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1.
PLoS One ; 17(2): e0259751, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35120132

RESUMO

BACKGROUND: Transgenic overexpression of apolipoprotein A-I (apoA1) has been shown to delay atherosclerosis lesion progression and promote lesion regression in mouse models; however, apoA1 is subject to oxidation by myeloperoxidase (MPO) and loss of function. The activity of oxidant resistant human apoA1 was compared to unmodified human apoA1 in mouse models of atherosclerosis progression and regression. METHODS AND RESULTS: Human apoA1 and the MPO oxidant resistant 4WF isoform transgenic mice were bred to LDL receptor deficient (LDLr KO) mice and fed a western-type diet. High level expression of these human apoA1 isoforms did not lead to increased HDL-cholesterol levels on the LDLr KO background. In males and females, lesion progression was studied over time, and both apoA1 and 4WF transgenic mice vs. LDLr KO mice had significant and similar delayed lesion progression and reduced non-HDL cholesterol. Using time points with equivalent lesion areas, lesion regression was initiated by feeding the mice a low-fat control diet containing a microsomal triglyceride transfer protein inhibitor for 7 weeks. Lesions regressed more in the male apoA1 and 4WF transgenics vs. the LDLr KO, but the 4WF isoform was not superior to the unmodified isoform in promoting lesion regression. CONCLUSIONS: Both human apoA1 and the 4WF MPO oxidant resistant apoA1 isoform delayed lesion progression and promoted lesion regression in LDLr KO mice, with more pronounced effects in males than females; moreover, the 4WF isoform functioned similarly to the unmodified human apoA1 isoform.


Assuntos
Apolipoproteína A-I
2.
Sci Rep ; 11(1): 10249, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986446

RESUMO

We have previously shown that the DBA/2J versus AKR/J mouse strain is associated with decreased autophagy-mediated lysosomal hydrolysis of cholesterol esters. Our objective was to determine differences in lysosome function in AKR/J and DBA/2J macrophages, and identify the responsible genes. Using a novel dual-labeled indicator of lysosome function, DBA/2J versus AKR/J bone marrow derived macrophages had significantly decreased lysosome function. We performed quantitative trait loci mapping of lysosome function in bone marrow macrophages from an AKR/J × DBA/2J strain intercross. Four distinct lysosome function loci were identified, which we named macrophage lysosome function modifier (Mlfm) Mlfm1 through Mlfm4. The strongest locus Mlfm1 harbors the Gpnmb gene, which has been shown to recruit autophagy protein light chain 3 to autophagosomes for lysosome fusion. The parental DBA/2J strain has a nonsense variant in Gpnmb. siRNA knockdown of Gpnmb in AKR/J macrophages decreased lysosome function, and Gpnmb deletion through CRISP/Cas9 editing in RAW 264.7 mouse macrophages also demonstrated a similar result. Furthermore, a DBA/2 substrain, called DBA/2J-Gpnmb+/SjJ, contains the wildtype Gpnmb gene, and macrophages from this Gpnmb-preserved DBA/2 substrain exhibited recovered lysosome function. In conclusion, we identified Gpnmb as a causal modifier gene of lysosome function in this strain pair.


Assuntos
Proteínas do Olho/genética , Lisossomos/metabolismo , Macrófagos/fisiologia , Glicoproteínas de Membrana/genética , Animais , Mapeamento Cromossômico/métodos , Proteínas do Olho/metabolismo , Feminino , Genes Modificadores/genética , Lisossomos/genética , Lisossomos/fisiologia , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos DBA , Locos de Características Quantitativas/genética
3.
Artigo em Inglês | MEDLINE | ID: mdl-33031913

