Inverse association of ApoB and HSP60 antibodies with coronary artery disease in Indian population.

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.

Comparative analysis of inflammatory gene expression levels in metabolic syndrome & coronary artery disease.

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.

Danger-recognizing proteins, ß-defensin-128 and histatin-3, as potential biomarkers of recurrent coronary events.

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.

Oral administration of recombinant Mycobacterium smegmatis expressing a tripeptide construct derived from endogenous and microbial antigens prevents atherosclerosis in ApoE(-/-) mice.

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.

Understanding the progression of atherosclerosis through gene profiling and co-expression network analysis in Apob(tm2Sgy)Ldlr(tm1Her) double knockout mice.

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.

Contribution of classical and emerging risk factors to coronary artery disease in Asian Indians.

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.

Regulating Inflammatory Immune Response to Atherogenic Antigens Prevents Development and Progression of Atherosclerosis in New Zealand White Rabbits.

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.

Essential Roles of Toll-Like Receptors in Atherosclerosis.

Atherosclerosis is driven by inflammation with an involvement of innate and adaptive immune responses. Toll-like receptors, the well-defined pattern recognition receptors of the immune system, play a central role in macrophage activation. Toll-like receptors recognize pathogen-associated molecular patterns expressed by a wide range of infectious agents and provide a strong link between local innate and adaptive immunity. Activation of these receptors triggers an intracellular signaling cascade mediated through myeloid differentiation factor 88 or toll/interleukin-1 receptor-domain-containing adapter-inducing interferon-ß, leading to the secretion of proand anti-inflammatory cytokines. Engagement of Toll-like receptors with their ligands induces leukocyte recruitment and enhances matrix metalloproteinase expression within atherosclerotic lesions. Recently certain Toll-like receptors have shown a protective role in atherosclerosis. TLRs, therefore, represent an important link between inflammation and atheroma, making them attractive targets for the treatment of atherosclerosis. This review will briefly describe the general biological structure and potential roles of Toll-like receptors as therapeutic targets for the treatment of atherosclerosis and highlight the potential challenges on Toll-like receptor- based therapy in cardiovascular disease.

Understanding gene expression in coronary artery disease through global profiling, network analysis and independent validation of key candidate genes.

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.

The role of interleukin 35 in atherosclerosis.

Atherosclerosis has been widely recognized as a slow progressing inflammatory disease of the arterial walls involving both inflammation and autoimmune processes with a complex etiology in which the immune system plays a key role. A hallmark of atherosclerosis is that the macrophages pick up the lipids to form the foam cells which build up the plaque in the arterial wall. Consequently, the arteries become narrowed. Plaque rupture can trigger thrombosis which is superimposed on atherosclerotic lesion. The activation of macrophages and T cells plays key roles in these lesions. Cells involved in the atherosclerotic process secrete soluble factors, known as cytokines. These cytokines can be further divided into two classes namely proinflammatory and anti-inflammatory cytokines based on their roles in inflammation. Among the cytokines, interleukin (IL)-35 is the one most recently discovered that suppresses inflammatory responses of immune cells. Accumulating evidence suggests that IL-35 represents an attractive target for antiatherosclerotic therapy based on its several atheroprotective features. In this review, we will provide a brief overview of IL-35 biology and the role of IL-35 in the development, or the progression of atherosclerosis.

The role of interleukin 35 in atherosclerosis.

Atherosclerosis has been widely recognized as a slow progressing inflammatory disease of the arterial walls involving both inflammation and autoimmune processes with a complex etiology in which the immune system plays a key role. A hallmark of atherosclerosis is that the macrophages pick up the lipids to form the foam cells which build up the plaque in the arterial wall. Consequently, the arteries become narrowed. Plaque rupture can trigger thrombosis which is superimposed on atherosclerotic lesion. The activation of macrophages and T cells plays key roles in these lesions. Cells involved in the atherosclerotic process secrete soluble factors, known as cytokines. These cytokines can be further divided into two classes namely proinflammatory and anti-inflammatory cytokines based on their roles in inflammation. Among the cytokines, interleukin (IL)-35 is the one most recently discovered that suppresses inflammatory responses of immune cells. Accumulating evidence suggests that IL-35 represents an attractive target for anti-atherosclerotic therapy based on its several atheroprotective features. In this review, we will provide a brief overview of IL-35 biology and the role of IL-35 in the development, or the progression of atherosclerosis.

Hypercholesterolemia Induced Immune Response and Inflammation on Progression of Atherosclerosis in Apob(tm2Sgy) Ldlr(tm1Her)/J Mice.

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.

Modulation of recombinant antigenic constructs containing multi-epitopes towards effective reduction of atherosclerotic lesion in B6;129S-Ldlr(tm1Her)Apob(tm2Sgy)/J mice.

