The MMP-9 promoter genetic variant rs3918242, mRNA and protein expression in advanced carotid plaque tissue.

BACKGROUND: The MMP-9 is a known player in atherosclerosis, yet associations of the MMP-9 -1562 C/T variant (rs3918242) with various atherosclerotic phenotypes and tissue mRNA expression are still contradictory. This study aimed to investigate the MMP-9 -1562 C/T variant, its mRNA and protein expression in carotid plaque (CP) tissue, as a risk factor for CP presence and as a marker of different plaque phenotypes (hyperechoic and hypoechoic) in patients undergoing carotid endarterectomy. The MnSOD as an MMP-9 negative regulator was also studied in relation to CP phenotypes. METHODS AND RESULTS: Genotyping of 770 participants (285 controls/485 patients) was done by tetra-primer ARMS PCR. The MMP-9 mRNA expression in 88 human CP tissues was detected by TaqMan® technology. The protein levels of MMP-9 and MnSOD were assessed by Western blot analysis. The MMP-9 -1562 C/T variant was not recognized as a risk factor for plaque presence or in predisposing MMP-9 mRNA and protein levels in plaque tissue. Patients with hypoechoic plaques had significantly lower MMP-9 mRNA and protein levels than those with hyperechoic plaque (p = 0.008, p = 0.003, respectively). MnSOD protein level was significantly higher in hypoechoic plaque compared to hyperechoic (p = 0.039). MMP-9 protein expression in CP tissue was significantly affected by sex and plaque type interaction (p = 0.009). CONCLUSIONS: Considering the differences of MMP-9 mRNA and protein expression in CP tissue regarding different plaque phenotypes and the observed sex-specific effect, the role of MMP-9 in human atherosclerotic plaques should be further elucidated.

Gender-Specific Association between Angiotensin II Type 2 Receptor -1332 A/G Gene Polymorphism and Advanced Carotid Atherosclerosis.

BACKGROUND: The angiotensin II type 2 receptor (AT2R) -1332 A/G polymorphism has been denoted as functional and associated with certain cardiovascular disease phenotypes. However, there are no studies considering the association of this gene polymorphism with carotid atherosclerosis (CA) and cerebrovascular events. Therefore, the aim of our study was to investigate a possible association of the AT2R -1332 A/G polymorphism with the occurrence of carotid plaques (CPs) and history of cerebrovascular insult (CVI) in advanced CA. METHODS: The study group included 381 controls and 509 patients with CA consecutively admitted for endarterectomy. Genotyping was determined by polymerase chain reaction-restriction fragment length polymorphism method. The association was analyzed separately for males and females because the AT2R gene is located on the X chromosome. RESULTS: The AT2R -1332 GG genotype was associated with the advanced CA in the female study group (recessive model of inheritance, AA+AG versus GG; adjusted odds ratio [OR] = 2.25; 95% confidence interval [CI] 1.17-4.33; P = .01). In the male subgroup of patients with CA, the significant overrepresentation of G/- hemizygote was detected in patients with CVI compared to male patients without this event (crude OR = 2.05, 95% CI 1.20-3.50, P = .008). CONCLUSIONS: This study suggests a gender-specific association between the AT2R -1332 A/G polymorphism and the occurrence of CP and the history of CVI in advanced CA, but further replication studies are needed.

Effects of glutathione S-transferase T1 and M1 deletions on advanced carotid atherosclerosis, oxidative, lipid and inflammatory parameters.

Glutathione S-transferases (GSTs) carry out a wide range of functions in cells, such as detoxification of endogenous compounds, removal of reactive oxygen species, and even catalysis of reactions in metabolic pathways beyond detoxification. Based on previous research, GSTM1 and GSTT1 might modify the risk of atherosclerosis. The aim of our study was to analyze the possible association of GSTM1 and GSTT1 gene polymorphisms with the occurrence of carotid plaque (CP); and biochemical parameters of oxidative stress, lipid profile and inflammation, in 346 consecutive patients with advanced atherosclerosis that underwent endarterectomy. A multiplex polymerase chain reaction (PCR) method was used to detect the deletions in GSTM1 and GSTT1 genes in the genomic DNA in 346 patients and 330 controls. The adjusted OR for CP presence (adjusted for age, gender, smoking, hypertension, BMI, HDLC, TG) was 0.24, 95 %CI 0.08-0.7, p < 0.01 for GSTT1 null and 1.13, 95 %CI 0.62-2.07, p = 0.7 for GSTM1 null genotype. We found significantly lower plasma lipoprotein (a) (Lp(a)) levels in GSTT1 null compared to wild-type genotype carriers in patient group (20.68 ± 26.02 mg/dl vs. 40.66 ± 42.89 mg/dl, mean ± SD, p = 0.04). The serum interleukin-6 (IL-6) values were significantly influenced by both GST polymorphisms in patients with CP. Our results, showing the significant reduction of GSTT1 deletions in patients with CP, suggest involvement of GSTs in carotid atherosclerosis. This study shows additional view of the possible role of GSTs in advanced chronic inflammatory disease of vascular system, but the confirmation in a larger studies in different populations are needed.