RESUMO

We previously demonstrated that AKR vs. DBA/2 mouse bone marrow derived macrophages have higher levels of free cholesterol and lower levels of esterified cholesterol after cholesterol loading, and that AKR, but not DBA/2, macrophages induced C/EBP homologous protein (CHOP) expression after cholesterol loading. We earlier determined that the free and esterified cholesterol level effect is due to a truncation in the sterol O-acyltransferase 1 (Soat1) gene, encoding acetyl-coenzyme A acetyltransferase 1 (ACAT1). Here we examined the mechanism for the differential induction of CHOP by cholesterol loading. CHOP was induced in both strains after incubation with tunicamycin, indicating both strains have competent endoplasmic reticulum stress pathways. CHOP was induced when DBA/2 macrophages were cholesterol loaded in the presence of an ACAT inhibitor, indicating that the difference in free cholesterol levels were responsible for this strain effect. This finding was confirmed in macrophages derived from DBA/2 embryonic stem cells. Cholesterol loading of Soat1 gene edited cells, mimicking the AKR allele, led to increased free cholesterol levels and restored CHOP induction. The upstream pathway of free cholesterol induced endoplasmic reticulum stress was investigated; and, RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1 α protein kinase (IRE1α) pathways were required for maximal CHOP expression.


Assuntos
Colesterol/farmacologia , Estresse do Retículo Endoplasmático/genética , Macrófagos/metabolismo , Esterol O-Aciltransferase/genética , Fator de Transcrição CHOP/genética , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Feminino , Fêmur/citologia , Fêmur/metabolismo , Regulação da Expressão Gênica , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos AKR , Camundongos Endogâmicos DBA , Camundongos Knockout para ApoE , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Especificidade da Espécie , Esterol O-Aciltransferase/metabolismo , Fator de Transcrição CHOP/metabolismo , Tunicamicina/farmacologia , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
4.
Atherosclerosis ; 286: 71-78, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31102955

RESUMO

BACKGROUND AND AIMS: We previously demonstrated that Apoe-/- mice on DBA/2 vs. AKR genetic background have >10-fold larger atherosclerotic lesions. Prior quantitative trait locus mapping via strain intercrossing identified a region on chromosome 17, Ath26, as the strongest atherosclerosis-modifying locus. We aimed to confirm Ath26, identify candidate genes, and validate the candidate gene effects on atherosclerosis. METHODS: We bred chromosome 17 interval congenic mice to confirm that Ath26 locus contains atherosclerosis modifying gene(s). Bone marrow derived macrophage transcriptomics was performed to identify candidate genes at this locus whose expression was correlated with lesions in a strain intercross. The Cyp4f13 candidate gene was tested via a gene knockout approach and in vivo and ex vivo phenotype analyses. RESULTS: A congenic mouse strain containing the DBA/2 interval on chromosome 17 on the AKR Apoe-/- background demonstrated that this interval conferred increased lesion area. Transcriptomic analysis of bone marrow macrophages identified that expression of the Cyp4f13 gene, mapping to this locus, was highly associated with lesion area in an F2 cohort. AKR vs. DBA/2 macrophages had less Cyp4f13 mRNA expression, and their livers had lower leukotriene B4 (LTB4) 20-hydroxylase enzymatic activity. A Cyp4f13 knockout allele was bred onto the DBA/2 Apoe-/- background and this conferred less enzymatic activity, decreased macrophage migration in response to LTB4, and smaller aortic root atherosclerotic lesions. CONCLUSIONS: Allelic differences in the Cyp4f13 gene may in part be responsible for the Ath26 QTL conferring larger lesions in DBA/2 vs. AKR Apoe-/- mice.


Assuntos
Aterosclerose/genética , Cromossomos de Mamíferos/genética , Loci Gênicos , Animais , Feminino , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos DBA
5.
Arterioscler Thromb Vasc Biol ; 38(2): 292-303, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29301789

RESUMO

There are many differences in arterial diseases between men and women, including prevalence, clinical manifestations, treatments, and prognosis. The new policy of the National Institutes of Health, which requires the inclusion of sex as a biological variable for preclinical studies, aims to foster new mechanistic insights and to enhance our understanding of sex differences in human diseases. The purpose of this statement is to suggest guidelines for designing and reporting sex as a biological variable in animal models of atherosclerosis, thoracic and abdominal aortic aneurysms, and peripheral arterial disease. We briefly review sex differences of these human diseases and their animal models, followed by suggestions on experimental design and reporting of animal studies for these vascular pathologies.


