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1.
Atherosclerosis ; 312: 1-7, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32942042

RESUMO

BACKGROUND AND AIMS: In focal areas of advanced human atherosclerotic lesions, the intimal fluid is acidic. An acidic medium impairs the ABCA1-mediated cholesterol efflux from macrophages, so tending to increase their content of free cholesterol, which is then available for esterification by the macrophage enzyme ACAT1. Here we investigated whether low extracellular pH would affect the activity of ACAT1. METHODS: - Human monocyte-derived macrophages were first incubated with acetyl-LDL at neutral and acidic conditions (pH 7.5, 6.5, and 5.5) to generate foam cells, and then the foam cells were incubated with [3H]oleate-BSA complexes, and the formation of [3H]oleate-labeled cholesteryl esters was measured. ACAT1 activity was also measured in cell-free macrophage extracts. RESULTS: - In acidic media, ACAT1-dependent cholesteryl [3H]oleate generation became compromised in the developing foam cells and their content of free cholesterol increased. In line with this finding, ACAT1 activity in the soluble cell-free fraction derived from macrophage foam cells peaked at pH 7, and gradually decreased under acidic pH with a rapid drop below pH 6.5. Incubation of macrophages under progressively more acidic conditions (until pH 5.5) lowered the cytosolic pH of macrophages (down to pH 6.0). Such intracellular acidification did not affect macrophage gene expression of ACAT1 or the neutral CEH. CONCLUSIONS: Exposure of human macrophage foam cells to acidic conditions lowers their intracellular pH with simultaneous decrease in ACAT1 activity. This reduces cholesterol esterification and thus leads to accumulation of potentially toxic levels of free cholesterol, a contributing factor to macrophage foam cell death.

2.
Arterioscler Thromb Vasc Biol ; 40(9): 2310-2321, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32611242

RESUMO

OBJECTIVE: Plant stanol ester supplementation (2-3 g plant stanols/d) reduces plasma LDL (low-density lipoprotein) cholesterol concentration by 9% to 12% and is, therefore, recommended as part of prevention and treatment of atherosclerotic cardiovascular disease. In addition to plasma LDL-cholesterol concentration, also qualitative properties of LDL particles can influence atherogenesis. However, the effect of plant stanol ester consumption on the proatherogenic properties of LDL has not been studied. Approach and Results: Study subjects (n=90) were randomized to consume either a plant stanol ester-enriched spread (3.0 g plant stanols/d) or the same spread without added plant stanol esters for 6 months. Blood samples were taken at baseline and after the intervention. The aggregation susceptibility of LDL particles was analyzed by inducing aggregation of isolated LDL and following aggregate formation. LDL lipidome was determined by mass spectrometry. Binding of serum lipoproteins to proteoglycans was measured using a microtiter well-based assay. LDL aggregation susceptibility was decreased in the plant stanol ester group, and the median aggregate size after incubation for 2 hours decreased from 1490 to 620 nm, P=0.001. Plant stanol ester-induced decrease in LDL aggregation was more extensive in participants having body mass index<25 kg/m2. Decreased LDL aggregation susceptibility was associated with decreased proportion of LDL-sphingomyelins and increased proportion of LDL-triacylglycerols. LDL binding to proteoglycans was decreased in the plant stanol ester group, the decrease depending on decreased serum LDL-cholesterol concentration. CONCLUSIONS: Consumption of plant stanol esters decreases the aggregation susceptibility of LDL particles by modifying LDL lipidome. The resulting improvement of LDL quality may be beneficial for cardiovascular health. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01315964.

3.
Hepatology ; 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31785104

RESUMO

BACKGROUND AND AIMS: Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species-related, physiological differences. APPROACH AND RESULTS: Fah-/- , Rag2-/- , and Il2rg-/- knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, characterized by a high ratio of low-density lipoprotein to high-density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low-density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human-like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level. CONCLUSIONS: LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.

