RESUMO
Aims: Low-density lipoprotein (LDL) particles cause atherosclerotic cardiovascular disease (ASCVD) through their retention, modification, and accumulation within the arterial intima. High plasma concentrations of LDL drive this disease, but LDL quality may also contribute. Here, we focused on the intrinsic propensity of LDL to aggregate upon modification. We examined whether inter-individual differences in this quality are linked with LDL lipid composition and coronary artery disease (CAD) death, and basic mechanisms for plaque growth and destabilization. Methods and results: We developed a novel, reproducible method to assess the susceptibility of LDL particles to aggregate during lipolysis induced ex vivo by human recombinant secretory sphingomyelinase. Among patients with an established CAD, we found that the presence of aggregation-prone LDL was predictive of future cardiovascular deaths, independently of conventional risk factors. Aggregation-prone LDL contained more sphingolipids and less phosphatidylcholines than did aggregation-resistant LDL. Three interventions in animal models to rationally alter LDL composition lowered its susceptibility to aggregate and slowed atherosclerosis. Similar compositional changes induced in humans by PCSK9 inhibition or healthy diet also lowered LDL aggregation susceptibility. Aggregated LDL in vitro activated macrophages and T cells, two key cell types involved in plaque progression and rupture. Conclusion: Our results identify the susceptibility of LDL to aggregate as a novel measurable and modifiable factor in the progression of human ASCVD.
Assuntos
Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/mortalidade , Lipoproteínas LDL/sangue , Lipoproteínas LDL/fisiologia , Adulto , Animais , Feminino , Humanos , Lipídeos , Masculino , Camundongos , Pessoa de Meia-Idade , Prognóstico , Medição de RiscoRESUMO
Cardiovascular disease (CVD) is the leading global cause of death, accounting for 17.3 million deaths per year. Preventive treatment that reduces CVD by even a small percentage can substantially reduce, nationally and globally, the number of people who develop CVD and the costs of caring for them. This American Heart Association presidential advisory on dietary fats and CVD reviews and discusses the scientific evidence, including the most recent studies, on the effects of dietary saturated fat intake and its replacement by other types of fats and carbohydrates on CVD. In summary, randomized controlled trials that lowered intake of dietary saturated fat and replaced it with polyunsaturated vegetable oil reduced CVD by ≈30%, similar to the reduction achieved by statin treatment. Prospective observational studies in many populations showed that lower intake of saturated fat coupled with higher intake of polyunsaturated and monounsaturated fat is associated with lower rates of CVD and of other major causes of death and all-cause mortality. In contrast, replacement of saturated fat with mostly refined carbohydrates and sugars is not associated with lower rates of CVD and did not reduce CVD in clinical trials. Replacement of saturated with unsaturated fats lowers low-density lipoprotein cholesterol, a cause of atherosclerosis, linking biological evidence with incidence of CVD in populations and in clinical trials. Taking into consideration the totality of the scientific evidence, satisfying rigorous criteria for causality, we conclude strongly that lowering intake of saturated fat and replacing it with unsaturated fats, especially polyunsaturated fats, will lower the incidence of CVD. This recommended shift from saturated to unsaturated fats should occur simultaneously in an overall healthful dietary pattern such as DASH (Dietary Approaches to Stop Hypertension) or the Mediterranean diet as emphasized by the 2013 American Heart Association/American College of Cardiology lifestyle guidelines and the 2015 to 2020 Dietary Guidelines for Americans.
