RESUMO
The citric acid cycle intermediate citrate plays a crucial role in metabolic processes such as fatty acid synthesis, glucose metabolism, and ß-oxidation. Citrate is imported from the circulation across the plasma membrane into liver cells mainly by the sodium-dependent citrate transporter (NaCT; SLC13A5). Deletion of NaCT from mice led to metabolic changes similar to caloric restriction; therefore, NaCT has been proposed as an attractive therapeutic target for the treatment of obesity and type 2 diabetes. In this study, we expressed mouse and human NaCT into Xenopus oocytes and examined some basic functional properties of those transporters. Interestingly, striking differences were found between mouse and human NaCT with respect to their sensitivities to citric acid cycle intermediates as substrates for these transporters. Mouse NaCT had at least 20- to 800-fold higher affinity for these intermediates than human NaCT. Mouse NaCT is fully active at physiologic plasma levels of citrate, but its human counterpart is not. Replacement of extracellular sodium by other monovalent cations revealed that human NaCT was markedly less dependent on extracellular sodium than mouse NaCT. The low sensitivity of human NaCT for citrate raises questions about the translatability of this target from the mouse to the human situation and raises doubts about the validity of this transporter as a therapeutic target for the treatment of metabolic diseases in humans.
Assuntos
Ciclo do Ácido Cítrico , Transportadores de Ácidos Dicarboxílicos/fisiologia , Simportadores/fisiologia , Animais , Cátions Monovalentes , Colina/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Feminino , Humanos , Lítio/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp , Potássio/metabolismo , Sódio/metabolismo , Especificidade por Substrato , Simportadores/genética , Xenopus laevisRESUMO
In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.
Assuntos
Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Zinco/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/síntese química , Histona Desacetilases/genética , Humanos , Modelos Moleculares , Estrutura Molecular , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Compostos Organometálicos/farmacologia , Oxidiazóis/química , Relação Estrutura-Atividade , Zinco/metabolismoRESUMO
OBJECTIVE: Acyl-CoA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters in plaque foam cells. Complete deficiency of macrophage ACAT has been shown to increase atherosclerosis in hypercholesterolemic mice because of cytotoxicity from free cholesterol accumulation, whereas we previously showed that partial ACAT inhibition by Fujirebio compound F1394 decreased early atherosclerosis development. In this report, we tested F1394 effects on preestablished, advanced lesions of apolipoprotein-E-deficient mice. METHODS AND RESULTS: Apolipoprotein-E-deficient mice on Western diet for 14 weeks developed advanced plaques, and were either euthanized (Baseline), or continued on Western diet with or without F1394 and euthanized after 14 more weeks. F1394 was not associated with systemic toxicity. Compared with the baseline group, lesion size progressed in both groups; however, F1394 significantly retarded plaque progression and reduced plaque macrophage, free and esterified cholesterol, and tissue factor contents compared with the untreated group. Apoptosis of plaque cells was not increased, consistent with the decrease in lesional free cholesterol. There was no increase in plaque necrosis and unimpaired efferocytosis (phagocytic clearance of apoptotic cells). The effects of F1394 were independent of changes in plasma cholesterol levels. CONCLUSIONS: Partial ACAT inhibition by F1394 lowered plaque cholesterol content and had other antiatherogenic effects in advanced lesions in apolipoprotein-E-deficient mice without overt systemic or plaque toxicity, suggesting the continued potential of ACAT inhibition for the clinical treatment of atherosclerosis, in spite of recent trial data.
