RESUMO
Chronic inflammation is a common feature of obesity, with elevated cytokines such as interleukin-1 (IL-1) in the circulation and tissues. Here, we report an unconventional IL-1R-MyD88-IRAK2-PHB/OPA1 signaling axis that reprograms mitochondrial metabolism in adipocytes to exacerbate obesity. IL-1 induced recruitment of IRAK2 Myddosome to mitochondria outer membranes via recognition by TOM20, followed by TIMM50-guided translocation of IRAK2 into mitochondria inner membranes, to suppress oxidative phosphorylation and fatty acid oxidation, thereby attenuating energy expenditure. Adipocyte-specific MyD88 or IRAK2 deficiency reduced high-fat-diet-induced weight gain, increased energy expenditure and ameliorated insulin resistance, associated with a smaller adipocyte size and increased cristae formation. IRAK2 kinase inactivation also reduced high-fat diet-induced metabolic diseases. Mechanistically, IRAK2 suppressed respiratory super-complex formation via interaction with PHB1 and OPA1 upon stimulation of IL-1. Taken together, our results suggest that the IRAK2 Myddosome functions as a critical link between inflammation and metabolism, representing a novel therapeutic target for patients with obesity.
Assuntos
Adipócitos/imunologia , Inflamação/imunologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-1/metabolismo , Membranas Mitocondriais/metabolismo , Obesidade/imunologia , Adipócitos/patologia , Animais , Células Cultivadas , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Masculino , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fosforilação Oxidativa , Proibitinas , Transporte Proteico , Receptores de Interleucina-1/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: To characterize the effects of CSL112 (human APOA1 [apolipoprotein A1]) on the APOA1 exchange rate (AER) and the relationships with specific HDL (high-density lipoprotein) subpopulations when administered in the 90-day high-risk period post-acute myocardial infarction. METHODS: A subset of patients (n=50) from the AEGIS-I (ApoA-I Event Reducing in Ischemic Syndromes I) study received either placebo or CSL112 post-acute myocardial infarction. AER was measured in AEGIS-I plasma samples incubated with lipid-sensitive fluorescent APOA1 reporter. HDL particle size distribution was assessed by native gel electrophoresis followed by fluorescent imaging and detection of APOA1 and SAA (serum amyloid A) by immunoblotting. RESULTS: CSL112 infusion increased AER peaking at 2 hours and returning to baseline 24 hours post-infusion. AER correlated with cholesterol efflux capacity (r=0.49), HDL-cholesterol (r=0.30), APOA1 (r=0.48), and phospholipids (r=0.48; all P<0.001) over all time points. Mechanistically, changes in cholesterol efflux capacity and AER induced by CSL112 reflected HDL particle remodeling resulting in increased small HDL species that are highly active in mediating ABCA1 (ATP-binding cassette transporter 1)-dependent efflux, and large HDL species with high capacity for APOA1 exchange. The lipid-sensitive APOA1 reporter predominantly exchanged into SAA-poor HDL particles and weakly incorporated into SAA-enriched HDL species. CONCLUSIONS: Infusion of CSL112 enhances metrics of HDL functionality in patients with acute myocardial infarction. This study demonstrates that in post-acute myocardial infarction patients, HDL-APOA1 exchange involves specific SAA-poor HDL populations. Our data suggest that progressive enrichment of HDL with SAA may generate dysfunctional particles with impaired HDL-APOA1 exchange capacity, and that infusion of CSL112 improves the functional status of HDL with respect to HDL-APOA1 exchange. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT02108262.
