RESUMO
A20 haploinsufficiency (HA20) is an autoinflammatory disease caused by heterozygous loss-of-function variations in TNFAIP3, the gene encoding the A20 protein. Diagnosis of HA20 is challenging due to its heterogeneous clinical presentation and the lack of pathognomonic symptoms. While the pathogenic effect of TNFAIP3 truncating variations is clearly established, that of missense variations is difficult to determine. Herein, we identified a novel TNFAIP3 variation, p.(Leu236Pro), located in the A20 ovarian tumor (OTU) domain and demonstrated its pathogenicity. In the patients' primary cells, we observed reduced A20 levels. Protein destabilization was predicted in silico for A20_Leu236Pro and enhanced proteasomal degradation was confirmed in vitro through a flow cytometry-based functional assay. By applying this approach to the study of another missense variant, A20_Leu275Pro, for which no functional characterization has been performed to date, we showed that this variant also undergoes enhanced proteasomal degradation. Moreover, we showed a disrupted ability of A20_Leu236Pro to inhibit the NF-κB pathway and to deubiquitinate its substrate TRAF6. Structural modeling revealed that two residues involved in OTU pathogenic missense variations (i.e. Glu192Lys and Cys243Tyr) establish common interactions with Leu236. Interpretation of newly identified missense variations is challenging, requiring, as illustrated here, functional demonstration of their pathogenicity. Together with functional studies, in silico structure analysis is a valuable approach that allowed us (i) to provide a mechanistic explanation for the haploinsufficiency resulting from missense variations and (ii) to unveil a region within the OTU domain critical for A20 function.
Assuntos
Mutação de Sentido Incorreto , NF-kappa B , Humanos , NF-kappa B/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genéticaRESUMO
OBJECTIVE: To determine the molecular and cellular bases of autoinflammatory syndromes in a multigenerational French family with Muckle-Wells syndrome and in a patient originating from Portugal with familial cold autoinflammatory syndrome. METHODS: Sequencing of NLRP3 exon 3 was performed in all accessible patients. Microsatellite and whole-genome single nucleotide polymorphism genotyping was used i) to test the intrafamilial segregation of the identified variant and ii) to look for a founder effect. Functional analyses included the study of i) apoptosis-associated speck-like protein containing a CARD (ASC) speck formation in HEK293T cells (stably expressing ASC-green fluorescent protein and pro-caspase 1-FLAG) transiently expressing the wild-type or mutated NLRP3 protein, ii) levels of IL-1ß secreted from transfected THP-1 cells, and iii) inflammasome-related gene expression and cytokine secretion from monocytes isolated from patients in crisis (probands from the two families), related patients out of crisis, and from controls. RESULTS: The same heterozygous mutation (c.1322C>T, p.A441V) located in the NACHT domain, segregating with the disease within the first family, was identified in the two families. This mutation was found to be associated with different core haplotypes. NLRP3-A441V led to increased ASC speck formation and high levels of secreted IL-1ß. Monocyte inflammasome-related gene expression and cytokine secretion, which were within the normal range in patients out of crisis, were found to be differentially regulated between the two probands, correlating with their phenotypic status. CONCLUSION: These molecular and cellular findings, which indicate a recurrent mutational event, clearly demonstrate the pathogenicity of the p.A441V missense mutation in NLRP3-associated autoinflammatory disease and point to the interest of studying patients' primary cells to assess disease activity.