Assuntos
Acetil-CoA C-Aciltransferase/antagonistas & inibidores , Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/tratamento farmacológico , Cicloexanos/farmacologia , Dioxanos/farmacologia , Inibidores Enzimáticos/farmacologia , Células Espumosas/efeitos dos fármacos , Acetil-CoA C-Aciltransferase/metabolismo , Animais , Aorta/enzimologia , Aorta/patologia , Doenças da Aorta/sangue , Doenças da Aorta/enzimologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apoptose/efeitos dos fármacos , Aterosclerose/sangue , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Colesterol/sangue , Dieta Aterogênica , Modelos Animais de Doenças , Progressão da Doença , Células Espumosas/enzimologia , Masculino , Camundongos , Camundongos Knockout , Necrose , Placa Aterosclerótica , Tromboplastina/metabolismoRESUMO
Growing evidence indicates that PPARγ agonists, including rosiglitazone (RSG), induce adipose mitochondrial biogenesis. By systematically analyzing mitochondrial gene expression in two common murine adipocyte models, the current study aimed to further establish the direct role of RSG and capture temporal changes in gene transcription. Microarray profiling revealed that in fully differentiated 3T3-L1 and C3H/10T1/2 adipocytes treated with RSG or DMSO vehicle for 1, 2, 4, 7, 24, and 48 hrs, RSG overwhelmingly increased mitochondrial gene transcripts time dependently. The timing of the increases was consistent with the cascade of organelle biogenesis, that is, initiated by induction of transcription factor(s), followed by increases in the biosynthesis machinery, and then by increases in functional components. The transcriptional increases were further validated by increased mitochondrial staining, citrate synthase activity, and O(2) consumption, and were found to be associated with increased adiponectin secretion. The work provided further insight on the mechanism of PPARγ-induced mitochondrial biogenesis in differentiated adipocytes.
RESUMO
HDL cholesterol (HDL-C) plasma levels are inversely related to cardiovascular disease risk. Previous studies have shown in animals and humans that HDL promotes regression of atherosclerosis. We hypothesized that this was related to an ability to promote the loss of monocyte-derived cells (CD68(+), primarily macrophages and macrophage foam cells) from plaques. To test this hypothesis, we used an established model of atherosclerosis regression in which plaque-bearing aortic arches from apolipoprotein E-deficient (apoE(-/-)) mice (low HDL-C, high non-HDL-C) were transplanted into recipient mice with differing levels of HDL-C and non-HDL-C: C57BL6 mice (normal HDL-C, low non-HDL-C), apoAI(-/-) mice (low HDL-C, low non-HDL-C), or apoE(-/-) mice transgenic for human apoAI (hAI/apoE(-/-); normal HDL-C, high non-HDL-C). Remarkably, despite persistent elevated non-HDL-C in hAI/apoE(-/-) recipients, plaque CD68(+) cell content decreased by >50% by 1 wk after transplantation, whereas there was little change in apoAI(-/-) recipient mice despite hypolipidemia. The decreased content of plaque CD68(+) cells after HDL-C normalization was associated with their emigration and induction of their chemokine receptor CCR7. Furthermore, in CD68(+) cells laser-captured from the plaques, normalization of HDL-C led to decreased expression of inflammatory factors and enrichment of markers of the M2 (tissue repair) macrophage state. Again, none of these beneficial changes were observed in the apoAI(-/-) recipients, suggesting a major requirement for reverse cholesterol transport for the beneficial effects of HDL. Overall, these results establish HDL as a regulator in vivo of the migratory and inflammatory properties of monocyte-derived cells in mouse atherosclerotic plaques, and highlight the phenotypic plasticity of these cells.
Assuntos
Aterosclerose/metabolismo , HDL-Colesterol/metabolismo , Células Espumosas/metabolismo , Monócitos/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Aorta/metabolismo , Aorta/transplante , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , HDL-Colesterol/genética , Modelos Animais de Doenças , Células Espumosas/patologia , Humanos , Camundongos , Camundongos Knockout , Monócitos/patologia , Receptores CCR7/genética , Receptores CCR7/metabolismoRESUMO
BACKGROUND: We previously showed that the progression of atherosclerosis in the Reversa mouse (Ldlr(-/-Apob100/100Mttpfl/fl) Mx1Cre(+/+)) was arrested when the hyperlipidemia was normalized by inactivating the gene for microsomal triglyceride transfer protein. Here, we tested whether atherosclerosis would regress if the lipid levels were reduced after advanced plaques formed. METHODS AND RESULTS: Reversa mice were fed an atherogenic diet for 16 weeks. Plasma lipid levels were then reduced. Within 2 weeks, this reduction led to decreased monocyte-derived (CD68(+)) cells in atherosclerotic plaques and was associated with emigration of these cells out of plaques. In addition, the fall in lipid levels was accompanied by lower plaque lipid content and by reduced expression in plaque CD68(+) cells of inflammatory genes and higher expression of genes for markers of antiinflammatory M2 macrophages. Plaque composition was affected more than plaque size, with the decreased content of lipid and CD68(+) cells balanced by a higher content of collagen. When the reduced lipid level was combined with the administration of pioglitazone to simulate the clinical aggressive lipid management and proliferator-activated receptor-γ agonist treatment, the rate of depletion of plaque CD68(+) cells was unaffected, but there was a further increase in their expression of antiinflammatory macrophage markers. CONCLUSION: The Reversa mouse is a new model of atherosclerosis regression. After lipid lowering, favorable changes in plaque composition were independent of changes in size. In addition, plaque CD68(+) cells became less inflammatory, an effect enhanced by treatment with pioglitazone.