4.
J Clin Lipidol ; 13(6): 910-919.e2, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31753722

RESUMO

BACKGROUND: South Asians are more prone to develop atherosclerotic cardiovascular disease (ASCVD) compared with white Caucasians, which is not fully explained by classical risk factors. We recently reported that the presence of aggregation-prone low-density lipoprotein (LDL) in the circulation is associated with increased ASCVD mortality. OBJECTIVE: We hypothesized that LDL of South Asians is more prone to aggregate, which may be explained by differences in their LDL lipid composition. METHODS: In this cross-sectional hypothesis-generating study, LDL was isolated from plasma of healthy South Asians (n = 12) and age- and BMI-matched white Caucasians (n = 12), and its aggregation susceptibility and lipid composition were analyzed. RESULTS: LDL from South Asians was markedly more prone to aggregate compared with white Caucasians. Among all measured lipids, sphingomyelin 24:0 and triacylglycerol 56:8 showed the highest positive correlation with LDL aggregation. In addition, LDL from South Asians was enriched in arachidonic acid containing phosphatidylcholine 38:4 and had less phosphatidylcholines and cholesteryl esters containing monounsaturated fatty acids. Interestingly, body fat percentage, which was higher in South Asians (+26%), positively correlated with LDL aggregation and highly positively correlated with triacylglycerol 56:8, sphingomyelin 24:0, and total sphingomyelin. CONCLUSIONS: LDL aggregation susceptibility is higher in healthy young South Asians compared with white Caucasians. This may be partly explained by the higher body fat percentage of South Asians, leading to sphingomyelin enrichment of LDL. We anticipate that the presence of sphingomyelin-rich, aggregation-prone LDL particles in young South Asians may increase LDL accumulation in the arterial wall and thereby contribute to their increased risk of developing ASCVD later in life.

5.
Sci Rep ; 9(1): 11235, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375727

RESUMO

Low-density lipoprotein (LDL) is considered the major risk factor for the development of atherosclerotic cardiovascular diseases (ASCVDs). A novel and rapid method for the isolation of LDL from human plasma was developed utilising affinity chromatography with monolithic stationary supports. The isolation method consisted of two polymeric monolithic disk columns, one immobilized with chondroitin-6-sulfate (C6S) and the other with apolipoprotein B-100 monoclonal antibody (anti-apoB-100 mAb). The first disk with C6S was targeted to remove chylomicrons, very-low-density lipoprotein (VLDL) particles, and their remnants including intermediate-density lipoprotein (IDL) particles, thus allowing the remaining major lipoprotein species, i.e. LDL, lipoprotein(a) (Lp(a)), and high-density lipoprotein (HDL) to flow to the anti-apoB-100 disk. The second disk captured LDL particles via the anti-apoB-100 mAb attached on the disk surface in a highly specific manner, permitting the selective LDL isolation. The success of LDL isolation was confirmed by different techniques including quartz crystal microbalance. In addition, the method developed gave comparable results with ultracentrifugation, conventionally used as a standard method. The reliable results achieved together with a short isolation time (less than 30 min) suggest the method to be suitable for clinically relevant LDL functional assays.

6.
Nat Rev Cardiol ; 16(7): 389-406, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30846875

RESUMO

Atherosclerosis is a lipid-driven inflammatory disease of the arterial intima in which the balance of pro-inflammatory and inflammation-resolving mechanisms dictates the final clinical outcome. Intimal infiltration and modification of plasma-derived lipoproteins and their uptake mainly by macrophages, with ensuing formation of lipid-filled foam cells, initiate atherosclerotic lesion formation, and deficient efferocytotic removal of apoptotic cells and foam cells sustains lesion progression. Defective efferocytosis, as a sign of inadequate inflammation resolution, leads to accumulation of secondarily necrotic macrophages and foam cells and the formation of an advanced lesion with a necrotic lipid core, indicative of plaque vulnerability. Resolution of inflammation is mediated by specialized pro-resolving lipid mediators derived from omega-3 fatty acids or arachidonic acid and by relevant proteins and signalling gaseous molecules. One of the major effects of inflammation resolution mediators is phenotypic conversion of pro-inflammatory macrophages into macrophages that suppress inflammation and promote healing. In advanced atherosclerotic lesions, the ratio between specialized pro-resolving mediators and pro-inflammatory lipids (in particular leukotrienes) is strikingly low, providing a molecular explanation for the defective inflammation resolution features of these lesions. In this Review, we discuss the mechanisms of the formation of clinically dangerous atherosclerotic lesions and the potential of pro-resolving mediator therapy to inhibit this process.