The sex-specific association of Met62Ile gene polymorphism in P-selectin glycoprotein ligand (PSGL-1) with carotid plaque presence: preliminary study.

Atherosclerosis is known as an inflammatory disease in which a recruitment of leukocytes to the endothelium wall represents a preliminary step of the initiation and the development of disease. The P-selectin glycoprotein ligand (PSGL-1) seems to be the major molecule mediating leukocyte-endothelium interactions and leukocyte rolling on stimulated endothelium. There are limited number of studies reporting on association of Met62Ile SNP in PSGL-1 gene and the risk for atherosclerosis. The aim of this study was to analyze possible association of this polymorphism with an advanced carotid atherosclerosis and biochemical markers of inflammation and haemostasis. The 275 patients consecutively admitted for carotid endarterectomy with stenosis >70% and 256 controls of the same ethnic origin were included in the study. The Met62Ile genotypes were determined by PCR RFLP. The Ile/Ile homozygotes had significantly higher CRP compared to the other genotypes in patients. Female patients had Ile allele dose-dependent association with the carotid plaque presence (Met/Met vs. Met/Ile vs. Ile/Ile; OR 1, OR 2.02, CI 1.0-4.08, OR 4.08, CI 1.0-16.81, respectively, p = 0.04). Our results suggest the impact of PSGL-1 Met62Ile polymorphism on inflammation in advanced atherosclerosis. We observed the sex-differential association of Met62Ile with advanced carotid atherosclerosis. Studies in larger and different populations should validate and further examine the suggested role of genetic variations in PSGL-1 with atherosclerosis and thrombosis.

The association of glutathione S-transferase T1 and M1 deletions with myocardial infarction.

Glutathione S-transferases (GSTs) are the family of enzymes involved in the second line of defense against oxidative stress (OS). The lack of GSTT1/GSTM1 enzyme quantity or activity, due to the presence of homozygous deletion compromises antioxidative defense resulting in OS. OS is the critical mechanism in the pathophysiology of atherosclerosis, coronary artery disease, and myocardial infarction (MI). The increase in reactive oxygen species together with the process of apoptosis plays a role in left ventricular remodeling (LVR) after MI. The associations of GSTT1 and GSTM1 gene polymorphisms with the risk of MI are inconsistent. The aim was to analyze the association of GSTT1/GSTM1 null genotypes with first MI and LVR 8 months after the MI. The study involved 330 controls and 438 consecutive patients with symptoms and signs of first MI. The subgroup of 150 MI patients was prospectively followed up for 6 months. Evidence of maladaptive LVR was obtained by 2D Doppler echocardiography 3-5 days and 6 months after the MI. A multiplex polymerase chain reaction was used to detect the deletion in GSTT1 and GSTM1 genes. GSTM1 null genotype was significantly and independently associated with first MI (adjusted OR = 1.45 95% CI 1.03-2.03, p = 0.03). Association of double null genotypes with maladaptive LVR in patients 6 months after the first MI was no longer significant after adjustment for factors that differed significantly between patients with and without maladaptive LVR. This study demonstrated the association of GSTM1 null genotypes with the risk of MI in the Serbian population.

Plasma levels of matrix metalloproteinase-8 in patients with carotid atherosclerosis.

