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1.  Human monocyte colony-stimulating factor stimulates the gene expression of monocyte chemotactic protein-1 and increases the adhesion of monocytes to endothelial monolayers. 
Journal of Clinical Investigation  1993;92(4):1745-1751.
The stimulation of the human umbilical vein endothelial cell (HUVEC) with recombinant human monocyte-derived colony-stimulating factor (MCSF) increased the gene expression of monocyte chemotactic protein (MCP-1). Northern blot analysis indicated that 50 U/ml of MCSF is the optimal concentration for this effect. The elevation of MCP-1 mRNA started as early as 1 h after stimulation and was maintained for at least 8 h. An increased MCP-1 level in MCSF-treated HUVEC was also demonstrated at the protein level by immunocytochemical staining using a polyclonal MCP-1-specific antibody. HUVEC activated by 50 U/ml of MCSF for 5 h showed a stronger immunofluorescence staining than control cells. Micropipette separation of THP-1 monocytes from HUVEC showed that the activation of both THP-1 and endothelium by MCSF led to an increase in the separation force by more than three times (36.2 +/- 6.7 x 10(-4) vs. 9.6 +/- 3.6 x 10(-4) dyn). An increased adhesiveness was also observed after MCSF activation of peripheral blood monocytes and HUVEC (16.7 +/- 2.7 x 10(-4) vs. 5.2 +/- 0.9 x 10(-4) dyn). The increased adhesive force in both systems was blocked by the use of anti-MCP-1 (5.5 +/- 0.8 x 10(-4) and 6.8 +/- 1.1 x 10(-4) dyn). Similar results were obtained in experiments in which only HUVEC, but not monocytes, were activated by MCSF. This increased adhesion of untreated monocytes to MCSF-activated HUVEC was also blocked by the addition of anti-MCP-1. In contrast, experiments in which only THP-1 or peripheral blood monocytes, but not HUVEC, were treated with MCSF did not show a significant increase of adhesion between these cells. These results indicate that MCSF augments monocyte-endothelium interaction primarily by its action on the endothelial cell and that this function is probably mediated through an increased expression of MCP-1. The MCSF/MCP-1-dependent adhesive mechanism might be operative in the arterial wall in vivo to lead to the trapping of the infiltrated monocyte-macrophage in the subendothelial space during atherogenesis.
PMCID: PMC288335  PMID: 8408626
2.  HMOX1 Gene Promoter Polymorphism is Not Associated with Coronary Artery Disease in Koreans 
Annals of Laboratory Medicine  2014;34(5):337-344.
The heme oxygenase-1 gene (HMOX1) promoter polymorphisms modulate its transcription in response to oxidative stress. This study screened for HMOX1 polymorphisms and investigated the association between HMOX1 polymorphisms and coronary artery disease (CAD) in the Korean population.
The study population consisted of patients with CAD with obstructive lesions (n=110), CAD with minimal or no lesions (n=40), and controls (n=107). Thirty-nine patients with CAD with obstructive lesions underwent follow-up coronary angiography after six months for the presence of restenosis. The 5'-flanking region containing (GT)n repeats of the HMOX1 gene was analyzed by PCR.
The numbers of (GT)n repeats in the HMOX1 promoter showed a bimodal distribution. The alleles were divided into two subclasses, S25 and L25, depending on whether there were less than or equal to and more than 25 (GT)n repeats, respectively. The allele and genotype frequencies among groups were statistically not different. More subjects in the S25-carrier group had the low risk levels of high sensitivity C-reactive protein (hsCRP) for the CAD than those in the non-S25 carrier group (P=0.034). Multivariate logistic regression analysis revealed that the genotypes of (GT)n repeats were not related to CAD status. The restenosis group in the coronary angiography follow-up did not show any significant difference in HMOX1 genotype frequency.
The HMOX1 genotypes were not found to be associated with CAD, but the short allele carrier group contained more individuals with hsCRP values reflecting low risk of cardiovascular disease in the Korean population.
PMCID: PMC4151001  PMID: 25187885
Coronary artery disease; Heme oxygenase-1; HMOX1 gene; Polymorphism
3.  Development of a blood-based gene expression algorithm for assessment of obstructive coronary artery disease in non-diabetic patients 
BMC Medical Genomics  2011;4:26.
Alterations in gene expression in peripheral blood cells have been shown to be sensitive to the presence and extent of coronary artery disease (CAD). A non-invasive blood test that could reliably assess obstructive CAD likelihood would have diagnostic utility.
Microarray analysis of RNA samples from a 195 patient Duke CATHGEN registry case:control cohort yielded 2,438 genes with significant CAD association (p < 0.05), and identified the clinical/demographic factors with the largest effects on gene expression as age, sex, and diabetic status. RT-PCR analysis of 88 CAD classifier genes confirmed that diabetic status was the largest clinical factor affecting CAD associated gene expression changes. A second microarray cohort analysis limited to non-diabetics from the multi-center PREDICT study (198 patients; 99 case: control pairs matched for age and sex) evaluated gene expression, clinical, and cell population predictors of CAD and yielded 5,935 CAD genes (p < 0.05) with an intersection of 655 genes with the CATHGEN results. Biological pathway (gene ontology and literature) and statistical analyses (hierarchical clustering and logistic regression) were used in combination to select 113 genes for RT-PCR analysis including CAD classifiers, cell-type specific markers, and normalization genes.
RT-PCR analysis of these 113 genes in a PREDICT cohort of 640 non-diabetic subject samples was used for algorithm development. Gene expression correlations identified clusters of CAD classifier genes which were reduced to meta-genes using LASSO. The final classifier for assessment of obstructive CAD was derived by Ridge Regression and contained sex-specific age functions and 6 meta-gene terms, comprising 23 genes. This algorithm showed a cross-validated estimated AUC = 0.77 (95% CI 0.73-0.81) in ROC analysis.
We have developed a whole blood classifier based on gene expression, age and sex for the assessment of obstructive CAD in non-diabetic patients from a combination of microarray and RT-PCR data derived from studies of patients clinically indicated for invasive angiography.
Clinical trial registration information
PREDICT, Personalized Risk Evaluation and Diagnosis in the Coronary Tree,, NCT00500617
PMCID: PMC3072303  PMID: 21443790
Atherosclerosis; gene expression; whole blood classifier
4.  Analysis of tear inflammatory mediators: A comparison between the microarray and Luminex methods 
Molecular Vision  2016;22:177-188.