Assuntos
Modelos Animais de Doenças , Genes de Troca/genética , Hipercolesterolemia/genética , Hipercolesterolemia/terapia , Macrófagos/patologia , Placa Aterosclerótica/genética , Placa Aterosclerótica/terapia , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Movimento Celular/genética , Marcação de Genes/métodos , Terapia Genética/métodos , Hipercolesterolemia/patologia , Inflamação/genética , Inflamação/patologia , Inflamação/terapia , Macrófagos/classificação , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microssomos/metabolismo , Monócitos/metabolismo , Monócitos/patologia , Placa Aterosclerótica/patologiaRESUMO
The objective of this study was to further establish and confirm the relationship of adipose mitochondrial biogenesis in diabetes/obesity and the effects of rosiglitazone (RSG), a peroxisome proliferator-activated receptor (PPAR) gamma agonist, by systematically analyzing mitochondrial gene expression and function in two mouse models of obesity and type 2 diabetes. Using microarray technology, adipose mitochondrial gene transcription was studied in db/db, high-fat diet-fed C57BL/6 (HFD) and respective control mice with or without RSG treatment. The findings were extended using mitochondrial staining, DNA quantification, and measurements of citrate synthase activity. In db/db and HFD mice, gene transcripts associated with mitochondrial ATP production, energy uncoupling, mitochondrial ribosomal proteins, outer and inner membrane translocases, and mitochondrial heat-shock proteins were decreased in abundance, compared with db/+ and standard-fat diet-fed control mice, respectively. RSG dose-dependently increased these transcripts in both db/db and HFD mice and induced transcription of mitochondrial structural proteins and cellular antioxidant enzymes responsible for removal of reactive oxygen species generated by increased mitochondrial activity. Transcription factors, including PPAR coactivator (PGC)-1beta, PGC-1alpha, estrogen-related receptor alpha, and PPARalpha, were suppressed in both models and induced by RSG. The effects of RSG on adipose mitochondrial genes were confirmed by quantitative RT-PCR and further supported by mitochondrial staining, mitochondrial DNA quantification, and citrate synthase activity. Adipose mitochondrial biogenesis was overwhelmingly suppressed in both mouse models of diabetes/obesity and globally induced by RSG. These findings suggest an important role of adipose mitochondria in diabetes/obesity and the potential for new treatment approaches targeting adipose mitochondria.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Mitocôndrias/efeitos dos fármacos , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Tecido Adiposo/metabolismo , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Gorduras na Dieta , Modelos Animais de Doenças , Expressão Gênica , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Obesidade/tratamento farmacológico , Rosiglitazona , Transcrição GênicaRESUMO
Central (visceral) obesity is more closely associated with insulin resistance, type 2 diabetes, and cardiovascular disease than peripheral (subcutaneous) obesity, however the underlying differences in morphology and pathophysiology between subcutaneous and visceral adipose are largely unknown. To evaluate the effects of diabetes and rosiglitazone (RSG) treatment, the expression of mitochondrial Hsp60, UCP-1 and F4/80 in inguinal subcutaneous (SC) fat, composed of white and brown adipose tissues, and epididymal (EP) fat, mainly white adipose tissue, were evaluated. In diabetic db/db mice, there was significant increased number of aggregated macrophage foci compared to db/+ mice, especially in EP fat. On the other hand, the expression of mitochondrial Hsp60 protein was suppressed in both SC and EP fat of db/db mice compared to db/+ mice, and the expression level of mitochondrial Hsp60 in db/+ mice was lower in EP fat compared with SC. In db/db mice, RSG suppressed the number of aggregated macrophage foci in EP fat, but not in SC fat. RSG ameliorated the mitochondrial Hsp60 expression and induced the expression of UCP-1 in both SC and EP fat. Taken together, these data suggest that differences exist in mitochondrial and macrophage content, and in the response to RSG between visceral and subcutaneous adipose tissue, and adipose type and distribution may be important for obesity-linked insulin resistance.
