Different Concentrations Of ox-LDL Research Articles

Profile of IL-6 and TNF in Foam Cell Formation: An Improved Method Using Fluorescein Isothiocyanate (FITC) Probe.

ResumoFundamento A formação de células espumosas ocorre devido ao aumento em lipoproteína plasmática de baixa densidade (LDL) e desregulação da inflamação, sendo importante para o desenvolvimento da aterosclerose.Objetivo Avaliar o perfil do fator de necrose tumoral alfa (TNF-α) e da interleucina-6 (IL-6) no método de formação da célula espumosa existente, otimizando esse protocolo.Métodos A LDL foi isolada, oxidada e marcada com sonda de isotiocianato de fluoresceína (FITC). As células espumosas foram geradas de célula derivada de monócitos humanos THP-1 e incubadas na ausência (controle) ou presença de FITC-ox-LDL (10, 50, 100, 150 ou 200 μg/mL), por 12, 24, 48 ou 72 horas. A FITC-ox-LDL na célula foi quantificada por microscopia. O ensaio de imunoabsorção enzimática foi avaliado para quantificar a IL-6 e o TNF-α, com um p <0,05 considerado significativo.Resultados Todas as concentrações de FITC-ox-LDL testadas apresentaram fluorescência mais alta em comparação com o controle, demonstrando maior acúmulo de lipoproteínas nas células. Quanto mais alta a concentração de FITC-ox-LDL, maior a produção de TNF-α e IL-6. A produção de IL-6 pelas células espumosas foi detectada até o valor de 150 µg/mL da LDL máxima de estímulo. Concentrações acima de 50 μg/mL de LDL estimularam maior liberação de TNF-α comparado ao controle.Conclusões Nosso modelo contribui para o entendimento da liberação de IL-6 e TNF-α em resposta a várias concentrações de ox-LDL usando o método otimizado para a formação de células espumosas.

ZAKβ Alleviates Oxidized Low-density Lipoprotein (ox-LDL)-Induced Apoptosis and B-type Natriuretic Peptide (BNP) Upregulation in Cardiomyoblast.

Oxidized low-density lipoprotein (ox-LDL) is a type of modified cholesterol that promotes apoptosis and inflammation and advances the progression of heart failure. Leucine-zipper and sterile-α motif kinase (ZAK) is a kinase of the MAP3K family which is highly expressed in the heart and encodes two variants, ZAKα and ZAKβ. Our previous study serendipitously found opposite effects of ZAKα and ZAKβ in which ZAKβ antagonizes ZAKα-induced apoptosis and hypertrophy of the heart. This study aims to test the hypothesis of whether ZAKα and ZAKβ are involved in the damaging effects of ox-LDL in the cardiomyoblast. Cardiomyoblast cells H9c2 were treated with different concentrations of ox-LDL. Cell viability and apoptosis were measured by MTT and TUNEL assay, respectively. Western blot was used to detect apoptosis, hypertrophy, and pro-survival signaling proteins. Plasmid transfection, pharmacological inhibition with D2825, and siRNA transfection were utilized to upregulate or downregulate ZAKβ, respectively. Ox-LDL concentration-dependently reduces the viability and expression of several pro-survival proteins, such as phospho-PI3K, phospho-Akt, and Bcl-xL. Furthermore, ox-LDL increases cleaved caspase-3, cleaved caspase-9 as indicators of apoptosis and increases B-type natriuretic peptide (BNP) as an indicator of hypertrophy. Overexpression of ZAKβ by plasmid transfection attenuates apoptosis and prevents upregulation of BNP. Importantly, these effects were abolished by inhibiting ZAKβ either by D2825 or siZAKβ application. Our results suggest that ZAKβ upregulation in response to ox-LDL treatment confers protective effects on cardiomyoblast.

Ox-LDL-Induced Endothelial Progenitor Cell Oxidative Stress via p38/Keap1/Nrf2 Pathway.