Inflammatory mediators have been shown to modulate dry eye (DE) disease and may correlate with disease severity, yet the methods used and the associated findings vary significantly in the literature. The goal of this research was to compare two methods, the quantitative microarray and the magnetic bead assay, for detecting cytokine levels in extracted tear samples across three subject groups.
Tears were collected from Schirmer strips of the right and left eyes of 20 soft contact lens wearers (CL), 20 normal non-contact lens wearers (NOR), and 20 DE subjects and stored at −80 °C. Tear proteins were eluted and precipitated using ammonium bicarbonate and acetone. The right and left eye samples were combined for each subject. Following the Bradford protein quantitation method, 10 µg of total protein was used for each of the two analyses, Quantibody® Human Inflammation Array 3 (RayBiotech) and High Sensitivity Human Cytokine Magnetic Bead Kit (Millipore). The assays were run using the GenePix® 4000B Scanner (Molecular Devices) or the Luminex MagPix® plate reader (Luminex), respectively. The data were then compared between the two instruments and the three subject groups
Of the 40 proteins on the Quantibody® microarray, seven had average expression levels above the lower limit of detection: ICAM-1, MCP-1, MIG, MCSF, TIMP-1, TIMP-2, and TNF-RI. Significant differences in expression levels (p<0.05) were detected between the CL and DE groups for MCSF, TIMP-1, and TNF R1, between the NOR and DE groups for ICAM-1, and between the CL and NOR groups for ICAM-1, MCP-1, MCSF, TIMP-1, TIMP-2, and TNF-R1 when using the Student t test. Of the 13 proteins tested with Luminex, IL-1β, IL-4, IL-6, IL-7, and IL-8 had expression levels above the minimum detectable level, and these were most often detected using the Luminex assay compared to the Quantibody® microarray. Contrarily, IL-2, IL-12, IL-13, INF-g, and GM-CSF were detected more frequently using the Quantibody® microarray than the Luminex assay. Significant differences in expression levels (p<0.05) were only detected between the CL and DE groups for IL-7 and IL-8 and between the CL and NOR subjects for IL-8.
In addition to detecting more significant differences between the subject groups, the Quantibody® microarray detected more inflammatory cytokines in total within the range of detection than the Luminex assay. Differences were also noted in the types of cytokines each assay could detect from the limited protein samples. Both methods offer advantages and disadvantages; therefore, these factors should be considered when determining the appropriate assay for analyzing tear protein samples.
PMCID: PMC4767412  PMID: 26957901
5.  Tumor necrosis factor alpha inhibits entry of human immunodeficiency virus type 1 into primary human macrophages: a selective role for the 75-kilodalton receptor. 
Journal of Virology  1996;70(11):7388-7397.
The proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) is readily detected after human immunodeficiency virus type 1 (HIV-1) infection of primary macrophages in vitro and is present in plasma and tissues of patients with AIDS. Previous studies have shown that human recombinant TNFalpha (hrTNFalpha) enhances HIV replication in both chronically infected promonocytic and T-lymphoid cell lines in vitro. We report here that in contrast to untreated tissue culture-differentiated macrophages (TCDM), in which the proviral long terminal repeat (LTR) could be detected as soon as 8 h postinfection by a PCR assay, TCDM pretreatment for 3 days by hrTNFalpha markedly delayed its appearance until 72 h after infection with the HIV-1 Ada monocytotropic strain. Moreover the inhibition of formation of the proviral LTR in HIV-1-infected TCDM was directly proportional to the concentration of hrTNFalpha used. To determine if the inhibition of LTR formation results from blockade of viral entry, we performed a reverse transcription PCR assay to detect intracellular genomic viral RNA as early as 2 h after infection. Pretreatment of primary TCDM by hrTNFalpha for 3 days and even for only 2 h inhibits 75% of the viral entry into the cells. The inhibition of viral entry by hrTNFalpha was totally abolished by the use of anti-human TNFalpha monoclonal antibody. By using TNFalpha mutants specific for each human TNFalpha receptor, we showed that the inhibition of HIV-1 entry into TCDM was mediated not through the 55-kDa TNF receptor but through the 75-kDa TNF receptor. Although prolonged (1 to 5 days) TNFalpha treatment can downregulate CD4 expression in primary human TCDM, surface CD4 levels were not reduced by 2 h of treatment and was therefore not a limiting step for HIV-1 entry. In contrast to the inhibition of viral entry into primary TCDM, pretreatment with hrTNFalpha did not modify HIV-1 entry into phytohemagglutinin A-activated peripheral blood lymphocytes. TNFalpha-pretreatment inhibited HIV-1 replication in primary TCDM but not in phytohemagglutinin A-activated peripheral blood lymphocytes as assessed by decreased reverse transcriptase activity in culture supernatants. These results demonstrate that TNFalpha is able to enhance host cellular resistance to HIV-1 infection and that selective inhibition of HIV-1 entry into primary TCDM by TNFalpha involves the 75-kDa TNF receptor but not the 55-kDa TNF receptor.
PMCID: PMC190806  PMID: 8892857
6.  The Cinnamyl Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns 
PLoS ONE  2014;9(7):e101730.
Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. However, little was known about CADs in melon. Five CAD-like genes were identified in the genome of melons, namely CmCAD1 to CmCAD5. The signal peptides analysis and CAD proteins prediction showed no typical signal peptides were found in all CmCADs and CmCAD proteins may locate in the cytoplasm. Multiple alignments implied that some motifs may be responsible for the high specificity of these CAD proteins, and may be one of the key residues in the catalytic mechanism. The phylogenetic tree revealed seven groups of CAD and melon CAD genes fell into four main groups. CmCAD1 and CmCAD2 belonged to the bona fide CAD group, in which these CAD genes, as representative from angiosperms, were involved in lignin synthesis. Other CmCADs were distributed in group II, V and VII, respectively. Semi-quantitative PCR and real time qPCR revealed differential expression of CmCADs, and CmCAD5 was expressed in different vegetative tissues except mature leaves, with the highest expression in flower, while CmCAD2 and CmCAD5 were strongly expressed in flesh during development. Promoter analysis revealed several motifs of CAD genes involved in the gene expression modulated by various hormones. Treatment of abscisic acid (ABA) elevated the expression of CmCADs in flesh, whereas the transcript levels of CmCAD1 and CmCAD5 were induced by auxin (IAA); Ethylene induced the expression of CmCADs, while 1-MCP repressed the effect, apart from CmCAD4. Taken together, these data suggested that CmCAD4 may be a pseudogene and that all other CmCADs may be involved in the lignin biosynthesis induced by both abiotic and biotic stresses and in tissue-specific developmental lignification through a CAD genes family network, and CmCAD2 may be the main CAD enzymes for lignification of melon flesh and CmCAD5 may also function in flower development.