Assuntos
Diabetes Mellitus/metabolismo , Gordura Intra-Abdominal/metabolismo , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Gordura Subcutânea/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Glicemia/metabolismo , Peso Corporal , Contagem de Células , Forma Celular , Tamanho Celular , Chaperonina 60/metabolismo , Diabetes Mellitus/patologia , Modelos Animais de Doenças , Gordura Intra-Abdominal/patologia , Canais Iônicos/metabolismo , Macrófagos/citologia , Masculino , Camundongos , Proteínas Mitocondriais/metabolismo , Rosiglitazona , Gordura Subcutânea/patologia , Tiazolidinedionas/farmacologia , Proteína Desacopladora 1RESUMO
OBJECTIVE: Studies in vitro and in vivo of macrophage foam cells have shown evidence of cytotoxicity after acyl-CoA:cholesterol acyltransferase (ACAT) inhibition. Foam cells of smooth muscle origin are also found in human and animal atherosclerotic lesions. METHODS AND RESULTS: To study whether cytotoxicity from ACAT inhibition is independent of cell type, we first established a protocol to conveniently induce aortic smooth muscle foam cell formation using cholesterol-cyclodextrin complexes (CCC). Rat aortic smooth muscle cells (ASMCs) treated for 48 hours with CCC (20 microg/mL) became foam cells by morphological (oil-red-O staining) and biochemical (approximately 1200% and approximately 180% increase in cellular esterified and free cholesterol, respectively) criteria. ACAT activity increased 500% (P<0.01 versus untreated). Similar results were obtained in human ASMC, but ACAT activity increased to an even greater extent (3200%; P<0.01 versus untreated). Western blots indicated that CCC treatment increased human (to 380+/-20% of untreated, P<0.001), but not rat, ACAT protein expression. ACAT inhibition by Fujirebio compound F1394 suppressed CCC-induced foam cell formation in rat and human ASMC, but, notably, did not induce significant cytotoxicity. CONCLUSIONS: ASMC might be more resistant to FC-induced adverse effects than are macrophages.
Assuntos
Aorta/enzimologia , Cicloexanos/toxicidade , Dioxanos/toxicidade , Células Espumosas/metabolismo , Miócitos de Músculo Liso/enzimologia , Esterol O-Aciltransferase/antagonistas & inibidores , Animais , Aorta/citologia , Extratos Celulares/química , Células Cultivadas , Colesterol/metabolismo , Colesterol/farmacologia , Ciclodextrinas/metabolismo , Ciclodextrinas/farmacologia , Cicloexanos/farmacologia , Dioxanos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/toxicidade , Células Espumosas/química , Células Espumosas/efeitos dos fármacos , Células Espumosas/enzimologia , Humanos , Peso Molecular , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , Ratos , Esterol O-Aciltransferase/química , Esterol O-Aciltransferase/metabolismo , Esterol O-Aciltransferase/fisiologiaRESUMO
High LDL and/or low HDL are risk factors for atherosclerosis and are also a common clinical feature in systemic lupus erythematosus, rheumatoid arthritis, and psoriasis. Here, we show that changes in lipid profiles that reflect atherosclerotic disease led to activation of skin murine dendritic cells (DCs) locally, promoted dermal inflammation, and induced lymph node hypertrophy. Paradoxically, DC migration to lymph nodes was impaired, suppressing immunologic priming. Impaired migration resulted from inhibitory signals generated by platelet-activating factor (PAF) or oxidized LDL that acts as a PAF mimetic. Normal DC migration and priming was restored by HDL or HDL-associated PAF acetylhydrolase (PAFAH), which mediates inactivation of PAF and oxidized LDL. Thus, atherosclerotic changes can sequester activated DCs in the periphery where they may aggravate local inflammation even as they poorly carry out functions that require their migration to lymph nodes. In this context, HDL and PAFAH maintain a normally functional DC compartment.