RESUMO
Circulating serum amyloid A (SAA) is increased in various inflammatory conditions. The human SAA protein family comprises the acute phase SAA1/SAA2, known to activate a large set of innate and adaptive immune cells, and the constitutive SAA4. The liver synthesis of SAA1/SAA2 is well-established but there is still an open debate on extrahepatic SAA expression especially in macrophages. We aimed to investigate the ability of human primary monocytes and monocyte-derived macrophages to express SAA1, SAA2 and SAA4 at both the transcriptional and protein levels, as previous studies almost exclusively dealt with monocytic cell lines. Monocytes and derived macrophages from healthy donors were stimulated under various conditions. In parallel with SAA, pro-inflammatory IL1A, IL1B and IL6 cytokine expression was assessed. While LPS alone was non-effective, a combined LPS/dexamethasone treatment induced SAA1 and to a lesser extent SAA2 transcription in human monocytes and macrophages. In contrast, as expected, pro-inflammatory cytokine expression was strongly induced following stimulation with LPS, an effect which was dampened in the presence of dexamethasone. Furthermore, in monocytes polarized towards a pro-inflammatory M1 phenotype, SAA expression in response to LPS/dexamethasone was potentiated; a result mainly seen for SAA1. However, a major discrepancy was observed between SAA mRNA and intracellular protein levels under the experimental conditions used. Our results demonstrate that human monocytes and macrophages can express SAA genes, mainly SAA1 in response to an inflammatory environment. While SAA is considered as a member of a large cytokine network, its expression in the monocytes-macrophages in response to LPS-dexamethasone is strikingly different from that observed for classic pro-inflammatory cytokines. As monocytes-macrophages are major players in chronic inflammatory diseases, it may be hypothesized that SAA production from macrophages may contribute to the local inflammatory microenvironment, especially when macrophages are compactly organized in granulomas as in sarcoidosis.
Assuntos
Inflamação/sangue , Proteína Amiloide A Sérica/genética , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Voluntários Saudáveis , Humanos , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/patologia , Interleucina-1alfa/sangue , Interleucina-1beta/sangue , Interleucina-6/sangue , Lipopolissacarídeos/toxicidade , Fígado/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismoRESUMO
Photoaging and epithelial skin tumorigenesis are complex processes triggered mainly by UV radiation from chronic sun exposure. This leads to DNA damage and reactive oxygen species (ROS) production, which initiate an inflammatory response that alters cell structure and function. Changes in cell homeostasis and ROS production activate intracellular multiprotein platforms called inflammasomes. Inflammasomes nucleate around cytoplasmic receptors mainly of the NLR (nucleotide-binding domain and leucine-rich repeat) family and regulate caspase-1-dependant secretion of pro-inflammatory interleukin (IL)1ß and IL18 cytokines, and an inflammatory form of death named pyroptosis. NLRP1 inflammasomes have taken centre stage in skin biology, as mutations in NLRP1 underlie the genetic etiology of dermatological diseases and increase the susceptibility to skin cancer. Targeting inflammasome(s) might be an important approach to improve skin inflammation, photoaging and reduce the risk of epithelial skin tumorigenesis. In this context, we discuss the potential implication of NLRP1 and NLRP3 inflammasomes.
Assuntos
Envelhecimento da Pele/efeitos da radiação , Neoplasias Cutâneas/metabolismo , Raios Ultravioleta/efeitos adversos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas NLR , Proteínas de Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Cutâneas/patologiaRESUMO
Inflammasomes are intracellular multiprotein signaling complexes, mainly present in myeloid cells. They commonly assemble around a cytoplasmic receptor of the nucleotide-binding leucine-rich repeat containing receptor (NLR) family, although other cytoplasmic receptors like pyrin have been shown to form inflammasomes. The nucleation of the multiprotein scaffolding platform occurs upon detection of a microbial, a danger or a homeostasis pattern by the receptor that will, most commonly, associate with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) through homotypic domain interactions resulting in recruitment of procaspase-1. This will lead to the autoproteolytic activation of caspase-1, which regulates the secretion of proinflammatory IL1ß and IL18 cytokines and pyroptosis, a caspase-1-mediated form of cell death. Pyroptosis occurs through cleavage of Gasdermin D, a membrane pore forming protein. Recently, non-canonical inflammasomes have been described, which directly sense intracellular pathogens through caspase-4 and -5 in humans, leading to pyroptosis. Inflammasomes are important in host defense; however, a deregulated activity is associated with a number of inflammatory, immune and metabolic disorders. Furthermore, mutations in inflammasome receptor coding genes are causal for an increasing number of rare autoinflammatory diseases. Biotherapies targeting the products of inflammasome activation as well as molecules that directly or indirectly inhibit inflammasome nucleation and activation are promising therapeutic areas. This review discusses recent advances in inflammasome biology, the molecular pathology of several inflammasomes, and current therapeutic approaches in autoinflammatory diseases and in selected common multifactorial inflammasome-mediated disorders.