Assuntos
American Heart Association , Doenças Cardiovasculares/dietoterapia , Doenças Cardiovasculares/epidemiologia , Gorduras Insaturadas na Dieta/administração & dosagem , Política Nutricional , Doenças Cardiovasculares/prevenção & controle , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/efeitos adversos , Gorduras Insaturadas na Dieta/efeitos adversos , Estilo de Vida Saudável , Humanos , Política Nutricional/tendências , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos , Estados Unidos/epidemiologiaRESUMO
RATIONALE: Cholesterol esters (CE), especially cholesterol oleate, generated by hepatic and intestinal sterol O-acyltransferase 2 (SOAT2) play a critical role in cholesterol homeostasis. However, it is unknown whether the contribution of intestine-derived CE from SOAT2 would have similar effects in promoting atherosclerosis progression as for liver-derived CE. OBJECTIVE: To test whether, in low-density lipoprotein receptor null (LDLr(-/-)) mice, the conditional knockout of intestinal SOAT2 (SOAT2(SI-/SI-)) or hepatic SOAT2 (SOAT2(L-/L-)) would equally limit atherosclerosis development compared with the global deletion of SOAT2 (SOAT2(-/-)). METHODS AND RESULTS: SOAT2 conditional knockout mice were bred with LDLr(-/-) mice creating LDLr(-/-) mice with each of the specific SOAT2 gene deletions. All mice then were fed an atherogenic diet for 16 weeks. SOAT2(SI-/SI-)LDLr(-/-) and SOAT2(-/-)LDLr(-/-) mice had significantly lower levels of intestinal cholesterol absorption, more fecal sterol excretion, and lower biliary cholesterol levels. Analysis of plasma LDL showed that all mice with SOAT2 gene deletions had LDL CE with reduced percentages of cholesterol palmitate and cholesterol oleate. Each of the LDLr(-/-) mice with SOAT2 gene deletions had lower accumulations of total cholesterol and CE in the liver compared with control mice. Finally, aortic atherosclerosis development was significantly lower in all mice with global or tissue-restricted SOAT2 gene deletions. Nevertheless, SOAT2(-/-)LDLr(-/-) and SOAT2(L-/L-)LDLr(-/-) mice had less aortic CE accumulation and smaller aortic lesions than SOAT2(SI-/SI-)LDLr(-/-) mice. CONCLUSIONS: SOAT2-derived CE from both the intestine and liver significantly contribute to the development of atherosclerosis, although the CE from the hepatic enzyme appeared to promote more atherosclerosis development.
Assuntos
Aorta/metabolismo , Aterosclerose/metabolismo , Ésteres do Colesterol/metabolismo , Absorção Intestinal/fisiologia , Fígado/metabolismo , Esterol O-Aciltransferase/deficiência , Animais , Aorta/patologia , Aterosclerose/sangue , Aterosclerose/patologia , Ésteres do Colesterol/sangue , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esterol O-Aciltransferase 2RESUMO
OBJECTIVE: To test the hypothesis that the attenuation of cholesterol oleate packaging into apoB-containing lipoproteins will arrest progression of pre-existing atherosclerotic lesions. APPROACH AND RESULTS: Atherosclerosis was induced in apoB-100 only, LDLr(-/-) mice by feeding a diet enriched in cis-monounsaturated fatty acids for 24 weeks. A subset of mice was then euthanized to quantify the extent of atherosclerosis. The remaining mice were continued on the same diet (controls) or assigned to the following treatments for 16 weeks: (1) a diet enriched in n-3 polyunsaturated fatty acids, (2) the cis-monounsaturated fatty acid diet plus biweekly injections of an antisense oligonucleotide specific to hepatic sterol-O-acyltransferase 2 (SOAT2); or (3) the cis-monounsaturated fatty acid diet and biweekly injections of a nontargeting hepatic antisense oligonucleotide. Extent of atherosclerotic lesions in the aorta was monitored morphometrically in vivo with magnetic resonance imaging and ex vivo histologically and immunochemically. Hepatic knockdown of SOAT2 via antisense oligonucleotide treatment arrested lesion growth and stabilized lesions. CONCLUSIONS: Hepatic knockdown of SOAT2 in apoB100-only, LDLr(-/-) mice resulted in remodeling of aortic atherosclerotic lesions into a stable phenotype, suggesting SOAT2 is a viable target for the treatment of atherosclerosis.