Matrix metalloproteinases (MMPs) are involved in the remodeling of the extracellular matrix (ECM) in the arterial wall during atherogenesis. Collagens are the most abundant proteins in the ECM. MMP-8 is expressed by cells associated with the development of the atherosclerotic plaque. It cleaves collagen type I three times more potently than two other interstitial collagenases MMP-1 and MMP-13. The aim of this study was to investigate whether plasma MMP-8 values are associated with occurrence of carotid plaque (CP) and possible correlations with clinical and biochemical parameters in carotid atherosclerosis (CA) patients. Total plasma MMP-8 levels were quantified by ELISA in 63 patients with ultrasonographic evidence of CP presence and 12 controls. Plasma MMP-8 values were significantly higher in patients with CA compared with controls (median 23.36 ng/ml vs. 13.02 ng/ml, P<0.001) but they did not differ significantly according to gender, smoking and hypertensive status, associated diseases, and use of statins. Statistically significant positive correlations were observed between MMP-8 plasma values and C reactive protein (r=0.41, P=0.001), urea (r=0.50, P<0.001), aspartate transaminase (r=0.48, P=0.001), and creatinine levels (r=0.38, P=0.006). These results suggest association of MMP-8 plasma levels with occurrence of CP and correlation with certain biochemical markers.

The Effects of Aronia melanocarpa Juice Consumption on the mRNA Expression Profile in Peripheral Blood Mononuclear Cells in Subjects at Cardiovascular Risk.

Foods and food products that contain polyphenols are proposed to modulate risk of cardiovascular disease. The aim of this three-arm, crossover, randomized, double-blind, placebo-controlled intervention study was to examine the impact of Aronia melanocarpa juice (AMJ), high-polyphenol (AMJ treatment, 1.17 g/100 mL polyphenols) and low-polyphenol (dAMJ treatment, 0.29 g/100 mL polyphenols) dose, on the transcriptome in peripheral blood mononuclear cells (PBMC) of 19 subjects at cardiovascular risk. Transcriptome data were obtained by microarray. Bioinformatic functional annotation analysis was performed on both the whole transcriptome datasets and the differentially expressed genes (DEGs). Expression of selected DEGs was validated by RT-qPCR. Administration of AMJ and dAMJ treatments during the two consecutive four-week treatment periods had additive effects on PBMC transcriptome profiles, with the most pronounced and specific effect noticed for AMJ in the last treatment period (TP3) of the trial. Between the high-dose and low-dose treatments in TP3, there was a multitude of overlapping DEGs and DEG-enriched biological processes and pathways, which primarily included immunomodulation and regulation of cell proliferation/death. Increased expression of TNF, IL1B, IL8, RGS1, OSM, and DUSP2 in TP3 was confirmed by RT-qPCR. The results suggest the immunomodulatory effects of prolonged habitual consumption of polyphenol-rich aronia juice in individuals at cardiovascular risk.

Angiotensin receptor type 1 polymorphism A1166C is associated with altered AT1R and miR-155 expression in carotid plaque tissue and development of hypoechoic carotid plaques.

BACKGROUND AND AIMS: The principal biologic effects of the renin-angiotensin system are mediated by activation of the AT1R receptor. The microRNA miR-155 regulates AT1R expression, with both its, and AT1R's activity, linked to atherosclerosis. Target sites for miR-155 lie within the 3' UTR of the human AT1R gene, and include the AT1R A1166C polymorphism. Thus far, only levels of circulating miR-155 have been investigated with respect to A1166C genotypes. We hypothesized that the A1166C polymorphism could correlate with different, ultra-sonographically defined plaque phenotypes, as well as with an altered expression of AT1R mRNA and protein in human carotid plaques (CP), and altered expression of miR-155 in patients with advanced atherosclerosis. METHODS: Our study cohort comprised 411 patients with advanced carotid atherosclerosis (298 hyperechoic; 113 hypoechoic plaques). PCR analyses identified A1166C genotypes; quantitative real-time PCR determined AT1R and miR-155 expression levels, with AT1R protein expression evaluated by western blot. RESULTS: Genotypes containing the C allele bore a significant association with the hypoechoic plaque phenotype (adjusted OR 1.87, 95% CI 1.16-3.00, p = 0.01). The expression of AT1R mRNA and miR-155 were significantly up-regulated in the CPs of CC genotype carriers compared to the AA/AC genotypes (p = 0.032, p = 0.015, respectively). AT1R protein expression was also significantly higher for CC genotypes (p < 0.01). CONCLUSION: Our results indicate that the AT1R A1166C polymorphism impacts an ultrasonographically-defined human plaque phenotype, with intra-plaque AT1R and miR-155 expression altered in advanced carotid atherosclerosis. Validation and replication of these data should contribute to an improved personalized therapy with which to prevent carotid atherosclerosis.

