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OBJECTIVE: Atherosclerosis is an autoimmune condition and the underlying cause of coronary artery disease (CAD). Circulating antibodies to self-antigens can have a pathogenic or protective function in atherosclerosis. The objective of the study was to understand the association of autoantibody levels with CAD and its correlation with circulating immune cells. METHODS: We assessed antigen concentration and antibodies to apolipoprotein B (ApoB) and heat shock protein (HSP)60 by ELISA in 252 acute coronary syndromes (ACS), 112 patients with stable angina (SA) and 203 healthy controls from Indian population. T cells in peripheral blood mononuclear cells (PBMC) were enumerated by flow cytometry. Cytokine concentrations were measured by multiplex assay. RESULTS: IgG and IgM antibodies to ApoB and HSP60 proteins were significantly lower in patients with ACS while only IgG levels to ApoB were lower in patients with SA, compared with control. Subjects in the highest tertile of antibodies showed significantly lower OR for ACS (IgG 0.52, 95% CI 0.31 to 0.88, p=0.02 and IgM 0.58, 95% CI 0.34 to 0.98, p=0.04), ApoB100 (IgG 0.52, 95% CI 0.31 to 0.88, p=0.02 and IgM 0.58, 95% CI 0.34 to 0.99, p=0.04) and HSP60, respectively. Interestingly, T helper 17 (TH17) cells showed an inverse relationship with ApoB and HSP60 IgG antibodies (r2=-0.17, p<0.001 and r2=-0.20, p<0.001, respectively), while interleukin 17 concentrations were negatively correlated with IgM antibodies to the proteins. CONCLUSION: This study shows that higher antibodies to ApoB and HSP60 proteins are less often associated with ACS and that these antibodies are inversely associated with inflammatory Th17 cells.
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BACKGROUND & OBJECTIVES: Metabolic syndrome (MetS) increases the likelihood of developing coronary artery disease (CAD), and inflammation is involved in the pathogenesis of both these conditions. The present work was conducted to examine the relative expression of 18 key inflammatory genes associated with MetS and incident CAD in a representative group of patients. METHODS: A total of 178 male patients, including 57 with CAD and 121 without CAD, were enrolled in the study. The participants without CAD were characterized for the presence of MetS using modified criteria specific for Asian Indians, which included a lower cut-off for waist circumference (≥90 cm for men). The expression of 18 inflammatory genes was evaluated in peripheral whole blood by quantitative polymerase chain reaction method. RESULTS: Of the 121 participants without CAD, 53 (43.8%) had three or more risk factors (MetS group), 50 (41.3%) had one or two risk factors (non-MetS group), while 18 (14.8%) did not have any risk factors (control group). High nuclear factor-kappa B (NF-κB) expression levels and low interleukin-10 (IL-10) levels were observed in MetS patients. Linear association was seen between NF-κB and vascular endothelial growth factor A (VEGFA) expression and with increase in MetS components. Comparison of gene expression pattern between CAD and MetS revealed significantly higher expression of leukotriene genes - arachidonate 5-lipoxygenase (ALOX5), arachidonate 5-lipoxygenase activating protein (ALOX5 AP), leukotriene A4 hydrolase (LTA4H) and leukotriene C4 synthase (LTC4S), and lower expression of NF-κB, interleukin 1 beta (IL-1ß), monocyte chemoattractant protein-1 (MCP-1/CCL2) and signal transducer and activator of transcription 3 (STAT3) genes in CAD. There was linear increase in expression of LTA4H, LTC4S, IL-8 and VEGFA genes across the four groups, namely from controls, non-MetS, MetS and CAD. INTERPRETATION & CONCLUSIONS: A distinct gene expression pattern was seen in MetS and CAD implying a well-orchestrated inflammatory and immune activity. Specifically, NF-κB might be playing an active role in MetS, allowing further expansion of the inflammatory process with resolution of inflammation in full-blown CAD, wherein other gene players such as leukotrienes may dominate.