Assuntos
Inflamassomos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Pirina/fisiologia , Animais , Humanos , Inflamação/genética , Inflamação/imunologiaRESUMO
Inflammasomes are multiprotein complexes nucleating around an NLR (Nucleotide-binding domain and Leucine-rich Repeat containing protein), which regulate the secretion of the pro-inflammatory interleukin (IL)-1ß and IL-18 cytokines. Monocytes and macrophages, the main cells expressing the inflammasome genes, adapt to their surrounding microenvironment by a phenotypic polarization towards a pro-inflammatory M1 phenotype that promotes inflammation or an anti-inflammatory M2 phenotype important for resolution of inflammation. Despite the importance of inflammasomes in health and disease, little is known about inflammasome gene expression in relevant human cells and the impact of monocyte and macrophage polarization in inflammasome gene expression. We examined the expression of several members of the NLR, caspase and cytokine family, and we studied the activation of the well-described NLRP3 inflammasome in an experimental model of polarized human primary monocytes and monocyte-derived macrophages (M1/M2 phenotypes) before and after activation with LPS, a well-characterized microbial pattern used in inflammasome activation studies. Our results show that the differentiation of monocytes to macrophages alters NLR expression. Polarization using IFN-γ (M1 phenotype), induces among the NLRs studied, only the expression of NOD2. One of the key results of our study is that the induction of NLRP3 expression by LPS is inhibited in the presence of IL-4+IL-13 (M2 phenotype) at both mRNA and protein level in monocytes and macrophages. Unlike caspase-3, the expression of inflammasome-related CASP1 (encodes caspase-1) and CASP4 (encodes caspase-4) is up-regulated in M1 but not in M2 cells. Interestingly, the presence of LPS marginally influenced IL18 mRNA expression and secretion, unlike its impact on IL1B. Our data provide the basis for a better understanding of the role of different inflammasomes within a given environment (M1 and M2) in human cells and their impact in the pathophysiology of several important inflammatory disorders.
Assuntos
Inflamassomos/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteínas NLR/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Caspases/genética , Caspases/imunologia , Polaridade Celular , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Regulação da Expressão Gênica , Humanos , Inflamassomos/genética , Lipopolissacarídeos/imunologia , Macrófagos/citologia , Macrófagos/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas NLR/genética , Proteína Adaptadora de Sinalização NOD2/genéticaRESUMO
We report on a familial Mediterranean fever (FMF) patient homozygous for p.M694V in the MEFV gene who developed chronic myelomonocytic leukemia (CMML) leading to an uncontrolled and fatal inflammatory syndrome. Plasma levels of IL-6 and IL-18 were found to be very high, as compared to healthy controls and CMML-free FMF patients.Our study unveils the interplay between two different disorders involving the same target cells, suggesting that in myelodysplasia with inflammatory manifestations, mutations in genes causing autoinflammatory syndromes, like MEFV, can be present and thus could be sought. Early chemotherapy with interleukin inhibitors could be proposed in such unusual situations.
Assuntos
Febre Familiar do Mediterrâneo/imunologia , Inflamação/etiologia , Inflamação/imunologia , Leucemia Mielomonocítica Crônica/complicações , Leucemia Mielomonocítica Crônica/imunologia , Idoso de 80 Anos ou mais , Proteínas do Citoesqueleto/genética , Febre Familiar do Mediterrâneo/sangue , Humanos , Inflamação/sangue , Interleucina-18/sangue , Interleucina-6/sangue , Leucemia Mielomonocítica Crônica/sangue , Masculino , Mutação , PirinaRESUMO
BACKGROUND: Observational studies report that secretory phospholipase A2 (sPLA2) activity is a marker for coronary heart disease (CHD) risk, and activity measures are thought to represent the composite activity of sPLA2-IIA, -V, and -X. The aim of this study was to use genetic variants of PLA2G10, encoding sPLA2-X, to investigate the contribution of sPLA2-X to the measure of sPLA2 activity and coronary heart disease (CHD) risk traits and outcome. METHODS AND RESULTS: Three PLA2G10 tagging single-nucleotide polymorphisms (rs72546339, rs72546340, and rs4003232) and a previously studied PLA2G10 coding single-nucleotide polymorphism rs4003228, R38C, were genotyped in a nested case: control cohort drawn from the prospective EPIC-Norfolk Study (2175 cases and 2175 controls). Meta-analysis of rs4003228 (R38C) and CHD was performed using data from the Northwick Park Heart Study II and 2 published cohorts AtheroGene and SIPLAC, providing in total an additional 1884 cases and 3119 controls. EPIC-Norfolk subjects in the highest tertile of sPLA2 activity were older and had higher inflammatory markers compared with those in the lowest tertile for sPLA2 activity. None of the PLA2G10 tagging single-nucleotide polymorphism nor R38C, a functional variant, were significantly associated with sPLA2 activity, intermediate CHD risk traits, or CHD risk. In meta-analysis, the summary odds ratio for R38C was odds ratio=0.97 (95% confidence interval, 0.77-1.22). CONCLUSIONS: PLA2G10 variants are not significantly associated with plasma sPLA2 activity or with CHD risk.