Renin-angiotensin system gene polymorphisms as risk factors for multiple sclerosis.

The components of renin-angiotensin system, such as angiotensin-converting enzyme (ACE), angiotensin II and angiotensin II receptor type 1 and 2 (AT1R and AT2R), are expressed in the central nervous system and leukocytes and proposed to be involved in the inflammation and pathogenesis of multiple sclerosis (MS). ACE I/D, AT1R 1166A/C and AT2R -1332A/G are functional polymorphisms associated with phenotypes of diverse chronic inflammatory diseases. The aim of this study was to investigate the association between ACE I/D, AT1R 1166A/C and AT2R -1332A/G gene polymorphisms and MS in Serbian population. A total of 470 MS patients and 478 controls participated in the study. Allele-specific polymerase chain reaction (PCR) was performed for genotyping of the ACE polymorphism. The AT1R and AT2R genotyping was done by duplex PCR and restriction fragment length polymorphism analysis. Both ACE homozygotes, II and DD, were significantly overrepresented in MS patients, compared to controls (χ(2) test p=0.03). Neither genotype nor allele frequencies of AT1R 1166A/C polymorphism were significantly different between patients and controls. Significant overrepresentation of AT2R -1332 AA genotype in female patients, compared to female controls, was detected (OR=1.67, 95%CI=1.13-2.49, χ(2) test p=0.01), suggesting that this genotype could be a gender-specific genetic risk factor for MS.

MiR-155 expression level changes might be associated with initial phases of breast cancer pathogenesis and lymph-node metastasis.

BACKGROUND: Breast carcinoma is heterogeneous disease. Understanding the process of invasion and metastasis and the selection of the therapy for patients with breast carcinomas still remains difficult. MicroRNAs are powerful gene expression regulators. Because of inconsistent findings, we have analyzed potential difference in miR-155 levels in three breast cancer groups. OBJECTIVES: Our goals were to examine miR-155 expression levels in normal tissue, non-invasive and invasive breast carcinomas, and their association with standard clinical and pathological parameters and oncomiR-21, and to investigate the ability of miR-155 to separate invasive breast carcinomas with non-invasive component from pure invasive. METHODS: In the group of 40 breast tissue samples, relative expression levels of miR-155 were examined with stem-loop quantitative real-time PCR using TaqMan technology. RESULTS: The significant difference among four examined groups of the breast tissue was detected (p = 0.001). In the group of pure invasive tumors, patients with positive nodal status had significantly higher miR-155 levels (p = 0.046). CONCLUSION: Our results suggest that miR-155 might be involved in breast cancer pathogenesis and in tumor spreading to the lymph nodes, and that it might be used as biomarker for additional stratification of patients with invasive breast carcinomas with non-invasive component.

The association of ACE I/D gene polymorphism with severe carotid atherosclerosis in patients undergoing carotid endarterectomy.

INTRODUCTION: The ACE I/D polymorphism was mostly investigated in association with intima-media thickness, rarely with severe atherosclerotic phenotype. MATERIALS AND METHODS: We investigated the association of I/D polymorphism with severe carotid atherosclerosis (CA) (stenosis > 70%) in asymptomatic and symptomatic patients undergoing carotid endarterectomy. The 504 patients subjected to endarterectomy and 492 healthy controls from a population in Serbia were investigated as a case-control study. RESULTS: The univariate logistic regression analysis revealed ACE DD as a significant risk factor for severe CA (odds ratio [OR] = 1.3, 95% confidence interval [CI] 1.0-1.7, p = 0.04). After adjustment for the common risk factors (age, hypertension, smoking, and HDL) ACE was no longer significant. However, we found a significant independent influence of DD genotype on plaque presence in a normotensive subgroup of patients (OR 1.8, CI 1.2-3.0, p = 0.01, corrected for multiple testing). In symptomatic patients D allele carriers were significantly more frequent compared with asymptomatic patients (OR 1.6 CI 1.0-2.6, p = 0.05). CONCLUSIONS: Our data suggests that ACE I/D is not an independent risk factor for severe CA. On the other hand, a significant independent genetic influence of ACE I/D appeared in normotensive and symptomatic patients with severe CA. This should be considered in further research toward resolving the complex genetic background of severe CA phenotype.