PMCID: PMC4096510  PMID: 25019207
7.  Correlation between genetic polymorphism of matrix metalloproteinase-9 in patients with coronary artery disease and cardiac remodeling 
To explore the correlation between genetic polymorphism of matrix metalloproteinase-9 (MMP-9) in patients with coronary artery disease (CAD) and cardiac remodeling.
A total of 272 subjects who received coronary angiography in our hospital from July 2008 to September 2013 were selected, including 172 CAD patients (CAD group) and another 100 ones (control group). Both groups were subjected to MMP-9 and ultrasonic detections to determine vascular remodeling and atherosclerotic plaques. C1562G polymorphism of MMP-9 gene was detected, and correlation with vascular remodeling and atherosclerotic plaque was analyzed.
Serum MMP-9 level of CAD group (330.87±50.39 ng/ml) was significantly higher than that of control group (134.87±34.02 ng/ml) (P<0.05). Compared with control group, CAD group had significantly higher intima-media thickness, and significantly lower systolic peak velocity, mean systolic velocity and end-diastolic velocity (P<0.05). Total area of stenotic blood vessels was 67.34±22.98 mm2, while that of control blood vessels was 64.00±20.83 mm2. G/G, G/C and C/C genotype frequencies of MMP-9 differed significantly in the two groups (P<0.05). G and C allele frequencies of CAD group (70.9% and 29.1%) were significantly different from those of control group (50.0% and 50.0%) (P<0.05). G/G, G/C and C/C genotypes were manifested as lipid-rich, fibrous and calcified or ulcerated plaques respectively. Total area of stenotic blood vessels of G/G genotype significantly exceeded those of G/C and C/C genotypes (P<0.05), whereas the latter two had no significant differences.
CAD promoted 1562C-G transformation of MMP-9 gene into genetic polymorphism, thus facilitating arterial remodeling and increasing unstable atherosclerotic plaques.
PMCID: PMC4485288  PMID: 26150861
Coronary artery disease; Matrix metalloproteinase-9; Genetic polymorphism; Cardiac remodeling
8.  Hepatic HMOX1 Expression Positively Correlates with Bach-1 and miR-122 in Patients with HCV Mono and HIV/HCV Coinfection 
PLoS ONE  2014;9(4):e95564.
To analyze the expression of HMOX1 and miR-122 in liver biopsy samples obtained from HCV mono-and HIV/HCV co-infected patients in relation to selected clinical parameters, histological examination and IL-28B polymorphism as well as to determine whether HMOX1 expression is dependent on Bach-1.
Materials and Methods
The study group consisted of 90 patients with CHC: 69 with HCV mono and 21 with HIV/HCV co-infection. RT-PCR was used in the analysis of HMOX1, Bach-1 and miR-122 expression in liver biopsy samples and in the assessment of IL-28B single-nucleotide polymorphism C/T (rs12979860) in the blood. Moreover in liver biopsy samples an analysis of HO-1 and Bach-1 protein level by Western Blot was performed.
HCV mono-infected patients, with lower grading score (G<2) and higher HCV viral load (>600000 IU/mL) demonstrated higher expression of HMOX1. In patients with HIV/HCV co-infection, the expression of HMOX1 was lower in patients with lower lymphocyte CD4 count and higher HIV viral load. IL28B polymorphism did not affect the expression of either HMOX1 or miR-122. Higher HMOX1 expression correlated with higher expression of Bach-1 (Spearman’s ρ = 0.586, p = 0.000001) and miR-122 (Spearman’s ρ = 0.270, p = 0.014059).
HMOX1 and miR-122 play an important role in the pathogenesis of CHC in HCV mono-and HIV/HCV co-infected patients. Reduced expression of HMOX1 in patients with HIV/HCV co-infection may indicate a worse prognosis in this group. Our results do not support the importance of Bach-1 in repression of HMOX1 in patients with chronic hepatitis C.
PMCID: PMC3994072  PMID: 24752012
9.  Neutrophil/Lymphocyte Ratio Is Associated with Non-Calcified Plaque Burden in Patients with Coronary Artery Disease 
PLoS ONE  2014;9(9):e108183.
Elevations in soluble markers of inflammation and changes in leukocyte subset distribution are frequently reported in patients with coronary artery disease (CAD). Lately, the neutrophil/lymphocyte ratio has emerged as a potential marker of both CAD severity and cardiovascular prognosis.
The aim of the study was to investigate whether neutrophil/lymphocyte ratio and other immune-inflammatory markers were related to plaque burden, as assessed by coronary computed tomography angiography (CCTA), in patients with CAD.
Twenty patients with non-ST-elevation acute coronary syndrome (NSTE-ACS) and 30 patients with stable angina (SA) underwent CCTA at two occasions, immediately prior to coronary angiography and after three months. Atherosclerotic plaques were classified as calcified, mixed and non-calcified. Blood samples were drawn at both occasions. Leukocyte subsets were analyzed by white blood cell differential counts and flow cytometry. Levels of C-reactive protein (CRP) and interleukin(IL)-6 were measured in plasma. Blood analyses were also performed in 37 healthy controls.
Plaque variables did not change over 3 months, total plaque burden being similar in NSTE-ACS and SA. However, non-calcified/total plaque ratio was higher in NSTE-ACS, 0.25(0.09–0.44) vs 0.11(0.00–0.25), p<0.05. At admission, levels of monocytes, neutrophils, neutrophil/lymphocyte ratios, CD4+ T cells, CRP and IL-6 were significantly elevated, while levels of NK cells were reduced, in both patient groups as compared to controls. After 3 months, levels of monocytes, neutrophils, neutrophil/lymphocyte ratios and CD4+ T cells remained elevated in patients. Neutrophil/lymphocyte ratios and neutrophil counts correlated significantly with numbers of non-calcified plaques and also with non-calcified/total plaque ratio (r = 0.403, p = 0.010 and r = 0.382, p = 0.024, respectively), but not with total plaque burden.
Among immune-inflammatory markers in NSTE-ACS and SA patients, neutrophil counts and neutrophil/lymphocyte ratios were significantly correlated with non-calcified plaques. Data suggest that these easily measured biomarkers reflect the burden of vulnerable plaques in CAD.
PMCID: PMC4182451  PMID: 25268632
10.  Relation of Subclinical Coronary Artery Atherosclerosis to Cerebral White Matter Disease in Healthy Individuals from Families with Early-Onset Coronary Artery Disease 
The American journal of cardiology  2013;112(6):747-752.