Assuntos
Arteriosclerose/imunologia , Movimento Celular/imunologia , Células Dendríticas/imunologia , Hiperlipidemias/imunologia , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Animais , Apolipoproteínas E/deficiência , Arteriosclerose/fisiopatologia , HDL-Colesterol/metabolismo , Feminino , Humanos , Hiperlipidemias/fisiopatologia , Imuno-Histoquímica , Lipoproteínas LDL/metabolismo , Linfonodos/citologia , Linfonodos/imunologia , Masculino , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Fator de Ativação de Plaquetas/metabolismo , Receptores de LDL/deficiência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia , Pele/imunologia , Pele/patologiaRESUMO
OBJECTIVE: Protective properties of high-density lipoproteins (HDL) may include reverse cholesterol transport and suppression of oxidation and inflammation. These were investigated in vivo, as were the effects of HDL on the characteristics of atherosclerotic lesions. METHODS AND RESULTS: Male apolipoprotein E knockout (apoE-/-) and apoE-/- mice expressing human apolipoprotein AI (hAI/apoE-/-) were studied up to 20 weeks after commencing a high-fat diet. Plasma HDL cholesterol was twice as high in hAI/apoE-/- mice. Over time, aortic root lesion area remained less in hAI/apoE-/- mice, although plaques became complex. In advanced lesions, plaque lipid was higher in apoE-/- mice, whereas plaque collagen and alpha actin were increased in hAI/apoE-/- mice. In nonlesional aorta, mRNA abundance for pro-inflammatory proteins (vascular cell adhesion molecule [VCAM]-1, intercellular adhesion molecule-1 [ICAM-1], monocyte chemoattractant protein-1 [MCP-1]) increased between 4 and 16 weeks in apoE-/- (but not wild-type) mice, and were not reduced by elevated HDL. Autoantibodies to malondialdehyde low-density lipoprotein (LDL) increased progressively in apoE-/- mice, with similar results in hAI/apoE-/- mice. CONCLUSIONS: HDL retarded plaque size progression despite greatly elevated plasma cholesterol. This effect was over a wide range of lesion severity. Expression of hAI reduced plaque lipid and increased the proportion of plaque occupied by collagen and smooth muscle cells, but did not affect indicators of inflammation or LDL oxidation.
Assuntos
Arteriosclerose/prevenção & controle , Lipoproteínas HDL/fisiologia , Neovascularização Fisiológica/fisiologia , Animais , Apolipoproteína A-I/biossíntese , Apolipoproteína A-I/sangue , Apolipoproteínas E , Autoanticorpos , Quimiocina CCL2/metabolismo , Colesterol/sangue , Humanos , Inflamação/patologia , Molécula 1 de Adesão Intercelular/metabolismo , Lipoproteínas LDL/imunologia , Masculino , Malondialdeído/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , RNA Mensageiro/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Mouse aortic smooth muscle cells (SMCs) were loaded for 72 h with cholesterol by using cholesterol:methyl-beta-cyclodextrin complexes, leading to approximately 2-fold and approximately 10-fold increases in the contents of total cholesterol and cholesteryl ester, respectively. Foam-cell formation was demonstrated by accumulation of intracellular, Oil Red O-stained lipid droplets. Immunostaining showed decreased protein levels of smooth muscle alpha-actin and alpha-tropomyosin and increased levels of macrophage markers CD68 and Mac-2 antigen. Quantitative real-time RT-PCR revealed that after cholesterol loading, the expression of SMC-related genes alpha-actin, alpha-tropomyosin, myosin heavy chain, and calponin H1 decreased (to 11.5 +/- 0.5%, 29.3 +/- 1.4%, 23.8 +/- 1.4%, and 3.8 +/- 0.5% of unloaded cells, respectively; P < 0.05 for all), whereas expression of macrophage-related genes CD68, Mac-2, and ABCA1 mRNA increased (to 709 +/- 84%, 330 +/- 11%, and 207 +/- 13% of unloaded cells, respectively; P < 0.05 for all), thereby demonstrating that the protein changes were regulated at the mRNA level. Furthermore, these changes were accompanied by a gain in macrophage-like function as assessed by phagocytotic activity. Expression of vascular cell adhesion molecule 1 and monocyte chemoattractant protein 1, known responders to inflammation, were not changed. In conclusion, cholesterol loading of SMC causes phenotypic changes regulated at the mRNA level that result in a transdifferentiation to a macrophage-like state. This finding suggests that not all foam cells in lesions may have a macrophage origin, despite what is indicated by immunostaining for macrophage-related markers. Furthermore, inflammatory changes in foam cells observed in vivo may not be simple consequences of cholesterol accumulation.
