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1.
Life Sci ; 239: 117000, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31654747

RESUMO

AIMS: ß2-glycoprotein I/anti-ß2-glycoprotein I antibody complex (ß2/aß2) could promote oxLDL-induced endothelial inflammation through Toll-like receptor 4 (TLR4), therefore accelerates atherosclerosis in patients with anti-phospholipid syndrome (APS). However, effects of ß2/aß2 and TLR4 on oxLDL-induced CD36 activation in macrophages remain to be elucidated and are currently under investigation. MATERIALS AND METHODS: THP-1 macrophages with or without the pre-treatment of TAK-242, a TLR4 inhibitor, were treated with RPMI 1640, oxLDL, oxLDL+ß2/aß2 or oxLDL + LPS.CD36 expression and subsequent intracellular lipid accumulation, cholesterol-transportation-related proteins (ACAT1, ABCG1 and ABCA1) expression, inflammatory cytokines (IL-1ß, TNF-α and IL-6) secretion, focal adhesion kinases (FAK) activation and matrix metalloproteinases (MMP-2 and MMP-9) expression by these THP-1 macrophages were evaluated. Moreover, effects of TLR4 on oxLDL+ß2/aß2-induced peroxisome proliferators-activated receptor-γ (PPAR-γ) expression and CD36 translocation have also been observed. KEY FINDINGS: Compared with oxLDL-treated ones, CD36 expression, intracellular lipid accumulation and FAK activation were inhibited, whereas the levels of inflammatory cytokines and MMPs were upregulated in THP-1 macrophages treated with oxLDL+ß2/aß2 (p < 0.05). Moreover, observed differences between oxLDL-treated and oxLDL+ß2/aß2-treated THP-1 macrophages could be reversed by TAK-242 pre-treatment (p < 0.05). Furthermore, oxLDL+ß2/aß2 promoted PPAR-γ expression and CD36 cytoplasmic translocation in THP-1 macrophages, these effects could also be attenuated by TAK-242 (p < 0.05). SIGNIFICANCE: Through a TLR4 dependent manner, ß2/aß2 inhibited oxLDL-induced CD36 expression, lipid accumulation and FAK activation, while promoted inflammatory cytokines and MMPs expression in THP-1 macrophages, indicating the novel dual roles played by ß2/aß2 in APS-related atherosclerosis.


Assuntos
Antígenos CD36/genética , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , beta 2-Glicoproteína I/antagonistas & inibidores , beta 2-Glicoproteína I/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Colesterol/metabolismo , Citocinas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Humanos , Macrófagos/metabolismo , Metaloproteinases da Matriz/metabolismo , PPAR gama/biossíntese , Sulfonamidas/farmacologia , Receptor 4 Toll-Like/antagonistas & inibidores , beta 2-Glicoproteína I/imunologia
2.
Artif Cells Nanomed Biotechnol ; 47(1): 2775-2782, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31284768

RESUMO

Atherosclerosis is a chronic inflammatory disease of the blood vasculature. Endothelial dysfunction is an early event in the development of atherosclerosis and the endothelium plays an important role in the innate immune defense in the pathology of cardiovascular diseases. New therapies are being developed based on the involvement of the immune system in atherosclerosis. In this study, we demonstrate that a commonly used anti-rheumatic drug, tofacitinib, possesses vascular protective properties in cultured primary human aortic endothelial cells (HAECs). Tofacitinib ameliorates oxidized low-density lipoprotein (ox-LDL)-induced adhesion of THP-1 cells to HAECs, suppresses the expression of vascular adhesion molecules and production of cytokines, including vascular cell adhesion molecule 1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). Moreover, tofacitinib inhibits elevation of endothelial lectin-like ox-LDL receptor-1 (LOX-1) and production of reactive oxygen species (ROS) triggered by ox-LDL. As a result, the presence of tofacitinib reduces ox-LDL-induced cytotoxicity and improves endothelial viability. Mechanistically, we demonstrate that tofacitinib suppresses ox-LDL-mediated activation of NF-κB inhibitor α (IκB-α), accumulation of nuclear p65 and activation of nuclear factor κB (NF-κB) promoter, indicating that tofacitinib inhibits NF-κB activation. Collectively, our data support that tofacitinib possesses a novel protective function in endothelial cells, implying that tofacitinib could have the therapeutic potential to modulate inflammation in cardiovascular diseases.