Background Nrf2 which was recently reported to regulate the antioxidant genes and cellular redox regulators was highly expressed in EPCs. However, its role in ox-LDL-induced EPC oxidative stress and apoptosis has not been fully illustrated. Methods EPCs isolated from human peripheral blood mononuclear cells were treated with different concentrations of ox-LDL, Keap1 siRNA, and a specific p38 MAPK inhibitor SB203580 and then used to assay the cytoplasmic Nrf2, nuclear Nrf2, NAD(P) H:quinone oxidoreductase 1 (NQO1) and Bax/Bcl-2 levels with Western blot, NQO1 mRNA levels with RT-PCR, ROS levels with H2DCF-DA, loss/disruption of mitochondrial membrane potential with JC-1, apoptosis with Annexin V and PI, migration with transwell chambers, and tube formation with Matrigel. Results ox-LDL decreased the nuclear Nrf2/Histone H3 to cytoplasmic Nrf2/GAPDH ratio, NQO1 mRNA, and protein levels. ox-LDL enhanced ROS production, induced the loss of membrane potential, and increased the cell shrinkage, pyknotic nuclei, and apoptosis of EPCs. Keap1 siRNA increased Nrf2 nuclear translocation, NQO1 mRNA transcription, and protein expression and prevented ROS generation and formation of JC-1 monomers. ox-LDL increased the activation of p38. SB203580 significantly eliminated ox-LDL induced inhibition of Nrf2 nuclear translocation, depression of NQO1 mRNA transcription, generation of ROS, and formation of JC-1 monomers in EPCs. Keap1 siRNA decreased the Bax/Bcl-2 ratio which was increased by ox-LDL in EPCs. ox-LDL decreased EPC migration and tube formation. Keap1 siRNA preserved the migration and tube formation of EPCs. Conclusion ox-LDL activated EPCs p38/Keap1/Nrf2 pathway and induced oxidative stress, dysfunction, and apoptosis of EPCs.

Downregulated lncRNA UCA1 accelerates proliferation and migration of vascular smooth muscle cells by epigenetic regulation of MMP9.

Function of long non-coding RNA urothelial carcinoma antigen 1 (lncRNA UCA1) in regulating the proliferative and migratory abilities of vascular smooth muscle cells (VSMCs) by mediating matrix metalloproteinase-9 (MMP9) level were elucidated. After treatment with different concentrations of ox-LDL for different time points, lncRNA UCA1 level in VSMCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Subcellular distribution of UCA1 was analyzed. Proliferative and migratory abilities of VSMCs transfected with pcDNA-UCA1 were assessed. Protein level of MMP9 in HA-VSMCs treated with different concentrations of ox-LDL for different time points was also determined. The potential interaction between UCA1 and enhancer of zeste homolog 2 (EZH2) was identified by RNA immunoprecipitation (RIP) assay. Recruitment ability of EZH2 to MMP9 promoter region influenced by UCA1 was determined by Chromatin immunoprecipitation (ChIP) assay. Finally, the potential function of MMP9 in UCA1-mediated cellular behavior of VSMCs was explored. UCA1 was time-dependently and dose-dependently upregulated in VSMCs by ox-LDL treatment. Proliferative and migratory abilities of VSMCs were enhanced by treatment of 100 mg/l ox-LDL for 48 h, which were further reduced after transfection of pcDNA-UCA1. Subcellular distribution analysis showed that UCA1 was mainly distributed in the nucleus. Protein level of MMP9 was gradually elevated with the treatment of increased concentrations of ox-LDL in VSMCs. Its level was downregulated by transfection of pcDNA-UCA1 in VSMCs. The interaction between UCA1 and EZH2 was confirmed by RIP assay. Transfection of pcDNA-UCA1 stimulated the binding of EZH2 on MMP9 promoter region. Finally, overexpression of MMP9 reversed the decreased proliferative and migratory abilities in ox-LDL-treated VSMCs overexpressing UCA1. Downregulated UCA1 accelerates VSMCs to proliferate and migrate through negatively regulating MMP9 level.