Assuntos
Apolipoproteína A-I , Infarto do Miocárdio , Humanos , Colesterol , Proteína Amiloide A Sérica , Síndrome , Lipoproteínas HDL , HDL-Colesterol , Infarto do Miocárdio/tratamento farmacológicoRESUMO
Macrophages are the principal component of the innate immune system. They play very crucial and multifaceted roles in the pathogenesis of inflammatory vascular diseases. There is an increasing recognition that transcriptionally dynamic macrophages are the key players in the pathogenesis of inflammatory vascular diseases. In this context, the accumulation and aberrant activation of macrophages in the subendothelial layers govern atherosclerotic plaque development. Macrophage-mediated inflammation is an explicitly robust biological response that involves broad alterations in inflammatory gene expression. Thus, cell-intrinsic negative regulatory mechanisms must exist which can restrain inflammatory response in a spatiotemporal manner. In this study, we identified CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2) as one such cell-intrinsic negative regulator of inflammation. Our in vivo studies show that myeloid-CITED2-deficient mice on the Apoe-/- background have larger atherosclerotic lesions on both control and high-fat/high-cholesterol diets. Our integrated transcriptomics and gene set enrichment analyses studies show that CITED2 deficiency elevates STAT1 and interferon regulatory factor 1 (IRF1) regulated pro-inflammatory gene expression in macrophages. At the molecular level, our studies identify that CITED2 deficiency elevates IFNγ-induced STAT1 transcriptional activity and STAT1 enrichment on IRF1 promoter in macrophages. More importantly, siRNA-mediated knockdown of IRF1 completely reversed elevated pro-inflammatory target gene expression in CITED2-deficient macrophages. Collectively, our study findings demonstrate that CITED2 restrains the STAT1-IRF1 signaling axis in macrophages and limits the development of atherosclerotic plaques.
Assuntos
Aterosclerose/genética , Fator Regulador 1 de Interferon/genética , Proteínas Repressoras/genética , Fator de Transcrição STAT1/genética , Transdução de Sinais/genética , Transativadores/genética , Animais , Feminino , Inflamação/genética , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Células RAW 264.7 , Transcrição Gênica/genéticaRESUMO
High-density lipoprotein (HDL) metabolism is facilitated in part by scavenger receptor class B, type 1 (SR-B1) that mediates HDL uptake into cells. Higher levels of HDL have been associated with protection in other diseases, however, its role in prostate cancer is not definitive. SR-B1 is up-regulated in prostate cancer tissue, suggesting a possible role of this receptor in tumor progression. Here, we report that knockout (KO) of SR-B1 in both human and mouse prostate cancer cell lines through CRISPR/Cas9-mediated genome editing reduces HDL uptake into the prostate cancer cells and reduces their proliferation in response to HDL. In vivo studies using syngeneic SR-B1 WT (SR-B1+/+) and SR-B1 KO (SR-B1-/-) prostate cancer cells in WT and apolipoprotein-AI KO (apoA1-KO) C57BL/6J mice revealed that WT hosts, containing higher levels of total and HDL-cholesterol, grew larger tumors than apoA1-KO hosts with lower levels of total and HDL-cholesterol. Furthermore, SR-B1-/- prostate cancer cells formed smaller tumors in WT hosts than SR-B1+/+ cells in the same host model. Increased tumor volume was overall associated with reduced survival. We conclude that knocking out SR-B1 in prostate cancer tumors reduces HDL-associated increases in prostate cancer cell proliferation and disease progression.
Assuntos
Progressão da Doença , Lipoproteínas HDL/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Receptores Depuradores Classe B/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Colesterol/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/genética , Regulação para Cima/genéticaRESUMO
Atrial fibrillation (AF) is strongly associated with risk of stroke and heart failure. AF promotes atrial remodeling that increases risk of stroke due to left atrial thrombogenesis, and increases energy demand to support high rate electrical activity and muscle contraction. While many transcriptomic studies have assessed AF-related changes in mRNA abundance, fewer studies have assessed proteomic changes. We performed a proteomic analysis on left atrial appendage (LAA) tissues from 12 patients with a history of AF undergoing elective surgery; atrial rhythm was documented at time of surgery. Proteomic analysis was performed using liquid chromatography with mass spectrometry (LC/MS-MS). Data-dependent analysis identified 3090 unique proteins, with 408 differentially expressed between sinus rhythm and AF. Ingenuity Pathway Analysis of differentially expressed proteins identified mitochondrial dysfunction, oxidative phosphorylation, and sirtuin signaling among the most affected pathways. Increased abundance of electron transport chain (ETC) proteins in AF was accompanied by decreased expression of ETC complex assembly factors, tricarboxylic acid cycle proteins, and other key metabolic modulators. Discordant changes were also evident in the contractile unit with both up and downregulation of key components. Similar pathways were affected in a comparison of patients with a history of persistent vs. paroxysmal AF, presenting for surgery in sinus rhythm. Together, these data suggest that while the LAA attempts to meet the energetic demands of AF, an uncoordinated response may reduce ATP availability, contribute to tissue contractile and electrophysiologic heterogeneity, and promote a progression of AF from paroxysmal episodes to development of a substrate amenable to persistent arrhythmia.