Assuntos
Aterosclerose/etiologia , Aterosclerose/fisiopatologia , Inflamação/etiologia , Inflamação/fisiopatologia , Macrófagos/fisiologia , Túnica Íntima/patologia , Animais , Apoptose , Aterosclerose/prevenção & controle , Humanos , Inflamassomos/metabolismo , Inflamação/tratamento farmacológico , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Transdução de Sinais
7.
Atherosclerosis ; 281: 56-61, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30658192

RESUMO

BACKGROUND AND AIMS: Omega-3 fatty acids are known to have several cardioprotective effects. Our aim was to investigate the effects of intakes of fish and Camelina sativa oil (CSO), rich in alpha-linolenic acid, on the atherogenic and anti-atherogenic functions of LDL and HDL particles. METHODS: Altogether, 88 volunteers with impaired glucose metabolism were randomly assigned to CSO (10 g of alpha-linolenic acid/day), fatty fish (4 fish meals/week), lean fish (4 fish meals/week) or control group for 12 weeks. 79 subjects completed the study. The binding of lipoproteins to aortic proteoglycans, LDL aggregation and activation of endothelial cells by LDL and cholesterol efflux capacity of HDL were determined in vitro. RESULTS: Intake of CSO decreased the binding of lipoproteins to aortic proteoglycans in a non-normalized model (p = 0.006). After normalizing with serum concentrations of non-HDL cholesterol, apolipoprotein B (apoB) or LDL cholesterol, which decreased in the CSO group, the change was no longer statistically significant. In the fish groups, there were no changes in the binding of lipoproteins to proteoglycans. Regarding other lipoprotein functions, there were no changes in any of the groups. CONCLUSIONS: Intake of CSO decreases the binding of lipoproteins to aortic proteoglycans by decreasing serum LDL cholesterol concentration, which suggests that the level of apoB-containing lipoproteins in the circulation is the main driver of lipoprotein retention within the arterial wall. Intake of fish or CSO has no effects on other lipoprotein functions.


Assuntos
Brassicaceae , Colesterol/sangue , Dieta Saudável , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácido Eicosapentaenoico/administração & dosagem , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Óleos Vegetais/administração & dosagem , Alimentos Marinhos , Adulto , Idoso , Aorta/metabolismo , Biomarcadores/sangue , Células Cultivadas , Suplementos Nutricionais/efeitos adversos , Ácidos Docosa-Hexaenoicos/efeitos adversos , Ácido Eicosapentaenoico/efeitos adversos , Células Endoteliais/metabolismo , Feminino , Finlândia , Humanos , Masculino , Pessoa de Meia-Idade , Óleos Vegetais/efeitos adversos , Ligação Proteica , Proteoglicanas/metabolismo , Recomendações Nutricionais
8.
Curr Med Chem ; 26(9): 1701-1710, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29848270

RESUMO

Apolipoprotein B -containing lipoproteins include triglyceride-rich lipoproteins (chylomicrons and their remnants, and very low-density lipoproteins and their remnants) and cholesterol-rich low-density lipoprotein particles. Of these, lipoproteins having sizes below 70-80 nm may enter the arterial wall, where they accumulate and induce the formation of atherosclerotic lesions. The processes that lead to accumulation of lipoprotein-derived lipids in the arterial wall have been largely studied with a focus on the low-density lipoprotein particles. However, recent observational and genetic studies have discovered that the triglyceriderich lipoproteins and their remnants are linked with cardiovascular disease risk. In this review, we describe the potential mechanisms by which the triglyceride-rich remnant lipoproteins can contribute to the development of atherosclerotic lesions, and highlight the differences in the atherogenicity between low-density lipoproteins and the remnant lipoproteins.


Assuntos
Artérias/metabolismo , Inflamação/metabolismo , Lipídeos/química , Lipoproteínas/metabolismo , Triglicerídeos/metabolismo , Animais , Artérias/química , Doença da Artéria Coronariana/metabolismo , Doença da Artéria Coronariana/patologia , Humanos , Lipoproteínas/química , Triglicerídeos/química
9.
Lipids Health Dis ; 17(1): 285, 2018 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-30545366