PP2A alleviates oxidized LDL-induced endothelial dysfunction by regulating LOX-1/ROS/MAPK axis

AimsThe purpose of this study is to investigate the effect of PP2A on the progression of AS and the special molecular mechanism. Main methodsThe expression of PP2A in Human umbilical vein endothelial cells (HUVECs) induced by different concentrations of Ox-LDL was measured by RT-PCR and Western blot. The binding activity of PP2A and LOX-1 was determined by CoIP assay. Western blot was used to measure the protein expression of VCAM-1, ICAM-1 and MCP-1. Key findingThe results revealed that the expression of PP2A was decreased with the increase of Ox-LDL concentration in HUVECs. Overexpression of PP2A alleviated Ox-LDL-induced dysfunction and inflammatory response in HUVECs. The results of Co-immunoprecipitation (CoIP) showed that PP2A had direct effect on LOX-1, and PP2A inhibited the expression of LOX-1. In addition, overexpression of LOX-1 reversed the inhibitory effect of PP2A on Ox-LDL-induced dysfunction and inflammatory response in HUVECs. What is more, PP2A inhibited LOX-1/ROS/MAPK axis. Significanceit suggests that PP2A alleviates Ox-LDL-induced dysfunction and inflammatory response of HUVECs potentially by regulating the LOX-1/ROS/MAPK axis，which suggests that PP2A has anti-inflammatory effect during the formation of as, and the molecular therapy of PP2A provides a theoretical basis.

Albiflorin inhibits the formation of THP-1-derived foam cells through the LOX-1/NF-κB pathway

Foam cells are characteristic pathologic cells of atherosclerosis (AS), they are lipid-loaded macrophages present on atherosclerotic lesions. A large number of studies has shown that the pathogenesis of AS is the result of interactions between the lipid metabolism disorders and chronic inflammatory responses in the body. Albiflorin can inhibit the inflammatory response and it has shown a therapeutic effect on certain inflammatory diseases. In this study, a human acute monocytic leukemia cell line (THP-1)-derived foam cell model was established via oxidized low-density lipoprotein (ox-LDL) to observe the effects of albiflorin on the AS-characteristic foam cells. Our results showed that, after the treatment with ox-LDL, macrophages induced by propylene glycol methyl ether acetate (PMA), presented large amounts of lipid deposition in their cytoplasm, indicating that the THP-1-derived foam cell model was successfully established. On the other hand, the same cells pretreated with albiflorin presented significantly reduced amounts of lipid deposition, and their contents of total cholesterol and triglyceride were also clearly lower. Besides, the expression levels of low-density lipoprotein receptor-1 (LOX-1) and nuclear factor-κB (NF-κB) were significantly decreased, and the expression levels of downstream factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also obviously decreased in the cells treated with albiflorin but not in the negative control cells. Moreover, after treatment of macrophages with different concentrations of ox-LDL, the expression levels of LOX-1 and NF-κB were up-regulated in an ox-LDL concentration-dependent manner, and so were the expression levels of IL-6 and TNF-α. And, it was found after treatment with LOX-1 neutralizing antibody or NF-κB inhibitor (during the foam cell formation induction via ox-LDL) that the lipid deposition in the cytoplasm of the cells was reduced, as in the cells treated with albiflorin. Taken together, our findings suggest that albiflorin decreases lipid deposition in the cytoplasm and blocks the foaming process by regulating the LOX-1/NF-κB signaling pathway.

PCSK9 Promotes oxLDL-Induced PC12 Cell Apoptosis Through the Bcl-2/Bax-Caspase 9/3 Signaling Pathway.