White matter disease (WMD) of the brain is associated with incident stroke. Similarly subclinical calcified coronary artery plaque has been associated with incident coronary artery disease (CAD) events. Although atherogenesis in both vascular beds may share some common mechanisms, the extent to which subclinical CAD is associated with WMD across age ranges in individuals with a family history of early onset CAD remains unknown. We screened 405 apparently healthy participants in the Genetic Study of Atherosclerotic Risk (GeneSTAR) for CAD risk factors, and for the presence of noncalcified and calcified coronary plaque using dual-source multi-detector cardiac CT angiography. The presence and volumes of WMD were assessed by 3 Tesla brain MRI. Participants were 60% female, 36% African American; mean age 51.6 ± 10.6 years. The prevalence of coronary plaque overall was 43.0%. Individuals with coronary plaque had significantly higher WMD volumes (median 1222 mm3, IQR [448 to 3871]) compared to those without coronary plaque (median 551 mm3, IQR [105 to 1523], p<0.001). In multivariable regression analysis, adjusting for age, sex, race, traditional risk factors, total brain volume, and intrafamilial correlations, the presence of coronary plaque was independently associated with WMD volume (p=0.05). This study shows a significant association between WMD and noncalcified and calcified coronary plaque in healthy individuals, independent of age and risk factors. In conclusion, these findings support the premise of possible shared causal pathways in two vascular beds in families at increased risk for early-onset vascular disease.
PMCID: PMC3759559  PMID: 23742943
coronary artery disease; brain white matter disease; subclinical
11.  Determination of Early and Late Endothelial Progenitor Cells in Peripheral Circulation and Their Clinical Association with Coronary Artery Disease 
The clinical implications of early and late endothelial progenitor cells (EPCs) in coronary artery disease (CAD) remain unclear. We investigated endothelial dysfunction in CAD by simultaneously examining early and late EPC colony formation and gene expression of specific surface markers in EPCs. EPCs were extracted from a total of 83 subjects with (n = 47) and without (n = 36) CAD. Early and late EPC colonies were formed from mononuclear cells extracted from peripheral blood. We found that fewer early EPC colonies were produced in the CAD group (7.2 ± 3.l/well) than those in the control group (12.4 ± 1.4/well, p < 0.05), and more late EPC colonies were produced in the CAD group (0.8 ± 0.2/well) than those in the control group (0.25 ± 0.02/well, p < 0.05). In the CAD group, the relative expression of CD31 and KDR of early and late EPCs was lower than in the control group. These results demonstrate that CAD patients could have increased late EPC density and that early and late EPCs in CAD patients exhibited immature endothelial characteristics. We suggest that changes in EPC colony count and gene expression of endothelial markers may have relation with development of CAD.
PMCID: PMC4588339  PMID: 26451256
12.  APJ receptor A445C gene polymorphism in Turkish patients with coronary artery disease 
Coronary artery disease (CAD) is a disease in which a waxy substance called plaque builds up inside the coronary arteries. Apelin is a novel endogenous peptide with inotropic and vasodilatory properties and is the ligand for the angiotensin receptor-like 1 (APJ) receptor. We aimed to determine genotype and allele frequencies of APJ receptor A445C gene polymorphism in Turkish patients with CAD and healthy controls by RFLP-PCR. This study was performed on 159 unrelated CAD patients and 62 healthy controls. We obtained AA, AC and CC genotype frequencies in CAD patients as 41.5%, 49.1% and 9.4%, respectively. In the control group, frequencies of genotypes were found as 35.5% for AA, 48.4% for AC and 16.1% for CC. We did not observe difference in APJ receptor A445C polymorphism between CAD patients and healthy controls (χ2 = 2.178; df = 2; P = 0.336). The A allele was encountered in 66% (210) of the CAD and 59.7% (74) of the controls. The C allele was seen in 34% (108) of the CAD and 40.3% (50) of the controls. Allele frequencies of interested genes were not significantly different between groups (χ2 = 1.57; df = 1; p = 0.225). The frequencies of APJ receptor A445C genotype were not significantly different between control and patients. None of the three APJ receptor A445C genotypes, AA, AC and CC displayed significant difference in CAD patients. We did not find any difference in the clinical parameters except for weight and diastolic blood pressure levels in the AA, AC and CC genotypes of patients. Individuals with CC genotypes had significantly higher weight, systolic and diastolic blood pressure levels and systolic blood pressure than other genotypes, P ≤ 0.05. In addition, HDL-C level was found decreased, but this reduction was not statistically significant. Contrarily, the low levels of weight, SBP, DBP and TC were statistically significant in the subjects with AA genotype in CAD. In conclusion, CC genotype carriers may have more risk than other genotypes in the development of hypertension in CAD, but not AAgenotype carriers. We suggest that this polymorphism may not be a marker of CAD, but it may cause useful in function of the apelin/APJ system and may be a genetic predisposing factor for diagnostic processes and can be helpfull in finding new treatment strategies. We think that it is required to further comprehensive studies in order to make clear this situation in CAD.
PMCID: PMC4694397  PMID: 26770497
APJ receptor A445C gene; coronary artery disease; polymorphism
13.  Procoagulant activity of circulating microparticles is associated with the presence of moderate calcified plaque burden detected by multislice computed tomography 
Circulating microparticles (MPs) have been reported to be associated with coronary artery disease (CAD). In this study, we explored the relationship between MPs procoagulant activity and characteristics of atherosclerotic plaque detected by 64-slice computed tomography angiography (CTA).
In 127 consecutive patients with CAD but without acute coronary syndrome and who underwent 64-slice CTA, MPs procoagulant activity in plasma (by a thrombin generation test), soluble form of lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1) and N(epsilon)-(carboxymethyl) lysine (CML) circulating levels (by ELISA) were measured. A quantitative volumetric analysis of the lumen and plaque burden of the vessel wall (soft and calcific components), for the three major coronary vessels, was performed. The patients were classified in three groups according to the presence of calcium volume: non-calcified plaque (NCP) group (calcium volume (%) = 0), moderate calcified plaque (MCP) group (0 < calcium volume (%) < 1), and calcified plaque (CP) group (calcium volume (%) ≥ 1).
MPs procoagulant activity and CML levels were higher in MCP group than in CP or NCP group (P = 0.009 and P = 0.027, respectively). MPs procoagulant activity was positively associated with CML (r = 0.317, P < 0.0001) and sLOX-1 levels (r = 0.216, P = 0.0025).