Assuntos
Aorta/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Miócitos de Músculo Liso/citologia , Actinas/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação Mielomonocítica/biossíntese , Compostos Azo/farmacologia , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Ésteres do Colesterol/metabolismo , Corantes/farmacologia , Ciclodextrinas/metabolismo , Relação Dose-Resposta a Droga , Células Espumosas , Galectina 3/biossíntese , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Fagócitos/metabolismo , Fagocitose , Fenótipo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Tropomiosina/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Excess cellular cholesterol induces apoptosis in macrophages, an event likely to promote progression of atherosclerosis. The cellular mechanism of cholesterol-induced apoptosis is unknown but had previously been thought to involve the plasma membrane. Here we report that the unfolded protein response (UPR) in the endoplasmic reticulum is activated in cholesterol-loaded macrophages, resulting in expression of the cell death effector CHOP. Cholesterol loading depletes endoplasmic reticulum calcium stores, an event known to induce the UPR. Furthermore, endoplasmic reticulum calcium depletion, the UPR, caspase-3 activation and apoptosis are markedly inhibited by selective inhibition of cholesterol trafficking to the endoplasmic reticulum, and Chop-/- macrophages are protected from cholesterol-induced apoptosis. We propose that cholesterol trafficking to endoplasmic reticulum membranes, resulting in activation of the CHOP arm of the UPR, is the key signalling step in cholesterol-induced apoptosis in macrophages.
Assuntos
Apoptose/fisiologia , Colesterol/toxicidade , Doença da Artéria Coronariana/metabolismo , Retículo Endoplasmático/metabolismo , Macrófagos/metabolismo , Dobramento de Proteína , Animais , Apoptose/efeitos dos fármacos , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Proteínas Estimuladoras de Ligação a CCAAT/deficiência , Proteínas Estimuladoras de Ligação a CCAAT/genética , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Colesterol/metabolismo , Doença da Artéria Coronariana/fisiopatologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Feminino , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Camundongos Knockout , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: LDL receptor-deficient "apolipoprotein (apo)-B100-only" mice (Ldlr-/-Apob100/100 have elevated LDL cholesterol levels on a chow diet and develop severe aortic atherosclerosis. We hypothesized that both the hypercholesterolemia and the susceptibility to atherosclerosis could be eliminated by switching off hepatic lipoprotein production. METHODS AND RESULTS: We bred Ldlr-/-Apob100/100 mice that were homozygous for a conditional allele for Mttp (the gene for microsomal triglyceride transfer protein) and the inducible Mx1-Cre transgene. In these animals, which we called "Reversa mice," the hypercholesterolemia could be reversed, without modifying the diet or initiating a hypolipidemic drug, by the transient induction of Cre expression in the liver. After Cre induction, hepatic Mttp expression was virtually eliminated (as judged by quantitative real-time PCR), hepatic lipoprotein secretion was abolished (as judged by electron microscopy), and LDLs were virtually eliminated from the plasma. Intestinal lipoprotein production was unaffected. In mice fed a chow diet, Cre induction reduced plasma cholesterol levels from 233.9+/-46.0 to 37.2+/-6.5 mg/dL. In mice fed a high-fat diet, cholesterol levels fell from 525.7+/-32.2 to 100.6+/-14.3 mg/dL. The elimination of hepatic lipoprotein production completely prevented both the development of atherosclerosis and the changes in gene expression that accompany atherogenesis. CONCLUSIONS: We developed mice in which hypercholesterolemia can be reversed with a genetic switch. These mice will be useful for understanding gene-expression changes that accompany the reversal of hypercholesterolemia and atherosclerosis.