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Proteínas Sanguíneas/genética , Enfermedad de la Arteria Coronaria/genética , Inflamación/genética , Síndrome Metabólico/genética , Adulto , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/patología , Femenino , Regulación de la Expresión Génica/genética , Humanos , Inflamación/sangre , Inflamación/patología , Masculino , Síndrome Metabólico/sangre , Síndrome Metabólico/patología , Persona de Mediana Edad , FN-kappa B/sangre , FN-kappa B/genética , Factores de RiesgoRESUMEN
Conventional risk factors have limited ability to predict recurrent events in subjects with first-time coronary artery disease (CAD). This aim of this study was to identify novel biomarkers using comparative global proteome analysis to improve the risk assessment for recurrent coronary events. We used samples from phase-I of the Indian Atherosclerosis Research Study (IARS), consisting of 2,332 subjects, of whom 772 were CAD-affected subjects, including 152 with recurrent events identified during a 5-year follow-up period. Global proteome analysis was performed on serum samples of 85 subjects with recurrent coronary events and 85 age- and gender-matched subjects with first-time CAD using surface-enhanced laser desorption ionization time-of-flight mass spectrometry with CM10 arrays. TagIdent was used for protein identification followed by validation by western blot analysis and ELISA. Data were analyzed by logistic analysis, Cox-regression, hazards ratio, C-statistics and combined-marker risk score using SPSS version-17 and R-package version-2.13.0 software. We identified 16 significantly differentially expressed protein peaks. Of these, 2 peaks corresponding to m/z 8588 and 1864 were identified as ß-defensin-128 and histatin-3, belonging to the danger-recognizing peptide family, which exhibited a significant independent association with recurrent events (odds ratios of 7.49 and 1.4, respectively). C-statistics improved significantly from 0.677 for conventional risk factors alone to 0.800 (p-value=0.001) in combination with ß-defensin-128 and histatin-3 with a hazards ratio of 1.833. A combined risk score of ß-defensin-128 and histatin-3 could reclassify 112 out of the 170 subjects into intermediate- and high-risk groups. On the whole, our data indicate that ß-defensin-128 and histatin-3 may be potential biomarkers whch may be used to improve risk the stratification for recurrent coronary events.
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Enfermedad de la Arteria Coronaria/sangre , Histatinas/sangre , beta-Defensinas/sangre , Biomarcadores/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Proteómica , RecurrenciaRESUMEN
INTRODUCTION: Immunotherapy by inducing oral tolerance to atherogenic self-antigens is gaining importance as an alternative treatment modality for atherosclerosis. The use of live bacterial vectors to express the recombinant antigen in vivo will obviate the need for large-scale purification of recombinant protein and may also augment the efficacy of oral tolerance induction. AIM: The objective of the study was to explore the use of recombinant Mycobacterium smegmatis as a live vector for oral delivery of antigens to induce immune tolerance. METHOD AND RESULTS: We developed a M. smegmatis vector to secrete a recombinant tripeptide construct (AHC; peptides from Apolipoprotein B, Heat-shock protein 60 and Chlamydia pneumoniae outer membrane protein) expressed in a dendroaspin protein scaffold in pJH154 background. Immune response and oral tolerance to the cloned peptides were studied in C57/BL6 mice. The efficacy of this live vaccine to control atherosclerosis was studied in ApoE(-/-) knockout mice in C57/BL6 background. Oral administration of M. smegmatis secreting the cloned AHC antigen was found to induce tolerance to cloned protein and reduce the development of atherosclerosis by 24.0% compared to control. Protection against atherosclerosis was associated with increase in expression of regulatory T cell-associated markers including CTLA4 (1.8-fold), Foxp3 (2.6-fold), TGF-ß (2.8-fold), IL10 (2.9-fold), and reduction in lipids, macrophage infiltration, and expression of inflammatory mediators in aorta. CONCLUSIONS: Our results suggest that M. smegmatis can be developed as an oral carrier of recombinant proteins to treat inflammatory autoimmune diseases.
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Antígenos/administración & dosificación , Enfermedades de la Aorta/prevención & control , Apolipoproteínas E/deficiencia , Aterosclerosis/prevención & control , Vectores Genéticos , Inmunoterapia/métodos , Mycobacterium smegmatis/genética , Oligopéptidos/administración & dosificación , Administración Oral , Animales , Antígenos/genética , Antígenos/inmunología , Antígenos/metabolismo , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Antígeno CTLA-4/inmunología , Antígeno CTLA-4/metabolismo , Modelos Animales de Enfermedad , Femenino , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Predisposición Genética a la Enfermedad , Tolerancia Inmunológica , Inmunización , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Metabolismo de los Lípidos , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium smegmatis/inmunología , Mycobacterium smegmatis/metabolismo , Oligopéptidos/genética , Oligopéptidos/inmunología , Oligopéptidos/metabolismo , Fenotipo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Vacunas Sintéticas/administración & dosificaciónRESUMEN
The objective of the study was to gain molecular insights into the progression of atherosclerosis in Apob(tm2Sgy)Ldlr(tm1Her) mice, using transcriptome profiles. Weighted gene co network analysis (WGCNA) and time course analysis using limma were used to study disease progression from 0 to 20weeks. Five co-expression modules were identified by WGCNA using the expression values of 2153 genes. Genes associated with autophagy, endoplasmic reticulum stress, inflammation and lipid metabolism were differentially expressed at early stages of atherosclerosis. Time course analysis highlighted activation of inflammatory gene signaling at 4weeks, cell proliferation and calcification at 8weeks, amyloid like structures and oxidative stress at 14weeks and enhanced production of inflammatory cytokines at 20weeks. Our results suggest that maximum gene perturbations occur during early atherosclerosis which could be the danger signals associated with subclinical disease. Understanding these genes and associated pathways can help in improvement of diagnostic and therapeutic targets for atherosclerosis.