MPs procoagulant activity was higher in the MCP patient group and correlated positively with sLOX-1 and CML levels, suggesting that it may characterize a state of blood vulnerability that may locally precipitate plaque instability and increase the risk of subsequent major cardiovascular events.
PMCID: PMC3981978  PMID: 24748876
Computed tomography; Microparticles; Low density lipoprotein; Lysine; Coronary artery disease
14.  Association Study between Coronary Artery Disease and rs1333049 and rs10757274 Polymorphisms at 9p21 Locus in South-West Iran  
Cell Journal (Yakhteh)  2015;17(1):89-98.
Coronary artery disease (CAD) is a multi-factorial and heterogenic disease with atherosclerosis plaques formation in internal wall of coronary artery. Plaque formation results to limitation of the blood reaching to myocardium leading to appearance of some problems, such as ischemia, sudden thrombosis veins and myocardial infarction (MI). Several environmental and genetic factors are involved in prevalence and incident of CAD as follows: hypertension, high low density lipoprotein-cholesterol (LDL-C), age, diabetes mellitus, family history of early-onset heart disease and smoking. According to genome wide association studies (GWAS), five polymorphisms in the 9p21 locus seem to be associated with the CAD. We aimed to evaluate the remarkable association of two polymorphisms at 9p21 locus, rs1333049 and rs10757274, with CAD.
Materials and Methods
This experimental study was conducted in Golestan, Aria Hospitals and Genetics Lab of Shahid Chamran University in the city of Ahvaz, Iran, in 2010- 2011. The collected blood samples belonging to 170 CAD patients (case group) and 100 healthy individuals (control group) were analyzed by tetra-primer amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR) technique. The results were analyzed using software package used for statistical analysis (SPSS; SPSS Inc., USA) version 16. A value of p<0.05 and an odd ratio (OR) with 95% confidence intervals (CI) were considered significant.
The frequencies of CC, CG and GG genotypes for rs1333049 polymorphism in patients were 18.2, 65.3 and 16.5%, while in controls, the related values were 25, 67 and 8%, respectively. GG genotypes of rs1333049 polymorphism in CAD patients were more than control cases (OR: 0.354, 95%CI: 0.138-0.912, p=0.032). The frequencies of AA, AG and GG genotypes for rs10757274 in CAD patients were 8.2, 58.3 and 33.5%, while in controls, the related values were 35, 63 and 2%, respectively. GG Genotype in rs10757274 polymorphism in CAD patients was found more than control cases (OR: 0.014, 95% CI: 0.003 -0.065, p=0.0001).
The rs1333049 polymorphism at 9p21 locus shows a weak association with CAD, whereas rs10757274 polymorphism reveals a significant association with CAD. These variants may help the identification of patients with increased risk for coronary artery disease.
PMCID: PMC4393676  PMID: 25870838
CAD; 9p21; Polymorphism
15.  Diagnostic Potential of Differentially Expressed Homer1, IL-1β, and TNF-α in Coronary Artery Disease 
Increasing evidences suggest that inflammation plays an important role in the pathogenesis of coronary artery disease (CAD). Numerous inflammatory cytokines and related genes mediate adverse cardiovascular events in patients with CAD, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and Homer in the present study. The study was carried out on 163 CAD patients at different stages and 68 controls. The gene expression of Homer1, Homer2, Homer3, IL-1β, and TNF-α in the peripheral blood leukocytes were measured by real-time polymerase chain reaction. The mRNA levels of Homer1, IL-1β, and TNF-α in CAD patients were significantly higher than those in the control group, but not Homer2 and Homer3. However, there was no considerable difference in the mRNA levels of Homer1, IL-1β, and TNF-α among AMI, UAP, and SAP three subgroups of CAD. The receiver operating characteristic (ROC) curves showed that Homer1 had a better diagnostic value for UAP patients compared with IL-1β and TNF-α. Like IL-1β and TNF-α, Homer1 may also be an important participant of atherosclerotic plaque development and eventually rupture. The results of the present study may provide an important basis for diagnosing CAD patients, and provide new therapeutic targets for CAD.
PMCID: PMC4307261  PMID: 25551602
coronary artery disease; gene expression; inflammation; Homer; IL-1β; TNF-α
16.  Number of circulating pro‐angiogenic cells, growth factor and anti‐oxidative gene profiles might be altered in type 2 diabetes with and without diabetic foot syndrome 
Type 2 diabetes is often complicated by diabetic foot syndrome (DFS). We analyzed the circulating stem cells, growth factor and anti‐oxidant gene expression profiles in type 2 diabetes patients without or with different forms of DFS.
Materials and Methods
Healthy volunteers (n = 13) and type 2 diabetes patients: (i) without DFS (n = 10); or with (ii) Charcot osteoneuropathy (n = 10); (iii) non‐infected (n = 17); (iv) infected (n = 11); and (v) healed ulceration were examined (n = 12). Peripheral blood endothelial progenitor cells (EPC), mesenchymal stem cells (MSC), hematopoietic stem cells (HSC) and very small embryonic‐like (VSEL) cells were phenotyped using flow cytometry. Plasma cytokine concentrations and gene expressions in blood cells were measured by Luminex and quantitative real‐time polymerase chain reaction assays, respectively.
Patients with non‐complicated type 2 diabetes showed reduced HMOX1 expression, accompanied by HMOX2 upregulation, and had less circulating EPC, MSC or HSC than healthy subjects. In contrast, VSEL cells were elevated in the type 2 diabetes group. However, subjects with DFS, even with healed ulceration, had fewer VSEL cells, more CD45‐CD29+CD90+MSC, and upregulated HMOX1 when compared with the type 2 diabetes group. Patients with Charcot osteopathy had lowered plasma fibroblast growth factor‐2. Elevated plasma tumor necrosis factor‐α and decreased catalase expression was found in all diabetic patients.
Patients with type 2 diabetes and different forms of DFS have an altered number of circulating stem cells. Type 2 diabetes might also be associated with a changed plasma growth factor and anti‐oxidant gene expression profile. Altogether, these factors could contribute to the pathogenesis of different forms of DFS.