Assuntos
Artérias/patologia , Pesquisa Biomédica/normas , Patologia/normas , Projetos de Pesquisa/normas , Doenças Vasculares/patologia , Animais , Pesquisa Biomédica/métodos , Consenso , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Fatores de Risco , Fatores Sexuais
6.
Arterioscler Thromb Vasc Biol ; 38(1): 83-91, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29097366

RESUMO

OBJECTIVE: Cholesterol metabolism is a dynamic process involving intracellular trafficking, cholesterol esterification, and cholesterol ester hydrolysis. Our objective was to identify genes that regulate macrophage cholesterol metabolism. APPROACHES AND RESULTS: We performed quantitative trait loci mapping of free and esterified cholesterol levels and the ratio of esterified to free cholesterol in acetylated low-density lipoprotein-loaded bone marrow-derived macrophages from an AKR×DBA/2 strain intercross. Ten distinct cholesterol modifier loci were identified, and bioinformatics was used to prioritize candidate genes. The strongest locus was located on distal chromosome 1, which we named Mcmm1 (macrophage cholesterol metabolism modifier 1). This locus harbors the Soat1 (sterol O-acyltransferase 1) gene, encoding Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), which esterifies free cholesterol. The parental AKR strain has an exon 2 deletion in Soat1, which leads to a 33 amino acid N-terminal truncation in ACAT1. CRISPR/Cas9 editing of DBA/2 embryonic stem cells was performed to replicate the AKR strain Soat1 exon 2 deletion, while leaving the remainder of the genome unaltered. DBA/2 stem cells and stem cells heterozygous and homozygous for the Soat1 exon 2 deletion were differentiated into macrophages and loaded with acetylated low-density lipoprotein. DBA/2 stem cell-derived macrophages accumulated less free cholesterol and more esterified cholesterol relative to cells heterozygous and homozygous for the Soat1 exon 2 deletion. CONCLUSIONS: A Soat1 deletion present in AKR mice, and resultant N-terminal ACAT1 truncation, was confirmed to be a significant modifier of macrophage cholesterol metabolism. Other Mcmm loci candidate genes were prioritized via bioinformatics.


Assuntos
Acetil-CoA C-Acetiltransferase/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Colesterol/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Genes Modificadores , Macrófagos/enzimologia , Locos de Características Quantitativas , Acetil-CoA C-Acetiltransferase/metabolismo , Animais , Proteína 9 Associada à CRISPR/metabolismo , Biologia Computacional , Cruzamentos Genéticos , Estudos de Associação Genética , Genótipo , Camundongos Endogâmicos AKR , Camundongos Endogâmicos DBA , Fenótipo , Especificidade da Espécie , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/metabolismo
7.
Autophagy ; 11(7): 1207-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26042659

RESUMO

Cholesterol confers unique biophysical properties to the plasma membrane bilayer that are essential for maintaining optimal membrane fluidity, which in turn regulate multiple physiological functions required to promote cellular integrity and viability. Conversely, excessive cholesterol causes pathological conditions such as atherosclerosis that can lead to heart attacks. Human atheroma macrophages carry a large burden of free cholesterol (FC) in addition to cholesterol esters. It is recognized that sterols can modulate the levels of other lipids to attain lipid homeostasis; thus, excess FC may play a role in modulating compensatory sphingolipid pathways. Recent studies have shown that excess lipids can cause ER stress and apoptosis. In contrast, autophagy may play a protective role by clearing excess lipids from macrophage foam cell lipid droplets. Interestingly, a macrophage study using a TLR4-specifc agonist showed that de novo sphingolipid biosynthesis is essential for autophagy induction, suggesting links between sphingolipid biosynthesis and autophagy. While the role of autophagy in removing excess lipids has been the focus of many studies, its role in fine-tuning cellular lipid homeostasis remains largely unexplored.


Assuntos
Autofagia , Colesterol/metabolismo , Proteínas de Membrana/metabolismo , Esfingomielinas/biossíntese , Modelos Biológicos
8.
Proc Natl Acad Sci U S A ; 112(12): 3728-33, 2015 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-25775599

