RESUMO
A common metabolic alteration in the tumor microenvironment (TME) is lipid accumulation, a feature associated with immune dysfunction. Here, we examined how CD8+ tumor infiltrating lymphocytes (TILs) respond to lipids within the TME. We found elevated concentrations of several classes of lipids in the TME and accumulation of these in CD8+ TILs. Lipid accumulation was associated with increased expression of CD36, a scavenger receptor for oxidized lipids, on CD8+ TILs, which also correlated with progressive T cell dysfunction. Cd36-/- T cells retained effector functions in the TME, as compared to WT counterparts. Mechanistically, CD36 promoted uptake of oxidized low-density lipoproteins (OxLDL) into T cells, and this induced lipid peroxidation and downstream activation of p38 kinase. Inhibition of p38 restored effector T cell functions in vitro, and resolution of lipid peroxidation by overexpression of glutathione peroxidase 4 restored functionalities in CD8+ TILs in vivo. Thus, an oxidized lipid-CD36 axis promotes intratumoral CD8+ T cell dysfunction and serves as a therapeutic avenue for immunotherapies.
Assuntos
Antígenos CD36/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Peroxidação de Lipídeos/fisiologia , Lipoproteínas LDL/metabolismo , Neoplasias/metabolismo , Receptores Depuradores/metabolismo , Animais , Transporte Biológico/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microambiente Tumoral/fisiologiaRESUMO
Tissue-resident and recruited macrophages contribute to both host defense and pathology. Multiple macrophage phenotypes are represented in diseased tissues, but we lack deep understanding of mechanisms controlling diversification. Here, we investigate origins and epigenetic trajectories of hepatic macrophages during diet-induced non-alcoholic steatohepatitis (NASH). The NASH diet induced significant changes in Kupffer cell enhancers and gene expression, resulting in partial loss of Kupffer cell identity, induction of Trem2 and Cd9 expression, and cell death. Kupffer cell loss was compensated by gain of adjacent monocyte-derived macrophages that exhibited convergent epigenomes, transcriptomes, and functions. NASH-induced changes in Kupffer cell enhancers were driven by AP-1 and EGR that reprogrammed LXR functions required for Kupffer cell identity and survival to instead drive a scar-associated macrophage phenotype. These findings reveal mechanisms by which disease-associated environmental signals instruct resident and recruited macrophages to acquire distinct gene expression programs and corresponding functions.
Assuntos
Microambiente Celular/genética , Reprogramação Celular/genética , Epigênese Genética , Regulação da Expressão Gênica , Células Mieloides/metabolismo , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Biomarcadores , Sequenciamento de Cromatina por Imunoprecipitação , Dieta , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Células de Kupffer/imunologia , Células de Kupffer/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/patologia , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Ligação Proteica , Transdução de Sinais , Análise de Célula ÚnicaRESUMO
BACKGROUND: Familial chylomicronemia syndrome is a genetic disorder associated with severe hypertriglyceridemia and severe acute pancreatitis. Olezarsen reduces the plasma triglyceride level by reducing hepatic synthesis of apolipoprotein C-III. METHODS: In a phase 3, double-blind, placebo-controlled trial, we randomly assigned patients with genetically identified familial chylomicronemia syndrome to receive olezarsen at a dose of 80 mg or 50 mg or placebo subcutaneously every 4 weeks for 49 weeks. There were two primary end points: the difference between the 80-mg olezarsen group and the placebo group in the percent change in the fasting triglyceride level from baseline to 6 months, and (to be assessed if the first was significant) the difference between the 50-mg olezarsen group and the placebo group. Secondary end points included the mean percent change from baseline in the apolipoprotein C-III level and an independently adjudicated episode of acute pancreatitis. RESULTS: A total of 66 patients underwent randomization; 22 were assigned to the 80-mg olezarsen group, 21 to the 50-mg olezarsen group, and 23 to the placebo group. At baseline, the mean (±SD) triglyceride level among the patients was 2630±1315 mg per deciliter, and 71% had a history of acute pancreatitis within the previous 10 years. Triglyceride levels at 6 months were significantly reduced with the 80-mg dose of olezarsen as compared with placebo (-43.5 percentage points; 95% confidence interval [CI], -69.1 to -17.9; P<0.001) but not with the 50-mg dose (-22.4 percentage points; 95% CI, -47.2 to 2.5; P = 0.08). The difference in the mean percent change in the apolipoprotein C-III level from baseline to 6 months in the 80-mg group as compared with the placebo group was -73.7 percentage points (95% CI, -94.6 to -52.8) and between the 50-mg group as compared with the placebo group was -65.5 percentage points (95% CI, -82.6 to -48.3). By 53 weeks, 11 episodes of acute pancreatitis had occurred in the placebo group, and 1 episode had occurred in each olezarsen group (rate ratio [pooled olezarsen groups vs. placebo], 0.12; 95% CI, 0.02 to 0.66). Adverse events of moderate severity that were considered by a trial investigator at the site to be related to the trial drug or placebo occurred in 4 patients in the 80-mg olezarsen group. CONCLUSIONS: In patients with familial chylomicronemia syndrome, olezarsen may represent a new therapy to reduce plasma triglyceride levels. (Funded by Ionis Pharmaceuticals; Balance ClinicalTrials.gov number, NCT04568434.).