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Apolipoproteínas B/genética , Aterosclerosis/genética , Inflamación/genética , Receptores de LDL/efectos de los fármacos , Animales , Aterosclerosis/patología , Autofagia/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Estrés del Retículo Endoplásmico/genética , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Inflamación/patología , Metabolismo de los Lípidos/genética , Ratones , Ratones Noqueados , Estrés Oxidativo/genéticaRESUMEN
BACKGROUND: The merits and demerits of classical risk factors in coronary artery disease (CAD) are widely debated. We analyzed the role of conventional (age, gender, diabetes, hypertension, smoking) and non-conventional risk factors (anthropometrics, fasting blood sugar, atherogenic index of plasma - AIP, family history) in Asian Indians with CAD. METHODS: Out of 11,164 subjects (4855 affected, 6309 unaffected) enrolled in the Indian Atherosclerosis Research Study (IARS), 269 unaffected individuals with abnormal electrocardiogram and seven underage were excluded. Around 10,888 subjects along with two subsets, including 9888 individuals having family history information and 1616 individuals with intermediate Framingham risk score (FRS), were statistically analyzed using SPSS version 17.0 and R software. RESULTS: A combination of classical risk factors showed good discrimination between affected and unaffected individuals (C>0.85). Hypertension (OR 3.79) or male gender (OR 5.31) showed significant association with CAD when lipids were included or excluded from the predictive model, followed by age, diabetes and smoking. Hypertension and diabetes frequencies were higher in older patients (>55years) while smoking was more prevalent in younger patients (<55years). Family history and AIP provided a modest increase in C index over the classical factors (0.864 to 0.873), with 7.1% net reclassification in the intermediate FRS group. In CAD patients, 4% were classified as high risk by FRS, 52% were classified as having metabolic syndrome with revised criteria and over 90% had a high AIP score. CONCLUSION: Addition of AIP and family history to conventional risk factors improved risk discrimination in Asian Indians with CAD.
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Enfermedad de la Arteria Coronaria/epidemiología , Diabetes Mellitus/epidemiología , Hipertensión/epidemiología , Fumar/epidemiología , Adulto , Pueblo Asiatico/estadística & datos numéricos , Enfermedad de la Arteria Coronaria/etnología , Femenino , Humanos , India/epidemiología , Masculino , Persona de Mediana Edad , Prevalencia , Factores de RiesgoRESUMEN
BACKGROUND: Inflammatory immune response to atherogenic self-antigens plays an important role in the development of atherosclerosis. We evaluated the role of oral tolerance to three peptides in controlling atherosclerosis in New Zealand white rabbits. METHODS: Peptides derived from apolipoprotein B (ApoB), heat shock protein 60, and outer membrane protein from Chlamydia pneumoniae were expressed as part of the dendroaspin protein scaffold (AHC). Groups of 3-month-old rabbits were dosed orally with purified AHC protein either before the onset of disease or 2 months after inducing atherosclerosis; they were euthanized at the age of 7 months to study disease development and progression. RESULTS: Oral treatment with AHC resulted in a marked increase in regulatory T cells in the lymphoid organs and reduced the development and progression of atherosclerosis by 48.6% and 28.4%, respectively (P < 0.05). Oral tolerance decreased plaque inflammation, enhanced expression of anti-inflammatory and regulatory markers in the aorta, and attenuated the adaptive immune response to self-antigens. AHC treatment in rabbits with established disease significantly decreased vascular cell adhesion molecule 1 (VCAM-1) (6.2 fold) and monocyte chemoattractant protein-1(MCP-1) (3 fold) expression and reduced the infiltration of macrophages into the aorta. Collagen content and the smooth muscle cell-to-macrophage ratio were higher in treated animals, whereas markers of plaque vulnerability, including matrix metalloproteinase expression, were reduced. CONCLUSIONS: Our results suggest that oral tolerance to multiantigenic AHC molecule restores the immune balance and induces markers of plaque stability in rabbits.
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Apolipoproteínas B/administración & dosificación , Aterosclerosis/inmunología , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Chaperonina 60/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Inmunidad Adaptativa , Animales , Aorta/citología , Aterosclerosis/patología , Quimiocina CCL2/metabolismo , Chlamydophila pneumoniae , Colágeno/metabolismo , Citocinas/sangre , Progresión de la Enfermedad , Venenos Elapídicos , Glutatión Sintasa/administración & dosificación , Tolerancia Inmunológica/inmunología , Ganglios Linfáticos/citología , Macrófagos/metabolismo , Miocitos del Músculo Liso/citología , Placa Aterosclerótica/inmunología , Placa Aterosclerótica/patología , Conejos , Bazo/citología , Linfocitos T/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Molecular mechanism underlying the patho-physiology of coronary artery disease (CAD) is complex. We used global expression profiling combined with analysis of biological network to dissect out potential genes and pathways associated with CAD in a representative case-control Asian Indian cohort. We initially performed blood transcriptomics profiling in 20 subjects, including 10 CAD patients and 10 healthy controls on the Agilent microarray platform. Data was analysed with Gene Spring Gx12.5, followed by network analysis using David v 6.7 and Reactome databases. The most significant differentially expressed genes from microarray were independently validated by real time PCR in 97 cases and 97 controls. A total of 190 gene transcripts showed significant differential expression (fold change>2,P<0.05) between the cases and the controls of which 142 genes were upregulated and 48 genes were downregulated. Genes associated with inflammation, immune response, cell regulation, proliferation and apoptotic pathways were enriched, while inflammatory and immune response genes were displayed as hubs in the network, having greater number of interactions with the neighbouring genes. Expression of EGR1/2/3, IL8, CXCL1, PTGS2, CD69, IFNG, FASLG, CCL4, CDC42, DDX58, NFKBID and NR4A2 genes were independently validated; EGR1/2/3 and IL8 showed >8-fold higher expression in cases relative to the controls implying their important role in CAD. In conclusion, global gene expression profiling combined with network analysis can help in identifying key genes and pathways for CAD.