Assuntos
Apolipoproteína B-100/sangue , DNA/genética , Regulação da Expressão Gênica , Fígado/enzimologia , Oligonucleotídeos Antissenso/genética , Placa Aterosclerótica/tratamento farmacológico , Esterol O-Aciltransferase/genética , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Modelos Animais de Doenças , Progressão da Doença , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Oligonucleotídeos Antissenso/farmacologia , Placa Aterosclerótica/sangue , Placa Aterosclerótica/genética , Esterol O-Aciltransferase/biossíntese , Esterol O-Aciltransferase/farmacologia , Esterol O-Aciltransferase 2RESUMO
Although APOL1 gene variants are associated with nephropathy in African Americans, little is known about APOL1 protein synthesis, uptake, and localization in kidney cells. To address these questions, we examined APOL1 protein and mRNA localization in human kidney and human kidney-derived cell lines. Indirect immunofluorescence microscopy performed on nondiseased nephrectomy cryosections from persons with normal kidney function revealed that APOL1 protein was markedly enriched in podocytes (colocalized with synaptopodin and Wilms' tumor suppressor) and present in lower abundance in renal tubule cells. Fluorescence in situ hybridization detected APOL1 mRNA in glomeruli (podocytes and endothelial cells) and tubules, consistent with endogenous synthesis in these cell types. When these analyses were extended to renal-derived cell lines, quantitative RT-PCR did not detect APOL1 mRNA in human mesangial cells; however, abundant levels of APOL1 mRNA were observed in proximal tubule cells and glomerular endothelial cells, with lower expression in podocytes. Western blot analysis revealed corresponding levels of APOL1 protein in these cell lines. To explain the apparent discrepancy between the marked abundance of APOL1 protein in kidney podocytes observed in cryosections versus the lesser abundance in podocyte cell lines, we explored APOL1 cellular uptake. APOL1 protein was taken up readily by human podocytes in vitro but was not taken up efficiently by mesangial cells, glomerular endothelial cells, or proximal tubule cells. We hypothesize that the higher levels of APOL1 protein in human cryosectioned podocytes may reflect both endogenous protein synthesis and APOL1 uptake from the circulation or glomerular filtrate.
Assuntos
Apolipoproteínas/metabolismo , Glomérulos Renais/metabolismo , Túbulos Renais Proximais/metabolismo , Rim/metabolismo , Lipoproteínas HDL/metabolismo , Células Mesangiais/metabolismo , RNA Mensageiro/metabolismo , Apolipoproteína L1 , Biópsia , Linhagem Celular , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Técnicas In Vitro , Rim/patologia , Rim/cirurgia , Glomérulos Renais/patologia , Túbulos Renais Proximais/patologia , Células Mesangiais/patologia , Microscopia de Fluorescência , Nefrectomia , Podócitos/metabolismo , Podócitos/patologiaRESUMO
In most organs, the bulk of cholesterol is unesterified, although nearly all possess a varying capability of esterifying cholesterol through the action of either sterol O-acyltransferase (SOAT) 1 or, in the case of hepatocytes and enterocytes, SOAT2. Esterified cholesterol (EC) carried in plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to whether pharmacological inhibition of SOAT2 might reduce tissue EC accretion in CESD. When weaned at 21 days, Lal(-/-) mice, of either gender, had a whole liver cholesterol content that was 12- to 13-fold more than that of matching Lal(+/+) littermates (23 versus 1.8 mg, respectively). In Lal(-/-) males given the selective SOAT2 inhibitor PRD125 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl-1,7,11-trideacetylpyripyropene A in their diet (â¼10 mg/day per kg body weight) from 21 to 53 days, whole liver cholesterol content was 48.6 versus 153.7 mg in untreated 53-day-old Lal(-/-) mice. This difference reflected a 59% reduction in hepatic EC concentration (mg/g), combined with a 28% fall in liver mass. The treated mice also showed a 63% reduction in plasma alanine aminotransferase activity, in parallel with decisive falls in hepatic mRNA expression levels for multiple proteins that reflect macrophage presence and inflammation. These data implicate SOAT2 as a potential target in CESD management.