Assuntos
Apolipoproteína C-III , Hiperlipoproteinemia Tipo I , Pancreatite , Triglicerídeos , Humanos , Pancreatite/tratamento farmacológico , Masculino , Feminino , Método Duplo-Cego , Apolipoproteína C-III/sangue , Pessoa de Meia-Idade , Adulto , Triglicerídeos/sangue , Hiperlipoproteinemia Tipo I/tratamento farmacológico , Hiperlipoproteinemia Tipo I/sangue , Hiperlipoproteinemia Tipo I/complicações , Doença Aguda , Oligonucleotídeos/uso terapêutico , Oligonucleotídeos/efeitos adversos , Idoso , Hipertrigliceridemia/tratamento farmacológico , Hipertrigliceridemia/sangue , Adulto JovemRESUMO
Liver X receptors (LXRs) are regulators of cholesterol metabolism that also modulate immune responses. Inactivation of LXR α and ß in mice leads to autoimmunity; however, how the regulation of cholesterol metabolism contributes to autoimmunity is unclear. Here we found that cholesterol loading of CD11c+ cells triggered the development of autoimmunity, whereas preventing excess lipid accumulation by promoting cholesterol efflux was therapeutic. LXRß-deficient mice crossed to the hyperlipidemic ApoE-deficient background or challenged with a high-cholesterol diet developed autoantibodies. Cholesterol accumulation in lymphoid organs promoted T cell priming and stimulated the production of the B cell growth factors Baff and April. Conversely, B cell expansion and the development of autoantibodies in ApoE/LXR-ß-deficient mice was reversed by ApoA-I expression. These findings implicate cholesterol imbalance as a contributor to immune dysfunction and suggest that stimulating HDL-dependent reverse cholesterol transport could be beneficial in the setting of autoimmune disease.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Doenças Autoimunes/imunologia , Colesterol/metabolismo , Hipercolesterolemia/metabolismo , Animais , Doenças Autoimunes/metabolismo , Antígeno CD11c/imunologia , Colesterol/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Perfilação da Expressão Gênica , Hipercolesterolemia/imunologia , Receptores X do Fígado/imunologia , Receptores X do Fígado/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , TranscriptomaRESUMO
BACKGROUND: Dyslipidemia increases cardiovascular disease risk, the leading cause of death worldwide. Under time-restricted feeding (TRF), wherein food intake is restricted to a consistent window of <12 hours, weight gain, glucose intolerance, inflammation, dyslipidemia, and hypercholesterolemia are all reduced in mice fed an obesogenic diet. LDLR (low-density lipoprotein receptor) mutations are a major cause of familial hypercholesterolemia and early-onset cardiovascular disease. METHODS: We subjected benchmark preclinical models, mice lacking LDLR-knockout or ApoE knockout to ad libitum feeding of an isocaloric atherogenic diet either ad libitum or 9 hours TRF for up to 13 weeks and assessed disease development, mechanism, and global changes in hepatic gene expression and plasma lipids. In a regression model, a subset of LDLR-knockout mice were ad libitum fed and then subject to TRF. RESULTS: TRF could significantly attenuate weight gain, hypercholesterolemia, and atherosclerosis in mice lacking the LDLR-knockout mice under experimental conditions of both prevention and regression. In LDLR-knockout mice, increased hepatic expression of genes mediating ß-oxidation during fasting is associated with reduced VLDL (very-low-density lipoprotein) secretion and lipid accumulation. Additionally, increased sterol catabolism coupled with fecal loss of cholesterol and bile acids contributes to the atheroprotective effect of TRF. Finally, TRF alone or combined with a cholesterol-free diet can reduce atherosclerosis in LDLR-knockout mice. However, mice lacking ApoE, which is an important protein for hepatic lipoprotein reuptake do not respond to TRF. CONCLUSIONS: In a preclinical animal model, TRF is effective in both the prevention and regression of atherosclerosis in LDLR knockout mice. The results suggest TRF alone or in combination with a low-cholesterol diet can be a lifestyle intervention for reducing cardiovascular disease risk in humans.