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Enfermedad de la Arteria Coronaria/genética , Expresión Génica/genética , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The effect of hypercholesterolemia induced immune response and inflammation on progression of atherosclerosis in ApoB(tm25gy) LDLr(tm1Her) mice, expressing only ApoB100 and deficient in the low density lipoprotein (LDL) receptor, thus closely resembling human cholesterol transport is not well defined. Atherosclerosis was induced by a high cholesterol diet and its progression was studied at 8, 14 and 20 weeks. Antibody response was determined by ELISA. Lymphocytes in spleen and aortic expression of inflammatory markers were studied by flow cytometry, and immunohistochemistry respectively. A rapid increase in plasma LDL levels in the first 8 weeks was followed by the exponential development of atherosclerosis between 8 and 14 weeks. Progression of the disease was accompanied by an accumulation of macrophages and increased expression of IL17 and IFN-γ in the aorta. Hypercholesterolemia resulted in increased immune response to modified lipids and aortic inflammation, with an expansion of Th17 cells in the spleen. Progression of atherosclerosis showed a positive correlation (r = 0.84, P < 0.001) with Th17 cells and a negative correlation with Treg cells (r = 0.83, P < 0.001). IgM antibodies to Ox-LDL and Th17 cells in spleen showed greatest association with disease development. Our results suggest that anti Ox-LDL IgM antibodies, Th17 cells could be developed as a potential marker to study disease progression and to study the effect of therapeutic regulation of inflammation.
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Inmunidad Adaptativa , Apolipoproteínas B/genética , Aterosclerosis/complicaciones , Hipercolesterolemia/complicaciones , Inflamación/complicaciones , Receptores de LDL/genética , Animales , Formación de Anticuerpos , Aorta/inmunología , Aorta/metabolismo , Aorta/patología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Progresión de la Enfermedad , Eliminación de Gen , Hipercolesterolemia/genética , Hipercolesterolemia/inmunología , Hipercolesterolemia/patología , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Lípidos/sangre , Lipoproteínas LDL/inmunología , Ratones , Ratones Endogámicos C57BL , Bazo/inmunología , Bazo/metabolismo , Bazo/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
BACKGROUND: Tissue factor (TF) and coagulation proteases are involved in promoting atherosclerosis, but the molecular and cellular bases for their involvement are unknown. METHODS AND RESULTS: We generated a new strain (ApX4) of apolipoprotein E-deficient mice expressing a membrane-tethered human tissue factor pathway inhibitor fusion protein on smooth muscle actin-positive cells, including vascular smooth muscle cells (SMCs). ApX4 mice developed little atherosclerosis on either a normal chow or high-fat diet. Lipid levels were similar to those in parental ApoE(-/-) mice, and there was no detectable difference in systemic (circulating) tissue factor pathway inhibitor levels or activity. The small lipid-rich lesions that developed had markedly reduced leukocyte infiltrates, and in contrast to ApoE(-/-) mice, SMCs did not express macrophage migratory inhibitory factor (MIF), including at sites distant from atheromatous lesions. Low levels of circulating MIF in ApX4 mice normalized to levels seen in ApoE(-/-) mice after injection of an inhibitory anti-human tissue factor pathway inhibitor antibody, which also led to MIF expression by tissue factor-positive medial SMCs. MIF production by SMCs in ApoE(-/-) mice in vitro and in vivo was shown to be dependent on tissue factor and protease-activated receptor signaling, which were inhibited in ApX4 mice. CONCLUSIONS: Our data indicate that tissue factor plays a hitherto unreported role in the generation of MIF by SMCs in atherosclerosis-prone ApoE(-/-) mice, inhibition of which significantly prevents the development of atherosclerosis, through inhibition of leukocyte recruitment. These data significantly enhance our understanding of the pathophysiology of this important pathology and suggest new potential translational strategies to prevent atheroma formation.