Assuntos
Ésteres do Colesterol/biossíntese , Fígado/efeitos dos fármacos , Piridinas/farmacologia , Sesquiterpenos/farmacologia , Esterol Esterase/genética , Esterol O-Aciltransferase/antagonistas & inibidores , Animais , Feminino , Hepatite/tratamento farmacológico , Hepatite/imunologia , Hepatite/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Fígado/fisiologia , Masculino , Camundongos , Camundongos Knockout , Tamanho do Órgão , Transaminases/sangue , Esterol O-Aciltransferase 2RESUMO
Sterol O-acyltransferase 2 (SOAT2; also known as ACAT2) is considered as a new therapeutic target for the treatment or prevention of hypercholesterolemia and atherosclerosis. Fungal pyripyropene A (PPPA: 1,7,11-triacyl type), the first SOAT2-selective inhibitor, proved orally active in vivo using atherogenic mouse models. The purpose of the present study was to demonstrate that the PPPA derivatives (PRDs) prove more effective in the mouse models than PPPA. Among 196 semisynthetic PPPA derivatives, potent, SOAT2-selective, and stable PRDs were selected. In vivo antiatherosclerotic activity of selected PRDs was tested in apolipoprotein E knockout (Apoe(-/-)) mice or low-density lipoprotein receptor knockout (Ldlr(-/-)) mice fed a cholesterol-enriched diet (0.2% cholesterol and 21% fat) for 12 weeks. During the PRD treatments, no detrimental side effects were observed. Among three PRDs, Apoe(-/-) mice treated with PRD125 (1-,11-O-benzylidene type) at 1 mg/kg/day had significantly lower total plasma cholesterol concentration by 57.9 ± 9.3%; further, the ratio of cholesteryl oleate to cholesteryl linoleate in low-density lipoprotein was lower by 55.6 ± 7.5%, respectively. The hepatic cholesteryl ester levels and SOAT2 activity in the small intestines and livers of the PRD-treated mice were selectively lowered. The atherosclerotic lesion areas in the aortae of PRD125-treated mice were significantly lower at 62.2 ± 13.1%, respectively. Furthermore, both PRDs were also orally active in atherogenic Ldlr(-/-) mice. Among the PRDs tested, PRD125 was the most potent in both mouse models. These results suggest that SOAT2-selective inhibitors such as PRD125 have a high potential as poststatin agents for treatment and/or prevention in patients with atherosclerosis and hypercholesterolemia.
Assuntos
Anticolesterolemiantes/química , Aterosclerose/tratamento farmacológico , Hipercolesterolemia/tratamento farmacológico , Piridinas/química , Sesquiterpenos/química , Esterol O-Aciltransferase/antagonistas & inibidores , Animais , Anticolesterolemiantes/síntese química , Anticolesterolemiantes/farmacologia , Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Células CHO , Colesterol/sangue , Ésteres do Colesterol/sangue , Cricetulus , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Gotículas Lipídicas/metabolismo , Lipoproteínas/sangue , Fígado/metabolismo , Masculino , Camundongos Knockout , Piridinas/síntese química , Piridinas/farmacologia , Receptores de LDL/genética , Sesquiterpenos/síntese química , Sesquiterpenos/farmacologia , Esterol O-Aciltransferase/metabolismo , Relação Estrutura-Atividade , Esterol O-Aciltransferase 2RESUMO
OBJECTIVE: Interindividual variation in pathways affecting cellular cholesterol metabolism can influence levels of plasma cholesterol, a well-established risk factor for cardiovascular disease. Inherent variation among immortalized lymphoblastoid cell lines from different donors can be leveraged to discover novel genes that modulate cellular cholesterol metabolism. The objective of this study was to identify novel genes that regulate cholesterol metabolism by testing for evidence of correlated gene expression with cellular levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) mRNA, a marker for cellular cholesterol homeostasis, in a large panel of lymphoblastoid cell lines. APPROACH AND RESULTS: Expression array profiling was performed on 480 lymphoblastoid cell lines established from participants of the Cholesterol and Pharmacogenetics (CAP) statin clinical trial, and transcripts were tested for evidence of correlated expression with HMGCR as a marker of intracellular cholesterol homeostasis. Of these, transmembrane protein 55b (TMEM55B) showed the strongest correlation (r=0.29; P=4.0E-08) of all genes not previously implicated in cholesterol metabolism and was found to be sterol regulated. TMEM55B knockdown in human hepatoma cell lines promoted the decay rate of the low-density lipoprotein receptor, reduced cell surface low-density lipoprotein receptor protein, impaired low-density lipoprotein uptake, and reduced intracellular cholesterol. CONCLUSIONS: Here, we report identification of TMEM55B as a novel regulator of cellular cholesterol metabolism through the combination of gene expression profiling and functional studies. The findings highlight the value of an integrated genomic approach for identifying genes that influence cholesterol homeostasis.
