml) treatment induced LOX-1 mRNA and protein expression in a dose-dependent manner. In addition, when HMCs were treated with IL-1β and Ox-LDL, the expression of LOX-1 was enhanced further. These results indicated that inhibiting LOX-1 expression or inhibiting the Ox-LDL/LOX-1 signaling axis may be a potential novel method for treating renal disease.

Abstract 94: Deficiency of Epsins in Macrophages Ameliorates Atherosclerosis by Attenuating Inflammation

Background: Atherosclerosis is caused by the immune and inflammatory cell infiltration of the vascular wall, leading to enhanced inflammation and lipid accumulation. Understanding the molecular mechanisms underlying this disease is critical for the development of new therapies. Our recently studies demonstrate that endothelial epsins, a family of ubiquitin-binding endocytic adaptors are critical regulators of atherosclerosis. However, whether epsins in macrophages play a role in regulating vascular inflammation is unknown. We hypothesize that epsins in macrophages promote inflammation to facilitate atherogenesis. Methods and Results: We engineered myeloid cell-specific epsins double knockout mice (MΦ-DKO) on an ApoE-/- background fed western diet. Strikingly, these mice exhibited reduced atherosclerotic lesion formation, diminished immune and inflammatory cell recruitment to aortas and reduced cleaved caspase 3 staining but increased α-SMA staining within aortic root sections. Epsin deficiency hindered foam cell formation, suppressed the pro-inflammatory M1 macrophage phenotype but increased the anti-inflammatory macrophage phenotype, and enhanced efferocytosis in primary macrophages. Mechanistically, we show that epsin loss specifically increases total and surface levels of LRP-1, a protein with anti-inflammatory properties without altering levels of LDL scavenger receptors. We further show that epsin and LRP-1 interact via epsin’s UIM domain. Oxidized LDL treatment increased LRP-1 ubiquitination and subsequent binding to epsin while mutation of cytoplasmic lysine residues attenuated LRP-1 ubiquitination, suggesting that epsin promotes the ubiquitin-dependent internalization and degradation of LRP-1. Importantly, MΦ-DKO/ApoE null mice on LRP-1 heterozygous background restored atherosclerosis, suggesting that epsin-mediated LRP-1 downregulation in macrophages plays a pivotal role in propelling atherogenesis. Conclusions: Macrophage epsins promote atherogenesis, in part, by facilitating pro-inflammatory macrophage recruitment and potentiating foam cell formation by downregulating LRP-1, implicating that targeting epsin in macrophages may serve as a novel therapeutic strategy to treat atheroma.

Oxidized Lipoprotein Uptake Through the CD36 Receptor Activates the NLRP3 Inflammasome in Human Retinal Pigment Epithelial Cells.

PurposeAccumulation of oxidized phospholipids/lipoproteins with age is suggested to contribute to the pathogenesis of AMD. We investigated the effect of oxidized LDL (ox-LDL) on human RPE cells.MethodsPrimary human fetal RPE (hf-RPE) and ARPE-19 cells were treated with different doses of LDL or ox-LDL. Assessment of cell death was measured by lactate dehydrogenase release into the conditioned media. Barrier function of RPE was assayed by measuring transepithelial resistance. Lysosomal accumulation of ox-LDL was determined by immunostaining. Expression of CD36 was determined by RT-PCR; protein blot and function was examined by receptor blocking. NLRP3 inflammasome activation was assessed by RT-PCR, protein blot, caspase-1 fluorescent probe assay, and inhibitor assays.ResultsTreatment with ox-LDL, but not LDL, for 48 hours caused significant increase in hf-RPE and ARPE-19 (P < 0.001) cell death. Oxidized LDL treatment of hf-RPE cells resulted in a significant decrease in transepithelial resistance (P < 0.001 at 24 hours and P < 0.01 at 48 hours) relative to LDL-treated and control cells. Internalized ox-LDL was targeted to RPE lysosomes. Uptake of ox-LDL but not LDL significantly increased CD36 protein and mRNA levels by more than 2-fold. Reverse transcription PCR, protein blot, and caspase-1 fluorescent probe assay revealed that ox-LDL treatment induced NLRP3 inflammasome when compared with LDL treatment and control. Inhibition of NLRP3 activation using 10 μM isoliquiritigenin significantly (P < 0.001) inhibited ox-LDL induced cytotoxicity.ConclusionsThese data are consistent with the concept that ox-LDL play a role in the pathogenesis of AMD by NLRP3 inflammasome activation. Suppression of NLRP3 inflammasome activation could attenuate RPE degeneration and AMD progression.