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Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Lipoproteínas/metabolismo , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Músculo Liso Vascular/metabolismo , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerosis/patología , Aterosclerosis/fisiopatología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Leucocitos/patología , Metabolismo de los Lípidos/fisiología , Lipoproteínas/genética , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Músculo Liso Vascular/patología , Transducción de Señal/fisiologíaRESUMEN
Inflammatory immune response to self-antigens plays an important role in the development of atherosclerosis. Restoring immune tolerance to self-proteins reduces the pro-inflammatory response. We previously showed that oral tolerance to a combination of two peptides is atheroprotective. In the present study we expressed epitopes from apolipoprotein B 100 (ApoB), human heat shock protein (HSP60) and Chlamydia pneumonia outer membrane protein (Cpn) in a single protein scaffold and used this multi-antigenic construct to induce tolerance to individual peptides by oral route in ApoBtm2Sgy/Ldlrtm1Her/J mice. Antigen specific tolerance to individual peptides was observed in treated animals as seen by an increase in regulatory T cells. Tolerance to the peptides resulted in a 46.5% (p=0.002) reduction in the development of atherosclerosis compared with control. Atheroprotection was associated with a significant (p<0.05) decrease in plaque inflammation and an increase in the expression of immune regulatory markers in the aorta. CD11c+ cells coexpressing CD11b and CD103 increased in lymphoid organs and were found to activate regulatory T cells and reduce effector T-cell response. Adoptive transfer of CD11c+ cells was atheroprotective. Our results suggest that atheroprotection by oral tolerance to a multi-antigenic construct is mediated by antigen specific regulatory T cells and CD11c+ cells with immune regulatory properties.
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Aterosclerosis/tratamiento farmacológico , Aterosclerosis/inmunología , Tolerancia Inmunológica/inmunología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Administración Oral , Animales , Chaperonina 60/administración & dosificación , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/administración & dosificación , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Network analysis is a novel method to understand the complex pathogenesis of inflammation-driven atherosclerosis. Using this approach, we attempted to identify key inflammatory genes and their core transcriptional regulators in coronary artery disease (CAD). Initially, we obtained 124 candidate genes associated with inflammation and CAD using Polysearch and CADgene database for which protein-protein interaction network was generated using STRING 9.0 (Search Tool for the Retrieval of Interacting Genes) and visualized using Cytoscape v 2.8.3. Based on betweenness centrality (BC) and node degree as key topological parameters, we identified interleukin-6 (IL-6), vascular endothelial growth factor A (VEGFA), interleukin-1 beta (IL-1B), tumor necrosis factor (TNF) and prostaglandin-endoperoxide synthase 2 (PTGS2) as hub nodes. The backbone network constructed with these five hub genes showed 111 nodes connected via 348 edges, with IL-6 having the largest degree and highest BC. Nuclear factor kappa B1 (NFKB1), signal transducer and activator of transcription 3 (STAT3) and JUN were identified as the three core transcription factors from the regulatory network derived using MatInspector. For the purpose of validation of the hub genes, 97 test networks were constructed, which revealed the accuracy of the backbone network to be 0.7763 while the frequency of the hub nodes remained largely unaltered. Pathway enrichment analysis with ClueGO, KEGG and REACTOME showed significant enrichment of six validated CAD pathways - smooth muscle cell proliferation, acute-phase response, calcidiol 1-monooxygenase activity, toll-like receptor signaling, NOD-like receptor signaling and adipocytokine signaling pathways. Experimental verification of the above findings in 64 cases and 64 controls showed increased expression of the five candidate genes and the three transcription factors in the cases relative to the controls (p<0.05). Thus, analysis of complex networks aid in the prioritization of genes and their transcriptional regulators in complex diseases.
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Biología Computacional , Enfermedad de la Arteria Coronaria/genética , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Transcripción Genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Humanos , Inflamación/genética , Mapas de Interacción de Proteínas , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Genetic regulation of plasma lipids has been shown to influence the risk of coronary artery disease (CAD). We analyzed the relationship between rs599839 and rs646776 single nucleotide polymorphisms (SNPs) present in the CELSR2-PSRC1-SORT1 gene cluster, candidate gene expression, and their association with CAD and circulating lipid levels in a representative cohort of Asian Indians selected from the Indian Atherosclerosis Research Study. METHODS: SNPs rs599839 and rs646776 were genotyped by Taqman assay in 1034 CAD patients (cases) and 1034 age- and gender-matched controls. Expression of CELSR2, PSRC1, and SORT1 genes was measured in 100 cases and 100 controls. Plasma levels of total cholesterol (TC), triglycerides, high-density lipoprotein-cholesterol, and low-density lipoprotein-cholesterol (LDL-c) were measured by enzymatic assay. RESULTS: Both rs646776 and rs599839 were in strong linkage disequilibrium (r = 0.98) and showed significant protective association with CAD (OR = 0.315, 95% CI 0.136-0.728, p<0.007 and OR = 0.422, 95% CI 0.181-0.981, p = 0.045, respectively). Haplotype TA showed 72% frequency and was associated with CAD (OR 0.77, 95% CI 0.67-0.88, p = 0.0002). PSRC1 gene expression was lower in the cases than in the controls (0.75 ± 0.405 versus 1.04 ± 0.622, p = 2.26 × 10(-4)). The homozygous variant and heterozygous genotypes showed 30% and 15% higher PSRC1 expression, respectively. Correspondingly, the minor alleles were associated with lower plasma TC and LDL-c levels. CONCLUSION: PSRC1 in the cholesterol gene cluster shows a significant association with CAD by virtue of the two SNPs, rs646776 and rs599839 that also regulate plasma cholesterol levels.