PMCID: PMC4025239  PMID: 24843745
Anti‐oxidant genes; Diabetic foot syndrome; Stem cells
17.  Gene expression profiling in whole blood of patients with coronary artery disease 
Owing to the dynamic nature of the transcriptome, gene expression profiling is a promising tool for discovery of disease-related genes and biological pathways. In the present study, we examined gene expression in whole blood of 12 patients with CAD (coronary artery disease) and 12 healthy control subjects. Furthermore, ten patients with CAD underwent whole-blood gene expression analysis before and after the completion of a cardiac rehabilitation programme following surgical coronary revascularization. mRNA and miRNA (microRNA) were isolated for expression profiling. Gene expression analysis identified 365 differentially expressed genes in patients with CAD compared with healthy controls (175 up- and 190 down-regulated in CAD), and 645 in CAD rehabilitation patients (196 up- and 449 down-regulated post-rehabilitation). Biological pathway analysis identified a number of canonical pathways, including oxidative phosphorylation and mitochondrial function, as being significantly and consistently modulated across the groups. Analysis of miRNA expression revealed a number of differentially expressed miRNAs, including hsa-miR-140-3p (control compared with CAD, P=0.017), hsa-miR-182 (control compared with CAD, P=0.093), hsa-miR-92a and hsa-miR-92b (post- compared with pre-exercise, P<0.01). Global analysis of predicted miRNA targets found significantly reduced expression of genes with target regions compared with those without: hsa-miR-140-3p (P=0.002), hsa-miR-182 (P=0.001), hsa-miR-92a and hsa-miR-92b (P=2.2×10−16). In conclusion, using whole blood as a ‘surrogate tissue’ in patients with CAD, we have identified differentially expressed miRNAs, differentially regulated genes and modulated pathways which warrant further investigation in the setting of cardiovascular function. This approach may represent a novel non-invasive strategy to unravel potentially modifiable pathways and possible therapeutic targets in cardiovascular disease.
PMCID: PMC2922838  PMID: 20528768
coronary artery disease (CAD); gene expression; microRNA (miRNA); mitochondrion; oxidative phosphorylation; rehabilitation programme; ATP5I, ATP synthase, H+ transporting, mitochondrial F0 complex, subunit E; ATP5L, ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G; CABG, coronary artery bypass graft; CAD, coronary artery disease; CASP3, caspase 3; COX7C, cytochrome c oxidase subunit VIIc, CRP, C-reactive protein; FDR, False Discovery Rate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miRNA, microRNA; NDUFA1, NADH dehydrogenase (ubiquinone) 1α subcomplex 1; NDUFB3, NADH dehydrogenase (ubiquinone) 1β subcomplex 3; qRT-PCR, quantitative real-time PCR; ROS, reactive oxygen species; UQCRQ, ubiquinol-cytochrome c reductase, complex III subunit VII; UTR, untranslated region
18.  HMOX1 Gene Promoter Alleles and High HO-1 Levels Are Associated with Severe Malaria in Gambian Children 
PLoS Pathogens  2012;8(3):e1002579.
Heme oxygenase 1 (HO-1) is an essential enzyme induced by heme and multiple stimuli associated with critical illness. In humans, polymorphisms in the HMOX1 gene promoter may influence the magnitude of HO-1 expression. In many diseases including murine malaria, HO-1 induction produces protective anti-inflammatory effects, but observations from patients suggest these may be limited to a narrow range of HO-1 induction, prompting us to investigate the role of HO-1 in malaria infection. In 307 Gambian children with either severe or uncomplicated P. falciparum malaria, we characterized the associations of HMOX1 promoter polymorphisms, HMOX1 mRNA inducibility, HO-1 protein levels in leucocytes (flow cytometry), and plasma (ELISA) with disease severity. The (GT)n repeat polymorphism in the HMOX1 promoter was associated with HMOX1 mRNA expression in white blood cells in vitro, and with severe disease and death, while high HO-1 levels were associated with severe disease. Neutrophils were the main HO-1-expressing cells in peripheral blood, and HMOX1 mRNA expression was upregulated by heme-moieties of lysed erythrocytes. We provide mechanistic evidence that induction of HMOX1 expression in neutrophils potentiates the respiratory burst, and propose this may be part of the causal pathway explaining the association between short (GT)n repeats and increased disease severity in malaria and other critical illnesses. Our findings suggest a genetic predisposition to higher levels of HO-1 is associated with severe illness, and enhances the neutrophil burst leading to oxidative damage of endothelial cells. These add important information to the discussion about possible therapeutic manipulation of HO-1 in critically ill patients.
Author Summary
HO-1 is an important anti-inflammatory enzyme induced by several stimuli associated with critical illness. In humans, the amount of HO-1 produced is influenced by a genetic polymorphism in the gene promoter region. Using Plasmodium falciparum malaria that can cause a sepsis-like syndrome as an example, we characterize the associations between the (GT)n polymorphism, HO-1 protein levels and HMOX1-mRNA expression with severity of malaria in 307 Gambian children. Our results support the functionality of this polymorphism, demonstrate that P. falciparum infections increase HO-1 levels, and indicate that a genetic predisposition to strongly upregulate HO-1 is associated with severe forms of malaria and increased risk of dying. We identify neutrophils as the main HO-1-producing blood cells, and provide evidence that hemin-mediated induction of HMOX1 in neutrophils in vitro enhances the oxidative burst. In this way sequestered neutrophils may contribute to oxidative damage of endothelial cells, which may be part of a causal pathway explaining the association between short (GT)n repeats and increased disease severity. Our findings imply that the beneficial effects of HO-1 may be limited to a narrow window of concentrations, which should be born in mind when considering the therapeutic potential of manipulating HO-1 induction in critically ill patients.
PMCID: PMC3305414  PMID: 22438807
19.  Impact of Glutathione S-Transferase M1 and T1 Gene Polymorphisms on the Smoking-Related Coronary Artery Disease 
Journal of Korean Medical Science  2008;23(3):365-372.
Glutathione S-transferase (GST) plays a key role in the detoxification of xenobiotic atherogen generated by smoking. To analyze the effect of GSTM1/T1 gene polymorphisms on the development of smoking-related coronary artery disease (CAD), 775 Korean patients who underwent coronary angiography were enrolled. The subjects were classified by luminal diameter stenosis into group A (>50%), B (20-50%), or C (<20%). GSTM1 and GSTT1 gene polymorphisms were analyzed using multiplex polymerase chain reaction (PCR) for GSTM1/T1 genes and CYP1A1 gene for internal control. Of 775 subjects, 403 patients belonged to group A. They had higher risk factors for CAD than group B (N=260) and group C (N=112). The genotype frequencies of null GSTM1 and GSTT1 showed no significant differences among 3 groups. Considering the effect of GSTM1 gene polymorphisms on the smoking-related CAD, smokers with GSTM1 null genotype had more increased risk for CAD than non-smoker with GSTM1 positive genotype (odds ratios [OR], 2.07, confidence interval [CI], 1.06-4.07). Also the effect of GSTT1 gene polymorphism on smoking-related CAD showed the same tendency as GSTM1 gene (OR, 2.00, CI, 1.05-3.84). This effect of GSTM1/T1 null genotype on smoking-related CAD was augmented when both gene polymorphisms were considered simultaneously (OR, 2.76, CI, 1.17-6.52). We concluded that GSTM1/T1 null genotype contributed to the pathogenesis of smoking-related CAD to some degree.