Assuntos
Doença das Coronárias , Fosfolipases A2 do Grupo X , Polimorfismo de Nucleotídeo Único , Característica Quantitativa Herdável , Idoso , Idoso de 80 Anos ou mais , Doença das Coronárias/sangue , Doença das Coronárias/enzimologia , Doença das Coronárias/genética , Feminino , Seguimentos , Fosfolipases A2 do Grupo X/sangue , Fosfolipases A2 do Grupo X/genética , Humanos , Masculino , Metanálise como Assunto , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de RiscoRESUMO
OBJECTIVE: Atherosclerosis is an inflammatory disease, where activated immunocompetent cells, including dendritic cells (DCs) and T cells are abundant in plaques. Low-density lipoprotein modified either by oxidation (oxLDL) or by human group X-secreted phospholipase A2 (LDLx) and heat shock proteins (HSP), especially HSP60 and 90, have been implicated in atherosclerosis. We previously reported that Annexin A5 inhibits inflammatory effects of phospholipids, decreases vascular inflammation and improves vascular function in apolipoprotein E(-/-) mice. Here, we focus on the LDLx effects on human DCs and T cells. APPROACH AND RESULTS: Human DCs were differentiated from peripheral blood monocytes, stimulated by oxLDL or LDLx. Naive autologous T cells were cocultured with pretreated DCs. oxLDL and LDLx, in contrast to LDL, induced DC-activation and T-cell proliferation. T cells exposed to LDLx-treated DCs produced interferon-γ, interleukin (IL)-17 but not IL-4 and IL-10. Annexin A5 abrogated LDLx effects on DCs and T cells and increased production of transforming growth factor-ß and IL-10. Furthermore, IL-10 producing T cells suppressed primary T-cell activation via soluble IL-10, transforming growth factor-ß, and cell-cell contact. Lentiviral-mediated shRNA knock-down HSP60 and 90 in DCs attenuated the effect of LDLx on DCs and subsequent T-cell proliferation. Experiments on DC and T cells derived from carotid atherosclerotic plaques gave similar results. CONCLUSIONS: Our data show that modified forms of LDL such as LDLx but not native LDL activate human T cells through DCs. HSP60 and 90 contribute to such T-cell activation. Annexin A5 promotes induction of regulatory T cells and is potentially interesting as a therapeutic agent.