Assuntos
Adesão Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Linhagem Celular , Citoproteção/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , L-Lactato Desidrogenase/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo
3.
Chin Med J (Engl) ; 132(14): 1713-1722, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31268904

RESUMO

BACKGROUND: Macrophage accumulation in the vascular wall is a hallmark of atherosclerosis. Studies showed that shifting of oxidized lipids-induced inflammatory macrophages towards an anti-inflammatory phenotype by promoting oxidative metabolism attenuated atherosclerosis progression. Therefore, this study aimed to investigate whether metformin, which has ameliorated atherosclerosis in animal models and clinical trials, modulated oxidized low-density lipoprotein (Ox-LDL) induced inflammatory status in macrophages by regulating cellular oxidative metabolism. METHODS: Murine raw264.7 macrophages were incubated with Ox-LDL (50 µg/mL) in the presence or absence of metformin (15 µmol/L) for 24 h. Real-time polymerase chain reaction was used to quantify the transcription of classically activated (M1) pro-inflammatory and alternatively activated (M2) anti-inflammatory markers and mitochondrial DNA copy numbers. Cellular reactive oxygen species (ROS) production and mitochondrial membrane potential were detected by immunofluorescence. Cellular adenosine triphosphate (ATP) synthesis, glucose uptake, and lactic acid production were measured by commercial kit and normalized to cellular lysates. Western blotting analysis was performed to detect the expression of mitochondrial fusion/fission related proteins, enzymes mediating lipid metabolism and signaling pathway of glucose transport. Differences between groups were analyzed using one-way analysis of variance. RESULTS: Metformin improved Ox-LDL-impaired anti-inflammatory phenotype in raw264.7 macrophages as shown by up-regulated transcription of anti-inflammatory markers including interleukin 10 (0.76 ±â€Š0.04 vs. 0.94 ±â€Š0.01, P = 0.003) and Resistin-like molecule alpha (0.67 ±â€Š0.08 vs. 1.78 ±â€Š0.34, P = 0.030). Conversely, Ox-LDL-diminished phosphorylation of Akt was up-regulated by metformin treatment (0.47 ±â€Š0.05 vs. 1.02 ±â€Š0.08, P = 0.040), associated with an improvement of mitochondrial function, characterized by decreased ROS generation (2.50 ±â€Š0.07 vs. 2.15 ±â€Š0.04, P = 0.040), increased lipid oxidation, and elevated cellular ATP production (0.026 ±â€Š0.001 vs. 0.035 ±â€Š0.003, P = 0.020). Moreover, metformin-mediated Akt activation increased Akt substrate of 160 kDa (AS160) phosphorylation (0.51 ±â€Š0.04 vs. 1.03 ±â€Š0.03, P = 0.0041), promoted membrane translocation of glucose transporter 1, and increased glucose influx into the cells (4.78 ±â€Š0.04 vs. 5.47 ±â€Š0.01, P < 0.001). CONCLUSION: This study suggested that targeting macrophage metabolism with new or existing drugs had therapeutic potential for the prevention and treatment of diabetes-accelerated atherosclerosis.


Assuntos
Lipoproteínas LDL/farmacologia , Metformina/farmacologia , Animais , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos
4.
Lipids Health Dis ; 18(1): 137, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182104

RESUMO

Breast cancer is a heterogeneous disease with increasing incidence and mortality and represents one of the most common cancer types worldwide. Low-density lipoprotein (LDL) is a complex particle composed of several proteins and lipids, which carries cholesterol into peripheral tissues and also affects the metabolism of fatty acids. Recent reports have indicated an emerging role of LDL in breast cancer, affecting cell proliferation and migration, thereby facilitating disease progression. However, controversy still exists among distinct types of breast cancer that can be affected by LDL. Classical therapeutic approaches, such as radiotherapy, chemotherapy, and lipid-lowering drugs were also reported as affecting LDL metabolism and content in breast cancer patients. Therefore, in this review we summarized and discussed the role of LDL in the development and treatment of breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Lipoproteínas LDL/metabolismo , Linhagem Celular Tumoral , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , Feminino , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos da radiação , Lipoproteínas LDL/farmacologia , Prognóstico
5.
Eur J Pharmacol ; 858: 172451, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31202806

RESUMO

Attachment of monocytes to endothelial cells is a major event in the pathogenesis of atherosclerosis and cardiovascular disease. As atherosclerosis is considered to be an inflammatory disease, increased expression of proinflammatory cytokines greatly contributes to endothelial dysfunction and atherogenesis. Additionally, attachment of monocytes to endothelial cells triggered by cellular adhesion molecules such as vascular cellular adhesion molecule 1 (VCAM-1) and E-selectin plays a vital role in the development of atherosclerotic plaques. Zinc therapy has been suggested as a potential strategy for countering atherosclerosis. In the present study, for the first time to our knowledge, we investigated the potential role of the GPR39 zinc-sensing receptor in mediating the adhesion of monocytes to endothelial cells, oxidative stress and inflammation in human aortic endothelial cells induced by oxidized low-density lipoprotein (ox-LDL). Our findings show that agonism of GPR39 by the selective agonist TC-G 1008 potently reversed the effects of ox-LDL including increased expression of proinflammatory cytokines and chemokines, markers of oxidative stress, and enhanced expression of cellular adhesion molecules. Importantly, we also show that this protective effect is mediated through the nuclear factor-κB (NF-κB) pathway. Taken together, our findings suggest a potential role of GPR39 as a novel therapeutic target for the treatment and prevention of atherosclerosis induced by ox-LDL.