Assuntos
Aterosclerose , Modelos Animais de Doenças , Fígado , Camundongos Knockout para ApoE , Receptores de LDL , Animais , Receptores de LDL/genética , Receptores de LDL/deficiência , Aterosclerose/prevenção & controle , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/etiologia , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fatores de Tempo , Jejum/sangue , Camundongos , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Hipercolesterolemia/complicações , Dieta Aterogênica , Aumento de Peso , Camundongos Knockout , Doenças da Aorta/prevenção & controle , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/metabolismo , Lipídeos/sangue , Apolipoproteínas ERESUMO
In this Letter, affiliation number 1 was originally missing from the HTML; the affiliations were missing for author Ming-Yow Hung in the HTML; and the Fig. 4 legend erroneously referred to panels a-h, instead of a-g. These errors have been corrected online.
RESUMO
Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.
Assuntos
Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Hipercolesterolemia/metabolismo , Inflamação/metabolismo , Fosfolipídeos/antagonistas & inibidores , Fosfolipídeos/metabolismo , Animais , Estenose da Valva Aórtica/tratamento farmacológico , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Apoptose , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Colesterol/administração & dosagem , Colesterol/farmacologia , Progressão da Doença , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Feminino , Hipercolesterolemia/patologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Imunoglobulina M/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/patologia , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxirredução , Fosfolipídeos/química , Fosfolipídeos/imunologia , Fosforilcolina/imunologia , Receptores de LDL/deficiência , Receptores de LDL/genética , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/uso terapêuticoRESUMO
Immunoglobulin M (IgM) autoantibodies to oxidation-specific epitopes (OSEs) can be present at birth and protect against atherosclerosis in experimental models. This study sought to determine whether high titers of IgM titers to OSE (IgM OSE) are associated with a lower risk of acute myocardial infarction (AMI) in humans. IgM to malondialdehyde (MDA)-LDL, phosphocholine-modified BSA, IgM apolipoprotein B100-immune complexes, and a peptide mimotope of MDA were measured within 24 h of first AMI in 4,559 patients and 4,617 age- and sex-matched controls in the Pakistan Risk of Myocardial Infarction Study. Multivariate-adjusted logistic regression was used to estimate odds ratio (OR) and 95% confidence interval for AMI. All four IgM OSEs were lower in AMI versus controls (P < 0.001 for all). Males, smokers and individuals with hypertension and diabetes had lower levels of all four IgM OSE than unaffected individuals (P < 0.001 for all). Compared to the lowest quintile, the highest quintiles of IgM MDA-LDL, phosphocholine-modified BSA, IgM apolipoprotein B100-immune complexes, and MDA mimotope P1 had a lower OR of AMI: OR (95% confidence interval) of 0.67 (0.58-0.77), 0.64 (0.56-0.73), 0.70 (0.61-0.80) and 0.72 (0.62-0.82) (P < 0.001 for all), respectively. Upon the addition of IgM OSE to conventional risk factors, the C-statistic improved by 0.0062 (0.0028-0.0095) and net reclassification by 15.5% (11.4-19.6). These findings demonstrate that IgM OSE provides clinically meaningful information and supports the hypothesis that higher levels of IgM OSE may be protective against AMI.