PMCID: PMC2526535  PMID: 18583868
Glutathione Transferase; Polymorphism, Genetic; Smoking; Coronary Artery Disease
20.  Association of Genetic Polymorphisms in Matrix Metalloproteinase-9 and Coronary Artery Disease in the Chinese Han Population: A Case–Control Study 
Objective: Matrix metalloproteinase-9 (MMP-9) plays an important role in inflammation and matrix degradation involved in atherosclerosis and plaque rupture. The T allele of rs3918242 has been reported to lead to a high promoter activity and associate with the extent of coronary artery disease (CAD). And some studies have reported that the G allele of rs17576 might be associated with CAD. The aim of this study was to assess the association between the polymorphisms of the MMP-9 gene and CAD in the Chinese Han population. Methods: This case–control study comprised 258 CAD cases and 153 controls from the Chinese Han Population. The genomic DNA of MMP-9 was isolated from whole blood. Polymerase chain reaction-based restriction fragment length polymorphism was used to determine the rs3918242 and rs17576 genotypes in the MMP-9 gene and the total serum levels of MMP-9 were measured using enzyme-linked immunosorbent assay in both case and control groups. Results: Analysis of MMP-9 gene polymorphisms showed that the frequencies of the T allele and CT+TT genotypes of rs3918242 were significantly higher in the case group than in the control group (p<0.05). However, the distribution of variant genotypes of rs17576 did not differ between the case and control groups (p>0.05). The total serum level of MMP-9 was significantly higher in the case group than in the control group (p<0.05). The subjects carrying T alleles in the CAD group had higher average serum MMP-9 levels compared with CC genotypes (p<0.05). Conclusions: Our results suggest that the single-nucleotide polymorphism of rs3918242 in the MMP-9 gene is associated with CAD and high serum levels of MMP-9 are also associated with CAD in the Chinese Han population. Therefore, genetic variation of rs3918242 may participate in the development of CAD through influencing MMP-9 expression.
PMCID: PMC3761435  PMID: 23819814
21.  CXCL4 induces a unique transcriptome in monocyte-derived macrophages 
In atherosclerotic arteries, blood monocytes differentiate to macrophages in the presence of growth factors like macrophage colony-stimulation factor (MCSF) and chemokines like platelet factor 4 (CXCL4). To compare the gene expression signature of CXCL4-induced macrophages with MCSF-induced macrophages or macrophages polarized with IFN-γ/LPS (M1) or IL-4 (M2), we cultured primary human peripheral blood monocytes for six days. mRNA expression was measured by Affymetrix gene chips and differences were analyzed by Local Pooled Error test, Profile of Complex Functionality and Gene Set Enrichment Analysis. 375 genes were differentially expressed between MCSF- and CXCL4-induced macrophages, 206 of them overexpressed in CXCL4 macrophages coding for genes implicated in the inflammatory/immune response, antigen processing/presentation, and lipid metabolism. CXCL4-induced macrophages overexpressed some M1 and M2 genes and the corresponding cytokines at the protein level, however, their transcriptome clustered with neither M1 nor M2 transcriptomes. They almost completely lost the ability to phagocytose zymosan beads. Genes linked to atherosclerosis were not consistently up- or downregulated. Scavenger receptors showed lower and cholesterol efflux transporters higher expression in CXCL4- than MCSF-induced macrophages, resulting in lower LDL content. We conclude that CXCL4 induces a unique macrophage transcriptome distinct from known macrophage types, defining a new macrophage differentiation that we propose to call M4.
PMCID: PMC3418140  PMID: 20335529
22.  Multi-Center Validation of the Diagnostic Accuracy of a Blood-based Gene Expression Test for Assessment of Obstructive Coronary Artery Disease in Non-Diabetic Patients 
Annals of internal medicine  2010;153(7):425-434.
Diagnosis of significant coronary artery disease (CAD) in at risk patients can be challenging, typically including non-invasive imaging modalities and ultimately the gold standard of coronary angiography. Previous studies suggested that peripheral blood gene expression can reflect the presence of CAD.
To validate a previously developed 23-gene expression-based classifier for diagnosis of obstructive CAD in non-diabetic patients.
Multi-center prospective trial with blood samples drawn prior to coronary angiography.
Thirty-nine US centers.
An independent validation cohort of 526 non-diabetic patients clinically-indicated for coronary angiography
Receiver-operator characteristics (ROC) analysis of classifier score measured by real-time polymerase chain reaction (RT-PCR), additivity to clinical factors, and reclassification of patient disease likelihood vs disease status defined by quantitative coronary angiography (QCA). Obstructive CAD defined as ≥50% stenosis in ≥1 major coronary artery by QCA.
The overall ROC curve area (AUC) was 0.70 ±0.02, (p<0.001); the classifier added to clinical variables (Diamond-Forrester method) (AUC 0.72 with classifier vs 0.66 without, p = 0.003). Net reclassification was improved by the classifier over Diamond-Forrester and an expanded clinical model (both p<0.001). At a score threshold corresponding to 20% obstructive CAD likelihood (14.75), the sensitivity and specificity were 85% and 43%, yielding NPV of 83% and PPV 46%, with 33% of patient scores below this threshold.
The study excluded patients with chronic inflammatory disorders, elevated white blood counts or cardiac protein markers, and diabetes.
This non-invasive whole blood test, based on gene expression and demographics, may be useful for assessment of obstructive CAD in non-diabetic patients without known CAD.
Primary Funding Source
CardioDx, Inc.
PMCID: PMC3786733  PMID: 20921541
23.  Adiponectin levels and expression of adiponectin receptors in isolated monocytes from overweight patients with coronary artery disease 
Adiponectin has insulin-sensitizing and anti-atherosclerotic effects, partly mediated through its action on monocytes. We aimed to determine adiponectin levels and expression of its receptors (AdipoR1 and AdipoR2) in peripheral monocytes from overweight and obese patients with coronary artery disease (CAD).