Assuntos
Adesão Celular/efeitos dos fármacos , Células Endoteliais/citologia , Lipoproteínas LDL/farmacologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Pirimidinas/farmacologia , Receptores Acoplados a Proteínas-G/agonistas , Sulfonamidas/farmacologia , Linhagem Celular Tumoral , Citocinas/metabolismo , Selectina E/metabolismo , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Monócitos/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Biochimie ; 163: 152-162, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31199942

RESUMO

Extra-cellular signal regulated kinase-5 (Erk-5), a transcriptional activator and regulator of endothelial cells (ECs) homeostasis, has been implicated in shear stress-induced endothelial dysfunction (ED), however its role in oxidized low-density lipoprotein (oxLDL)- induced ED during metabolic stress is not known. Herein, regulation and function of Erk-5 in oxLDL-induced EC death, inflammation and dysfunction has been investigated. Primary Human Umbilical Vein Endothelial Cells (pHUVECs) were stimulated with oxLDL. MTT and Trypan blue exclusion assays to assess cell viability, RT-qPCR and Western blotting assays to determine expression of endothelial and inflammatory markers and ED mediators at mRNA and protein levels, respectively were performed. Monocyte adhesion assay was performed to examine monocytes adherence to oxLDL-stimulated pHUVECs. The exposure of oxLDL induced a dose- and time-dependent decrease in pHUVECs viability, which concurred with decreased Erk-5 expression. Further, oxLDL (100 µg/ml) decreased the expression of endothelial markers eNOS and vWF, and increased the expression of ICAM-1, at both mRNA and protein levels. SiRNA-mediated silencing of Erk-5 or its inhibition showed that changes in eNOS, vWF and ICAM-1 expression could be mediated through Erk-5. Furthermore, oxLDL decreased the levels of Erk-5's upstream regulator MEK5 and downstream regulators Mef2c and KLF2, which were similar to their expressions in Erk-5 silenced cells. Fisetin, a phytochemical and bioflavonoid, could reduce the effect of oxLDL in ECs by upregulating the expression of endothelial markers including Erk-5, and downregulating the expression of inflammation markers. These results suggest that Erk-5 could be a critical regulator of oxLDL-induced EC death, inflammation and dysfunction via downregulation of Erk-5/Mef2c-KLF2 signaling pathway, which can be ameliorated by a bioflavonoid, fisetin.


Assuntos
Flavonoides/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lipoproteínas LDL/toxicidade , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Monócitos/fisiologia , Transdução de Sinais , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/prevenção & controle , Células Cultivadas , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/prevenção & controle , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Lipoproteínas LDL/farmacologia , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Substâncias Protetoras/farmacologia
7.
Artif Cells Nanomed Biotechnol ; 47(1): 1839-1845, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31066305

RESUMO

Atherosclerosis is the chronic inflammatory disease, and inflammation-elicited endothelial activation is an early event in the development of atherosclerosis. The P2Y11 receptor is a purinergic receptor and a member of the P2 family of G coupled protein which has been shown to modulate vascular function. Progress in the study of purine receptors has been tremendous and these receptors have become pharmacological targets for various diseases. In this study, we show that the P2Y11R antagonist NF157 can mitigate oxidized LDL (ox-LDL)-induced endothelial inflammation. Our study demonstrates that P2Y11R is expressed to a fair degree in human aortic endothelial cells and is induced by treatment with ox-LDL. Blockage of P2Y11R by its selective antagonist NF157 ameliorates ox-LDL-induced adhesion of THP-1 monocytes to endothelial cells. NF157 inhibits ox-LDL-induced expression of adhesion molecules including E-selectin and VCAM-1. NF157 also suppresses ox-LDL-associated ROS production and induction of the NADPH oxidase subunit NOX-4. Moreover, NF157 has an inhibitory effect on the production of major cytokines including IL-6 and TNF-α. Mechanistically, we show that NF157 mitigates ox-LDL-induced phosphorylation of MAPK kinase p38 and NF-κB activation. Our findings indicate that blockage of P2Y11R signalling by its antagonist NF157 may protect endothelial cells from ox-LDL-induced endothelial inflammation. Therefore, NF157 may have therapeutic implications in the modulation of atherosclerosis-associated inflammation.


Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Receptores Purinérgicos P2/metabolismo , Suramina/análogos & derivados , Adesão Celular/efeitos dos fármacos , Selectina E/genética , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Inflamação/tratamento farmacológico , Interleucina-6/biossíntese , Monócitos/citologia , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Suramina/farmacologia , Suramina/uso terapêutico , Fator de Necrose Tumoral alfa/biossíntese , Molécula 1 de Adesão de Célula Vascular/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
J Microbiol ; 57(8): 711-716, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31089970

RESUMO

Low-density lipoprotein (LDL) was recently reported to be an opsonin, enhancing the phagocytosis of group A Streptococcus (GAS) by human monocytic leukemia U937 cells due to the binding of LDL to some GAS strains. We postulated that LDL might also promote the opsonophagocytosis of Pseudomonas aeruginosa by U937 cells since this bacterium interacts with LDL. In this study, P. aeruginosa (CMCC10104), U937 cells, and human LDL were used in phagocytosis assays to test our hypothesis. Escherichia coli strain BL21, which does not interact with LDL, was used as a negative control. Colony counting and fluorescence microscopy were used to determine the bacterial quantity in the opsonophagocytosis assays. After incubation of U937 cells and P. aeruginosa with LDL (100 µg/ml) for 15 and 30 min, phagocytosis was observed to be increased by 22.71% and 32.90%, respectively, compared to that seen in the LDL-free group. However, LDL did not increase the phagocytosis of E. coli by U937 cells. In addition, we identified CD36 as a major opsonin receptor on U937 cells, since an anti-CD36 monoclonal antibody, but not an anti-CD4 monoclonal antibody, almost completely abolished the opsonophagocytosis of P. aeruginosa by U937 cells.


Assuntos
Anticorpos Monoclonais/imunologia , Lipoproteínas LDL , Proteínas Opsonizantes/imunologia , Fagocitose , Pseudomonas aeruginosa/imunologia , Escherichia coli/metabolismo , Humanos , Lipoproteínas LDL/imunologia , Lipoproteínas LDL/farmacologia , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Células U937
9.
Reprod Domest Anim ; 54(8): 1131-1138, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31145501

RESUMO

The current study aimed to explore the potential usefulness of liquid or lyophilized egg yolk plasma (EYP) as a substitute for low-density lipoproteins (LDL) for cryopreservation of canine spermatozoa. In the first experiment, a total of 20 ejaculates harvested from six Beagles were frozen in extenders containing 6% LDL (control) or liquid or lyophilized EYP at one of three concentrations (20%, 40% or 60%). Motility parameters were assessed 10 min after thawing using computer-assisted sperm analysis. For both liquid and lyophilized EYP, the 40% concentration yielded motility similar (p > 0.05) to that observed with the control extender. In the second experiment, 12 ejaculates collected from the same six dogs were frozen in 6% LDL (Control), 40% liquid EYP or 40% lyophilized EYP extenders. Spermatozoal membrane integrity (hypo-osmotic swelling test [HOSt] and SYBR14/propidium iodide [PI] staining), acrosome integrity (FITC-Pisum sativum agglutinin staining) and DNA integrity (acridine orange staining) characteristics were evaluated 10 min after thawing. Both liquid and lyophilized 40% EYP-based extenders successfully preserved all assessed integrity parameters as efficiently as the control. Results of this study suggest that lyophilized EYP is a viable alternative to LDL in freezing extenders for dog semen.


Assuntos
Criopreservação/veterinária , Cães , Gema de Ovo/química , Lipoproteínas LDL/farmacologia , Preservação do Sêmen/veterinária , Espermatozoides/efeitos dos fármacos , Animais , Crioprotetores/farmacologia , Liofilização , Congelamento , Masculino , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Motilidade Espermática/efeitos dos fármacos
10.
Mol Biol Rep ; 46(4): 3809-3816, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31004300

RESUMO

The purpose of our research is to elucidate whether oxLDL activates P2X7R in cultured human podocytes and if the activation of P2X7R leads to podocyte apoptosis. Additionally, we explore the underlying mechanism involved in podocyte apoptosis. Immortalized human podocytes were incubated with oxLDL (80 µg/ml), P2X7R antagonist A438079 (10 µM), or the compound of A438079 and oxLDL for 48 h, respectively. Cellular apoptosis and ROS were evaluated using flow cytometer. P2X7R, Bax, and Caspase-3 protein expression were detected by western blot and immunofluorescence analysis.The expression of P2X7R, ROS, Bax, and Caspase-3 in human podocytes incubated with oxLDL was significantly up-regulated and was found to have higher intracellular lipid accumulation and podocyte apoptosis compared with the NC group. However, co-administration with A438079, ROS, Bax, and Caspase-3 expression both significantly down-regulate as well as lower lipid accumulation and cellular apoptosis in the oxLDL-induced podocyte group. We revealed that P2X7R is involved in the regulation of oxLDL-treated podocytes. Additionally, we found that the anti-apoptotic effect of A438079 is correlated with ROS, Bax, and Caspase-3 expression down-regulated.