RESUMO

Eukaryotic cells have evolved robust mechanisms to counter excess cholesterol including redistribution of lipids into different compartments and compensatory up-regulation of phospholipid biosynthesis. We demonstrate here that excess cellular cholesterol increased the activity of the endoplasmic reticulum (ER) enzyme serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme in sphingomyelin synthesis. This increased SPT activity was not due to altered levels of SPTLC1 or SPTLC2, the major subunits of SPT. Instead, cholesterol loading decreased the levels of ORMDL1, a negative regulator of SPT activity, due to its increased turnover. Several lines of evidence demonstrated that free-cholesterol-induced autophagy, which led to increased turnover of ORMDL1. Cholesterol loading induced ORMDL1 redistribution from the ER to cytoplasmic p62 positive autophagosomes. Coimmunoprecipitation analysis of cholesterol-loaded cells showed increased association between ORMDL1 and p62. The lysosomal inhibitor chloroquine or siRNA knockdown of Atg7 inhibited ORMDL1 degradation by cholesterol, whereas proteasome inhibitors showed no effect. ORMDL1 degradation was specific to free-cholesterol loading as autophagy induced by serum starvation or general ER stress did not lead to ORMDL1 degradation. ORMDL proteins are thus previously unidentified responders to excess cholesterol, exiting the ER to activate SPT and increase sphingomyelin biosynthesis, which may buffer excess cellular cholesterol.


Assuntos
Autofagia , Orosomucoide/metabolismo , Animais , Asma/metabolismo , Aterosclerose/metabolismo , Transporte Biológico , Linhagem Celular , Colesterol/metabolismo , Cicloeximida/química , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Eosinófilos/metabolismo , Homeostase , Lipídeos/química , Macrófagos/metabolismo , Microdomínios da Membrana/química , Proteínas de Membrana , Camundongos , Transporte Proteico , Serina C-Palmitoiltransferase/química , Esfingolipídeos/química , Esfingomielinas/química
9.
Cardiovasc Res ; 102(1): 157-65, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24525840

RESUMO

AIMS: The goal of this study was to determine whether the A1 adenosine receptor (AR) plays a role in atherosclerosis development and to explore its potential mechanisms. METHODS AND RESULTS: Double knockout (DKO) mice, deficient in the genes encoding A1 AR and apolipoprotein E (apoE), demonstrated reduced atherosclerotic lesions in aortic arch (en face), aortic root, and innominate arteries when compared with apoE-deficient mice (APOE-KO) of the same age. Treating APOE-KO with an A1 AR antagonist (DPCPX) also led to a concentration-dependent reduction in lesions. The total plasma cholesterol and triglyceride levels were not different between DKO and APOE-KO; however, higher triglyceride was observed in DKO fed a high-fat diet. DKO also had higher body weights than APOE-KO. Plasma cytokine concentrations (IL-5, IL-6, and IL-13) were significantly lower in DKO. Proliferating cell nuclear antigen expression was also significantly reduced in the aorta from DKO. Despite smaller lesions in DKO, the composition of the innominate artery lesion and cholesterol loading and efflux from bone marrow-derived macrophages of DKO were not different from APOE-KO. CONCLUSION: The A1 AR may play a role in the development of atherosclerosis, possibly due to its pro-inflammatory and mitogenic properties.


Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/metabolismo , Colesterol/metabolismo , Receptor A1 de Adenosina/metabolismo , Animais , Aorta/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor A1 de Adenosina/deficiência
10.
Arterioscler Thromb Vasc Biol ; 34(3): 533-42, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24436370

RESUMO

OBJECTIVE: Unresolved inflammatory response of macrophages plays a pivotal role in the pathogenesis of atherosclerosis. Previously we showed that ribosomal protein L13a-dependent translational silencing suppresses the synthesis of a cohort of inflammatory proteins in monocytes and macrophages. We also found that genetic abrogation of L13a expression in macrophages significantly compromised the resolution of inflammation in a mouse model of lipopolysaccharide-induced endotoxemia. However, its function in the pathogenesis of atherosclerosis is not known. Here, we examine whether L13a in macrophage has a protective role against high-fat diet-induced atherosclerosis. APPROACH AND RESULTS: We bred the macrophage-specific L13a knockout mice L13a Flox(+/+) Cre(+/+) onto apolipoprotein E-deficient background and generated the experimental double knockout mice L13a Flox(+/+) Cre(+/+) apolipoprotein E deficient (apoE(-/-)). L13a Flox(+/+) Cre(-/-) mice on apolipoprotein E-deficient background were used as controls. Control and knockout mice were subjected to high-fat diet for 10 weeks. Evaluation of aortic sinus sections and entire aorta by en face showed significantly higher atherosclerosis in the knockout mice. Severity of atherosclerosis in knockout mice was accompanied by thinning of the smooth muscle cell layer in the media, larger macrophage area in the intimal plaque region and higher plasma levels of inflammatory cytokines. In addition, macrophages isolated from knockout mice had higher polyribosomal abundance of several target mRNAs, thus showing defect in translation control. CONCLUSIONS: Our data demonstrate that loss of L13a in macrophages increases susceptibility to atherosclerosis in apolipoprotein E-deficient mice, revealing an important role of L13a-dependent translational control as an endogenous protection mechanism against atherosclerosis.