Assuntos
Complexo Antígeno-Anticorpo , Infarto do Miocárdio , Masculino , Recém-Nascido , Humanos , Epitopos , Fosforilcolina , Autoanticorpos , Imunoglobulina M , Apolipoproteínas , Lipoproteínas LDLRESUMO
BACKGROUND: Lipoprotein(a) levels are genetically determined and, when elevated, are a risk factor for cardiovascular disease and aortic stenosis. There are no approved pharmacologic therapies to lower lipoprotein(a) levels. METHODS: We conducted a randomized, double-blind, placebo-controlled, dose-ranging trial involving 286 patients with established cardiovascular disease and screening lipoprotein(a) levels of at least 60 mg per deciliter (150 nmol per liter). Patients received the hepatocyte-directed antisense oligonucleotide AKCEA-APO(a)-LRx, referred to here as APO(a)-LRx (20, 40, or 60 mg every 4 weeks; 20 mg every 2 weeks; or 20 mg every week), or saline placebo subcutaneously for 6 to 12 months. The lipoprotein(a) level was measured with an isoform-independent assay. The primary end point was the percent change in lipoprotein(a) level from baseline to month 6 of exposure (week 25 in the groups that received monthly doses and week 27 in the groups that received more frequent doses). RESULTS: The median baseline lipoprotein(a) levels in the six groups ranged from 204.5 to 246.6 nmol per liter. Administration of APO(a)-LRx resulted in dose-dependent decreases in lipoprotein(a) levels, with mean percent decreases of 35% at a dose of 20 mg every 4 weeks, 56% at 40 mg every 4 weeks, 58% at 20 mg every 2 weeks, 72% at 60 mg every 4 weeks, and 80% at 20 mg every week, as compared with 6% with placebo (P values for the comparison with placebo ranged from 0.003 to <0.001). There were no significant differences between any APO(a)-LRx dose and placebo with respect to platelet counts, liver and renal measures, or influenza-like symptoms. The most common adverse events were injection-site reactions. CONCLUSIONS: APO(a)-LRx reduced lipoprotein(a) levels in a dose-dependent manner in patients who had elevated lipoprotein(a) levels and established cardiovascular disease. (Funded by Akcea Therapeutics; ClinicalTrials.gov number, NCT03070782.).
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Hipolipemiantes/administração & dosagem , Lipoproteína(a)/sangue , Oligonucleotídeos/administração & dosagem , Adulto , Idoso , Doenças Cardiovasculares/sangue , Colesterol/sangue , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipolipemiantes/efeitos adversos , Hipolipemiantes/uso terapêutico , Análise dos Mínimos Quadrados , Masculino , Pessoa de Meia-Idade , Oligonucleotídeos/efeitos adversos , Fatores de RiscoRESUMO
BACKGROUND: Pelacarsen decreases plasma levels of lipoprotein(a) [Lp(a)] and oxidized phospholipids (OxPL). It was previously reported that pelacarsen does not affect the platelet count. We now report the effect of pelacarsen on on-treatment platelet reactivity. METHODS: Subjects with established cardiovascular disease and screening Lp(a) levels ≥60 mg per deciliter (~ ≥150 nmol/L) were randomized to receive pelacarsen (20, 40, or 60 mg every 4 weeks; 20 mg every 2 weeks; or 20 mg every week), or placebo for 6-12 months. Aspirin Reaction Units (ARU) and P2Y12 Reaction Units (PRU) were measured at baseline and the primary analysis timepoint (PAT) at 6 months. RESULTS: Of the 286 subjects randomized, 275 had either an ARU or PRU test, 159 (57.8%) were on aspirin alone and 94 (34.2%) subjects were on dual anti-platelet therapy. As expected, the baseline ARU and PRU were suppressed in subjects on aspirin or on dual anti-platelet therapy, respectively. There were no significant differences in baseline ARU in the aspirin groups or in PRU in the dual anti-platelet groups. At the PAT there were no statistically significant differences in ARU in subjects on aspirin or PRU in subjects on dual anti-platelet therapy among any of the pelacarsen groups compared to the pooled placebo group (p > 0.05 for all comparisons). CONCLUSION: Pelacarsen does not modify on-treatment platelet reactivity through the thromboxane A2 or P2Y12 platelet receptor pathways.