Assuntos
Apoptose/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Podócitos/efeitos dos fármacos , Piridinas/farmacologia , Receptores Purinérgicos P2X7/metabolismo , Tetrazóis/farmacologia , Caspase 3/metabolismo , Linhagem Celular , Humanos , Podócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2X7/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo
11.
Eur J Pharmacol ; 858: 172338, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31029709

RESUMO

Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) has been validated as a potent protective agent of Atherosclerosis (AS). Here, we explored the molecular mechanism of MALAT1 exerting protective function in oxidized low-density lipoprotein (ox-LDL)-stimulated human umbilical vein endothelial cells (HUVECs). qRT-PCR assay was used to assess the expression of MALAT1 and miR-216a-5p. Western blot analysis was performed to detect LC3-I, LC3-II, Beclin-1 and p62 levels. The autophagosome formation was analyzed by immunofluorescence analysis. Cell apoptosis was measured by flow cytemotry and caspase 3 activity. Dual-luciferase reporter assay or RNA immunorecipitation assay was performed to verify the targeted interrelation between MALAT1 and miR-216a-5p, or miR-216a-5p and Beclin-1. Our data revealed that MALAT1 was upregulated and miR-216a-5p was downregulated in serum of AS patients and ox-LDL-treated HUVECs. ox-LDL treatment induced HUNECs autophagy. Moreover, MALAT1 enhanced autophagy and survival in ox-LDL-treated HUVECs. MALAT1 directly binded to miR-216a-5p and MALAT1 enhanced Beclin-1 expression by sponging miR-216a-5p. Also, miR-216-5p-mediated repressive effect on autophagy and survival was abrogated by MALAT1. Additionally, Beclin-1 knockdown inhibited autophagy and survival in ox-LDL-treated HUVECs. In all, our data suggested that MALAT1 might play a protective role at least partly by sponging miR-216a-5p and regulating Beclin-1, highlighting that MALAT1 might be a potential therapeutic target of AS.


Assuntos
Autofagia/efeitos dos fármacos , Proteína Beclina-1/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , MicroRNAs/genética , RNA Longo não Codificante/genética , Aterosclerose/genética , Aterosclerose/patologia , Autofagia/genética , Proteína Beclina-1/deficiência , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Feminino , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade
12.
EMBO J ; 38(11)2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31028084

RESUMO

Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL-4-activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL-4-activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti-inflammatory to a pro-inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo Altogether, we identified that MSR1 signals through JNK via K63 polyubiquitylation and provides evidence for the receptor's involvement in macrophage polarization.


Assuntos
Inflamação , Interleucina-4/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Ativação de Macrófagos , Receptores Depuradores Classe A/agonistas , Receptores Depuradores Classe A/genética , Animais , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/genética , Células Cultivadas , Feminino , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Lipólise/efeitos dos fármacos , Lipólise/genética , Lipoproteínas LDL/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/genética , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose/efeitos dos fármacos , Fagocitose/genética , Polissacarídeos/farmacologia , Processamento de Proteína Pós-Traducional/genética , Células RAW 264.7 , Receptores Depuradores Classe A/química , Receptores Depuradores Classe A/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ubiquitinação/genética
13.
Acta Biochim Biophys Sin (Shanghai) ; 51(5): 471-483, 2019 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-30950489

RESUMO

Sortilin is closely associated with hyperlipidemia and the risk of atherosclerosis (AS). The role of sortilin and the underlying mechanism in peripheral macrophage are not fully understood. In this study, we investigated the effect of macrophage sortilin on ATP-binding cassette transporter A1 (ABCA1) expression, ABCA1-mediated cholesterol efflux, and aortic AS. Macrophage sortilin expression was upregulated by oxidized low-density lipoproteins (ox-LDLs) in both concentration- and time-dependent manners. Its expression reached the peak level when cells were incubated with 50 µg/ml ox-LDL for 24 h. Overexpression of sortilin in macrophage reduced cholesterol efflux, leading to an increase in intracellular total cholesterol, free cholesterol, and cholesterol ester. Sortilin was found to bind with ABCA1 protein and suppress macrophage ABCA1 expression, resulting in a decrease in cholesterol efflux from macrophages. The inhibitory effect of sortilin in cholesterol efflux was partially reversed by treatment with chloroquine, a lysosomal inhibitor. On the contrary, the ABCA1 protein level and ABCA1-mediated cholesterol efflux is increased by sortilin short hairpin RNA transfection. The fecal and biliary cholesterol 3H-sterol from cholesterol-laden mouse peritoneal macrophage was reduced by sortilin overexpression through lentivirus vector (LV)-sortilin in low-density lipoprotein receptor knockout mice, which was prevented by co-treatment with chloroquine. Treatment with LV-sortilin reduced plasma high-density lipoprotein and increased plasma ox-LDL levels. Accordingly, aortic lipid deposition and plaque area were exacerbated, and ABCA1 expression was reduced in mice in response to infection with LV-sortilin alone. These effects of LV-sortilin were partially reversed by chloroquine. Sortilin enhances lysosomal degradation of ABCA1 protein and suppresses ABCA1-mediated cholesterol efflux from macrophages, leading to foam cell formation and AS development.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aterosclerose/metabolismo , Colesterol/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Aterosclerose/genética , Células Cultivadas , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Interferência de RNA , Receptores de LDL/genética , Receptores de LDL/metabolismo , Células THP-1
14.
Int J Mol Sci ; 20(6)2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893912