Assuntos
Colesterol/metabolismo , Linfócitos/metabolismo , Receptores de LDL/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Perfilação da Expressão Gênica , Células Hep G2 , Hepatócitos/metabolismo , Homeostase , Humanos , Hidroximetilglutaril-CoA Redutases/biossíntese , Hidroximetilglutaril-CoA Redutases/genética , Líquido Intracelular/metabolismo , Metabolismo dos Lipídeos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
Intestinal cholesterol absorption involves the chylomicron and HDL pathways and is dependent on microsomal triglyceride transfer protein (MTP) and ABCA1, respectively. Chylomicrons transport free and esterified cholesterol, whereas HDLs transport free cholesterol. ACAT2 esterifies cholesterol for secretion with chylomicrons. We hypothesized that free cholesterol accumulated during ACAT2 deficiency may be secreted with HDLs when chylomicron assembly is blocked. To test this, we studied cholesterol absorption in mice deficient in intestinal MTP, global ACAT2, and both intestinal MTP and global ACAT2. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their transport with chylomicrons. In contrast, global ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption with chylomicrons and increased cellular free cholesterol. Their combined deficiency reduced cholesterol secretion with both chylomicrons and HDLs. Thus, contrary to our hypothesis, free cholesterol accumulated in the absence of MTP and ACAT2 is unavailable for secretion with HDLs. Global ACAT2 deficiency causes mild hypertriglyceridemia and reduces hepatosteatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in global ACAT2-deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid hepatosteatosis in cholesterol-fed animals. Therefore, ACAT2 inhibition might avert hepatosteatosis associated with high cholesterol diets by increasing hepatic MTP expression and lipoprotein production.
Assuntos
Acetil-CoA C-Acetiltransferase/deficiência , Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Quilomícrons/metabolismo , Absorção Intestinal , Mucosa Intestinal/metabolismo , Lipoproteínas HDL/metabolismo , Acetil-CoA C-Acetiltransferase/genética , Animais , Proteínas de Transporte/genética , Colesterol/sangue , Colesterol na Dieta/metabolismo , Dieta Ocidental/efeitos adversos , Gorduras na Dieta/metabolismo , Enterócitos/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Lipoproteínas HDL/sangue , Fígado/metabolismo , Masculino , Camundongos , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Plant sterols, or phytosterols, are very similar in structure to cholesterol and are abundant in typical diets. The reason for poor absorption of plant sterols by the body is still unknown. Mutations in the ABC transporters G5 and G8 are known to cause an accumulation of plant sterols in blood and tissues (sitosterolemia). To determine the significance of phytosterol exclusion from the body, we fed wild-type and ABCG5/G8 knockout mice a diet enriched with plant sterols. The high-phytosterol diet was extremely toxic to the ABCG5/G8 knockout mice but had no adverse effects on wild-type mice. ABCG5/G8 knockout mice died prematurely and developed a phenotype that included high levels of plant sterols in many tissues, liver abnormalities, and severe cardiac lesions. This study is the first to report such toxic effects of phytosterol accumulation in ABCG5/G8 knockout mice. We believe these new data support the conclusion that plant sterols are excluded from the body because they are toxic when present at high levels.
Assuntos
Transportadores de Cassetes de Ligação de ATP/deficiência , Comportamento Alimentar/efeitos dos fármacos , Lipoproteínas/deficiência , Fitosteróis/toxicidade , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Dieta , Eritrócitos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatomegalia/sangue , Hepatomegalia/genética , Hepatomegalia/patologia , Lipoproteínas/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Tamanho do Órgão/efeitos dos fármacos , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Esplenomegalia/sangue , Esplenomegalia/genética , Esplenomegalia/patologia , Aumento de Peso/efeitos dos fármacosRESUMO
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
Several studies in humans and animals suggest that LDL particle core enrichment in cholesteryl oleate (CO) is associated with increased atherosclerosis. Diet enrichment with MUFAs enhances LDL CO content. Steroyl O-acyltransferase 2 (SOAT2) is the enzyme that catalyzes the synthesis of much of the CO found in LDL, and gene deletion of SOAT2 minimizes CO in LDL and protects against atherosclerosis. The purpose of this study was to test the hypothesis that the increased atherosclerosis associated with LDL core enrichment in CO results from an increased affinity of the LDL particle for arterial proteoglycans. ApoB-100-only Ldlr(-/-) mice with and without Soat2 gene deletions were fed diets enriched in either cis-MUFA or n-3 PUFA, and LDL particles were isolated. LDL:proteogylcan binding was measured using surface plasmon resonance. Particles with higher CO content consistently bound with higher affinity to human biglycan and the amount of binding was shown to be proportional to the extent of atherosclerosis of the LDL donor mice. The data strongly support the thesis that atherosclerosis was induced through enhanced proteoglycan binding of LDL resulting from LDL core CO enrichment.