Assuntos
Inibidores da Agregação Plaquetária , Tromboxanos , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Clopidogrel/farmacologia , Estudos Prospectivos , Plaquetas , Aspirina/uso terapêutico , Testes de Função Plaquetária , Resultado do Tratamento , Antagonistas do Receptor Purinérgico P2Y/uso terapêuticoRESUMO
AIMS: Hypertriglyceridaemia is associated with increased risk of cardiovascular events. This clinical trial evaluated olezarsen, an N-acetyl-galactosamine-conjugated antisense oligonucleotide targeted to hepatic APOC3 mRNA to inhibit apolipoprotein C-III (apoC-III) production, in lowering triglyceride levels in patients at high risk for or with established cardiovascular disease. METHODS AND RESULTS: A randomized, double-blind, placebo-controlled, dose-ranging study was conducted in 114 patients with fasting serum triglycerides 200-500 mg/dL (2.26-5.65 mmol/L). Patients received olezarsen (10 or 50 mg every 4 weeks, 15 mg every 2 weeks, or 10 mg every week) or saline placebo subcutaneously for 6-12 months. The primary endpoint was the percent change in fasting triglyceride levels from baseline to Month 6 of exposure. Baseline median (interquartile range) fasting triglyceride levels were 262 (222-329) mg/dL [2.96 (2.51-3.71) mmol/L]. Treatment with olezarsen resulted in mean percent triglyceride reductions of 23% with 10 mg every 4 weeks, 56% with 15 mg every 2 weeks, 60% with 10 mg every week, and 60% with 50 mg every 4 weeks, compared with increase by 6% for the pooled placebo group (P-values ranged from 0.0042 to <0.0001 compared with placebo). Significant decreases in apoC-III, very low-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol, and apolipoprotein B were also observed. There were no platelet count, liver, or renal function changes in any of the olezarsen groups. The most common adverse event was mild erythema at the injection site. CONCLUSION: Olezarsen significantly reduced apoC-III, triglycerides, and atherogenic lipoproteins in patients with moderate hypertriglyceridaemia and at high risk for or with established cardiovascular disease. TRIAL REGISTRATION NUMBER: NCT03385239.
Assuntos
Doenças Cardiovasculares , Hipertrigliceridemia , Apolipoproteína C-III , Doenças Cardiovasculares/prevenção & controle , Colesterol , Fatores de Risco de Doenças Cardíacas , Humanos , Hipertrigliceridemia/complicações , Hipertrigliceridemia/tratamento farmacológico , Lipoproteínas/uso terapêutico , Fatores de Risco , TriglicerídeosRESUMO
The burden of nonalcoholic fatty liver disease (NAFLD) is rising globally. Cardiovascular disease is the leading cause of death in patients with NAFLD. Nearly half of individuals with NAFLD have coronary heart disease, and more than a third have carotid artery atherosclerosis. Individuals with NAFLD are at a substantially higher risk of fatal and nonfatal cardiovascular events. NAFLD and cardiovascular disease share multiple common disease mechanisms, such as systemic inflammation, insulin resistance, genetic risk variants, and gut microbial dysbiosis. In this review, we discuss the epidemiology of cardiovascular disease in NAFLD, and highlight common risk factors. In addition, we examine recent advances evaluating the shared disease mechanisms between NAFLD and cardiovascular disease. In conclusion, multidisciplinary collaborations are required to further our understanding of the complex relationship between NAFLD and cardiovascular disease and potentially identify therapeutic targets.