Assuntos
Apêndice Atrial/metabolismo , Fibrilação Atrial/metabolismo , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , ProteômicaRESUMO
Multiple eukaryotic ribosomal proteins (RPs) are co-opted for extraribosomal "moonlighting" activities, but paradoxically, RPs exhibit rapid turnover when not ribosome-bound. In one illustrative case of a functional extraribosomal RP, interferon (IFN)-γ induces ribosome release of L13a and assembly into the IFN-gamma-activated inhibitor of translation (GAIT) complex for translational control of a subset of inflammation-related proteins. Here we show GAPDH functions as a chaperone, shielding newly released L13a from proteasomal degradation. However, GAPDH protective activity is lost following cell treatment with oxidatively modified low density lipoprotein and IFN-γ. These agonists stimulate S-nitrosylation at Cys(247) of GAPDH, which fails to interact with L13a, causing proteasomal degradation of essentially the entire cell complement of L13a and defective translational control. Evolution of extraribosomal RP activities might require coevolution of protective chaperones, and pathological disruption of either protein, or their interaction, presents an alternative mechanism of diseases due to RP defects, and targets for therapeutic intervention.
Assuntos
Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Marcha/genética , Inativação Gênica , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Células Mieloides/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Células U937 , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Altered lipid metabolism and inflammation are involved in the pathogenesis of both nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). Even though high-density lipoprotein (HDL), a CVD protective marker, is decreased, whether HDL metabolism and function are perturbed in NAFLD are currently unknown. We examined the effect of NAFLD and disease severity on HDL metabolism and function in patients with biopsy-proven simple steatosis (SS), nonalcoholic steatohepatitis (NASH), and healthy controls. HDL turnover and HDL protein dynamics in SS (n = 7), NASH (n = 8), and healthy controls (n = 9) were studied in vivo. HDL maturation and remodeling, antioxidant, cholesterol efflux properties, and activities of lecithin-cholesterol ester acyltransferase and cholesterol ester transfer protein (CETP) were quantified using in vitro assays. All patients with NAFLD had increased turnover of both HDL cholesterol (HDLc; 0.16 ± 0.09 vs. 0.34 ± 0.18 days, P < 0.05) and apolipoprotein A1 (ApoAI) (0.26 ± 0.04 vs. 0.34 ± 0.06 days, P < 0.005) compared with healthy controls. The fractional catabolic rates of other HDL proteins, including ApoAII (and ApoAIV) were higher (P < 0.05) in patients with NAFLD who also had higher CETP activity, ApoAI/HDLc ratio (P < 0.05). NAFLD-induced alterations were associated with lower antioxidant (114.2 ± 46.6 vs. 220.5 ± 48.2 nmol·mL-1·min-1) but higher total efflux properties of HDL (23.4 ± 1.3% vs. 25.5 ± 2.3%) (both P < 0.05), which was more pronounced in individuals with NASH. However, no differences were observed in either HDL turnover, antioxidant, and cholesterol efflux functions of HDL or HDL proteins' turnover between subjects with SS and subjects with NASH. Thus, HDL metabolism and function are altered in NAFLD without any significant differences between SS and NASH.