Assuntos
Aterosclerose/metabolismo , Ésteres do Colesterol/metabolismo , LDL-Colesterol/metabolismo , Proteoglicanas/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Animais , Artérias/metabolismo , Biglicano/metabolismo , Humanos , CamundongosRESUMO
Reverse cholesterol transport (RCT) can proceed through the classic hepatobiliary route or through the nonbiliary transintestinal cholesterol efflux (TICE) pathway. Scavenger receptor class B type I (SR-BI) plays a critical role in the classic hepatobiliary route of RCT. However, the role of SR-BI in TICE has not been studied. To examine the role of intestinal SR-BI in TICE, sterol balance was measured in control mice and mice transgenically overexpressing SR-BI in the proximal small intestine (SR-BI(hApoCIII-ApoAIV-Tg)). SR-BI(hApoCIII-ApoAIV-Tg) mice had significantly lower plasma cholesterol levels compared with wild-type controls, yet SR-BI(hApoCIII-ApoAIV-Tg) mice had normal fractional cholesterol absorption and fecal neutral sterol excretion. Both in the absence or presence of ezetimibe, intestinal SR-BI overexpression had no impact on the amount of cholesterol excreted in the feces. To specifically study effects of intestinal SR-BI on TICE we crossed SR-BI(hApoCIII-ApoAIV-Tg) mice into a mouse model that preferentially utilized the TICE pathway for RCT (Niemann-Pick C1-like 1 liver transgenic), and likewise found no alterations in cholesterol absorption or fecal sterol excretion. Finally, mice lacking SR-BI in all tissues also exhibited normal cholesterol absorption and fecal cholesterol disposal. Collectively, these results suggest that SR-BI is not rate limiting for intestinal cholesterol absorption or for fecal neutral sterol loss through the TICE pathway.
Assuntos
Colesterol/metabolismo , Absorção Intestinal/fisiologia , Mucosa Intestinal/metabolismo , Receptores Depuradores Classe B/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Azetidinas/farmacologia , Colesterol/genética , Ezetimiba , Absorção Intestinal/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Receptores Depuradores Classe B/genéticaRESUMO
In an effort to develop potent and selective inhibitors toward ACAT2, structure-activity relationship studies were carried out using derivatives based on pyripyropene A (PPPA, 1). We have successfully developed novel PPPA derivatives with a 7-O-substituted benzoyl substituent that significantly exhibit more potent ACAT2 inhibitory activity and higher ACAT2 isozyme selectivity than 1.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Piridinas/química , Piridinas/farmacologia , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Esterol O-Aciltransferase/antagonistas & inibidores , Animais , Inibidores Enzimáticos/síntese química , Piridinas/síntese química , Sesquiterpenos/síntese química , Relação Estrutura-Atividade , Esterol O-Aciltransferase 2RESUMO
Synthesis and structure-activity relationships of 7-O-p-cyanobenzoyl pyripyropene A derivatives with modification at C1 and 11 are described. Regioselective mono-deprotection of di-tert-butylsilylene acetal was critical in their synthesis.
Assuntos
Inibidores Enzimáticos/síntese química , Piridinas/química , Sesquiterpenos/química , Esterol O-Aciltransferase/antagonistas & inibidores , Butanos/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ligação Proteica , Piridinas/síntese química , Piridinas/metabolismo , Sesquiterpenos/síntese química , Sesquiterpenos/metabolismo , Silanos/química , Estereoisomerismo , Esterol O-Aciltransferase/metabolismo , Relação Estrutura-Atividade , Esterol O-Aciltransferase 2RESUMO
In an effort to develop potent and selective inhibitors toward ACAT2, structure-activity relationship studies were carried out using derivatives based on pyripyropene A (PPPA, 1). In particular, we investigated the possibility of introducing appropriate 1,11-O-benzylidene and 7-O-substituted benzoyl moieties into PPPA (1). The new o-substituted benzylidene derivatives showed higher selectivity for ACAT2 than PPPA (1). Among them, 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl PPPA derivative 7q and 1,11-O-o,o-dimethylbenzylidene-7-O-p-cyanobenzoyl PPPA derivative 7z proved to be potent ACAT2 inhibitors with unprecedented high isozyme selectivity.