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Proteínas Adaptadoras del Transporte Vesicular/genética , Cadherinas/genética , Colesterol/sangre , Enfermedad de la Arteria Coronaria/genética , Expresión Génica , Fosfoproteínas/genética , Anciano , Alelos , Pueblo Asiatico , Estudios de Cohortes , Haplotipos , Humanos , India , Desequilibrio de Ligamiento/genética , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , RiesgoRESUMEN
AIM: Leukotrienes are important lipid inflammatory mediators that play a pivotal role in the pathogenesis of atherosclerosis. We aimed to construct a network of interactions between leukotrienes and inflammatory biomarkers and evaluate the expression of key members of the leukotriene pathway and leukotriene-induced inflammatory molecules in patients with coronary artery disease (CAD) and healthy controls. METHODS: Leukotrienes and their regulatory inflammatory molecules reported in the literature were used to construct a biological network employing Gene spring GX v12.5. Key leukotriene genes and their closely interacting members were selected for expression study in 64 patients and 64 matched controls. Four single nucleotide polymorphisms(SNPs) (rs6538697, rs2660898, rs17525495 and rs1978331) in the leukotriene A4 hydrolase(LTA4H) gene were genotyped using SYBR green method, and plasma leukotriene B4 (LTB4) levels were measured using ELISA. RESULTS: The expression levels of arachidonate 5-lipoxygenase(ALOX5), LTA4H, tumor necrosis factor (TNF) and interleukin-8 (IL-8) genes were significantly higher in patients than in the controls(pï¼0.05). IL-8(r=0.35-0.47) and TNF (r=0.42-0.53) expression levels exhibited strong correlations with the leukotriene genes. The SNPs rs17525495 and rs1978331 were associated with LTA4H mRNA expression, while LTA4H and IL-8 levels were associated with CAD. The addition of these two markers to the conventional risk factors improved the c-statistics(area under the receiver operating characteristic(ROC) curve) from 0.75 to 0.93(pï¼0.01), with a Net Reclassification Index of 0.45(pï¼0.01) and Integrated Discrimination Improvement of 0.26(pï¼0.01). CONCLUSIONS: Leukotrienes and inflammatory genes, in particular, LTA4H and IL-8, exhibit close association in subjects with cardiovascular disease. Assessing these markers may provide incremental value for predicting cardiovascular risk beyond that obtained with classical risk factors.
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Enfermedad de la Arteria Coronaria/metabolismo , Leucotrienos/metabolismo , Anciano , Algoritmos , Araquidonato 5-Lipooxigenasa/metabolismo , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Epóxido Hidrolasas/metabolismo , Femenino , Perfilación de la Expresión Génica , Genotipo , Humanos , Inflamación , Interleucina-8/metabolismo , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
INTRODUCTION: A close association exists between oral health and cardiovascular disease. Periodontal disease induces early vascular changes while oral pathogens have been detected in sub gingival and atheromatous plaques. We examined the interrelationship between Periodontal disease, oral bacteria, surrogate sub-clinical markers and coronary artery disease (CAD) in a representative Asian Indian cohort. MATERIALS AND METHODS: 532 Gingivitis cases and 282 Periodontitis cases were assessed for early peripheral vascular changes, namely pulse wave velocity (PWV), arterial stiffness index (ASI) and ankle brachial index (ABI) using computerized oscillometry method. Relative quantitation (RQ) of Porphyromonas gingivalis (Pg) was estimated in saliva samples of 54 Periodontitis, 25 Gingivitis and 51 CAD cases (38 also had oral disease) by Taqman assay by amplifying pathogen-specific gene targets, 16srRNA and IktA, respectively, and 16s universal bacterial rRNA as endogenous control. RESULTS: PWV and ASI were elevated in Periodontitis compared to Gingivitis cases (p<0.0001) and in those with diabetes and hypertension. Cases with Periodontitis showed higher mean expression of Pg than Gingivitis (0.37±0.05 versus 0.15±0.04, p<0.0001), while CAD patients with oral disease (N=38) showed lower mean Pg expression than those without oral disease (N=13) (0.712±0.119 versus 1.526±0.257, p=0.008). Higher Pg expression was recorded in subjects with diabetes and hypertension. CONCLUSION: Oral disease induces early changes in the peripheral blood vessels. Further, common presence of Pg in subjects with oral disease, in those with established cardiovascular risk factors and in patients with symptomatic CAD reflects the importance of oral hygiene in the development of Coronary Artery Disease in Asian Indians.