RESUMO

Age-related macular degeneration is the main cause of vision loss in the aged population worldwide. Drusen, extracellular lesions formed underneath the retinal pigment epithelial (RPE) cells, are a clinical feature of AMD and associated with AMD progression. RPE cells support photoreceptor function by providing nutrition, phagocytosing outer segments and removing metabolic waste. Dysfunction and death of RPE cells are early features of AMD. The translocator protein, TSPO, plays an important role in RPE cholesterol efflux and loss of TSPO results in increased intracellular lipid accumulation and reactive oxygen species (ROS) production. This study aimed to investigate the impact of TSPO knockout on RPE cellular metabolism by identifying the metabolic differences between wildtype and knockout RPE cells, with or without treatment with oxidized low density lipoprotein (oxLDL). Using liquid chromatography mass spectrometry (LC/MS), we differentiated several metabolic pathways among wildtype and knockout cells. Lipids amongst other intracellular metabolites were the most influenced by loss of TSPO and/or oxLDL treatment. Glucose, amino acid and nucleotide metabolism was also affected. TSPO deletion led to up-regulation of fatty acids and glycerophospholipids, which in turn possibly affected the cell membrane fluidity and stability. Higher levels of glutathione disulphide (GSSG) were found in TSPO knockout RPE cells, suggesting TSPO regulates mitochondrial-mediated oxidative stress. These data provide biochemical insights into TSPO-associated function in RPE cells and may shed light on disease mechanisms in AMD.


Assuntos
Células Epiteliais/metabolismo , Deleção de Genes , Metabolômica , Receptores de GABA/genética , Epitélio Pigmentado da Retina/citologia , Linhagem Celular , Análise Discriminante , Células Epiteliais/efeitos dos fármacos , Glucose/metabolismo , Dissulfeto de Glutationa/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Metaboloma/efeitos dos fármacos , Nucleotídeos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Análise de Componente Principal , Receptores de GABA/metabolismo
15.
Mol Biol Rep ; 46(3): 3487-3496, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30847850

RESUMO

Bone abnormalities as a consequence of osteoblast deregulation are associated with several diseases such as diabetes and chronic kidney disease. Important role for oxidized low density lipoproteins (oxLDL) in the pathophysiology of bone disorders has been reported. However, little is known about the effects and mechanisms of oxLDL on the process of osteoblastogenesis in human mesenchymal stem cells (MSCs). We show that oxLDL concentrations of ~ 10-25 µg protein (0.43-1.0 µM MDA/mg protein) inhibited the differentiation of MSCs to osteoblasts. We demonstrate that the underlying mechanism entails the suppression of the Wnt signaling through the down-regulation of ß-catenin. Further, we show the association of scavenger receptor CD36 with the receptors LRP5/6 and Frizzled in mediating the oxLDL effects on the differentiation of MSCs to pre-osteoblasts. Inhibiting CD36 restored osteoblasts differentiation in the presence of oxLDL. Our findings suggest that oxLDL interferes with the canonical Wnt signaling pathway in a CD36 dependent manner leading to an inhibition of osteoblastogenesis.


Assuntos
Antígenos CD36/metabolismo , Lipoproteínas LDL/farmacologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lipoproteínas LDL/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Proteínas Wnt/metabolismo
16.
Nano Lett ; 19(4): 2562-2567, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30848605