Hyperlipidemia is a risk factor for neurodegenerative diseases. Proprotein convertase subtilisin / Kexin type 9 (PCSK9) degrades hepatic low-density lipoprotein receptor (LDLR) to regulate lipid metabolism. It is unclear if PCSK9 plays a role in neurodegenerative diseases. This study was designed to determine whether PCSK9 is crucial between hyperlipidemia and Alzheimer's disease. The interrelationship between PCSK9 and neuronal apoptosis was explored in PC12 cells in response to treatment with oxidized low-density lipoprotein (oxLDL). Cultured PC12 cells were serum-starved and incubated with different concentrations of oxLDL for 24 h. Intracytoplasmic lipid droplets were observed by oil red O staining. Morphological assessment of apoptotic cells was performed using Hoechst 33258 staining and flow cytometry analysis. The expression of mRNA and protein was detected by reverse-transcription polymerase chain reaction (RT-PCR) and western blot analyses, respectively. Transfection of small interfering RNA (siRNA) into PC12 cells was conducted using HiperFect Transfection Reagent. Concentrations of Aβ40 and Aβ42 were detected by enzyme-linked immunosorbent assay (ELISA) kit. Intracellular lipid content, the number of apoptotic cells, and PCSK9 expression were increased in PC12 cells after oxLDL treatment. Transfection with PCSK9 siRNA reduced the oxLDL-induced apoptosis of PC12 cells. We further confirmed the involvement of Bcl-2/Bax-Caspase (9, 3) signaling pathway in the regulation of PC12 cells apoptosis.β-Secretase 1, another target gene of PCSK9, was downregulated in PC12 cells in response to oxLDL treatment. Aβ40 and Aβ42 contents were also decreased. PCSK9 promotes oxLDL-induced PC12 cell apoptosis through the Bcl-2/Bax-Caspase 9/3 signaling pathway.

Th17/Treg cell expression in children with primary nephritic syndrome and the effects of ox-LDL on Th17/Treg cells.

To investigate the role of T-helper cells/Treg (Th17/Treg) and morbidity factors related to primary nephritic syndrome (PNS) in children, as well as the influence of ox-low density lipoprotein (ox-LDL) on Th17/Treg expression in children with PNS. To clarify the pathogenesis of PNS in children, 50 children with PNS treated in our hospital were enrolled in the study group. Additionally, 20 healthy children who came to our hospital for physical examination during the same period were enrolled in the control group. Th17 and Treg cells in children belonging to the two groups were detected by flow cytometry; the numbers of Th17/Treg cells in peripheral blood mononuclear cells at different concentrations of ox-LDL were detected simultaneously. Ox-LDL can affect the number of Th17/Treg cells in peripheral blood mononuclear cells, and both cell types decreased with increasing concentration of ox-LDL, with the numbers being significantly lower in the control group. However, the decrease in the number of Th17 cells was statistically insignificant (P > 0.05), whereas the decrease in Treg cells was more obvious and statistically significant (P < 0.05). The effect of ox-LDL the number of Treg cells was stronger than that on Th17 cells. We concluded that the imbalance of Th17/Treg cells influenced by high and low ox-LDL concentrations in children with PNS might be the immunological basis of the disease.

LOX-1 in macrophage migration in response to ox-LDL and the involvement of calpains

Previous studies have shown that oxidized low-density lipoprotein (ox-LDL) inhibits macrophage migration, but the precise mechanisms remain unclear. Lectin-like ox-LDL receptor-1 (LOX-1) is a scavenger receptor that is expressed in macrophages and binds ox-LDL. Calpains, a family of calcium-dependent proteases, influence several aspects of cell migration. In this study, we investigated the role of LOX-1 in macrophage migration in response to ox-LDL and the involvement of calpains in this process. Peritoneal macrophages from wild type C57BL/6 mice were exposed to different concentrations of ox-LDL (1–20 μg/mL), and expression of LOX-1 and calpain-1 and -2, cell migration and intracellular calcium (Ca2+in) were measured. Our results showed that ox-LDL stimulated LOX-1 and calpain-2 expression, and inhibited calpain-1 expression in a dose- and time-dependent manner. Further, ox-LDL inhibited macrophage migration and increased Ca2+in concentration in macrophages. To further elucidate the role of LOX-1 in ox-LDL-impaired macrophage migration, we isolated peritoneal macrophages from LOX-1 knockout mice, and treated them with ox-LDL. Interestingly, calpain-1 expression was much higher, and calpain-2 expression was lower in LOX-1 knockout macrophages than in wild-type macrophages following exposure to ox-LDL. LOX-1 deletion significantly improved macrophage migration and decreased Ca2+in concentration. These data indicate that LOX-1 is, at least in part, responsible for the inhibitory effect of ox-LDL on macrophage migration and this process involves calpain-1 and -2.