RESUMO

BACKGROUND: The focus of studies on high-density lipoproteins (HDL) has shifted from HDL-cholesterol (HDL-C) to HDL function. We recently demonstrated that low USF1 expression in mice and humans associates with high plasma HDL-C and low triglyceride levels, as well as protection against obesity, insulin resistance, and atherosclerosis. Here, we studied the impact of USF1 deficiency on HDL functional capacity and macrophage atherogenic functions, including inflammation, cholesterol efflux, and cholesterol accumulation. METHODS: We used a congenic Usf1 deficient mice in C57Bl/6JRccHsd background and blood samples were collected to isolate HDL for structural and functional studies. Lentiviral preparations containing the USF1 silencing shRNA expression vector were used to silence USF1 in human THP-1 and Huh-7 cells. Cholesterol efflux from acetyl-LDL loaded THP-1 macrophages was measured using HDL and plasma as acceptors. Gene expression analysis from USF1 silenced peritoneal macrophages was carried out using Affymetrix protocols. RESULTS: We show that Usf1 deficiency not only increases HDL-C levels in vivo, consistent with elevated ABCA1 protein expression in hepatic cell lines, but also improves the functional capacity of HDL particles. HDL particles derived from Usf1 deficient mice remove cholesterol more efficiently from macrophages, attributed to their higher contents of phospholipids. Furthermore, silencing of USF1 in macrophages enhanced the cholesterol efflux capacity of these cells. These findings are consistent with reduced inflammatory burden of USF1 deficient macrophages, manifested by reduced secretion of pro-inflammatory cytokines MCP-1 and IL-1ß and protection against inflammation-induced macrophage cholesterol accumulation in a cell-autonomous manner. CONCLUSIONS: Our findings identify USF1 as a novel factor regulating HDL functionality, showing that USF1 inactivation boosts cholesterol efflux, reduces macrophage inflammation and attenuates macrophage cholesterol accumulation, linking improved macrophage cholesterol metabolism and inflammatory pathways to the antiatherogenic function of USF1 deficiency.


Assuntos
HDL-Colesterol/genética , Colesterol/genética , Lipoproteínas HDL/genética , Fatores Estimuladores Upstream/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Quimiocina CCL2/genética , Colesterol/sangue , Expressão Gênica/genética , Humanos , Inflamação/sangue , Inflamação/genética , Inflamação/patologia , Resistência à Insulina/genética , Lipoproteínas HDL/sangue , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Knockout , Obesidade/sangue , Obesidade/genética , Obesidade/patologia
10.
Front Immunol ; 9: 2701, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30519244

RESUMO

The alternative pathway (AP) of complement is constantly active in plasma and can easily be activated on self surfaces and trigger local inflammation. Host cells are protected from AP attack by Factor H (FH), the main AP regulator in plasma. Although complement is known to play a role in atherosclerosis, the mechanisms of its contribution are not fully understood. Since FH via its domains 5-7 binds apoliporotein E (apoE) and macrophages produce apoE we examined how FH could be involved in the antiatherogenic effects of apoE. We used blood peripheral monocytes and THP-1 monocyte/macrophage cells which were also loaded with acetylated low-density lipoprotein (LDL) to form foam cells. Binding of FH and apoE on these cells was analyzed by flow cytometry. High-density lipoprotein (HDL)-mediated cholesterol efflux of activated THP-1 cells was measured and transcriptomes of THP-1 cells using mRNA sequencing were determined. We found that binding of FH to human blood monocytes and cholesterol-loaded THP-1 macrophages increased apoE binding to these cells. Preincubation of fluorescent cholesterol labeled THP-1 macrophages in the presence of FH increased cholesterol efflux and cholesterol-loaded macrophages displayed reduced transcription of proinflammatory/proatherogenic factors and increased transcription of anti-inflammatory/anti-atherogenic factors. Further incubation of THP-1 cells with serum reduced C3b/iC3b deposition. Overall, our data indicate that apoE and FH interact with monocytic cells in a concerted action and this interaction reduces complement activation and inflammation in the atherosclerotic lesions. By this way FH may participate in mediating the beneficial effects of apoE in suppressing atherosclerotic lesion progression.


Assuntos
Apolipoproteínas E/imunologia , Aterosclerose/imunologia , Fator H do Complemento/imunologia , Células Espumosas/imunologia , Monócitos/imunologia , Aterosclerose/patologia , Complemento C3b/imunologia , Células Espumosas/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Lipoproteínas HDL/imunologia , Monócitos/patologia , Células THP-1 , Transcrição Genética/imunologia
11.
J Am Heart Assoc ; 7(24): e009876, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30561264