Assuntos
Lipoproteínas HDL/sangue , Hepatopatia Gordurosa não Alcoólica/sangue , Adulto , Idoso , Antioxidantes/metabolismo , Apolipoproteína A-II/metabolismo , Biomarcadores/sangue , Colesterol/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosfatidilcolina-Esterol O-Aciltransferase/metabolismo , ProteômicaRESUMO
Objective- We have shown that ABCA1 (ATP-binding cassette protein A1) mediates unfolding of the apoA1 (apolipoprotein A1) N-terminal helical hairpin during apoA1 lipidation. Others have shown that an acidic pH exposes the hydrophobic surface of apoA1. We postulated that the V-ATPase (vacuolar ATPase) proton pump facilitates apoA1 unfolding and promotes ABCA1-mediated cholesterol efflux. Approach and Results- We found that V-ATPase inhibitors dose-dependently decreased ABCA1-mediated cholesterol efflux to apoA1 in baby hamster kidney cells and RAW264.7 cells; and similarly, siRNA knockdown of ATP6V0C inhibited ABCA1-mediated cholesterol efflux to apoA1 in RAW264.7 cells. Although ABCA1 expression did not alter total cellular levels of V-ATPase, ABCA1 increased the cell surface levels of the V0A1 and V1E1 subunits of V-ATPase. We generated a fluorescein isothiocyanate/Alexa647 double-labeled fluorescent ratiometric apoA1 pH indicator whose fluorescein isothiocyanate/Alexa647 emission ratio decreased as the pH drops. We found that ABCA1 induction in baby hamster kidney cells led to acidification of the cell-associated apoA1 pH indicator, compared with control cells without ABCA1 expression. The V-ATPase inhibitor bafilomycin A1 dose-dependently inhibited the apoA1 pH shift in ABCA1-expressing cells, without affecting the levels of cell-associated apoA1. However, we were not able to detect ABCA1-mediated extracellular proton release. We showed that acidic pH facilitated apoA1 unfolding, apoA1 solubilization of phosphatidycholine:phosphatidyserine liposomes, and increased lipid fluidity of these liposomes. Conclusions- Our results support a model that ABCA1 recruits V-ATPase to the plasma membrane where V-ATPase mediates apoA1 acidification and membrane remodeling that promote apoA1 unfolding and ABCA1-mediated HDL (high-density lipoprotein) biogenesis and lipid efflux.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Macrófagos/enzimologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Animais , Apolipoproteína A-I/química , Membrana Celular/enzimologia , Cricetinae , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Macrolídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Transporte Proteico , Desdobramento de Proteína , Células RAW 264.7 , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
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/metabolismoRESUMO
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 SexuaisRESUMO
Macrophage accumulation is a critical step during development of chronic inflammation, initiating progression of many devastating diseases. Leukocyte-specific integrin αDß2 (CD11d/CD18) is dramatically upregulated on macrophages at inflammatory sites. Previously we found that CD11d overexpression on cell surfaces inhibits in vitro cell migration due to excessive adhesion. In this study, we have investigated how inflammation-mediated CD11d upregulation contributes to macrophage retention at inflammatory sites during atherogenesis. Atherosclerosis was evaluated in CD11d-/-/ApoE-/- mice after 16 wk on a Western diet. CD11d deficiency led to a marked reduction in lipid deposition in aortas and isolated macrophages. Macrophage numbers in aortic sinuses of CD11d-/- mice were reduced without affecting their apoptosis and proliferation. Adoptive transfer of fluorescently labeled wild-type and CD11d-/- monocytes into ApoE-/- mice demonstrated similar recruitment from circulation, but reduced accumulation of CD11d-/- macrophages within the aortas. Furthermore, CD11d expression was significantly upregulated on macrophages in atherosclerotic lesions and M1 macrophages in vitro. Interestingly, expression of the related ligand-sharing integrin CD11b was not altered. This difference defines their distinct roles in the regulation of macrophage migration. CD11d-deficient M1 macrophages demonstrated improved migration in a three-dimensional fibrin matrix and during resolution of peritoneal inflammation, whereas migration of CD11b-/- M1 macrophages was not affected. These results prove the contribution of high densities of CD11d to macrophage arrest during atherogenesis. Because high expression of CD11d was detected in several inflammation-dependent diseases, we suggest that CD11d/CD18 upregulation on proinflammatory macrophages may represent a common mechanism for macrophage retention at inflammatory sites, thereby promoting chronic inflammation and disease development.