Oxidized LDL Induces Apoptosis of Human Retinal Pigment Epithelium Through Activation of ERK-Bax/Bcl-2 Signaling Pathways

Purpose: Retinal pigment epithelium (RPE) cell dysfunction and death play a vital role in the pathogenesis of age-related macular degeneration (AMD). We previously reported that oxidized low-density lipoprotein (OX-LDL) induces retinal degeneration in vivo. In this study, we investigated the role of the ERK-Bax/Bcl-2 signaling pathways in OX-LDL-induced apoptosis in human RPE.Methods: ARPE-19 cells were incubated with 10–100 mg/mL n-LDL or OX-LDL for 24 h. Cell viability was assessed using the Cell Titer 96 Aqueous One Solution cell proliferation assay. RPE apoptosis was measured with a flow cytometer. Reverse transcription polymerase chain reaction was used to detect Bcl-2 and Bax mRNA levels in RPE cells. Bcl-2 and Bax protein expression was measured by western blotting. Activation of extracellular signal-regulated kinase (ERK) protein was evaluated by western blot analysis. One-way analysis of variance was used to compare differences.Results: OX-LDL treatment decreased ARPE-19 cell viability in a dose-dependent manner, whereas n-LDL had no effect. Compared with the control group, OX-LDL significantly increased the apoptosis of RPE, 10 mg/mL, 50 mg/mL, 100 mg/mL apoptosis rate was 6.43 ± 0.19%, 5.12 ± 0.27%, 5.53 ± 0.35%, respectively. OX-LDL also increased Bcl-2 expression and decreased Bax expression significantly. The Bcl-2 to Bax ratio was elevated after OX-LDL treatment. Inhibition of ERK downregulated Bax and was associated with RPE apoptosis.Conclusions: Our data suggest that apoptosis induced by OX-LDL in RPE partly depends on Erk-Bax/Bcl-2 signaling pathway activation. These results may provide further information regarding the effects of OX-LDL in human RPE and their potential role in AMD pathogenesis.

Ceramide Mediates Ox-LDL-Induced Human Vascular Smooth Muscle Cell Calcification via p38 Mitogen-Activated Protein Kinase Signaling

Vascular calcification is associated with significant cardiovascular morbidity and mortality, and has been demonstrated as an actively regulated process resembling bone formation. Oxidized low density lipoprotein (Ox-LDL) has been identified as a regulatory factor involved in calcification of vascular smooth muscle cells (VSMCs). Additionally, over-expression of recombinant human neutral sphingomyelinase (N-SMase) has been shown to stimulate VSMC apoptosis, which plays an important role in the progression of vascular calcification. The aim of this study is to investigate whether ceramide regulates Ox-LDL-induced calcification of VSMCs via activation of p38 mitogen-activated protein kinase (MAPK) pathway. Ox-LDL increased the activity of N-SMase and the level of ceramide in cultured VSMCs. Calcification and the osteogenic transcription factor, Msx2 mRNA expression were reduced by N-SMase inhibitor, GW4869 in the presence of Ox-LDL. Usage of GW4869 inhibited Ox-LDL-induced apoptosis in VSMCs, an effect which was reversed by C2-ceramide. Additionally, C2-ceramide treatment accelerated VSMC calcification, with a concomitant increase in ALP activity. Furthermore, C2-ceramide treatment enhanced Ox-LDL-induced VSMC calcification. Addition of caspase inhibitor, ZVAD-fmk attenuated Ox-LDL-induced calcification. Both Ox-LDL and C2-ceramide treatment increased the phosphorylation of p38 MAPK. Inhibition of p38 MAPK by SB203580 attenuated Ox-LDL-induced calcification of VSMCs. These data suggest that Ox-LDL activates N-SMase-ceramide signaling pathway, and stimulates phosphorylation of p38 MAPK, leading to apoptosis in VSMCs, which initiates VSMC calcification.