RESUMO

Background In randomized trials (SHARP [Study of Heart and Renal Protection], IMPROVE -IT [Improved Reduction of Outcomes: Vytorin Efficacy International Trial]), combination of statin and ezetimibe resulted in additional reduction of cardiovascular events. The reduction was greater in patients with type 2 diabetes mellitus (T2 DM ), where elevated remnant cholesterol and high cardiovascular disease risk is characteristic. To evaluate possible causes behind these results, 40 patients eligible for cholecystectomy, randomized to simvastatin, ezetimibe, combined treatment (simvastatin+ezetimibe), or placebo treatment during 4 weeks before surgery, were studied. Methods and Results Fasting blood samples were taken before treatment start and at the end (just before surgery). Bile samples and liver biopsies were collected during surgery. Hepatic gene expression levels were assessed with qPCR . Lipoprotein, apolipoprotein levels, and content of cholesterol, cholesteryl ester, and triglycerides were measured after lipoprotein fractionation. Lipoprotein subclasses were analyzed by nuclear magnetic resonance. Apolipoprotein affinity for human arterial proteoglycans ( PG ) was measured. Biomarkers of cholesterol biosynthesis and intestinal absorption and bile lipid composition were analyzed using mass spectrometry. Combined treatment caused a statistically significant decrease in plasma remnant particles and apolipoprotein B (ApoB)/lipoprotein content of cholesterol, cholesteryl esters, and triglycerides. All treatments reduced ApoB-lipoprotein PG binding. Simvastatin and combined treatment modified the composition of lipoproteins. Changes in biomarkers of cholesterol synthesis and absorption and bile acid synthesis were as expected. No adverse events were found. Conclusions Combined treatment caused atheroprotective changes on ApoB-lipoproteins, remnant particles, bile components, and in ApoB-lipoprotein affinity for arterial PG . These effects might explain the decrease of cardiovascular events seen in the SHARP and IMPROVE - IT trials. Clinical Trial Registration URL : www.clinicaltrialsregister.eu . Unique identifier: 2006-004839-30).


Assuntos
Bile/metabolismo , Colesterol/sangue , Remanescentes de Quilomícrons/sangue , Dislipidemias/tratamento farmacológico , Combinação Ezetimiba e Simvastatina/uso terapêutico , Cálculos Biliares/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Apolipoproteína B-100/sangue , Biomarcadores/sangue , Colecistectomia , Dislipidemias/sangue , Dislipidemias/diagnóstico , Combinação Ezetimiba e Simvastatina/efeitos adversos , Feminino , Cálculos Biliares/diagnóstico , Cálculos Biliares/cirurgia , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Método Simples-Cego , Fatores de Tempo , Resultado do Tratamento
12.
Eur Heart J ; 39(27): 2562-2573, 2018 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-29982602

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 Risco
13.
Atherosclerosis ; 275: 390-399, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29703634

RESUMO

BACKGROUND AND AIMS: Subendothelial interaction of LDL with extracellular matrix drives atherogenesis. This interaction can be strengthened by proteolytic modification of LDL. Mast cells (MCs) are present in atherosclerotic lesions, and upon activation, they degranulate and release a variety of neutral proteases. Here we studied the ability of MC proteases to cleave apoB-100 of LDL and affect the binding of LDL to proteoglycans. METHODS: Mature human MCs were differentiated from human peripheral blood-derived CD34+ progenitors in vitro and activated with calcium ionophore to generate MC-conditioned medium. LDL was incubated in the MC-conditioned medium or with individual MC proteases, and the binding of native and modified LDL to isolated human aortic proteoglycans or to human atherosclerotic plaques ex vivo was determined. MC proteases in atherosclerotic human coronary artery lesions were detected by immunofluorescence and qPCR. RESULTS: Activated human MCs released the neutral proteases tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B. Of these, cathepsin G degraded most efficiently apoB-100, induced LDL fusion, and enhanced binding of LDL to isolated human aortic proteoglycans and human atherosclerotic lesions ex vivo. Double immunofluoresence staining of human atherosclerotic coronary arteries for tryptase and cathepsin G indicated that lesional MCs contain cathepsin G. In the lesions, expression of cathepsin G correlated with the expression of tryptase and chymase, but not with that of neutrophil proteinase 3. CONCLUSIONS: The present study suggests that cathepsin G in human atherosclerotic lesions is largely derived from MCs and that activated MCs may contribute to atherogenesis by enhancing LDL retention.