Assuntos
Doenças da Aorta/etiologia , Aterosclerose/etiologia , Inflamação/genética , Macrófagos/metabolismo , Biossíntese de Proteínas , Proteínas Ribossômicas/fisiologia , Animais , Doenças da Aorta/sangue , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Quimiotaxia de Leucócito , Colesterol/sangue , Citocinas/sangue , Gorduras na Dieta/toxicidade , Progressão da Doença , Predisposição Genética para Doença , Inflamação/complicações , Camundongos , Camundongos Knockout , Peritônio/patologia , Polirribossomos/metabolismo , RNA Mensageiro/análise , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/genética , Túnica Média/patologia
11.
PLoS One ; 8(5): e65003, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23705026

RESUMO

Cholesterol loaded macrophages in the arterial intima are the earliest histological evidence of atherosclerosis. Studies of mouse models of atherosclerosis have shown that the strain background can have a significant effect on lesion development. We have previously shown that DBA/2 ApoE(-/-) mice have aortic root lesions 10-fold larger than AKR ApoE(-/-) mice. The current study analyzes the response to cholesterol loading of macrophages from these two strains. Macrophages from the atherosclerosis susceptible DBA/2 strain had significantly higher levels of total and esterified cholesterol compared to atherosclerosis resistant AKR macrophages, while free cholesterol levels were higher in AKR cells. Gene expression profiles were obtained and data were analyzed for strain, cholesterol loading, and strain-cholesterol loading interaction effects by a fitted linear model. Pathway and transcriptional motif enrichment were identified by gene set enrichment analysis. In addition to observed strain differences in basal gene expression, we identified many transcripts whose expression was significantly altered in response to cholesterol loading, including P2ry13 and P2ry14, Trib3, Hyal1, Vegfa, Ccr5, Ly6a, and Ifit3. Eight pathways were significantly enriched in transcripts regulated by cholesterol loading, among which the lysosome and cytokine-cytokine receptor interaction pathways had the highest number of significantly regulated transcripts. Of the differentially regulated transcripts with a strain-cholesterol loading interaction effect, we identified three genes known to participate in the endoplasmic reticulum (ER) stress response, Ddit3, Trib3 and Atf4. These three transcripts were highly up-regulated by cholesterol in AKR and either down-regulated or unchanged in loaded DBA/2 macrophages, thus associating a robust ER stress response with atherosclerosis resistance. We identified significant transcripts with strain, loading, or strain-loading interaction effect that reside within previously described quantitative trait loci as atherosclerosis modifier candidate genes. In conclusion, we characterized several strain and cholesterol induced differences that may lead to new insights into cellular cholesterol metabolism and atherosclerosis.


Assuntos
Aterosclerose/genética , Colesterol/farmacologia , Estresse do Retículo Endoplasmático/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Lisossomos/metabolismo , Macrófagos/metabolismo , Animais , Aterosclerose/patologia , Western Blotting , Células da Medula Óssea/citologia , Análise por Conglomerados , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estudos de Associação Genética , Lipoproteínas LDL/metabolismo , Lisossomos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos DBA , Análise de Sequência com Séries de Oligonucleotídeos , Locos de Características Quantitativas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
12.
Arterioscler Thromb Vasc Biol ; 33(5): 903-10, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23493286