Assuntos
Inibidores Enzimáticos/farmacologia , Piridinas/farmacologia , Sesquiterpenos/farmacologia , Esterol O-Aciltransferase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Sesquiterpenos/síntese química , Sesquiterpenos/química , Esterol O-Aciltransferase/metabolismo , Relação Estrutura-Atividade , Esterol O-Aciltransferase 2RESUMO
The hypothesis tested in this study was that cholesterol esterification by ACAT2 would increase cholesterol absorption efficiency by providing cholesteryl ester (CE) for incorporation into chylomicrons. The assumption was that absorption would be proportional to Acat2 gene dosage. Male ACAT2âº/âº, ACAT2âº/â», and ACAT2â»/â» mice were fed a diet containing 20% of energy as palm oil with 0.2% (w/w) cholesterol. Cholesterol absorption efficiency was measured by fecal dual-isotope and thoracic lymph duct cannulation (TLDC) methods using [³H]sitosterol and [¹4C]cholesterol tracers. Excellent agreement among individual mice was found for cholesterol absorption measured by both techniques. Cholesterol absorption efficiency in ACAT2â»/â» mice was 16% compared with 46-47% in ACAT2âº/⺠and ACAT2âº/â» mice. Chylomicrons from ACAT2âº/⺠and ACAT2âº/â» mice carried â¼80% of total sterol mass as CE, whereas ACAT2â»/â» chylomicrons carried >90% of sterol mass in the unesterified form. The total percentage of chylomicron mass as CE was reduced from 12% in the presence of ACAT2 to â¼1% in ACAT2â»/â» mice. Altogether, the data demonstrate that ACAT2 increases cholesterol absorption efficiency by providing CE for chylomicron transport, but one copy of the Acat2 gene, providing â¼50% of ACAT2 mRNA and enzyme activity, was as effective as two copies in promoting cholesterol absorption.
Assuntos
Colesterol/metabolismo , Quilomícrons/metabolismo , Absorção Intestinal , Esterol O-Aciltransferase/metabolismo , Animais , Ésteres do Colesterol/metabolismo , Esterificação , Masculino , Camundongos , Tamanho da Partícula , Ducto Torácico/metabolismo , Esterol O-Aciltransferase 2RESUMO
The metabolic fate of newly absorbed cholesterol and phytosterol is orchestrated through adenosine triphosphate-binding cassette transporter G5 and G8 heterodimer (G5G8), and acyl CoA:cholesterol acyltransferase 2 (ACAT2). We hypothesized that intestinal G5G8 limits sterol absorption by reducing substrate availability for ACAT2 esterification and have attempted to define the roles of these two factors using gene deletion studies in mice. Male ACAT2(-/-), G5G8(-/-), ACAT2(-/-)G5G8(-/-) (DKO), and wild-type (WT) control mice were fed a diet with 20% of energy as palm oil and 0.2% (w/w) cholesterol. Sterol absorption efficiency was directly measured by monitoring the appearance of [(3)H]sitosterol and [(14)C]cholesterol tracers in lymph after thoracic lymph duct cannulation. The average percentage (± SEM) absorption of [(14)C]cholesterol after 8 h of lymph collection was 40.55 ± 0.76%, 19.41 ± 1.52%, 32.13 ± 1.60%, and 21.27 ± 1.35% for WT, ACAT2(-/-), G5G8(-/-), and DKO mice, respectively. [(3)H]sitosterol absorption was <2% in WT and ACAT2(-/-) mice, whereas it was up to 6.8% in G5G8(-/-) and DKO mice. G5G8(-/-) mice also produced chylomicrons with â¼70% less cholesterol ester mass than WT mice. In contrast to expectations, the data demonstrated that the absence of G5G8 led to decreased intestinal cholesterol esterification and reduced cholesterol transport efficiency. Intestinal G5G8 appeared to limit the absorption of phytosterols; ACAT2 more efficiently esterified cholesterol than phytosterols. The data indicate that handling of sterols by the intestine involves both G5G8 and ACAT2 but that an additional factor (possibly Niemann-Pick C1-like 1) may be key in determining absorption efficiency.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Cateterismo , Lipoproteínas/metabolismo , Vasos Linfáticos/enzimologia , Multimerização Proteica , Esterol O-Aciltransferase/metabolismo , Tórax , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Colesterol/metabolismo , Esterificação , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Absorção Intestinal , Lipoproteínas/química , Lipoproteínas/deficiência , Lipoproteínas/genética , Masculino , Camundongos , Estrutura Quaternária de Proteína , Esterol O-Aciltransferase/deficiência , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase 2RESUMO