Assuntos
Arteriosclerose/prevenção & controle , Proteínas de Transporte/genética , Lipoproteínas/metabolismo , Fígado/metabolismo , Animais , Apolipoproteína B-100 , Apolipoproteínas B/genética , Artérias/metabolismo , Arteriosclerose/etiologia , Arteriosclerose/metabolismo , Colesterol/sangue , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Hipercolesterolemia/sangue , Hipercolesterolemia/terapia , Integrases/genética , Integrases/metabolismo , Lipídeos/sangue , Lipoproteínas/química , Lipoproteínas/ultraestrutura , Camundongos , Tamanho da Partícula , Poli I-C/farmacologia , Receptores de LDL/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
PURPOSE: To test the ability of serial, in vivo magnetic resonance microscopy (MRM) to detect the development of atherosclerosis and quantify its progression in apolipoprotein E-deficient mice. MATERIALS AND METHODS: The abdominal aortae of six ApoE(-/-) and three wild-type (WT) control mice were imaged by MRM at 9.4T. Proton density weighted images were obtained (TR = 2000, TE = 9 msec) using four signal averages. The image resolution was 109 x 109 x 500 microm(3). The six ApoE(-/-) mice underwent serial MRM three to five times over a period < or = 44 weeks. Multiple, anatomically aligned MRM slices (N = 6-11 per time point, total 202) were compared serially in each animal. RESULTS: The abdominal aorta remained free of atherosclerosis until 20 weeks of age but thereafter, atherosclerosis was identified in all ApoE(-/-) mice (P < 0.05 to P < 0.001), but no WT controls. Lesion progression was accompanied by positive remodeling in which atherosclerosis within the aortic wall was accommodated by an increase in total cross sectional area (P < 0.01), while lumen area was unchanged. CONCLUSION: Serial MRM demonstrated the development and progression of atherosclerosis in mouse aorta. Importantly, progression of atherosclerosis could be identified within individual animals. By following the same aortic lesions over time, MRM demonstrated that progression of atherosclerosis in mice is associated with positive arterial remodeling.
Assuntos
Apolipoproteínas E/deficiência , Arteriosclerose/diagnóstico , Espectroscopia de Ressonância Magnética , Animais , Aorta Abdominal/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: Monocyte chemoattractant protein (MCP)-1 is a proatherogenic factor that is responsible for approximately 60% of plaque macrophages in mouse models of atherosclerosis. We investigated whether lysophosphatidylcholine (LPC), enriched in oxidized low density lipoprotein, can modulate the expression of MCP-1 in arterial wall cells. METHODS AND RESULTS: LPC induced a 3-fold increase in MCP-1 mRNA in rat vascular smooth muscle cells (VSMCs) in a time- and dose-dependent manner. Nuclear runon analysis showed that this increase was attributable to increased MCP-1 gene transcription. There was a 2-fold increase in MCP-1 protein in the conditioned media of cells treated with LPC. LPC-associated increases of MCP-1 mRNA and protein were similar to those produced by platelet-derived growth factor-BB, a known inducer of MCP-1. Analyses of the MCP-1 promoter in transiently transfected VSMCs indicated an LPC-responsive element(s) between base pairs -146 and -261 (relative to transcription initiation). Further studies suggested that LPC-induced MCP-1 expression partially involves mitogen-activated protein kinase/extracellular signal-regulated kinase, a tyrosine kinase(s), and (to a lesser extent) protein kinase C but not the activation of the platelet-derived growth factor receptor. CONCLUSIONS: LPC stimulates MCP-1 expression at the transcriptional level in VSMCs, suggesting a molecular mechanism by which LPC contributes to the atherogenicity of oxidized low density lipoprotein.