RESUMO

The fundamental task of lipoprotein particles is extracellular transport of cholesterol, lipids, and fatty acids. Besides, cholesterol-rich apoB-containing lipoprotein particles (i.e., low density lipoprotein LDL) are key players in progression of atherosclerotic cardiovascular disease and are associated with familial hypercholesterolemia (FH). So far, lipoprotein particle binding to the cell membrane and subsequent cargo transfer is directly linked to the lipoprotein receptors on the target cell surface. However, our observations showed that lipoprotein particle cargo transport takes place even in the absence of the receptor. This finding suggests that an alternative mechanism for lipoprotein-particle/membrane interaction, besides the receptor-mediated one, exists. Here, we combined several complementary biophysical techniques to obtain a comprehensive view on the nonreceptor mediated LDL-particle/membrane. We applied a combination of atomic force and single-molecule-sensitive fluorescence microscopy (AFM and SMFM) to investigate the LDL particle interaction with membranes of increasing complexity. We observed direct transfer of fluorescently labeled amphiphilic lipid molecules from LDL particles into the pure lipid bilayer. We further confirmed cargo transfer by fluorescence cross-correlation spectroscopy (FCCS) and spectral imaging of environment-sensitive probes. Moreover, the integration of the LDL particle into the membranes was directly visualized by high-speed atomic force microscopy (HS-AFM) and cryo-electron microscopy (cryo-EM). Overall, our data show that lipoprotein particles are able to incorporate into lipid membranes upon contact to transfer their cargo in the absence of specific receptors.


Assuntos
Membrana Celular/ultraestrutura , Doença da Artéria Coronariana/patologia , Hiperlipoproteinemia Tipo II/metabolismo , Lipoproteínas LDL/química , Apolipoproteínas B/química , Fenômenos Biofísicos , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Doença da Artéria Coronariana/metabolismo , Microscopia Crioeletrônica , Progressão da Doença , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Humanos , Hiperlipoproteinemia Tipo II/patologia , Bicamadas Lipídicas/química , Lipoproteínas LDL/farmacologia , Lipoproteínas LDL/ultraestrutura , Microscopia de Força Atômica
17.
Eur J Pharmacol ; 852: 99-106, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30851273

RESUMO

Metformin has been suggested to have cardiovascular protective effects. Previous researches showed that metformin activates Adenosine Monophosphate Activated Protein Kinase (AMPK) and Protein Phosphatase 2A (PP2A). This research aimed to elucidate whether and how metformin affects NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activity in oxidized low-density lipoprotein (ox-LDL) stimulated macrophages. Macrophages were treated with ox-LDL and metformin in a continuous manner, and NLRP3 inflammasome activation was evaluated by detecting IL-1ß and caspase-1 p10 release by ELISA and western blot, respectively. AMPK α1 and α2 gene expression was silenced in macrophages by siRNA transduction. Expression of NLRP3 and pro-IL-1ß was monitored by RT-qPCR and western blot. PP2A activity was inhibited by LB-100 treatment. Activation of NF-κB signaling was evaluated by detecting the nuclear accumulation of p65 and phosphorylation of IκBα by western blot. Activation of Tristetraprolin was evaluated by detecting its serine phosphorylation level by immunoprecipitation and western blot. In the results, upregulation of NLRP3 protein expression and NLRP3 inflammasome activation induced by ox-LDL treatment in macrophages were significantly attenuated by metformin treatment. AMPK gene silencing partially rescued NLRP3 inflammasome activation. Inhibition of PP2A significantly restored NLRP3 and pro-IL-1ß protein expression level downregulated by metformin in ox-LDL-stimulated macrophages. PP2A catalytic activity was required for NF-κB inhibition and Tristetraprolin activation induced by metformin in ox-LDL-stimulated macrophages. Our data showed Metformin reduced NLRP3 protein expression and NLRP3 inflammasome activation in ox-LDL-stimulated macrophages through AMPK and PP2A.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Inflamassomos/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Metformina/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína Fosfatase 2/metabolismo , Linhagem Celular , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos
18.
Lipids Health Dis ; 18(1): 62, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30871555

RESUMO

BACKGROUND: Endothelial-to-mesenchymal transition (EndMT) plays significant roles in atherosclerosis, but the regulatory mechanisms involving lncRNAs remain to be elucidated. Here we sort to identify the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in ox-LDL-induced EndMT. METHODS: The atherosclerosis model was established by feeding ApoE-/- mice with high-fat diet, and the levels of lncRNA MALAT1 in mouse arterial tissue were detected by RT-qPCR. Cell model was established by treating human umbilical vein endothelial cells (HUVECs) with ox-LDL, and the levels of EndMT markers, such as CD31, vWF, α-SMA and Vimentin and lncRNA MALAT1 levels were detected and their correlations were analyzed. The role of MALAT1 in EndMT and its dependence on Wnt/ß-catenin signaling pathway was further detected by knocking down or overexpressing MALAT1. RESULTS: MALAT1 was upregulated in high-fat food fed ApoE-/- mice. HUVECs treated with ox-LDL showed a significant decrease in expression of CD31 and vWF, a significant increase in expression of α-SMA and vimentin, and upregulated MALAT1. An increased MALAT1 level facilitated the nuclear translocation of ß-catenin induced by ox-LDL. Inhibition of MALAT1 expression reversed nuclear translocation of ß-catenin and EndMT. Moreover, overexpression of MALAT1 enhanced the effects of ox-LDL on HUVEC EndMT and Wnt/ß-catenin signaling activation. CONCLUSIONS: Our study revealed that the pathological EndMT required the activation of the MALAT1-dependent Wnt/ß-catenin signaling pathway, which may be important for the onset of atherosclerosis. TRIAL REGISTRATION: Not applicable.