RESUMO

OBJECTIVE: DBA/2 apoE(-/-) mice have ≈10-fold larger lesions than AKR apoE(-/-) mice. The objective of this study was to determine whether macrophages from these 2 strains had altered cholesterol metabolism that might play a role in their divergent atherosclerosis susceptibility. APPROACH AND RESULTS: AKR and DBA/2 macrophages incubated with acetylated low-density lipoprotein resulted in higher cholesterol ester (CE) and lower free cholesterol accumulation in the DBA/2 cells. However, these strains had equivalent acetylated low-density lipoprotein uptake and cholesterol esterification activity. Cholesterol efflux from unloaded cells to apolipoprotein A-I or high-density lipoprotein was similar in the 2 strains. However, on acetylated low-density lipoprotein loading, cholesterol efflux was impaired in the DBA/2 cells, but this impairment was corrected by loading in the presence of an inhibitor of cholesterol esterification. Thus, the cholesterol efflux capabilities are similar in these strains, but there seemed to be a defect in lipid droplet-stored CE mobilization in DBA/2 cells. Lalistat 1, a specific inhibitor of lysosomal acid lipase, completely blocked the hydrolysis of lipid droplet-stored CE, implying that lipid droplet autophagy is responsible for CE turnover in these cells. CE turnover was 2-fold slower in DBA/2 versus AKR cells. Autophagic flux, estimated by a fluorescent light chain 3-II reporter and the increase in p62 levels after chloroquine treatment, was higher in AKR versus DBA/2 macrophages, which had an apparent decrease in autophagosome fusion with lysosomes. When autophagy was activated by amino acid starvation, CE levels decreased in DBA/2 cells. CONCLUSIONS: Physiological regulation of autophagy in macrophages controls CE accumulation and may modify atherosclerosis susceptibility.


Assuntos
Autofagia , Ésteres do Colesterol/metabolismo , Macrófagos/metabolismo , Animais , Colesterol/metabolismo , Hidrólise , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos DBA , Especificidade da Espécie , Esterol O-Aciltransferase/fisiologia
13.
Cancer Res ; 73(3): 1211-8, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23233737

RESUMO

Recent epidemiologic data show that low serum cholesterol level as well as statin use is associated with a decreased risk of developing aggressive or advanced prostate cancer, suggesting a role for cholesterol in aggressive prostate cancer development. Intracellular cholesterol promotes prostate cancer progression as a substrate for de novo androgen synthesis and through regulation of AKT signaling. By conducting next-generation sequencing-based DNA methylome analysis, we have discovered marked hypermethylation at the promoter of the major cellular cholesterol efflux transporter, ABCA1, in LNCaP prostate cancer cells. ABCA1 promoter hypermethylation renders the promoter unresponsive to transactivation and leads to elevated cholesterol levels in LNCaP. ABCA1 promoter hypermethylation is enriched in intermediate- to high-grade prostate cancers and not detectable in benign prostate. Remarkably, ABCA1 downregulation is evident in all prostate cancers examined, and expression levels are inversely correlated with Gleason grade. Our results suggest that cancer-specific ABCA1 hypermethylation and loss of protein expression direct high intracellular cholesterol levels and hence contribute to an environment conducive to tumor progression.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Colesterol/metabolismo , Homeostase , Neoplasias da Próstata/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Linhagem Celular Tumoral , Metilação de DNA , Humanos , Masculino , Regiões Promotoras Genéticas
14.
J Immunol ; 186(5): 2871-80, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21278342

RESUMO

Exciting discoveries related to IL-1R/TLR signaling in the development of atherosclerosis plaque have triggered intense interest in the molecular mechanisms by which innate immune signaling modulates the onset and development of atherosclerosis. Previous studies have clearly shown the definitive role of proinflammatory cytokine IL-1 in the development of atherosclerosis. Recent studies have provided direct evidence supporting a link between innate immunity and atherogenesis. Although it is still controversial about whether infectious pathogens contribute to cardiovascular diseases, direct genetic evidence indicates the importance of IL-1R/TLR signaling in atherogenesis. In this study, we examined the role of IL-1R-associated kinase 4 (IRAK4) kinase activity in modified low-density lipoprotein (LDL)-mediated signaling using bone marrow-derived macrophage as well as an in vivo model of atherosclerosis. First, we found that the IRAK4 kinase activity was required for modified LDL-induced NF-κB activation and expression of a subset of proinflammatory genes but not for the activation of MAPKs in bone marrow-derived macrophage. IRAK4 kinase-inactive knockin (IRAK4KI) mice were bred onto ApoE(-/-) mice to generate IRAK4KI/ApoE(-/-) mice. Importantly, the aortic sinus lesion formation was impaired in IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Furthermore, proinflammatory cytokine production was reduced in the aortic sinus region of IRAK4KI/ApoE(-/-) mice compared with that in ApoE(-/-) mice. Taken together, our results indicate that the IRAK4 kinase plays an important role in modified LDL-mediated signaling and the development of atherosclerosis, suggesting that pharmacological inhibition of IRAK4 kinase activity might be a feasible approach in the development of antiatherosclerosis drugs.