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.

THE EFFECT OF OXIDISED LOW-DENSITY LIPOPROTEIN ON NOTCH

ObjectivesTo explore the expression of Notch signal and cytokines by oxidised low-density lipoprotein (ox-LDL) in macrophages of human acute monocytic leukaemia cell line (THP1) and to search for possible mechanism...

THE EFFECT OF OXIDISED LOW-DENSITY LIPOPROTEIN ON NOTCH EXPRESSION IN THP1 MACROPHAGES

ObjectivesTo explore the expression of Notch signal and cytokines by oxidised low-density lipoprotein (ox-LDL) in macrophages of human acute monocytic leukaemia cell line (THP1) and to search for possible mechanism...

Elevated Circulating Interleukin-27 in Patients with Coronary Artery Disease Is Associated with Dendritic Cells, Oxidized Low-Density Lipoprotein, and Severity of Coronary Artery Stenosis

Coronary artery disease (CAD) is an immune-mediated chronic inflammatory disease mainly caused by atherosclerosis. The aims of this study were to investigate the role of interleukin-27 (IL-27) in patients with CAD and the severity of coronary artery lesions, which was evaluated by Gensini score and to investigate the biosynthesis of IL-27 and oxidized low-density lipoprotein (ox-LDL) in vitro using monocyte-derived dendritic cells (DCs). To this aim, plasma levels of IL-27, ox-LDL, and Gensini score were analyzed in patients with CAD (n = 136) and normal subjects (controls, n = 29). IL-27 concentration of the supernatant and the mRNA expression levels of p28 and ebi3, subunits of IL-27, from cultured immature DCs incubated with different concentrations of ox-LDL for 24 h were also analyzed. We found that circulating IL-27 levels were significantly elevated in patients with CAD than in controls (P < 0.01), and positively correlated to ox-LDL and Gensini score. ox-LDL dose-dependently upregulated expression of both IL-27 protein and IL-27 (p28 and EBI3) mRNA in vitro, indicating that ox-LDL can stimulate DCs to produce IL-27. These results demonstrate that IL-27 might regulate the network of immunity and inflammation in the pathogenesis of atherosclerosis.

JAK-STAT SIGNALLING PATHWAY PROTEINS ARE OVER EXPRESSED IN ARTERIAL HUMAN ENDOTHELIAL CELLS EXPOSED TO OXIDIZED LOW DENSITY LIPOPROTEINS: PP.6.243

Objective: Oxidative modification of low density lipoprotein (oxLDL) is one of the earliest events in atherosclerosis. This pathology is known as a chronic and inflammatory process that is characterized by the accumulation of lipids, inflammatory cells, and fibrous tissue. Our aim was to analyze the effect of oxLDL on gene expression profile in human umbilical artery endothelial cells (HUAEC), looking for new genes involved in the endothelial immflamatory process. Design and Method: Genome U133 Plus 2.0 microarray analysis were performed in HUAEC exposed to oxLDL (100 μg/ml) for 24 hours. Data were analyzed with Gepas, Bioconductor, and FatiScan software, and biological processes were obtained with Ingenuity Pathway Analysis and Pathway Studio. Microarrays validation experiments were performed with RT-PCR and Western blot analysis in cells exposed to different concentrations of oxLDL or native LDL (nLDL) for different incubation times (up to 24 hours). Results: Treatment with oxLDL affected the expression of 201 probes: 142 were up-regulated and 59 down-regulated. The most altered categories were lipid metabolism, immune response and atherosclerosis induction functions. Many of the most up-regulated genes were part of the JAK-STAT signalling pathway and interferon induced proteins. RT-PCR and Western blot confirmed a dose-dependent and time-dependent induction of JAK-STAT signalling pathway by oxLDL, whereas remained unaffected when exposed to nLDL. Conclusions: In this study we provide solid evidences about the gene expression regulation modulated by oxLDL in arterial endothelial cells. Moreover, we described the importance of the JAK-STAT pathway in endothelial cell biology altered after exposure to oxLDL, which in turn could lead to increased cell apoptosis.