Assuntos
Doenças Cardiovasculares , Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Hepatopatia Gordurosa não Alcoólica/complicações , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/etiologia , Fatores de Risco , Inflamação/complicaçõesRESUMO
The study aims were to develop a new isoform-independent enzyme-linked immunoassay (ELISA) for the measurement of lipoprotein(a) [Lp(a)], validate its performance characteristics, and demonstrate its accuracy by comparison with the gold-standard ELISA method and an LC-MS/MS candidate reference method, both developed at the University of Washington. The principle of the new assay is the capture of Lp(a) with monoclonal antibody LPA4 primarily directed to an epitope in apolipoprotein(a) KIV2 and its detection with monoclonal antibody LPA-KIV9 directed to a single antigenic site present on KIV9. Validation studies were performed following the guidelines of the Clinical Laboratory Improvement Amendments and the College of American Pathologists. The analytical measuring range of the LPA4/LPA-KIV9 ELISA is 0.27-1,402 nmol/L, and the method meets stringent criteria for precision, linearity, spike and recovery, dilutability, comparison of plasma versus serum, and accuracy. Method comparison with both the gold-standard ELISA and the LC-MS/MS method performed in 64 samples with known apolipoprotein(a) isoforms resulted in excellent correlation with both methods (r=0.987 and r=0.976, respectively). Additionally, the variation in apolipoprotein(a) size accounted for only 0.2% and 2.2% of the bias variation, respectively, indicating that the LPA4/LPA-KIV9 ELISA is not affected by apolipoprotein(a) size polymorphism. Peptide mapping and competition experiments demonstrated that the measuring monoclonal antibodies used in the gold-standard ELISA (a-40) and in the newly developed ELISA (LPA-KIV9) are directed to the same epitope, 4076LETPTVV4082, on KIV9. In conclusion, no statistically or clinically significant bias was observed between Lp(a) measurements obtained by the LPA4/LPA-KIV9 ELISA and those obtained by the gold-standard ELISA or LC-MS/MS, and therefore, the methods are considered equivalent.
Assuntos
Anticorpos Monoclonais , Lipoproteína(a) , Apolipoproteínas A , Apoproteína(a) , Cromatografia Líquida , Ensaio de Imunoadsorção Enzimática , Epitopos , Humanos , Isoformas de Proteínas , Espectrometria de Massas em TandemRESUMO
BACKGROUND: LNK/SH2B3 inhibits Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling by hematopoietic cytokine receptors. Genome-wide association studies have shown association of a common single nucleotide polymorphism in LNK (R262W, T allele) with neutrophilia, thrombocytosis, and coronary artery disease. We have shown that LNK(TT) reduces LNK function and that LNK-deficient mice display prominent platelet-neutrophil aggregates, accelerated atherosclerosis, and thrombosis. Platelet-neutrophil interactions can promote neutrophil extracellular trap (NET) formation. The goals of this study were to assess the role of NETs in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency. METHODS: We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme PAD4 (peptidyl arginine deiminase 4) and transplanted their bone marrow into Ldlr-/- mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2V617F). RESULTS: Lnk-deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated Lnk-/- platelets promoted increased NETosis when incubated with Lnk-/- neutrophils compared with wild-type platelets or wild-type neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from Lnk-/- platelets, as well as increased priming and response of Lnk-/- neutrophils to OxPL. To counteract the effects of OxPL, we introduced a transgene expressing the single-chain variable fragment of E06 (E06-scFv). E06-scFv reversed accelerated NETosis, atherosclerosis, and thrombosis in Lnk-/- mice. We also showed increased NETosis when human induced pluripotent stem cell-derived LNK(TT) neutrophils were incubated with LNK(TT) platelet/megakaryocytes, but not in isogenic LNK(CC) controls, confirming human relevance. Using data from the UK Biobank, we found that individuals with the JAK2VF mutation only showed increased risk of coronary artery disease when also carrying the LNK R262W allele. Mice with hematopoietic Lnk+/- and Jak2VF clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with Jak2VFLnk+/+ controls. CONCLUSIONS: Hematopoietic Lnk deficiency promotes NETosis and arterial thrombosis in an OxPL-dependent fashion. LNK(R262W) reduces LNK function in human platelets and neutrophils, promoting NETosis, and increases coronary artery disease risk in humans carrying Jak2VF mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Plaquetas/metabolismo , Neutrófilos/metabolismo , Fosfolipídeos/metabolismo , Agregação Plaquetária , Trombose/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Artérias/metabolismo , Camundongos , Camundongos Knockout , Oxirredução , Fosfolipídeos/genética , Trombose/genéticaRESUMO