Assuntos
Apolipoproteína B-100/metabolismo , Aterosclerose/enzimologia , Doenças das Artérias Carótidas/enzimologia , Catepsina G/metabolismo , Doença da Artéria Coronariana/enzimologia , Lipoproteínas LDL/metabolismo , Mastócitos/enzimologia , Proteoglicanas/metabolismo , Aterosclerose/patologia , Doenças das Artérias Carótidas/patologia , Degranulação Celular , Células Cultivadas , Doença da Artéria Coronariana/patologia , Ativação Enzimática , Humanos , Placa Aterosclerótica , Ligação Proteica , Proteólise
14.
Atherosclerosis ; 269: 159-165, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29366988

RESUMO

BACKGROUND AND AIMS: While inhibition of proprotein convertase subtilisin/kexin type 9 (PCSK9) is known to result in dramatic lowering of LDL-cholesterol (LDL-C), it is poorly understood how it affects other lipid species and their metabolism. The aim of this study was to characterize the alterations in the lipidome of plasma and lipoprotein particles after administration of PCSK9 inhibiting antibody to patients with established coronary heart disease. METHODS: Plasma samples were obtained from patients undergoing a randomized placebo-controlled phase II trial (EQUATOR) for the safe and effective use of RG7652, a fully human monoclonal antibody inhibiting PCSK9 function. Lipoprotein fractions were isolated by sequential density ultracentrifugation, and both plasma and major lipoprotein classes (VLDL-IDL, LDL, HDL) were subjected to mass spectrometric lipidomic profiling. RESULTS: PCSK9 inhibition significantly decreased plasma levels of several lipid classes, including sphingolipids (dihydroceramides, glucosylceramides, sphingomyelins, ceramides), cholesteryl esters and free cholesterol. Previously established ceramide ratios predicting cardiovascular mortality, or inflammation related eicosanoid lipids, were not altered. RG7652 treatment also affected the overall and relative distribution of lipids in lipoprotein classes. An overall decrease of total lipid species was observed in LDL and VLDL + IDL particles, while HDL-associated phospholipids increased. Following the treatment, LDL displayed reduced lipid cargo, whereas relative lipid proportions of the VLDL + IDL particles were mostly unchanged, and there were relatively more lipids carried in the HDL particles. CONCLUSIONS: Administration of PCSK9 antibody significantly alters the lipid composition of plasma and lipoprotein particles. These changes further shed light on the link between anti-PCSK9 therapies and cardiovascular risk.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticolesterolemiantes/uso terapêutico , Doença das Coronárias/tratamento farmacológico , Lipídeos/sangue , Lipoproteínas/sangue , Pró-Proteína Convertase 9/antagonistas & inibidores , Idoso , Anticorpos Monoclonais Humanizados , Biomarcadores/sangue , Centrifugação com Gradiente de Concentração , Doença das Coronárias/sangue , Doença das Coronárias/diagnóstico , Feminino , Finlândia , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Pró-Proteína Convertase 9/metabolismo , Fatores de Tempo , Resultado do Tratamento
15.
Am J Pathol ; 188(2): 525-538, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29154769

RESUMO

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been lipolytically modified in vitro induced intracellular lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.


Assuntos
Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/metabolismo , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos/fisiologia , Artérias Carótidas/ultraestrutura , Doenças das Artérias Carótidas/patologia , Doenças das Artérias Carótidas/cirurgia , Células Cultivadas , Colesterol/metabolismo , Endarterectomia das Carótidas , Espaço Extracelular/metabolismo , Humanos , Imageamento Tridimensional/métodos , Inflamassomos/metabolismo , Lipólise/fisiologia , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Microscopia Eletrônica de Transmissão/métodos
16.
Anal Biochem ; 518: 25-34, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27984014

RESUMO

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low-Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool.


Assuntos
Anticorpos Monoclonais Murinos/química , Apolipoproteína B-100/química , Modelos Químicos , Termodinâmica , Animais , Humanos , Cinética , Camundongos
17.
Anal Biochem ; 514: 12-23, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27623434