Assuntos
Aterosclerose/imunologia , Vasos Sanguíneos/patologia , Antígenos CD11/genética , Antígenos CD18/genética , Cadeias alfa de Integrinas/genética , Macrófagos/imunologia , Animais , Aorta/imunologia , Aorta/patologia , Apolipoproteínas E/deficiência , Aterosclerose/etiologia , Aterosclerose/patologia , Vasos Sanguíneos/imunologia , Antígenos CD11/imunologia , Antígenos CD18/imunologia , Dieta Ocidental , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Cadeias alfa de Integrinas/deficiência , Cadeias alfa de Integrinas/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Knockout , Peritonite/imunologia , Peritonite/patologia , Ativação Transcricional , Regulação para CimaRESUMO
This paper explores the crucial part that faith-based organisations (FBOs) play in acting as intermediaries between international donors and local faith communities (LFCs) implementing humanitarian relief projects for Syrian refugees. Humanitarian responses to the mounting Syrian refugee crisis have coincided with greater collaboration between international donors and LFCs. This cooperation often is facilitated by a complex web of non-state intermediaries at the international, national, and local level. This study probes the breadth of roles of these intermediaries, drawing on primary data from case studies of two Christian intermediaries supporting Christian LFCs as they deliver aid primarily to Muslim Syrian refugees in Jordan and Lebanon. The results of the study are connected to the wider literature on LFCs in humanitarian response, revealing how intermediaries address issues of accountability, capacity-building, impartiality, neutrality, and professionalism. The paper concludes by offering suggestions for further research on intermediaries as key actors in the localisation of humanitarian assistance.
Assuntos
Organizações Religiosas , Cooperação Internacional , Refugiados , Socorro em Desastres/organização & administração , Humanos , Jordânia , Líbano , Síria/etnologiaRESUMO
The electrocardiographic QRS duration, a measure of ventricular depolarization and conduction, is associated with cardiovascular mortality. While single nucleotide polymorphisms (SNPs) associated with QRS duration have been identified at 22 loci in populations of European descent, the genetic architecture of QRS duration in non-European populations is largely unknown. We therefore performed a genome-wide association study (GWAS) meta-analysis of QRS duration in 13,031 African Americans from ten cohorts and a transethnic GWAS meta-analysis with additional results from populations of European descent. In the African American GWAS, a single genome-wide significant SNP association was identified (rs3922844, P = 4 × 10-14) in intron 16 of SCN5A, a voltage-gated cardiac sodium channel gene. The QRS-prolonging rs3922844 C allele was also associated with decreased SCN5A RNA expression in human atrial tissue (P = 1.1 × 10-4). High density genotyping revealed that the SCN5A association region in African Americans was confined to intron 16. Transethnic GWAS meta-analysis identified novel SNP associations on chromosome 18 in MYL12A (rs1662342, P = 4.9 × 10-8) and chromosome 1 near CD1E and SPTA1 (rs7547997, P = 7.9 × 10-9). The 22 QRS loci previously identified in populations of European descent were enriched for significant SNP associations with QRS duration in African Americans (P = 9.9 × 10-7), and index SNP associations in or near SCN5A, SCN10A, CDKN1A, NFIA, HAND1, TBX5 and SETBP1 replicated in African Americans. In summary, rs3922844 was associated with QRS duration and SCN5A expression, two novel QRS loci were identified using transethnic meta-analysis, and a significant proportion of QRS-SNP associations discovered in populations of European descent were transferable to African Americans when adequate power was achieved.
