Regulation of hepatic Na+/K+-ATPase in obese female and male rats: involvement of ERK1/2, AMPK, and Rho/ROCK.

In this study, we assessed whether the disturbed regulation of sodium/potassium-adenosine-triphosphatase (Na+/K+-ATPase) occurs as a consequence of obesity-induced IR in sex-specific manner. We also assessed whether alterations of IRS/PI3K/Akt, ERK1/2, AMPKα, and RhoA/ROCK signaling cascades have an important role in this pathology. Female and male Wistar rats (150-200 g, 8 weeks old) were fed a standard laboratory diet or a high-fat (HF) diet (42% fat) for 10 weeks. The activity of hepatic Na+/K+-ATPase and Rho, and the association of IRS-1/p85 were assessed in liver. Furthermore, the protein level of α1 Na+/K+-ATPase in plasma membrane fractions, and protein levels of IRS-1, PI3K-p85, -p110, RhoA, ROCK1, ROCK2, ERK1/2, AMPKα, ERα, and ERß in liver lysates were assessed. The expression of hepatic α1 Na+/K+-ATPase mRNA was also analyzed by qRT-PCR. The results show that HF-fed female rats exhibited an increase in hepatic ERK1/2 (p < 0.05) and AMPKα (p < 0.05) phosphorylation levels, unchanged level of Na+/K+-ATPase α1 mRNA, decreased level of Na+/K+-ATPase activity (p < 0.05), and decreased α1 Na+/K+-ATPase protein expression (p < 0.01). In liver of HF-fed male rats, results show decreased levels of Na+/K+-ATPase activity (p < 0.01), both protein and mRNA of α1 subunit (p < 0.05), but significant increase in Rho activity (p < 0.05). Our results indicate significant sex differences in α1 Na+/K+-ATPase mRNA expression and activation of ERK1/2, AMPKα, and Rho in the liver. Exploring the sex-specific factors and pathways that promote obesity-related diseases may lead to a better understanding of pathogenesis and discovering new therapeutic targets.

Left ventricular remodeling after the first myocardial infarction in association with LGALS-3 neighbouring variants rs2274273 and rs17128183 and its relative mRNA expression: a prospective study.

Post-infarct left ventricular remodeling (LVR) process increases the risk of heart failure (HF). Circulating galectin-3 has been associated with fibrosis, inflammation and cardiac dysfunction during the remodeling process after myocardial infarction (MI). The aims of this prospective case study were to investigate the association of potentially functional variants in the vicinity of LGALS-3 locus, rs2274273 and rs17128183 with maladaptive LVR and whether these variants could affect LGALS-3 mRNA expression in peripheral blood mononuclear cells of patients 6 months after the first MI. This study encompassed 167 patients with acute MI that were followed up for 6 months. Evidence of LVR was obtained by repeated 2D Doppler echocardiography. Rs2274273, rs17128183 and LGALS-3 mRNA expression were detected by TaqMan® technology. Rs2274273 and rs17128183 rare allele bearing genotypes, according to the dominant model (CT+TT vs. CC and AG+GG vs. AA, respectively), were significantly and independently associated with maladaptive LVR (adjusted OR = 3.02, P = 0.016; adjusted OR = 3.14, P = 0.019, respectively) and higher LGALS-3 mRNA expression (fold induction 1.203, P = 0.03 and 1.214, P = 0.03, respectively). Our exploratory results suggest that rs2274273 and rs17128183 variants affect LGALS-3 mRNA and bear the risk for maladaptive LVR post-MI remodeling. Further replication and validation in a larger group of patients is inevitable.

Changes in cardiac Na+/K+-ATPase expression and activity in female rats fed a high-fat diet.

The aim of this study was to investigate whether the presence of endogenous estradiol alters the effects of a high-fat (HF) diet on activity/expression of the cardiac Na+/K+-ATPase, via PI3K/IRS and RhoA/ROCK signalling cascades in female rats. For this study, female Wistar rats (8 weeks old, 150-200 g) were fed a standard diet or a HF diet (balanced diet for laboratory rats enriched with 42% fat) for 10 weeks. The results show that rats fed a HF diet exhibited a decrease in phosphorylation of the α1 subunit of Na+/K+-ATPase by 30% (p < 0.05), expression of total α1 subunit of Na+/K+-ATPase by 31% (p < 0.05), and association of IRS1 with p85 subunit of PI3K by 42% (p < 0.05), while the levels of cardiac RhoA and ROCK2 were significantly increased by 84% (p < 0.01) and 62% (p < 0.05), respectively. Our results suggest that a HF diet alters cardiac Na+/K+-ATPase expression via molecular mechanisms involving RhoA/ROCK and IRS-1/PI3K signalling in female rats.

Transcriptome-wide based identification of miRs in congenital anomalies of the kidney and urinary tract (CAKUT) in children: the significant upregulation of tissue miR-144 expression.