Assuntos
Anexina A5/metabolismo , Comunicação Celular , Chaperonina 60/metabolismo , Células Dendríticas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Lipoproteínas LDL/metabolismo , Ativação Linfocitária , Proteínas Mitocondriais/metabolismo , Subpopulações de Linfócitos T/metabolismo , Doenças das Artérias Carótidas/imunologia , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Chaperonina 60/genética , Técnicas de Cocultura , Citocinas/metabolismo , Células Dendríticas/imunologia , Fosfolipases A2 do Grupo X/metabolismo , Proteínas de Choque Térmico HSP90/genética , Humanos , Mediadores da Inflamação/metabolismo , Proteínas Mitocondriais/genética , Placa Aterosclerótica , Interferência de RNA , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , TransfecçãoRESUMO
OBJECTIVE: Increased secreted phospholipase A(2) (sPLA(2)) activity has been documented in several inflammatory disorders. Among sPLA(2)s, the human group X (hGX)-sPLA(2) has the highest catalytic activity towards phosphatidylcholine (PC), the major phospholipid of cell membranes and blood lipoproteins. hGX-sPLA(2) has been detected in human atherosclerotic lesions, indicating that sPLA(2)s are an important link between lipids and inflammation, both involved in atherosclerosis. The presence of dendritic cells (DC), the most potent antigen presenting cells, in atherosclerotic lesions has raised the question about their role in disease progression. METHODS AND RESULTS: In this study, we show that hGX-sPLA(2)-treated LDL induces human monocyte-derived DC maturation, resulting in a characteristic mature DC phenotype and enhanced DC ability to activate IFNγ secretion from T cells. hGX-sPLA(2) phospholipolysis of LDL produces high levels of lipid mediators, such as lysophosphatidylcholine (LPC) and free fatty acids (FFAs), which also modulate DC maturation. The major molecular species of LPC containing a palmitic or stearic acid esterified in the sn-1 position induce DC maturation, whereas the FFAs can positively or negatively modulate DC maturation depending on their nature. hGX-sPLA(2) added alone can also activate DC in vitro through the hydrolysis of the DC membrane phospholipids leading, however, to a different cytokine profile secretion pattern than the one observed with hGX-sPLA(2)-phospholipolysed LDL. CONCLUSION: hGX-sPLA(2) secreted in inflamed tissues can contribute to local DC maturation, resulting in pro-Th1 cells, through the production of various lipid mediators from hydrolysis of either LDL and/or cell plasma membrane.
Assuntos
Aterosclerose/enzimologia , Células Dendríticas/enzimologia , Fosfolipases A2 do Grupo X/metabolismo , Lipoproteínas LDL/metabolismo , Aterosclerose/imunologia , Técnicas de Cocultura , Células Dendríticas/imunologia , Ácidos Graxos não Esterificados/metabolismo , Humanos , Hidrólise , Interferon gama/metabolismo , Lisofosfatidilcolinas/metabolismo , Fenótipo , Fosfatidilcolinas/metabolismo , Células Th1/imunologiaRESUMO
Secreted phospholipases A2 (sPLA2s) are present in atherosclerotic plaques and are now considered novel attractive therapeutic targets and potential biomarkers as they contribute to the development of atherosclerosis through lipoprotein-dependent and independent mechanisms. We have previously shown that hGX-sPLA2-phospholipolyzed LDL (LDL-X) induces proinflammatory responses in human umbilical endothelial cells (HUVECs); here we explore the molecular mechanisms involved. Global transcriptional gene expression profiling of the response of endothelial cells exposed to either LDL or LDL-X revealed that LDL-X activates multiple distinct cellular pathways including the unfolded protein response (UPR). Mechanistic insight showed that LDL-X activates UPR through calcium depletion of intracellular stores, which in turn disturbs cytoskeleton organization. Treatment of HUVECs and aortic endothelial cells (HAECs) with LDL-X led to activation of all 3 proximal initiators of UPR: eIF-2alpha, IRE1alpha, and ATF6. In parallel, we observed a sustained phosphorylation of the p38 pathway resulting in the phosphorylation of AP-1 downstream targets. This was accompanied by significant production of the proinflammatory cytokines IL-6 and IL-8. Our study demonstrates that phospholipolyzed LDL uses a range of molecular pathways including UPR to initiate endothelial cell perturbation and thus provides an LDL oxidation-independent mechanism for the initiation of vascular inflammation in atherosclerosis.