Assuntos
Doenças Cardiovasculares/genética , Estudo de Associação Genômica Ampla , Ventrículos do Coração/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Negro ou Afro-Americano/genética , Alelos , Doenças Cardiovasculares/mortalidade , Doenças Cardiovasculares/fisiopatologia , Eletrocardiografia , Feminino , Genótipo , Humanos , Masculino , Miocárdio/patologia , Polimorfismo de Nucleotídeo Único/genética , População Branca/genéticaRESUMO
RATIONALE: The molecular mechanism by which ATP-binding cassette transporter A1 (ABCA1) mediates cellular binding of apolipoprotein A-I (apoA1) and nascent high-density lipoprotein (HDL) assembly is not well understood. OBJECTIVE: To determine the cell surface lipid that mediates apoA1 binding to ABCA1-expressing cells and the role it plays in nascent HDL assembly. METHODS AND RESULTS: Using multiple biochemical and biophysical methods, we found that apoA1 binds specifically to phosphatidylinositol (4,5) bis-phosphate (PIP2). Flow cytometry and PIP2 reporter-binding assays demonstrated that ABCA1 led to PIP2 redistribution from the inner to the outer leaflet of the plasma membrane. Enzymatic cleavage of cell surface PIP2 or decreased cellular PIP2 by knockdown of phosphatidylinositol-5-phosphate 4-kinase impaired apoA1 binding and cholesterol efflux to apoA1. PIP2 also increased the spontaneous solubilization of phospholipid liposomes by apoA1. Using site-directed mutagenesis, we found that ABCA1's PIP2 and phosphatidylserine translocase activities are independent from each other. Furthermore, we discovered that PIP2 is effluxed from cells to apoA1, where it is associated with HDL in plasma, and that PIP2 on HDL is taken up by target cells in a scavenger receptor-BI-dependent manner. Mouse plasma PIP2 levels are apoA1 gene dosage-dependent and are >1 µM in apoA1 transgenic mice. CONCLUSIONS: ABCA1 has PIP2 floppase activity, which increases cell surface PIP2 levels that mediate apoA1 binding and lipid efflux during nascent HDL assembly. We found that PIP2 itself is effluxed to apoA1 and it circulates on plasma HDL, where it can be taken up via the HDL receptor scavenger receptor-BI.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Membrana Celular/metabolismo , Lipoproteínas HDL/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Transporte Biológico/fisiologia , Cricetinae , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfatos de Fosfatidilinositol/farmacologia , Ligação Proteica/fisiologiaRESUMO
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/químicaRESUMO
Importance: Atrial fibrillation (AF) is the most common arrhythmia affecting 1% of the population. Young individuals with AF have a strong genetic association with the disease, but the mechanisms remain incompletely understood. Objective: To perform large-scale whole-genome sequencing to identify genetic variants related to AF. Design, Setting, and Participants: The National Heart, Lung, and Blood Institute's Trans-Omics for Precision Medicine Program includes longitudinal and cohort studies that underwent high-depth whole-genome sequencing between 2014 and 2017 in 18â¯526 individuals from the United States, Mexico, Puerto Rico, Costa Rica, Barbados, and Samoa. This case-control study included 2781 patients with early-onset AF from 9 studies and identified 4959 controls of European ancestry from the remaining participants. Results were replicated in the UK Biobank (346â¯546 participants) and the MyCode Study (42â¯782 participants). Exposures: Loss-of-function (LOF) variants in genes at AF loci and common genetic variation across the whole genome. Main Outcomes and Measures: Early-onset AF (defined as AF onset in persons <66 years of age). Due to multiple testing, the significance threshold for the rare variant analysis was P = 4.55 × 10-3. Results: Among 2781 participants with early-onset AF (the case group), 72.1% were men, and the mean (SD) age of AF onset was 48.7 (10.2) years. Participants underwent whole-genome sequencing at a mean depth of 37.8 fold and mean genome coverage of 99.1%. At least 1 LOF variant in TTN, the gene encoding the sarcomeric protein titin, was present in 2.1% of case participants compared with 1.1% in control participants (odds ratio [OR], 1.76 [95% CI, 1.04-2.97]). The proportion of individuals with early-onset AF who carried a LOF variant in TTN increased with an earlier age of AF onset (P value for trend, 4.92 × 10-4), and 6.5% of individuals with AF onset prior to age 30 carried a TTN LOF variant (OR, 5.94 [95% CI, 2.64-13.35]; P = 1.65 × 10-5). The association between TTN LOF variants and AF was replicated in an independent study of 1582 patients with early-onset AF (cases) and 41â¯200 control participants (OR, 2.16 [95% CI, 1.19-3.92]; P = .01). Conclusions and Relevance: In a case-control study, there was a statistically significant association between an LOF variant in the TTN gene and early-onset AF, with the variant present in a small percentage of participants with early-onset AF (the case group). Further research is necessary to understand whether this is a causal relationship.