Assuntos
Aterosclerose/genética , Transição Epitelial-Mesenquimal/genética , RNA Longo não Codificante/metabolismo , Animais , Apolipoproteínas E/genética , Aterosclerose/etiologia , Dieta Hiperlipídica/efeitos adversos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipoproteínas LDL/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
19.
Front Immunol ; 10: 13, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30728822

RESUMO

Objective: Damage and pathogen associated molecular patterns such as oxidized low-density lipoprotein (oxLDL) or bacillus Calmette-Guerin (BCG) vaccine can induce long term pro-inflammatory priming in monocytes and macrophages due to metabolic and epigenetic reprogramming-an emerging new concept called trained innate immunity. Vascular smooth muscle cells express pattern recognition receptors involved in trained innate immunity in monocytes. Here we investigated whether the mechanisms of trained innate immunity also control a proinflammatory phenotype in human coronary smooth muscle cells. Methods: Human coronary smooth muscle cells were primed with oxLDL or BCG for 24 h. After a resting time of 4 to 7 days, the cells were restimulated with either PAM3cys4, LPS or TNFα and cytokine production or mRNA expression were measured. Then, mechanisms of monocyte trained innate immunity were analyzed in smooth muscle cells, including receptors, intracellular pathways as well as metabolic and epigenetic reprogramming. Results: Priming with oxLDL or BCG lead to a significantly increased production of IL6, IL8 and MCP-1 following restimulation. OxLDL priming had little effect on the expression of macrophage or SMC marker genes. Proinflammatory priming of smooth muscle cells induced mTOR-HIF1α-signaling and could be blocked by mTOR-, TLR2-, and TLR4-inhibition. Finally, metabolic and epigenetic mechanisms of trained innate immunity in monocytes could be replicated in smooth muscle cells, including increased glucose consumption, lactate production, responsiveness to 6-fluoromevalonate and mevalonate treatment and inhibition of priming by the histone methyltransferase inhibitor methylthioadenosine (MTA). Conclusion: We demonstrate for the first time that mechanisms of the so called trained innate immunity control a proinflammatory phenotype in non-immune cells of the vascular wall. Our findings warrant further research into the specificity of trained innate immunity as an immune cell response as well as the mechanisms of vascular smooth muscle cells inflammation.


Assuntos
Imunidade Inata , Lipoproteínas LDL/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Vacina BCG/imunologia , Biomarcadores , Vasos Coronários , Citocinas/metabolismo , Expressão Gênica , Glucose/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Ácido Láctico/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/imunologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo
20.
Mol Nutr Food Res ; 63(10): e1800887, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30762936

RESUMO

SCOPE: HDL cholesterol is inversely related to the incidence of atherosclerosis. Polyphenols including ellagitannins have been shown to exert antiatherogenic properties. Urolithin B is formed from ellagitannins by components of the gut microbiota, and urolithins might be involved in beneficial effects against cardiovascular diseases in vitro. In this study, the influence of urolithin B on several parameters involved in the lipid plaque deposition and the reverse cholesterol transport is investigated. METHODS AND RESULTS: In apoE-/- mice and two different macrophage cell lines, the influence of urolithin B and its phase II conjugated metabolite on lipid plaque deposition, cholesterol uptake, and expression of ABCA1 and SR-BI is tested. It is shown that urolithin B decreases lipid plaque deposition, both urolithin B and urolithin B sulfate modulate expression of SR-BI and ABCA1, and cholesterol efflux increases from cholesterol laden macrophages to HDL particles as well as to reverse lipid uptake by stimulated THP-1 macrophages. CONCLUSIONS: Urolithin B can decrease lipid plaque deposition, and urolithin B and urolithin B sulfate are able to induce reverse cholesterol transport by influencing expression of key proteins of this pathway. Urolithin B may represent the basis for development of new drugs for prevention and treatment of atherosclerosis in humans.


Assuntos
Aterosclerose/prevenção & controle , Colesterol/metabolismo , Cumarínicos/farmacologia , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Placa Aterosclerótica/tratamento farmacológico , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Transporte Biológico , Linhagem Celular , Humanos , Metabolismo dos Lipídeos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Knockout para ApoE , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Receptores Depuradores Classe B/metabolismo
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