RESUMO

Immunoaffinity procedure was developed for isolation of low density lipoprotein (LDL) from biological samples by using silica-derived immunoaffinity sorbent. Sorbent was prepared by immobilization of monoclonal anti-apoB-100 antibody onto macroporous silica particles, using carefully optimized binding chemistry. Binding capacity of the sorbent towards LDL was determined by batch extraction experiments with solutions of isolated LDL in phosphate-buffered saline, and found to be 8 mg LDL/g. The bound LDL fraction was readily released from the sorbent by elution with ammonia at pH 11.2. The total time needed for isolation procedure was less than 1 h, with LDL recoveries being essentially quantitative for samples containing less than 0.3 mg LDL/mL. With higher concentrations, recoveries were less favorable, most probably due to irreversible adsorption caused by LDL aggreggation. However, reusability studies with isolated LDL at concentration 0.2 mg/mL indicate that the developed immunoaffinity material may be used for multiple binding-release cycles, with minor losses in binding capacity. Finally, the sorbent was successfully applied to isolation of LDL from diluted plasma. Apart from its practical implications for LDL isolation, this study provides crucial insights into issues associated with LDL-sorbent interactions, and may be useful in future efforts directed to development of lipoprotein isolation approaches.


Assuntos
Apolipoproteína B-100 , Técnicas de Imunoadsorção , Lipoproteínas LDL/isolamento & purificação , Apolipoproteína B-100/imunologia , Calibragem , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas de Imunoadsorção/instrumentação , Lipoproteínas LDL/metabolismo , Compostos de Silício/química , Dióxido de Silício
18.
Arterioscler Thromb Vasc Biol ; 36(9): 1937-46, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27417584

RESUMO

OBJECTIVE: Activation of the inflammasome pathway in macrophages results in the secretion of 2 potent proinflammatory and proatherogenic cytokines, interleukin (IL)-1ß, and IL-18. Atherosclerotic lesions are characterized by the presence of various endogenous activators of the NLR family pyrin domain containing 3 (NLRP3) inflammasome, including cholesterol crystals and extracellular ATP. The aim of this study was to comprehensively characterize the expression of inflammasome pathway components and regulators in human atherosclerotic lesions. APPROACH AND RESULTS: Twenty human coronary artery RNA samples from 10 explanted hearts were analyzed using an inflammasome pathway-focused quantitative polymerase chain reaction array. Advanced atherosclerotic plaques, when compared with early-to-intermediate lesions from the same coronary trees, displayed significant upregulation of 12 target genes, including the key inflammasome components apoptosis-associated speck-like protein containing a CARD domain, caspase-1, and IL-18. Immunohistochemical stainings of the advanced plaques revealed macrophage foam cells positive for NLRP3 inflammasome components around the necrotic lipid cores. The polymerase chain reaction array target p38δ mitogen-activated protein kinase was upregulated in advanced plaques and strongly expressed by lesional macrophage foam cells. In cultured human monocyte-derived macrophages, the p38δ mitogen-activated protein kinase was activated by intracellular stress signals triggered during ATP- and cholesterol crystal-induced NLRP3 inflammasome activation and was required for NLRP3-mediated IL-1ß secretion. CONCLUSIONS: Increased expression of the key inflammasome components in advanced coronary lesions implies enhanced activity of the inflammasome pathway in progression of coronary atherosclerosis. The p38δ mitogen-activated protein kinase was identified as a novel regulator of NLRP3 inflammasome activation in primary human macrophages, and thus, represents a potential target for modulation of atherosclerotic inflammation.


Assuntos
Doença da Artéria Coronariana/enzimologia , Vasos Coronários/enzimologia , Células Espumosas/enzimologia , Inflamassomos/metabolismo , Proteína Quinase 13 Ativada por Mitógeno/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Placa Aterosclerótica , Trifosfato de Adenosina/metabolismo , Células Cultivadas , Colesterol/metabolismo , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Vasos Coronários/patologia , Cristalização , Ativação Enzimática , Células Espumosas/patologia , Perfilação da Expressão Gênica/métodos , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Inflamassomos/genética , Masculino , Pessoa de Meia-Idade , Proteína Quinase 13 Ativada por Mitógeno/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Necrose , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Cultura Primária de Células , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Fatores de Tempo , Transativadores/metabolismo , Regulação para Cima
19.
Arterioscler Thromb Vasc Biol ; 36(2): 274-84, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26681753