Assuntos
Fibrilação Atrial/genética , Conectina/genética , Mutação com Perda de Função , Adulto , Idade de Início , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Controle de QualidadeRESUMO
Metabolomics studies hold promise for the discovery of pathways linked to disease processes. Cardiovascular disease (CVD) represents the leading cause of death and morbidity worldwide. Here we used a metabolomics approach to generate unbiased small-molecule metabolic profiles in plasma that predict risk for CVD. Three metabolites of the dietary lipid phosphatidylcholine--choline, trimethylamine N-oxide (TMAO) and betaine--were identified and then shown to predict risk for CVD in an independent large clinical cohort. Dietary supplementation of mice with choline, TMAO or betaine promoted upregulation of multiple macrophage scavenger receptors linked to atherosclerosis, and supplementation with choline or TMAO promoted atherosclerosis. Studies using germ-free mice confirmed a critical role for dietary choline and gut flora in TMAO production, augmented macrophage cholesterol accumulation and foam cell formation. Suppression of intestinal microflora in atherosclerosis-prone mice inhibited dietary-choline-enhanced atherosclerosis. Genetic variations controlling expression of flavin monooxygenases, an enzymatic source of TMAO, segregated with atherosclerosis in hyperlipidaemic mice. Discovery of a relationship between gut-flora-dependent metabolism of dietary phosphatidylcholine and CVD pathogenesis provides opportunities for the development of new diagnostic tests and therapeutic approaches for atherosclerotic heart disease.
Assuntos
Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/microbiologia , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Fosfatidilcolinas/metabolismo , Animais , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/microbiologia , Betaína/sangue , Betaína/metabolismo , Biomarcadores/sangue , Biomarcadores/metabolismo , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/diagnóstico , HDL-Colesterol/sangue , Colina/administração & dosagem , Colina/sangue , Colina/metabolismo , Colina/farmacologia , Dieta/efeitos adversos , Gorduras na Dieta/sangue , Gorduras na Dieta/metabolismo , Gorduras na Dieta/farmacologia , Feminino , Regulação da Expressão Gênica , Vida Livre de Germes , Humanos , Fígado/enzimologia , Macrófagos/metabolismo , Metabolômica , Metilaminas/sangue , Metilaminas/metabolismo , Metilaminas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Oxigenases/genética , Oxigenases/metabolismo , Fenótipo , Fosfatidilcolinas/administração & dosagem , Fosfatidilcolinas/sangue , Fosfatidilcolinas/farmacologia , Receptores Depuradores/metabolismo , Medição de RiscoRESUMO
Nonalcoholic fatty liver disease (NAFLD) is associated with an increased risk of cardiovascular disease. Because the liver is the major source of circulatory proteins, it is not surprising that hepatic disease could lead to alterations in the plasma proteome, which are therein implicated in atherosclerosis. The current study used low-density lipoprotein receptor-deficient (LDLR(-/-)) mice to examine the impact of Western diet (WD)-induced NAFLD on plasma proteome homeostasis. Using a (2)H2O-metabolic labeling method, we found that a WD led to a proinflammatory distribution of circulatory proteins analyzed in apoB-depleted plasma, which was attributed to an increased production. The fractional turnover rates of short-lived proteins that are implicated in stress-response, lipid metabolism, and transport functions were significantly increased with WD (P < 0.05). Pathway analyses revealed that alterations in plasma proteome dynamics were related to the suppression of hepatic PPARα, which was confirmed based on reduced gene and protein expression of PPARα in mice fed a WD. These changes were associated with â¼4-fold increase (P < 0.0001) in the proinflammatory property of apoB-depleted plasma. In conclusion, the proteome dynamics method reveals proinflammatory remodeling of the plasma proteome relevant to liver disease. The approach used herein may provide a useful metric of in vivo liver function and better enable studies of novel therapies surrounding NAFLD and other diseases.