Fifty-five overweight/obese patients, suspected for CAD, underwent coronary angiography: 31 were classified as CAD patients (stenosis ≥ 50% in at least one main vessel) and 24 as nonCAD. Quantitative RT-PCR and flow cytometry were used for determining mRNA and protein surface expression of adiponectin receptors in peripheral monocytes. A high sensitivity multiplex assay (xMAP technology) was used for the determination of plasma adiponectin and interleukin-10 (IL-10) secreted levels.
Plasma adiponectin levels were decreased in CAD compared to nonCAD patients (10.9 ± 3.1 vs. 13.8 ± 5.8 μg/ml respectively, p = 0.033). In multivariable analysis, Matsuda index was the sole independent determinant of adiponectin levels. AdipoR1 and AdipoR2 protein levels were decreased in monocytes from CAD compared to nonCAD patients (59.5 ± 24.9 vs. 80 ± 46 and 70.7 ± 39 vs. 95.6 ± 47.8 Mean Fluorescence Intensity Arbitrary Units respectively, p < 0.05). No significant differences were observed concerning the mRNA levels of the adiponectin receptors between CAD and nonCAD patients. AdipoR2 protein levels were positively correlated with plasma adiponectin and Matsuda index (r = 0.36 and 0.31 respectively, p < 0.05 for both). Furthermore, basal as well as adiponectin-induced IL-10 release was reduced in monocyte-derived macrophages from CAD compared to nonCAD subjects.
Overweight patients with CAD compared to those without CAD, had decreased plasma adiponectin levels, as well as decreased surface expression of adiponectin receptors in peripheral monocytes. This fact together with the reduced adiponectin-induced IL-10 secretion from CAD macrophages could explain to a certain extent, an impaired atheroprotective action of adiponectin.
PMCID: PMC3042923  PMID: 21284833
24.  Multi-Organ Expression Profiling Uncovers a Gene Module in Coronary Artery Disease Involving Transendothelial Migration of Leukocytes and LIM Domain Binding 2: The Stockholm Atherosclerosis Gene Expression (STAGE) Study 
PLoS Genetics  2009;5(12):e1000754.
Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD). The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE) study was to determine whether there are functionally associated genes (rather than individual genes) important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue) and atherosclerotic and unaffected arterial wall (n = 40/tissue) isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes) in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes). In the second step (performed within tissue clusters), one atherosclerotic lesion (n = 49/48) and one visceral fat (n = 59) cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015). The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54) relating to carotid stenosis (P = 0.04), 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10−27and−30). Genes in the transendothelial migration of leukocytes (TEML) pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module). In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004). The transcription co-factor LIM domain binding 2 (LDB2) was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2, and by the expression of 13 TEML genes in Ldb2–deficient arterial wall. Thus, the A-module appears to be important for atherosclerosis development and, together with LDB2, merits further attention in CAD research.
Author Summary
The WHO predicts that coronary artery disease (CAD) will become the leading cause of death worldwide in 2010. Currently, major research efforts are focused on understanding the genetics of CAD through multi-center, genome-wide association studies of tens of thousands of patients and controls. Such studies can identify common variants of general importance throughout the entire population, which are likely relatively few. The number of rare genetic variants and variants that act in the context of environmental risk factors for CAD is probably much higher. We performed whole-genome expression analyses in several organs to identify functionally associated genes important for CAD development. We found an atherosclerosis module (A-module) consisting of 128 genes, enriched with genetic risk for CAD, involving transendothelial migration of leukocytes (TEML) and LIM domain binding 2 (LDB2) as its high-hierarchy regulator. Our study design represents a novel way of understanding the molecular underpinnings of CAD, focusing on genome-wide expression sensing both environmental and genetic influences. Investigating the relative enrichment of genetic CAD risk in functional groups (modules and networks) is an alternative approach to extract additional relevant information from genome-wide association studies. The A-module and LDB2 are attractive targets for treatments to modulate TEML and atherosclerosis development.
PMCID: PMC2780352  PMID: 19997623
25.  Increasing Maternal Body Mass Index Is Associated with Systemic Inflammation in the Mother and the Activation of Distinct Placental Inflammatory Pathways1 
Biology of Reproduction  2014;90(6):129, 1-9.
Obese pregnant women have increased levels of proinflammatory cytokines in maternal circulation and placental tissues. However, the pathways contributing to placental inflammation in obesity are largely unknown. We tested the hypothesis that maternal body mass index (BMI) was associated with elevated proinflammatory cytokines in maternal and fetal circulations and increased activation of placental inflammatory pathways. A total of 60 women of varying pre-/early pregnancy BMI, undergoing delivery by Cesarean section at term, were studied. Maternal and fetal (cord) plasma were collected for analysis of insulin, leptin, IL-1beta, IL-6, IL-8, monocyte chemoattractant protein (MCP) 1, and TNFalpha by multiplex ELISA. Activation of the inflammatory pathways in the placenta was investigated by measuring the phosphorylated and total protein expression of p38-mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinase (JNK)-MAPK, signal transducer-activated transcription factor (STAT) 3, caspase-1, IL-1beta, IkappaB-alpha protein, and p65 DNA-binding activity. To determine the link between activated placental inflammatory pathways and elevated maternal cytokines, cultured primary human trophoblast (PHT) cells were treated with physiological concentrations of insulin, MCP-1, and TNFalpha, and inflammatory signaling analyzed by Western blot. Maternal BMI was positively correlated with maternal insulin, leptin, MCP-1, and TNFalpha, whereas only fetal leptin was increased with BMI. Placental phosphorylation of p38-MAPK and STAT3, and the expression of IL-1beta protein, were increased with maternal BMI; phosphorylation of p38-MAPK was also correlated with birth weight. In contrast, placental NFkappaB, JNK and caspase-1 signaling, and fetal cytokine levels were unaffected by maternal BMI. In PHT cells, p38-MAPK was activated by MCP-1 and TNFalpha, whereas STAT3 phosphorylation was increased following TNFalpha treatment. Maternal BMI is associated with elevated maternal cytokines and activation of placental p38-MAPK and STAT3 inflammatory pathways, without changes in fetal systemic inflammatory profile. Activation of p38-MAPK by MCP-1 and TNFalpha, and STAT3 by TNFalpha, suggests a link between elevated proinflammatory cytokines in maternal plasma and activation of placental inflammatory pathways. We suggest that inflammatory processes associated with elevated maternal BMI may influence fetal growth by altering placental function.
PMCID: PMC4094003  PMID: 24759787
cytokines; innate immune response; obesity

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