Assuntos
Retículo Endoplasmático/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Lipoproteínas LDL/farmacologia , Fator 6 Ativador da Transcrição/metabolismo , Western Blotting , Células Cultivadas , Endorribonucleases/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Imunofluorescência , Inativação Gênica , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipoproteínas LDL/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfolipases A2 Secretórias , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Veias Umbilicais/citologia , Resposta a Proteínas não DobradasRESUMO
Phospholipases A2 (PLA2) are a family of enzymes that catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids liberating lysophospholipids and free fatty acids; important second messengers involved in atherogenesis. Plasma PAF-acetylhydrolase (PAF-AH) or Lp-PLA2 is a Ca(2+)-independent PLA2 which is produced by monocyte-derived macrophages and by activated platelets, and circulates in plasma associated with lipoproteins. PAF-AH catalyzes the removal of the acetyl/short acyl group at the sn-2 position of PAF and oxidized phospholipids produced during inflammation and oxidative stress. In humans, PAF-AH is mainly associated with small dense LDL and to a lesser extent with HDL and with lipoprotein(a). PAF-AH is N-glycosylated prior to secretion which diminishes its association with HDL raising the question of its distribution between the proatherogenic LDL vs the antiatherogenic HDL. Hypercholesterolemic patients have higher plasma PAF-AH activity which is reduced upon hypolipidemic therapy. PAF-AH specific inhibitor darapladib stabilizes human and swine plaques, therefore challenging the antiatherogenic potential of PAF-AH shown in small animal models. Among secreted PLA2s (sPLA2), the group X sPLA2 (PLA2GX), due to its very high activity towards phosphatidylcholine the main phospholipid of LDL, became an attractive target in atherosclerosis. We showed that PLA2GX is present in human atherosclerotic lesions and that the PLA2GX-phospholipolysed LDL triggers human macrophage-foam cell formation. In contrast to other sPLA2s, including group IB, IIA and V, PLA2GX can efficiently hydrolyze PAF present in lipoproteins or vesicles indicating that PLA2GX may be a novel player in PAF regulation upon inflammatory processes. By a genetic approach we uncovered a relatively rare polymorphism (Arg38Cys) which produces a catalytically inactive PLA2GX; although no association was observed with cardiovascular risk factors in the AtheroGene study, this result should be replicated in cohorts of other inflammatory diseases. We anticipate that mores studies will be necessary to sort out the exact role of extracellular PLA2 family members in atherosclerosis initiation and progression.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Aterosclerose/enzimologia , Animais , Aterosclerose/metabolismo , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Lipoproteínas/metabolismoRESUMO
OBJECTIVE: To study the association of PAF-acetyl hydrolase (PAFAH) activity with inflammation, oxidative stress, and atherosclerosis in hypercholesterolemic swine. METHODS AND RESULTS: Cholesterol-rich diet feeding of miniature pigs was associated with an increase in PAFAH activity and an increase of the PAFAH to PON1 ratio. PLA2G7 RNA (coding for PAFAH) expression was increased in blood monocytes and plaque macrophages. Increased PAFAH activity was associated with higher plasma lysophosphatidylcholine and correlated with oxidized LDL. In THP1 monocytes and macrophages and in human blood-derived macrophages, oxidized LDL induced PLA2G7 RNA expression. Atherogenic diet feeding induced the accumulation of macrophages and oxidized LDL in the arterial wall leading to atherosclerosis. PAFAH activity correlated positively with plaque size and TNFalpha expression in plaque macrophages. CONCLUSIONS: We demonstrated that an increase in PAFAH activity was associated with increased levels of lysophosphatidylcholine, oxidized LDL, and inflammation, resulting in accelerated atherosclerosis in hypercholesterolemic minipigs. The significant correlation between PLA2G7 RNA expression in plaque macrophages and plasma PAFAH activity suggests that the latter is a consequence, rather than a cause of macrophage accumulation. Our cell experiments suggest that oxidized LDL can induce PAFAH, resulting in accumulation of lysophosphatidylcholine that increases the inflammatory action of oxidized LDL.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Aterosclerose/etiologia , Aterosclerose/metabolismo , Hipercolesterolemia/complicações , Hipercolesterolemia/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Metabolismo dos Lipídeos , 1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Sequência de Aminoácidos , Animais , Arildialquilfosfatase/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Vasos Coronários/patologia , Dieta Aterogênica , Expressão Gênica , Humanos , Hipercolesterolemia/genética , Inflamação/genética , Macrófagos/metabolismo , Macrófagos/patologia , Dados de Sequência Molecular , Oxirredução , Estresse Oxidativo , RNA/genética , RNA/metabolismo , Suínos , Porco MiniaturaRESUMO
Among secreted phospholipases A2 (sPLA2s), human group X sPLA2 (hGX sPLA2) is emerging as a novel attractive therapeutic target due to its implication in inflammatory diseases. To elucidate whether hGX sPLA2 plays a causative role in coronary artery disease (CAD), we screened the human PLA2G10 gene to identify polymorphisms and possible associations with CAD end-points in a prospective study, AtheroGene. We identified eight polymorphisms, among which, one non-synonymous polymorphism R38C in the propeptide region of the sPLA2. The T-512C polymorphism located in the 5' untranslated region was associated with a decreased risk of recurrent cardiovascular events during follow-up. The functional analysis of the R38C polymorphism showed that it leads to a profound change in expression and activity of hGX sPLA2, although there was no detectable impact on CAD risk. Due to the potential role of hGX sPLA2 in inflammatory processes, these polymorphisms should be investigated in other inflammatory diseases.