Mitofusin2 decreases intracellular cholesterol of oxidized LDL-induced foam cells from rat vascular smooth muscle cells

Mitofusin2 (Mfn2) plays a pivotal role in the proliferation and apoptosis of vascular smooth muscle cells (VSMCs). The purpose of this study was to investigate the effects of Mfn2 on the trafficking of intracellular cholesterol in the foam cells derived from rat VSMCs (rVSMCs) and also to investigate the effects of Mfn2 on the expression of adenosine triphosphate-binding cassette subfamily A member 1 (ABCA1), adenosine triphosphate-binding cassette subfamily G member 1 (ABCG1) and peroxisome proliferator-activated receptor gamma (PPARγ). The rVSMCs were co-cultured with oxidized low density lipoprotein (LDL, 80 μg/mL) to produce foam cells and cholesterol accumulation in cells. Before oxidized LDL treatment, different titers (20, 40 and 60 pfu/cell) of recombinant adenovirus containing Mfn2 gene (Adv-Mfn2) were added into the culture medium for 24 h to transfect the Mfn2 gene into the rVSMCs. Then the cells were harvested for analyses. The protein expression of Mfn2 was significantly higher in Adv-Mfn2-transfected group than in untransfected group (P<0.05), and the expression levels significantly increased when the titer of Adv-Mfn2 increased (P<0.05). At 24 or 48 h after oxidized LDL treatment, rVSMCs became irregular and their nuclei became larger, and their plasma abounded with red lipid droplets. However, the number of red lipid droplets was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group. At 48 h after oxidized LDL treatment, the intracellular cholesterol in rVSMCs was significantly increased (P<0.05), but it was significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05), and it also significantly decreased when the titer of Adv-Mfn2 increased (P<0.05). The mRNA and protein expression levels of ABCA1 and ABCG1 were significantly increased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05). Though the mRNA and protein expression levels of PPARγ was not significantly increased (P>0.05), the phosporylation levels of PPARγ were significantly decreased in Adv-Mfn2-transfected group as compared with untransfected group (P<0.05). These results suggest that the transfection of Adv-Mfn2 can significantly reduce intracellular cholesterol in oxidized LDL-induced rVSMCs possibly by decreasing PPARγ phosporylation and then increasing protein expression levels of ABCA1 and ABCG1, which may be helpful to suppress the formation of foam cells.

Oxidized LDL and lysophosphatidylcholine stimulate plasminogen activator inhibitor-1 expression through reactive oxygen species generation and ERK1/2 activation in 3T3-L1 adipocytes

Plasminogen activator inhibitor-1 (PAI-1) is secreted from adipose tissue and is considered to be a risk factor for both atherosclerosis and insulin resistance. Here we report for the first time that PAI-1 expression is enhanced by oxidized low-density lipoprotein (OxLDL) and its lipid component lysophosphatidylcholine (LPC) in mouse 3T3-L1 adipocytes. In fully differentiated 3T3-L1 cells, OxLDL treatment increased the mRNA expression and protein secretion of PAI-1 in a dose- and time-dependent manner, whereas native LDL had no effect. The addition of an anti-CD36 antibody suppressed OxLDL-stimulated PAI-1 expression by 50%, suggesting that adipose-derived CD36 contributes to roughly half of the PAI-1 expression stimulated by OxLDL. In addition, pharmacological experiments showed that the OxLDL-stimulated enhancement in PAI-1 expression was mediated through the generation of reactive oxygen species (ROS) and phosphorylation of extracellular signal-regulated kinase 1/2. Furthermore, LPC, a major lipid component of OxLDL, was responsible for the enhanced expression of PAI-1 as phospholipase A 2-treated acetyl LDL, which generates LPC, strongly stimulated PAI-1 expression, whereas acetyl LDL itself had no such activity. These data demonstrate that the uptake of OxLDL and, in particular, its lipid component LPC into adipocytes triggers aberrant ROS-mediated PAI-1 expression, which may be involved in the pathogenesis of metabolic syndrome.