Atherosclerosis is increasingly recognized as a complex chronic inflammatory disease. Many more studies have extended vaccination against atherosclerosis by using epitopes from self-antigens or beyond and demonstrated that vaccination with antigens or derivatives could reduce the extent of the lesions in atherosclerosis-prone mice. Our previous study has demonstrated that construct AHHC [ApoB100688-707 + hHSP60303-312 + hHSP60153-163 + Cpn derived peptide (C)] significantly reduced atherosclerotic lesion. The aim of this study was to investigate whether AHHC can be modulated towards increased lesion reduction in mice by creating two other derivatives with a sequential epitope-substitution named RHHC in which A was replaced by an "R" (C5aR1-31) and RPHC with a further "H" (hHSP60303-312) conversion into "P" (protease-activated receptor-142-55) in mice. Antigenic epitopes were incorporated into a dendroaspin scaffold. Immunization of B6;129S-Ldlrtm1HerApobtm2Sgy/J mice with three constructs elicited production of high levels of antibodies against each epitope (apart from hHSP60153-163 and P which induced a low antibody response). Histological analyses demonstrated that the mice immunized with either RPHC or RHHC showed significant reductions in the size of atherosclerostic lesions compared to those with AHHC (69.5±1.1% versus 55.7±3.4%, P<0.01 or 65.6±1.3% versus 55.7±3.4%, P<0.01). Reduction of plaque size in the aortic sinus and descending aorta correlated with alterations in cellular immune responses when compared with controls. We conclude that a recombinant construct RPHC may provide new antigenic and structural features which are favorable for significant reduction in atherosclerotic lesion formation. This approach offers a novel strategy for developing anti-atherosclerotic agents.

Expression of human tissue factor pathway inhibitor on vascular smooth muscle cells inhibits secretion of macrophage migration inhibitory factor and attenuates atherosclerosis in ApoE-/- mice.

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.

Oral dosing with multi-antigenic construct induces atheroprotective immune tolerance to individual peptides in mice.

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.

Impact of MCP-1 in atherosclerosis.

Monocyte chemotactic protein-1 (MCP-1) (also referred to as chemokine (C-C motif) ligand 2 (CCL2) is expressed by mainly inflammatory cells and endothelial cells. The expression level is upregulated after proinflammatory stimuli and tissue injury which are associated with atherosclerotic lesion. Atherosclerosis is a progressive disease starting with accumulation of lipids, lipoproteins, and immune cells in the arterial wall. MCP-1 has been reported to play an important role in the pathogenesis of atherosclerosis and considerable evidence supports that the monocyte containing MCPs and macrophage influences the growth of other cell types within the atherosclerotic lesion. This review will focus on the general structure features of MCP-1 and its role in atherosclerosis.

Network analysis of inflammatory genes and their transcriptional regulators in coronary artery disease.

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.

CELSR2-PSRC1-SORT1 gene expression and association with coronary artery disease and plasma lipid levels in an Asian Indian cohort.

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.

Impact of matrix metalloproteinases on atherosclerosis.

Atherosclerosis is now widely recognized as a chronic inflammatory disease that involves innate and adaptive immune responses. Both cellular and humoral components of the immune system have been implicated in atherogenesis. Growing evidence suggests that immune cells play crucial roles in atherogenic plaque formation. Vulnerability of the plaque probably plays an important role in rupture. Most ruptures occur at the periphery of the fibrous cap that covers the lipid-rich core-points where the cap is usually thinnest and most heavily infiltrated by macrophage foam cells. Sudden rupture of a plaque triggers unstable angina, acute myocardial infarction, and sudden cardiac death. Initiation of collagen breakdown in plaques requires matrix metalloproteinase (MMP) family members including MMP-1, MMP-8, and MMP- 13. In addition, other MMPs such as MMP-2, -3, -9, -10 and -12 have also been reported to play roles in atherosclerosis. This review aims to focus on description of general structural features of MMPs and their roles in atherosclerosis.

Genetic analysis of the 9p21.3 CAD risk locus in Asian Indians.

The 9p21.3 locus is the best replicated region to date for coronary artery disease (CAD). We investigated the association of 9p21.3 common variants with CAD, candidate gene expression including ANRIL, a non-coding RNA, followed by in vitro validation. Five variants, rs10757278, rs10757274, rs2383206, rs1333049 and rs4977574 were genotyped in 1,034 cases and 1,034 controls. Gene expression of C9orf5, MTAP1, MTAP 2, p16INK4a, p14ARF, p15INK4b and two ANRIL splice variants, NR_003529 and EU741058, were measured in 100 cases and 100 controls. Human aortic smooth muscle cells (HuAoSMCs) were transfected with siRNA targeting ANRIL exon 19 (siRNA-1) or exon 2 (siRNA-2) and consequent effect determined. rs2383206 showed the highest association with CAD (odds ratio [OR] 2.02, 95% confidence interval [CI] 1.56 -2.62) and an adjusted OR of 2.55, 1.33-2.88 along with rs10757278. Conventional risk factors (conventional RFs), rs2383206 and rs10757278 variants together yielded a higher c index (OR 0.790, 95% CI 0.770 -0.810) as compared to conventional RFs (OR 0.783, 95% CI 0.763-0.803) or genetic variants (OR 0.561, 95% CI 0.536-0.586) alone. GAAAA haplotype showed significant protective association with CAD compared to CGGGG risk haplotype (OR 0.45, 95% CI 0.27-0.77). Expression of p16INK4a, p14ARF and p15INK4b as well as plasma CDKN2A levels were lower in cases than controls. GG genotype was associated with higher EU741058 expression and lower p16INK4a expression. HuAoSMCs transfected with siRNA-1 showed lower NR_003529, p16INK4aand p14ARFexpression. Our study provides further evidence on the significance of 9p21.3 locus for CAD wherein the risk allele regulate the expression of ANRIL and adjacent tumour suppressor genes which in turn alter smooth muscle proliferation, a fundamental process in atherosclerosis.