Acyl-CoA:cholesterol acyltransferase 2 (ACAT2) generates cholesterol esters (CE) for packaging into newly synthesized lipoproteins and thus is a major determinant of blood cholesterol levels. ACAT2 is expressed exclusively in the small intestine and liver, but the relative contributions of ACAT2 expression in these tissues to systemic cholesterol metabolism is unknown. We investigated whether CE derived from the intestine or liver would differentially affect hepatic and plasma cholesterol homeostasis. We generated liver-specific (ACAT2(L-/L-)) and intestine-specific (ACAT2(SI-/SI-)) ACAT2 knockout mice and studied dietary cholesterol-induced hepatic lipid accumulation and hypercholesterolemia. ACAT2(SI-/SI-) mice, in contrast to ACAT2(L-/L-) mice, had blunted cholesterol absorption. However, specific deletion of ACAT2 in the intestine generated essentially a phenocopy of the conditional knockout of ACAT2 in the liver, with reduced levels of plasma very low-density lipoprotein and hepatic CE, yet hepatic-free cholesterol does not build up after high cholesterol intake. ACAT2(L-/L-) and ACAT2(SI-/SI-) mice were equally protected from diet-induced hepatic CE accumulation and hypercholesterolemia. These results suggest that inhibition of intestinal or hepatic ACAT2 improves atherogenic hyperlipidemia and limits hepatic CE accumulation in mice and that depletion of intestinal ACAT2 is sufficient for most of the beneficial effects on cholesterol metabolism. Inhibitors of ACAT2 targeting either tissue likely would be beneficial for atheroprotection.
Assuntos
Colesterol/metabolismo , Dieta/efeitos adversos , Técnicas de Inativação de Genes , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Esterol O-Aciltransferase/deficiência , Esterol O-Aciltransferase/genética , Alelos , Animais , Sistema Biliar/metabolismo , Colesterol/sangue , Ésteres do Colesterol/metabolismo , Feminino , Hipercolesterolemia/etiologia , Hipercolesterolemia/metabolismo , Hipercolesterolemia/prevenção & controle , Absorção Intestinal , Intestino Delgado/metabolismo , Camundongos , Especificidade de Órgãos , Esterol O-Aciltransferase 2RESUMO
OBJECTIVE: Pyripyropene A (PPPA) of fungal origin is the first compound that has been found to strongly and selectively inhibit acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) isozyme activity in vitro. The purpose of the present study was to investigate in vivo efficacy of the ACAT2-selective inhibitor in atherosclerosis. METHODS AND RESULTS: PPPA treatment (10 to 100 mg/kg) caused 30.5±4.7% to 55.8±3.3% inhibition of the cholesterol absorption from the mouse intestine. When PPPA (10 to 50 mg/kg per day) was orally administered to apolipoprotein E-knockout mice for 12 weeks, the levels of plasma cholesterol, very-low-density lipoprotein (VLDL), and low-density lipoprotein (LDL) and hepatic cholesterol content were lowered. Furthermore, the ratio of cholesteryl oleate (exclusively synthesized in hepatic ACAT2) to cholesteryl linoleate in VLDL- and LDL-derived cholesteryl ester decreased, indicating that hepatic ACAT2 activity was inhibited by PPPA. PPPA-treated mice had reduced atherogenic lesion areas that were lowered by 26.2±3.7% to 46±3.8% in the aortae and by 18.9±3.6% to 37.6±6.0% in the hearts. CONCLUSIONS: Our findings indicate that ACAT2-selective inhibition in the intestine and the liver can be effective against atherosclerosis and that PPPA appears to be a potential antiatherogenic lead compound. This study is the first demonstration of the in vivo efficacy of PPPA, an ACAT2-selective inhibitor, in atherosclerosis. PPPA-treated atherogenic mice showed a decrease in intestinal cholesterol absorption and cholesterol and cholesteryl oleate levels in both LDL and VLDL, resulting in protection of atherosclerosis development.