Oxidized low density lipoprotein inhibits phosphate signaling and phosphate-induced mineralization in osteoblasts. Involvement of oxidative stress

Background It is well admitted that oxidized LDL (OxLDL) plays a major role in the generation and progression of atherosclerosis. Since atherosclerosis is often accompanied by osteoporosis, the effects of OxLDL on phosphate-induced osteoblast mineralization were investigated. Methods Calcium deposition, expression of osteoblast markers and inorganic phosphate (Pi) signaling were determined under OxLDL treatment. Results OxLDL, within the range of 10–50 μg protein/ml, inhibited Pi-induced UMR106 rat osteoblast mineralization. In parallel, the expression of Cbfa1/Runx2 transcription factor was decreased, and the intracellular level of the osteoblast marker osteopontin (OPN) was reduced. The extracellular level of another marker, receptor activator of nuclear factor kappa B ligand (RANKL), was also diminished. OxLDL inhibited Pi signaling via ERK/JNK kinases and AP1/CREB transcription factors. OxLDL triggered the generation of reactive oxygen species (ROS), either in the absence or presence of Pi. Furthermore, the effects of OxLDL on Pi-induced mineralization, generation of ROS and extracellular level OPN were reproduced by the lipid extract of the particle, whereas the antioxidant vitamin E prevented them. Conclusions This work demonstrates that OxLDL, by generation of an oxidative stress, inhibits of Pi signaling and impairs Pi-induced osteoblast differentiation. General significance This highlights the role of OxLDL in bone remodeling and in degenerative disorders other than atherosclerosis, especially in osteoporosis.

LOX-1-dependent transcriptional regulation in response to oxidized LDL treatment of human aortic endothelial cells

Oxidized low-density lipoprotein (OxLDL) has been implicated as a proatherogenic factor with a pathological role in the induction of endothelial dysfunction. Endothelial cells bind and uptake OxLDL primarily through the scavenger receptor lectin-like oxidized-low-density lipoprotein receptor-1 (LOX-1), which is believed to mediate critical effects of OxLDL in endothelial cells. To examine the biological events following LOX-1 activation by OxLDL, we used cDNA microarray analysis to globally analyze gene expression changes induced by OxLDL treatment of human aortic endothelial cell line (HAECT) cells overexpressing LOX-1. Consistent with reported functions of OxLDL, in control HAECT cells, OxLDL elicited gene changes in the oxidative stress pathway and other signaling pathways related to OxLDL. With OxLDL treatment, LOX-1-dependent gene expression changes associated with inflammation, cell adhesion, and signal transduction were observed. The transcripts of a number of cytokines and chemokines were induced, which included interleukin-8, CXCL2, CXCL3, and colony-stimulating factor-3. The secretion of these cytokines was confirmed by enzyme-linked immunosorbent assay analysis. In addition, our data revealed a novel link between LOX-1 and a number of genes, including Delta/notch-like epidermal growth factor repeat containing, stanniocalcin-1, cAMP response element modulator, and dual specificity phosphatase 1. Promoter analysis on the genes that changed as a result of LOX-1 activation by OxLDL allowed us to identify early growth response 1 and cAMP response element-binding protein as potential novel transcription factors that function downstream of LOX-1. Our study has enabled us to elucidate the gene expression changes following OxLDL activation of LOX-1 in endothelial cells and discover novel downstream targets for LOX-1.