BACKGROUND: Familial chylomicronemia syndrome is a rare genetic disorder that is caused by loss of lipoprotein lipase activity and characterized by chylomicronemia and recurrent episodes of pancreatitis. There are no effective therapies. In an open-label study of three patients with this syndrome, antisense-mediated inhibition of hepatic APOC3 mRNA with volanesorsen led to decreased plasma apolipoprotein C-III and triglyceride levels. METHODS: We conducted a phase 3, double-blind, randomized 52-week trial to evaluate the safety and effectiveness of volanesorsen in 66 patients with familial chylomicronemia syndrome. Patients were randomly assigned, in a 1:1 ratio, to receive volanesorsen or placebo. The primary end point was the percentage change in fasting triglyceride levels from baseline to 3 months. RESULTS: Patients receiving volanesorsen had a decrease in mean plasma apolipoprotein C-III levels from baseline of 25.7 mg per deciliter, corresponding to an 84% decrease at 3 months, whereas patients receiving placebo had an increase in mean plasma apolipoprotein C-III levels from baseline of 1.9 mg per deciliter, corresponding to a 6.1% increase (P<0.001). Patients receiving volanesorsen had a 77% decrease in mean triglyceride levels, corresponding to a mean decrease of 1712 mg per deciliter (19.3 mmol per liter) (95% confidence interval [CI], 1330 to 2094 mg per deciliter [15.0 to 23.6 mmol per liter]), whereas patients receiving placebo had an 18% increase in mean triglyceride levels, corresponding to an increase of 92.0 mg per deciliter (1.0 mmol per liter) (95% CI, -301.0 to 486 mg per deciliter [-3.4 to 5.5 mmol per liter]) (P<0.001). At 3 months, 77% of the patients in the volanesorsen group, as compared with 10% of patients in the placebo group, had triglyceride levels of less than 750 mg per deciliter (8.5 mmol per liter). A total of 20 of 33 patients who received volanesorsen had injection-site reactions, whereas none of the patients who received placebo had such reactions. No patients in the placebo group had platelet counts below 100,000 per microliter, whereas 15 of 33 patients in the volanesorsen group had such levels, including 2 who had levels below 25,000 per microliter. No patient had platelet counts below 50,000 per microliter after enhanced platelet-monitoring began. CONCLUSIONS: Volanesorsen lowered triglyceride levels to less than 750 mg per deciliter in 77% of patients with familial chylomicronemia syndrome. Thrombocytopenia and injection-site reactions were common adverse events. (Funded by Ionis Pharmaceuticals and Akcea Therapeutics; APPROACH Clinical Trials.gov number, NCT02211209.).
Assuntos
Apolipoproteína C-III/antagonistas & inibidores , Hiperlipoproteinemia Tipo I/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , RNA Mensageiro/antagonistas & inibidores , Trombocitopenia/induzido quimicamente , Triglicerídeos/sangue , Adulto , Idoso , Análise de Variância , Apolipoproteína C-III/sangue , Apolipoproteína C-III/genética , Método Duplo-Cego , Feminino , Humanos , Hiperlipoproteinemia Tipo I/sangue , Injeções Subcutâneas/efeitos adversos , Masculino , Pessoa de Meia-Idade , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/efeitos adversos , Contagem de Plaquetas , Adulto JovemRESUMO
RATIONALE: Patients with elevated levels of lipoprotein(a) [Lp(a)] are hallmarked by increased metabolic activity in the arterial wall on positron emission tomography/computed tomography, indicative of a proinflammatory state. OBJECTIVE: We hypothesized that Lp(a) induces endothelial cell inflammation by rewiring endothelial metabolism. METHODS AND RESULTS: We evaluated the impact of Lp(a) on the endothelium and describe that Lp(a), through its oxidized phospholipid content, activates arterial endothelial cells, facilitating increased transendothelial migration of monocytes. Transcriptome analysis of Lp(a)-stimulated human arterial endothelial cells revealed upregulation of inflammatory pathways comprising monocyte adhesion and migration, coinciding with increased 6-phophofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB)-3-mediated glycolysis. ICAM (intercellular adhesion molecule)-1 and PFKFB3 were also found to be upregulated in carotid plaques of patients with elevated levels of Lp(a). Inhibition of PFKFB3 abolished the inflammatory signature with concomitant attenuation of transendothelial migration. CONCLUSIONS: Collectively, our findings show that Lp(a) activates the endothelium by enhancing PFKFB3-mediated glycolysis, leading to a proadhesive state, which can be reversed by inhibition of glycolysis. These findings pave the way for therapeutic agents targeting metabolism aimed at reducing inflammation in patients with cardiovascular disease.
Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Glicólise , Leucócitos/metabolismo , Lipoproteína(a)/metabolismo , Migração Transendotelial e Transepitelial , Idoso , Idoso de 80 Anos ou mais , Animais , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Apolipoproteínas A/genética , Apolipoproteínas A/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/terapia , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais/patologia , Feminino , Humanos , Mediadores da Inflamação , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/patologia , Lipoproteína(a)/genética , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , Oligonucleotídeos Antissenso/uso terapêutico , Fosfofrutoquinase-2/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genéticaRESUMO
Current methods for determining "LDL-C" in clinical practice measure the cholesterol content of both LDL and lipoprotein(a) [Lp(a)-C]. We developed a high-throughput, sensitive, and rapid method to quantitate Lp(a)-C and improve the accuracy of LDL-C by subtracting for Lp(a)-C (LDL-Ccorr). Lp(a)-C is determined following isolation of the Lp(a) on magnetic beads linked to monoclonal antibody LPA4 recognizing apolipoprotein(a). This Lp(a)-C assay does not detect cholesterol in plasma samples lacking Lp(a) and is linear up to 747 nM Lp(a). To validate this method clinically over a wide range of Lp(a) (9.0-822.8 nM), Lp(a)-C and LDL-Ccorr were determined in 21 participants receiving an Lp(a)-specific lowering antisense oligonucleotide and in eight participants receiving placebo at baseline, at 13 weeks during peak drug effect, and off drug. In the groups combined, Lp(a)-C ranged from 0.6 to 35.0 mg/dl and correlated with Lp(a) molar concentration (r = 0.76; P < 0.001). However, the percent Lp(a)-C relative to Lp(a) mass varied from 5.8% to 57.3%. Baseline LDL-Ccorr was lower than LDL-C [mean (SD), 102.2 (31.8) vs. 119.2 (32.4) mg/dl; P < 0.001] and did not correlate with Lp(a)-C. It was demonstrated that three commercially available "direct LDL-C" assays also include measures of Lp(a)-C. In conclusion, we have developed a novel and sensitive method to quantitate Lp(a)-C that provides insights into the Lp(a) mass/cholesterol relationship and may be used to more accurately report LDL-C and reassess its role in clinical medicine.
Assuntos
LDL-ColesterolRESUMO
RATIONALE: B-1 cell-derived natural IgM antibodies against oxidation-specific epitopes on low-density lipoprotein are anti-inflammatory and atheroprotective. Bone marrow (BM) B-1a cells contribute abundantly to IgM production, yet the unique repertoire of IgM antibodies generated by BM B-1a and the factors maintaining the BM B-1a population remain unexplored. CXCR4 (C-X-C motif chemokine receptor 4) has been implicated in human cardiovascular disease and B-cell homeostasis, yet the role of B-1 cell CXCR4 in regulating atheroprotective IgM levels and human cardiovascular disease is unknown. OBJECTIVE: To characterize the BM B-1a IgM repertoire and to determine whether CXCR4 regulates B-1 production of atheroprotective IgM in mice and humans. METHODS AND RESULTS: Single-cell sequencing demonstrated that BM B-1a cells from aged ApoE-/- mice with established atherosclerosis express a unique repertoire of IgM antibodies containing increased nontemplate-encoded nucleotide additions and a greater frequency of unique heavy chain complementarity determining region 3 sequences compared with peritoneal cavity B-1a cells. Some complementarity determining region 3 sequences were common to both compartments suggesting B-1a migration between compartments. Indeed, mature peritoneal cavity B-1a cells migrated to BM in a CXCR4-dependent manner. Furthermore, BM IgM production and plasma IgM levels were reduced in ApoE-/- mice with B-cell-specific knockout of CXCR4, and overexpression of CXCR4 on B-1a cells increased BM localization and plasma IgM against oxidation specific epitopes, including IgM specific for malondialdehyde-modified LDL (low-density lipoprotein). Finally, in a 50-subject human cohort, we find that CXCR4 expression on circulating human B-1 cells positively associates with plasma levels of IgM antibodies specific for malondialdehyde-modified LDL and inversely associates with human coronary artery plaque burden and necrosis. CONCLUSIONS: These data provide the first report of a unique BM B-1a cell IgM repertoire and identifies CXCR4 expression as a critical factor selectively governing BM B-1a localization and production of IgM against oxidation specific epitopes. That CXCR4 expression on human B-1 cells was greater in humans with low coronary artery plaque burden suggests a potential targeted approach for immune modulation to limit atherosclerosis.