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Enfermedad de la Arteria Coronaria/epidemiología , Boca/microbiología , Enfermedades Periodontales/complicaciones , Enfermedades Periodontales/microbiología , Porphyromonas gingivalis/aislamiento & purificación , Resistencia Vascular , Adulto , Infecciones por Bacteroidaceae/complicaciones , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/fisiopatología , Femenino , Gingivitis/complicaciones , Gingivitis/microbiología , Gingivitis/fisiopatología , Humanos , India/epidemiología , Masculino , Persona de Mediana Edad , Enfermedades Periodontales/fisiopatología , Factores de RiesgoRESUMEN
Oral tolerance to auto antigens reduces the development of atherosclerosis in mouse models. However, the effect of immune tolerance to multiple self antigenic peptides in plaque progression and stabilization is not known. We studied the protective effect of mucosal tolerance to peptides from apolipoprotein B (ApoB; 661-680) and heat shock protein 60 (HSP60; 153-163), in combination with diet, in the prevention of atherosclerotic lesion progression and plaque stabilization in ApoB(tm25gy)LDLr(tm1Her) mice. We found that oral administration of five doses of a combination of ApoB and HSP60 peptides (20 µg/mice/dose) induced tolerance to both the peptides and reduced early plaque development by 39.9% better than the individual peptides (ApoBâ=â28.7%;HSP60â=â26.8%)(P<0.001). Oral tolerance to combination of peptides along with diet modification arrested plaque progression by 37.6% which was associated with increases in T-regulatory cell and transforming growth factor-ß expression in the plaque and peripheral circulation. Reduced macrophage infiltration and tumor necrosis factor-α expression in the plaque was also observed. Tolerance with continued hypercholesterolemia resulted in 60.8% reduction in necrotic core area suggesting plaque stabilization, which was supported by reduction in apoptosis and increased efferocytosis demonstrated by greater expression of receptor tyrosine kinase Mer (MerTK) in the plaque. Tolerance to the two peptides also reduced the expression of matrix metalloproteinase 9, tissue factor, calprotectin, and increased its collagen content. Our study suggests that oral tolerance to ApoB and HSP60 peptide combination induces CD4(+) CTLA4(+) Tregs and CD4(+)CD25(+)Foxp3(+) Tregs secreting TGF-ß, which inhibit pathogenic T cell response to both peptides thus reducing the development and progression of atherosclerosis and provides evidence for plaque stabilization in ApoB(tm25gy)LDLr(tm1Her) mice.
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Apolipoproteínas B/química , Chaperonina 60/química , Tolerancia Inmunológica , Membrana Mucosa/inmunología , Péptidos/inmunología , Placa Aterosclerótica/inmunología , Administración Oral , Animales , Apolipoproteínas B/genética , Apolipoproteínas B/inmunología , Apoptosis/inmunología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Autoantígenos/química , Autoantígenos/inmunología , Chaperonina 60/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Ratones , Ratones Noqueados , Péptidos/administración & dosificación , Placa Aterosclerótica/patologíaRESUMEN
OBJECTIVE: Antigen-specific immune modulation is an attractive approach to atherosclerosis treatment. The aim of this study was to develop an in-vitro assay to screen peptide molecules for their inflammatory propensity. MATERIALS: Human dendritic cells derived from CD14(+) monocytes were activated using peptides derived from apolipoprotein B100 (ApoB), heat shock protein 60 (HSP60) and complement cascade (peptide A) in vitro, and used for priming autologous T cells. Proliferation of T cells, their differentiation to regulatory cells (Treg) and their cytokine profile were studied. The efficacy of the peptides in preventing atherosclerosis was studied in ApoB(tm2Sgy)/Ldlr(tm1Her/J) knockout mice. RESULTS AND CONCLUSION: ApoB and HSP60 peptides induced T-cell proliferation and expansion of regulatory T cells with interleukin-10 and transforming growth factor-ß secretion. In comparison, peptide A was a poor stimulator of T cells and was found to induce tumor necrosis factor-α secretion by activated T cells. ApoB and HSP60 peptides were found to reduce early atherosclerotic lesion formation in mice by 32.1 and 33.5 %, respectively, while the reduction with peptide A was 5.7 %. Thus the in-vitro assay shows an apparent correlation with in-vivo activity and can be developed as a screening assay to prioritize the candidate molecules for animal efficacy testing.