Assuntos
Aterosclerose/enzimologia , Aterosclerose/imunologia , Regulação da Expressão Gênica/imunologia , Mediadores da Inflamação/administração & dosagem , Quinases Associadas a Receptores de Interleucina-1/fisiologia , Lipoproteínas LDL/administração & dosagem , NF-kappa B/metabolismo , Acetilação , Animais , Aorta Torácica/enzimologia , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/patologia , LDL-Colesterol/metabolismo , LDL-Colesterol/fisiologia , Regulação da Expressão Gênica/genética , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/deficiência , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/fisiologia
15.
J Lipid Res ; 51(11): 3364-9, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20688754

RESUMO

A precise and sensitive method for measuring cellular free and esterified cholesterol is required in order to perform studies of macrophage cholesterol loading, metabolism, storage, and efflux. Until now, the use of an enzymatic cholesterol assay, commonly used for aqueous phase plasma cholesterol assays, has not been optimized for use with solid phase samples such as cells, due to inefficient solubilization of total cholesterol in enzyme compatible solvents. We present an efficient solubilization protocol compatible with an enzymatic cholesterol assay that does not require chemical saponification or chromatographic separation. Another issue with enzyme compatible solvents is the presence of endogenous peroxides that interfere with the enzymatic cholesterol assay. We overcame this obstacle by pretreatment of the reaction solution with the enzyme catalase, which consumed endogenous peroxides resulting in reduced background and increased sensitivity in our method. Finally, we demonstrated that this method for cholesterol quantification in macrophages yields results that are comparable to those measured by stable isotope dilution gas chromatography with mass spectrometry detection. In conclusion, we describe a sensitive, simple, and high-throughput enzymatic method to quantify cholesterol in complex matrices such as cells.


Assuntos
Colesterol/metabolismo , Enzimas/metabolismo , Células Espumosas/metabolismo , Macrófagos/metabolismo , Animais , Bovinos , Colesterol/isolamento & purificação , Fluorometria , Cromatografia Gasosa-Espectrometria de Massas , Reprodutibilidade dos Testes , Solventes/química
16.
Traffic ; 7(7): 811-23, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16787396

RESUMO

Cholesterol is one of the most essential membrane components in mammalian cells and plays a critical role in several cellular functions. It is now established that intracellular cholesterol transport contributes to the regulation of cellular cholesterol homeostasis by mechanisms that are yet poorly defined. In this study, we examined the role of clathrin- and dynamin-dependent trafficking on the regulatory machinery involved in cholesterol homeostasis. Thus, expression levels of three major sterol-sensitive genes, that is sterol-regulatory element binding protein 2 (SREBP-2), hydroxymethylglutaryl-coenzyme A (HMGCoA) reductase and low-density lipoprotein (LDL) receptor, were monitored to study the cell response to the addition of LDL-derived cholesterol. We found that inhibition of clathrin-dependent endocytosis had no effect on the intracellular distribution of cholesterol and the regulation of sterol-sensitive genes. In contrast, inhibition of dynamin activity resulted in the lack of regulation of SREBP-2, HMGCoA reductase and LDL receptor genes. Immunolocalization studies along with the measure of free and esterified cholesterol indicated that dynamin inactivation led to the accumulation of free cholesterol (FC) within the late endosomal (LE)/lysosomal compartment resulting in insufficient delivery of regulatory cholesterol to the endoplasmic reticulum (ER) where the transcriptional control of sterol-sensitive genes occurs. Our data therefore indicate that dynamin plays a critical role in the delivery of cholesterol from the LE/lysosomal network to the ER and highlight the importance of LE trafficking in cholesterol homeostasis.