Assuntos
Doença da Artéria Coronariana/enzimologia , Doença da Artéria Coronariana/genética , Predisposição Genética para Doença , Fosfolipases A2 do Grupo X/genética , Fosfolipases A2 do Grupo X/metabolismo , Polimorfismo Genético/genética , Regiões 5' não Traduzidas/genética , Adulto , Idoso , Feminino , Fluorimunoensaio , Predisposição Genética para Doença/genética , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Mutagênese Sítio-DirigidaRESUMO
Increasing evidence suggests that secreted phospholipases A2 (sPLA2s) play an important role in the pathophysiology of atherosclerosis. Among sPLA2s, the human group X (hGX) enzyme has the highest catalytic activity toward phosphatidylcholine, one of the major phospholipid species of cell membranes and low-density lipoprotein (LDL). Our study examined the presence of hGX sPLA2 in human atherosclerotic lesions and investigated the ability of hGX modified LDL to alter human endothelial cell (HUVEC) function. Our results show that hGX sPLA2 is present in human atherosclerotic lesions and that the hydrolysis of LDL by hGX sPLA2 results in a modified particle that induces lipid accumulation in human monocyte-derived macrophages. Acting on endothelial cells, hGX-modified LDL activates the MAP kinase pathway, which leads to increased arachidonic acid release, increased expression of adhesion molecules on the surface of HUVEC, and increased adhesion of monocytes to HUVEC monolayers. Together, our data suggest that LDL modified by hGX, rather than hGX itself may have strong proinflammatory and proatherogenic properties, which could play an important role in the propagation of atherosclerosis.
Assuntos
Aterosclerose/metabolismo , LDL-Colesterol/metabolismo , Células Endoteliais/metabolismo , Fosfolipases A/metabolismo , Artérias/citologia , Aterosclerose/patologia , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Fosfolipases A2 do Grupo X , Humanos , Macrófagos/metabolismo , Fosfolipases A2 , Transporte Proteico , RNA Mensageiro/metabolismo , Veias/citologiaRESUMO
Platelet Activating Factor (PAF) is a potent mediator of inflammation whose biological activity depends on the acetyl group esterified at the sn-2 position of the molecule. PAF-acetylhydrolase (PAF-AH), a secreted calcium-independent phospholipase A(2), is known to inactivate PAF by formation of lyso-PAF and acetate. However, PAF-AH deficient patients are not susceptible to the biological effects of inhaled PAF in airway inflammation, suggesting that other enzymes may regulate extracellular levels of PAF. We therefore examined the hydrolytic activity of the recently described human group X secreted phospholipase A(2) (hGX sPLA(2)) towards PAF. Among different sPLA(2)s, hGX sPLA(2) has the highest affinity towards phosphatidylcholine (PC), the major phospholipid of cellular membranes and plasma lipoproteins. Our results show that unlike group IIA, group V, and the pancreatic group IB sPLA(2), recombinant hGX sPLA(2) can efficiently hydrolyze PAF. The hydrolysis of PAF by hGX sPLA(2) rises abruptly when the concentration of PAF passes through its critical micelle concentration suggesting that the enzyme undergoes interfacial binding and activation to PAF. In conclusion, our study shows that hGX sPLA(2) may be a novel player in PAF regulation during inflammatory processes.