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Apolipoproteínas B , Chaperonina 60 , Epítopos/farmacología , Péptidos/farmacología , Receptores de Complemento , Adulto , Animales , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/inmunología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Dieta Alta en Grasa , Epítopos/uso terapéutico , Humanos , Ratones , Ratones Noqueados , Péptidos/uso terapéutico , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Adiponectin and leptin link metabolic disorders and coronary artery disease (CAD). We analysed their relationship with CAD, classical risk factors and biomarkers in 287 CAD patients (cases) and 477 unaffected family members (controls) selected from the Indian Atherosclerosis Research Study (IARS). Classical risk factors included diabetes, hypertension, dyslipidaemia and obesity markers. Novel biomarkers were measured according to manufacturer recommendations. Adverse clinical events were recorded through telephonic follow-up. Cases showed lower adiponectin levels (4684.62 ± 190.73 ng/ml) than controls (5768.86 ± 152.87 ng/ml) (p=1.58X10(-5)); Leptin levels were higher in affected males (12.47 ± 1.32 ng/ml) than in male controls (9.53 ± 1.19 ng/ml, p=0.017). Adiponectin 1st quartile showed significant protection against CAD in females when compared to 3rd (odds ratio [OR] 0.39, 0.16-0.92, p=0.032) or 4th (OR 0.32, 0.14-0.72; p=0.006) quartile group. Leptin 3rd quartile showed higher CAD risk in males as compared to 1st quartile group (OR 2.09, 1.09-4.01, p=0.028). Subjects with metabolic syndrome showed low adiponectin and high leptin levels. Adipokines showed opposing association trend with lipids, inflammatory and coagulation markers and strong correlation (r=-0.14 to 0.52) with obesity markers. Cases with recurrent event and controls who developed new cardiac event during follow up showed high adiponectin levels (p<0.05). A model that combined adiponectin, leptin and conventional risk factors yielded the best 'C' index (0.890, 0.067-0.912). CAD patients in the top adiponectin tertile showed relatively poor survival curve as compared to the bottom Adiponectin tertile group. In conclusion, our findings strengthen the reported association between low adiponectin, high leptin, obesity-related metabolic disturbances and incident CAD in Asian Indians.
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Adiponectina/sangre , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/etiología , Leptina/sangre , Adulto , Aterosclerosis/sangre , Aterosclerosis/etiología , Biomarcadores/sangre , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/etiología , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , India , Masculino , Síndrome Metabólico/sangre , Persona de Mediana Edad , Modelos Cardiovasculares , Obesidad/sangre , Pronóstico , Factores de RiesgoRESUMEN
Atherosclerosis, the underlying cause of cardiovascular disease, is characterized by chronic inflammation and altered immune response. Cholesterol is a well-known risk factor associated with the development of cardiovascular diseases. Elevated serum cholesterol is unique because it can lead to development of atherosclerosis in animals and humans even in the absence of other risk factors. Modifications of low-density lipoproteins mediated by oxidation, enzymatic degradation, and aggregation result in changes in their function and activate both innate and adaptive immune system. Oxidized low-density lipoprotein (LDL) has been identified as one of the most important autoantigens in atherosclerosis. This escape from self-tolerance is dependent on the formation of oxidized phospholipids. The emerging understanding of the importance of immune responses against oxidized LDL in atherosclerosis has focused attention on the possibility of development of novel therapy for atherosclerosis. This review provides an overview of immune response to lipoproteins and the fascinating possibility of developing an immunomodulatory therapy for atherosclerosis.
RESUMEN
BACKGROUND: Coronary artery disease (CAD) occurs at an earlier age in South Asians compared with other ethnic groups. Infection and inflammation show a positive association with the disease. OBJECTIVE: To investigate the association of infection and inflammatory markers with premature CAD in the Indian Atherosclerosis Research Study population. METHODS: Antibody titres for Chlamydia pneumoniae, cytomegalovirus (CMV), Helicobacter pylori, herpes simplex virus and levels of interleukin-6 (IL-6), high-sensitivity C-reactive protein (hsCRP), fibrinogen and secretory phospholipase A2, were measured in 866 individuals (433 CAD patients and matched controls). All individuals were followed-up for recurrent cardiac events for four years. ANOVA was used to study the association of infection and inflammation with CAD. RESULTS: The present study found that the odds of CAD occurrence was 2.42 (95% CI 1.26 to 4.64; P<0.008), with all four infections and increased in the presence of hsCRP (OR 4.67 [95% CI 1.43 to 15.25]); P=0.011). Only anti-CMV antibody levels were a significant risk factor for CAD occurrence (OR 2.23 [95% CI 1.20 to 4.15]; P=0.011) and recurrent cardiac events (OR 1.94 [95% CI 0.85 to 4.45]; P=0.015). Mean values of the inflammatory biomarkers IL-6 (P=0.035), fibrinogen (P=0.014), hsCRP (P=0.010) and secretory phospholipase A2 (P=0.002) increased with CMV antibody levels. Incorporating hsCRP and IL-6 in the risk prediction models significantly increased the OR to 2.56 (95% CI 1.16 to 5.63; P=0.019) with a c statistic of 0.826. CONCLUSIONS: Pathogen burden, especially CMV infection in combination with inflammatory markers, is a significant predictor of CAD risk in the young Indian population.