Influence of oxidized low-density lipoprotein on siglec-1 expression and cytokine secretion by U937 cells

Objective:To explore the role of sialic acid-binding immunoglobulin-like lectin-one ( Siglec-1) in oxidized low-density lipoprotein ( ox-LDL)-induced atherosclerotic inflammation. Methods:Ox-LDL was prepared by oxidization of native LDL; different concentrations of ox-LDL were used to treat U937 cells for 48 h. Cells and supernatants were collected. The expression of Siglec-1 protein and mRNA was examined by flow cytometry ( FCM) and real-time quantitative RT-PCR,respectively. The levels of MIP-1α,MCP-1 and IL -8 in the supernatants were determined by ELISA. Results:Stimulation with ox-LDL significantly increased Siglec-1 protein and mRNA in U937 cells compared with the control group( P 0. 01). Meanwhile,the levels of MIP-1α,MCP-1 and IL-8 in the supernatants were also significantly increased in a concentration-dependent manner compared with those in the control group( P 0. 01). Conclusion:The proatherogenic effect of ox-LDL may be partly through up-regulating Siglec-1 expression in macrophages and enhancing the expression of inflammatory cytokines and chemokines. The activated macrophages recruit more macrophages and lymphocytes to the site of atherosclerotic plaques and exacerbate the inflammatory responses.

Oxidized low density lipoprotein‐induced transdifferentiation of bone marrow‐derived smooth muscle‐like cells into foam‐like cells in vitro

Oxidized-low density lipoprotein (ox-LDL) is believed to contribute to atherogenesis in part by being taken up into smooth muscle cells (SMC) via specific scavenger receptors; however, it is not clear whether ox-LDL receptor(s) are expressed in bone marrow-derived smooth muscle-like cells (SMLCs) and whether they play a role in the process of SMLC development. Therefore, we examined the ox-LDL-induced transdifferentiation of SMLCs that is mediated by lectin-like ox-LDL receptor-1 (LOX-1). Smooth muscle progenitor cells (SMPCs) from bone marrow mesenchymal stem cells (BMSCs) were isolated using a tissue-specific promoter sorting method with a mouse SM22_ promoter (_480 bp)/green fluorescent protein recombinant plasmid. The SMPCs were myocardin+CD105+KDR+CD45(-)CD34(-), but did not express SMC-specific markers alpha-smooth muscle actin (alpha-SMA), SM22, smooth muscle myosin heavy chain (SM-MHC) and smoothelin. After long-term culture with platelet-derived growth factor-BB (PDGF-BB), SMPCs expressed alpha-SMA, SM22 and SM-MHC and differentiated into SMLCs. When SMLCs were incubated with different concentrations of ox-LDL, LOX-1 expression on the surface of SMLCs gradually increased with the increase of the ox-LDL concentration, but myocardin and SMC-specific marker genes decreased, accordingly. Furthermore, receptor-mediated endocytosis was enhanced and lipid droplets accumulated in the cytoplasm of SMLCs. A subpopulation of myocardin+CD105+KDR+CD45(-)CD34(-) SMPCs exist in BMSCs that can differentiate into SMLCs under induction with PDGF-BB. Moreover, LOX-1 contributes to the ox-LDL-induced transdifferentiation of bone marrow-derived SMLCs into foam-like cells.