Assuntos
Fosfolipases A/química , Fator de Ativação de Plaquetas/química , Animais , Fosfolipases A2 do Grupo V , Fosfolipases A2 do Grupo X , Humanos , Hidrólise , Lipoproteínas LDL/química , Fosfatidilcolinas/química , Fosfolipases A2 , Proteínas Recombinantes/química , Especificidade por Substrato , SuínosRESUMO
Plasma Platelet-activating-Factor (PAF)-acetylhydrolase (PAF-AH also named lipoprotein-PLA(2) or PLA(2)G7 gene) is secreted by macrophages, it degrades PAF and oxidation products of phosphatidylcholine produced upon LDL oxidation and/or oxidative stress, and thus is considered as a potentially anti-inflammatory enzyme. Cloning of PAF-AH has sustained tremendous promises towards the use of PAF-AH recombinant protein in clinical situations. The reason for that stems from the numerous animal models of inflammation, atherosclerosis or sepsis, where raising the levels of circulating PAF-AH either through recombinant protein infusion or through the adenoviral gene transfer showed to be beneficial. Unfortunately, neither in human asthma nor in sepsis the recombinant PAF-AH showed sufficient efficacy. One of the most challenging questions nowadays is as to whether PAF-AH is pro- or anti-atherogenic in humans, as PAF-AH may possess a dual pro- and anti-inflammatory role, depending on the concentration and the availability of potential substrates. It is equally possible that the plasma level of PAF-AH is a diagnostic marker of ongoing atherosclerosis.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Aterosclerose/enzimologia , Macrófagos/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase/uso terapêutico , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/uso terapêutico , Asma/tratamento farmacológico , Asma/enzimologia , Aterosclerose/diagnóstico , Aterosclerose/tratamento farmacológico , Biomarcadores/metabolismo , Modelos Animais de Doenças , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Lipoproteínas LDL/metabolismo , Oxirredução/efeitos dos fármacos , Fosfatidilcolinas/metabolismo , Fator de Ativação de Plaquetas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Sepse/tratamento farmacológico , Sepse/enzimologiaRESUMO
Platelet-activating-Factor (PAF) and its structural analogues formed upon low density lipoprotein oxidation are involved in atherosclerotic plaque formation and may signal through PAF-receptor (PAF-R) expressed in human macrophages and in certain smooth muscle cells (SMCs) in the media, but rarely in the intima of human plaques. Our aim was to determine which SMC phenotype expresses PAF-R and whether this receptor is functional in cell migration. Circulating SMC progenitors and two phenotypically distinct clones of proliferative, epithelioid phenotype vs contractile, spindle-shaped SMCs from the media of adult internal thoracic artery were studied for the presence of PAF-receptor (PAF-R). The levels of specific mRNA were obtained by reverse transcription/real-time PCR, the protein expression was deduced from immunohistochemistry staining, and the functional transmigration assay was performed by Boyden chamber-type chemotaxis assay. Only SMCs of spindle-shape and synthetic phenotype expressed both mRNA and PAF-R protein and in the functional test migrated at low concentrations of PAF. Two unrelated, specific PAF-R antagonists inhibited PAF-induced migration, but did not modify the migration initiated by PDGF. The presence of functional PAF-R in arterial spindle-shaped SMCs of synthetic phenotype may be important for their migration from the media into the intima and atherosclerotic plaques formation.
Assuntos
Movimento Celular , Células Clonais/citologia , Artéria Torácica Interna/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Clonais/metabolismo , Regulação da Expressão Gênica , Humanos , Fenótipo , Glicoproteínas da Membrana de Plaquetas/genética , Receptores Acoplados a Proteínas G/genéticaRESUMO
Human serum paraoxonase (PON1) has been implicated to play an important role in cardiovascular disease and diabetes. Studies in the literature indicate that PON1 has two different enzyme activities, i.e., esterase and hydroperoxide reducing activities. The objective of this study was to establish the importance of these two activities and to distinguish between them. As the addition of copper immediately inactivated the enzyme, we used auto-oxidation as the model system. Auto-oxidation of HDL resulted in more than 80% reduction of the esterolytic activity, which was protected by antioxidants, Vitamin E (50%) and PDTC (95%) and completely by 1 M glucose. In contrast, the hydroperoxide reducing activity, using unesterified hydroperoxides remained unaffected with time. We also used pNPHPODE (novel substrate) to establish that hydrolysis might be a prerequisite for the enzyme to act on the esterified hydroperoxide. The results indicated that the hydrolysis of the substrate was inhibited under oxidizing conditions with no reduction of the hydroperoxide. Overall, our findings suggest that protecting the esterolytic activity of PON1 by antioxidants might be important in preserving its action on phospholipid peroxides and a concerted reaction involving the esterolytic and hydroperoxide reducing activities might be suggested for the action of PON1.