Assuntos
Colesterol/metabolismo , Dinamina I/metabolismo , Endocitose , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , Transporte Biológico , Colesterol/farmacologia , Clatrina/metabolismo , Dinamina I/genética , Esterificação , Regulação da Expressão Gênica , Células HeLa , Homeostase , Humanos , Mutação/genética
17.
Atherosclerosis ; 171(2): 287-93, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14644399

RESUMO

Since elevated plasma triglycerides (TGs) are an independent cardiovascular risk factor, we have compared the cholesterol efflux potential of sera from asymptomatic hypertriglyceridemic (HTG) type IIb, type IV or normolipidemic (NLP) individuals using two different cell systems. In both type IIb and IV HTG, the efflux of cholesterol from SR-BI-rich Fu5AH cells was similar to that obtained with NLP. The maintenance of efflux efficiency in spite of reduced HDL-cholesterol levels can be mainly attributed to the relative enrichment of HDL with phospholipid. In the J774 macrophage cell system, pretreatment with cAMP, which upregulates ABCA1, induced a markedly higher increase in efflux to type IV sera compared with type IIb or NLP. In addition, type IV sera exhibited two-fold higher pre-beta HDL relative concentration (percentage of total apo AI) compared with NLP. Moreover, positive correlations were established between ABCA1-mediated efflux and the serum pre-beta HDL levels or TG concentrations. Thus, the hyperTGemia is associated with a higher fraction of apo AI recovered as pre-beta HDL which appear to be partly responsible for enhanced efflux obtained upon the cAMP stimulation of J774 cells. In conclusion, we demonstrated for the first time that the ABCA1-expressing J774 cell system is responsive to the percent of apo AI present in human serum as pre-beta HDL. Our results suggest that high-plasma TG, accompanied by low HDL may not result in an impaired cholesterol efflux capacity.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/metabolismo , AMP Cíclico/metabolismo , Hipertrigliceridemia/sangue , Macrófagos/metabolismo , Transportadores de Cassetes de Ligação de ATP/análise , Adulto , Idoso , Animais , Transporte Biológico/efeitos dos fármacos , Western Blotting , Estudos de Casos e Controles , Movimento Celular , Células Cultivadas , AMP Cíclico/farmacologia , Humanos , Hipertrigliceridemia/diagnóstico , Lipoproteínas HDL/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Probabilidade , RNA Mensageiro/análise , Estudos de Amostragem , Sensibilidade e Especificidade , Regulação para Cima
18.
Atherosclerosis ; 168(2): 381-7, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12801623

RESUMO

Sterol regulatory element binding proteins (SREBPs) are membrane-bound transcription factors that control the metabolism of cholesterol and fatty acids in mammalian cells. We postulated that polymorphisms (SNPs) in SREBP-2 gene might influence lipid parameters and the risk of coronary atherosclerosis. PCR-SSCP analysis and direct sequencing of DNA from 64 asymptomatic hypercholesterolemic men revealed seven genetic SREBP-2 SNPs. The genotype distribution of four of these SNPs (1668G>T, 1784G>C, 3474T>C and 3705C>T), and their influence on plasma lipid values and clinical parameters was studied in 655 asymptomatic men previously selected for the presence of at least one cardiovascular risk factor (hypertension, hypercholesterolemia, tobacco consumption). No significant relation was found with lipid parameters but there was a significant association between the 1784G>C polymorphism and intima-media thickness (IMT) measured in 497 subjects. Thus, a common variation in the SREBP-2 gene is related with early-stage carotid atherosclerosis in subjects with a risk of cardiovascular events without detectable change in plasma lipid levels.


Assuntos
Doenças das Artérias Carótidas/genética , Proteínas de Ligação a DNA/genética , Arteriosclerose Intracraniana/genética , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Adulto , Artérias Carótidas/diagnóstico por imagem , Artéria Femoral/diagnóstico por imagem , Frequência do Gene , Genótipo , Humanos , Hipercolesterolemia/genética , Hipertensão/genética , Desequilíbrio de Ligação , Lipídeos/sangue , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Fumar/genética , Proteína de Ligação a Elemento Regulador de Esterol 2 , Túnica Íntima/diagnóstico por imagem , Túnica Média/diagnóstico por imagem , Ultrassonografia
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