Assuntos
Quimiocina CCL2/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Músculo Liso Vascular/química , Músculo Liso Vascular/metabolismo , Animais , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Quimiocina CCL2/biossíntese , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/fisiologia , Lisofosfatidilcolinas/toxicidade , Mitocôndrias/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/fisiologia , Proteínas Tirosina Quinases/fisiologia , RNA Mensageiro/biossíntese , Ratos , Receptores do Fator de Crescimento Derivado de Plaquetas/fisiologia , Elementos de Resposta/efeitos dos fármacos , Elementos de Resposta/genética , Elementos de Resposta/fisiologia , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologiaRESUMO
We have previously shown that magnetic resonance microscopy (MRM) accurately quantifies atherosclerosis in Apolipoprotein E deficient (ApoE(-/-)) mice aged 36-84 weeks. The present study tests MRM in the quantification of aortic atherosclerosis over a broader range of lesion severity. Younger mice with less advanced disease were imaged in order to evaluate sensitivity, specificity and maximum practical resolution of MRM. Nineteen mice underwent in vivo MRM. Wall area measurements by MRM and light microscopy (LM) (n=43) were highly correlated (r=0.85, slope=0.88, P<0.0001). Wall areas by MRM ranged from 0.114 to 0.934 (median, 0.334) mm(2). A threshold of 0.35 mm(2), for the upper limit of normal, gave MRM positive predictive value (PPV) for detecting abnormally thickened arteries=89.5% and negative predictive value (NPV)=75%, referred to LM. Lesion shape assessed by LM and MRM were also well correlated (r=0.72, P<0.001). Increased wall area in atherosclerosis was found by MRM (P=0.01) and LM (P<0.0001) to be accommodated entirely by 'positive remodeling', confirming the importance of determining plaque size directly. MRM accurately quantifies mouse aortic atherosclerosis and will enhance studies in this important animal model.
Assuntos
Doenças da Aorta/diagnóstico , Apolipoproteínas E/deficiência , Arteriosclerose/diagnóstico , Imageamento por Ressonância Magnética , Animais , Aorta Abdominal/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Apolipoproteínas E/genética , Arteriosclerose/genética , Arteriosclerose/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Índice de Gravidade de DoençaRESUMO
Macrophage foam cells are integral in the development of atherosclerotic lesions. Gene expression analysis of lesional macrophage foam cells is complicated by the cellular heterogeneity of atherosclerotic plaque and the presence of lesions of various degrees of severity. To overcome these limitations, we tested the ability of laser capture microdissection (LCM) and real-time quantitative reverse transcription PCR to selectively analyze RNA from lesional macrophages of apolipoprotein E (apoE)-deficient mice. Proximal aortic tissue sections were immunostained for macrophagespecific CD68/macrosialin by a rapid (approximately 15-min) protocol. Alternating sections from each animal were used to isolate RNA either from entire sections (analogous to isolation from whole tissue) or by LCM selection of CD68-positive cells. We measured the mRNA levels of CD68, a macrophage-specific marker, alpha-actin, a smooth muscle cell marker, and cyclophilin A, a control gene. Compared with whole sections, CD68 mRNA levels were greatly enriched (33.6-fold) in the laser-captured lesional macrophages. In contrast to whole sections, LCM-derived RNA had undetectable levels of alpha-actin. To illustrate the ability of this method to measure changes in lesional macrophage gene expression, we injected 100 microg of lipopolysaccharide i.p. into apoE-deficient mice and detected in laser-captured lesional macrophages increased mRNA expression for vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, and monocyte chemoattractant protein-1 (11.9-, 32.5-, and 31.0-fold, respectively). By selectively enriching foam cell RNA, LCM provides a powerful approach to study the in situ expression and regulation of atherosclerosis-related genes. This approach will allow the study of macrophage gene expression under various conditions of plaque formation, regression, and response to genetic and environmental perturbations.