RESUMO

OBJECTIVE: Apolipoprotein A-I (apoA-I) has been shown to possess several atheroprotective functions, including inhibition of inflammation. Protease-secreting activated mast cells reside in human atherosclerotic lesions. Here we investigated the effects of the neutral proteases released by activated mast cells on the anti-inflammatory properties of apoA-I. APPROACH AND RESULTS: Activation of human mast cells triggered the release of granule-associated proteases chymase, tryptase, cathepsin G, carboxypeptidase A, and granzyme B. Among them, chymase cleaved apoA-I with the greatest efficiency and generated C-terminally truncated apoA-I, which failed to bind with high affinity to human coronary artery endothelial cells. In tumor necrosis factor-α-activated human coronary artery endothelial cells, the chymase-cleaved apoA-I was unable to suppress nuclear factor-κB-dependent upregulation of vascular cell adhesion molecule-1 (VCAM-1) and to block THP-1 cells from adhering to and transmigrating across the human coronary artery endothelial cells. Chymase-cleaved apoA-I also had an impaired ability to downregulate the expression of tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interleukin-8 in lipopolysaccharide-activated GM-CSF (granulocyte-macrophage colony-stimulating factor)- and M-CSF (macrophage colony-stimulating factor)-differentiated human macrophage foam cells and to inhibit reactive oxygen species formation in PMA (phorbol 12-myristate 13-acetate)-activated human neutrophils. Importantly, chymase-cleaved apoA-I showed reduced ability to inhibit lipopolysaccharide-induced inflammation in vivo in mice. Treatment with chymase blocked the ability of the apoA-I mimetic peptide L-4F, but not of the protease-resistant D-4F, to inhibit proinflammatory gene expression in activated human coronary artery endothelial cells and macrophage foam cells and to prevent reactive oxygen species formation in activated neutrophils. CONCLUSIONS: The findings identify C-terminal cleavage of apoA-I by human mast cell chymase as a novel mechanism leading to loss of its anti-inflammatory functions. When targeting inflamed protease-rich atherosclerotic lesions with apoA-I, infusions of protease-resistant apoA-I might be the appropriate approach.


Assuntos
Apolipoproteína A-I/metabolismo , Aterosclerose/enzimologia , Quimases/metabolismo , Células Endoteliais/metabolismo , Inflamação/enzimologia , Mastócitos/enzimologia , Apolipoproteína A-I/farmacologia , Aterosclerose/imunologia , Aterosclerose/prevenção & controle , Adesão Celular , Linhagem Celular Tumoral , Colesterol/metabolismo , Técnicas de Cocultura , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Espumosas/imunologia , Células Espumosas/metabolismo , Humanos , Inflamação/imunologia , Inflamação/prevenção & controle , Mediadores da Inflamação/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , NF-kappa B/metabolismo , Ativação de Neutrófilo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Peptídeos/farmacologia , Estrutura Terciária de Proteína , Proteólise , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Migração Transendotelial e Transepitelial , Molécula 1 de Adesão de Célula Vascular/metabolismo
20.
J Lipid Res ; 56(6): 1206-21, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25861792

RESUMO

Lipolytic modification of LDL particles by SMase generates LDL aggregates with a strong affinity for human arterial proteoglycans and may so enhance LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. LDL particles with and without 4F were incubated with SMase, after which their aggregation, structure, and proteoglycan binding were analyzed. At a molar ratio of L-4F to apoB-100 of 2.5 to 20:1, 4F dose-dependently inhibited SMase-induced LDL aggregation. At a molar ratio of 20:1, SMase-induced aggregation was fully blocked. Binding of 4F to LDL particles inhibited SMase-induced hydrolysis of LDL by 10% and prevented SMase-induced LDL aggregation. In addition, the binding of the SMase-modified LDL particles to human aortic proteoglycans was dose-dependently inhibited by pretreating LDL with 4F. The 4F stabilized apoB-100 conformation and inhibited SMase-induced conformational changes of apoB-100. Molecular dynamic simulations showed that upon binding to protein-free LDL surface, 4F locally alters membrane order and fluidity and induces structural changes to the lipid layer. Collectively, 4F stabilizes LDL particles by preventing the SMase-induced conformational changes in apoB-100 and so blocks SMase-induced LDL aggregation and the resulting increase in LDL retention.


Assuntos
Apolipoproteína A-I/farmacologia , Apolipoproteína B-100/metabolismo , Lipoproteínas LDL/metabolismo , Peptídeos/farmacologia , Esfingomielina Fosfodiesterase/metabolismo , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/patologia , Apolipoproteína A-I/metabolismo , Biomimética , Humanos , Lipólise/efeitos dos fármacos , Peptídeos/metabolismo , Esfingomielina Fosfodiesterase/antagonistas & inibidores
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