BACKGROUND: The genetic cause of most congenital anomalies of the kidney and urinary tract (CAKUT) cases remains unknown, therefore the novel approaches in searching for the common disease denominators are required. miRs regulate gene expression in humans and therefore have potentially therapeutic and biomarker properties. No studies thus far have attempted to explore the miRs in human CAKUT. We applied a new strategy to identify most specific miRs associated with CAKUT, in pediatric patients. METHODS: Data from the whole genome expression, gathered from ureter tissue samples of 19 patients and 7 controls, were used for the bioinformatic prediction of miRs activity in CAKUT. We integrated microarray gene expression data and miR target predictions from multiple prediction algorithms using Co-inertia analysis (CIA) in conjunction with correspondence analysis and between group analysis, to produce a ranked list of miRs associated with CAKUT. The CIA included five different sequence based miR target prediction algorithms and the Co-expression Meta-analysis of miR Targets. For the experimental validation of expression of miRs identified by the CIA we used tissue from 36 CAKUT patients and 9 controls. The results of gene ontology (GO) analysis on co-expressed targets of miRs associated with CAKUT were used for the selection of putative biological processes relevant to CAKUT. RESULTS: We identified 7 miRs with a potential role in CAKUT. The top ranked miRs from miRCos communities 4, 1 and 7 were chosen for experimental validation of expression in CAKUT tissue. The 5.7 fold increase of hsa-miR-144 expression in human tissue from CAKUT patients compared to controls (p = 0.005) was observed. From the GO we selected 7 biological processes that could contribute to CAKUT, which genes are potentially influenced by hsa-miR-144. The hsa-miR-200a, hsa-miR-183 and hsa-miR-375 weren't differentially expressed in CAKUT. CONCLUSIONS: This study shows that integrative approach applied here was useful in identification of the miRs associated with CAKUT. The hsa-miR-144, first time identified in CAKUT, could be connected with biological processes crucial for normal development of kidney and urinary tract. Further functional analysis must follow to reveal the impact of hsa-miR-144 on CAKUT occurrence.

Genetic Variants in the Vicinity of LGALS-3 Gene and LGALS-3 mRNA Expression in Advanced Carotid Atherosclerosis: An Exploratory Study.

BACKGROUND: Previous research has shown that there is an association between galectin-3 (gal-3) protein and cardiovascular pathology. The aim of this study was to investigate the effects of rs2274273 and rs17128183 on genetic susceptibility to advanced carotid atherosclerosis (CA) and its complications. The rs2274273 has been singled out as the lead SNP of the haplotype block containing LGALS-3 (gal-3 gene) associated with gal-3 circulating levels, while rs17128183 constitutes a potentially functional SNP of the same hap-block. We further sought to determine whether these genetic variants have an impact on the expression of LGALS-3 mRNA in human carotid atherosclerotic plaque tissue. METHODS: The study encompassed 300 control subjects and 485 patients with advanced CA who had undergone carotid endarterectomy. Rs2274273, rs17128183, and LGALS-3 relative mRNA expression was detected by means of real-time PCR (TaqMan® technology). RESULTS: There were no statistically significant associations of the investigated genetic variants with susceptibility to advanced CA, nor did we find any associations in terms of ultrasonographically defined plaque phenotypes. The relative expression of LGALS-3 mRNA proved to be significantly higher in carriers of the rare alleles (P = 0.039) for both genetic variants. CONCLUSION: Our exploratory results suggest that while rs2274273 and rs17128183 bear no association with the risk of advanced CA or CA-related complications, these genetic variants are likely to affect LGALS-3 expression levels. In order to reach a definitive conclusion on the role played by rs2274273 and rs17128183 in advanced CA, our results should be further validated.

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.

Association of PHACTR1 intronic variants with the first myocardial infarction and their effect on PHACTR1 mRNA expression in PBMCs.

BACKGROUND: Myocardial infarction (MI) and underlining atherosclerosis are the main causes of death worldwide. Phosphatase and actin regulator 1 (PHACTR1) variants have been associated with early onset MI, coronary artery disease and carotid dissection. PHACTR1 mRNA expression has been detected in tissues and cells related to atherosclerosis. Nonetheless, the true effect of PHACTR1 on vascular diseases is still unknown. Our aim was to examine the association of PHACTR1 intronic variants, rs9349379, rs2026458 and rs2876300, with MI and multi-vessel disease (MVD) and to assess their effect on PHACTR1 and EDN1 mRNA expression in PBMCs of patients six months after MI. METHODS: The study enrolled 537 patients with the first MI and 310 controls. Gene expression was assessed in 74 patients six months after MI and 37 healthy controls. Rs9349379, rs2026458, rs2876300 and relative mRNA expressions were detected by TaqMan® technology. RESULTS: The significant association between PHACTR1 variants and MI was not found, either individually or in haplotype. A higher frequency of rs2876300G-allele in MVD was rendered not significant after Bonferroni correction. PHACTR1 mRNA was significantly increased in PBMCs of patients six months after MI compared to controls (p = 0.02). Patients that carry ACG haplotype have increased PHACTR1 mRNA expression in PBMCs (p = 0.04). There was no effect of PHACTR1 variants on EDN1 mRNA expression. CONCLUSION: Our findings suggest that PHACTR1 intronic variants may have a role in severity and progression of coronary atherosclerosis. Future research is needed to clarify the mechanism underlying the role of PHACTR1 in coronary atherosclerosis and MI.

Interleukin 7 receptor alpha polymorphism rs6897932 and susceptibility to multiple sclerosis in the Western Balkans.

The interleukin 7 receptor alpha single nucleotide polymorphism rs6897932 was identified as a multiple sclerosis susceptibility-modifying polymorphism in genome-wide and gene scan studies, mainly in populations in western countries. The aim of this study was to investigate the association of interleukin 7 receptor alpha rs6897932 with multiple sclerosis in populations from the Western Balkans: Serbia, Croatia, and Slovenia. A total of 678 unrelated white patients and 597 unrelated, ethnically matched healthy controls were included in the study. Genotyping was performed by real-time polymerase chain reaction. We found no significant difference in genotype or allele frequencies between controls and patients with multiple sclerosis either separately in Serbian, Croatian, and Slovenian populations or in the whole sample from the Western Balkans. The odds ratio for multiple sclerosis in this study was 1.04 (0.86-1.25) for the C allele. It is known that demographic as well as environmental factors have a substantial role in multiple sclerosis development, as well as population genetic background. The results of this study indicate that other types of genome variants should be required for the development and/or progression of multiple sclerosis, which may vary among populations.

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.

PHACTR1 haplotypes are associated with carotid plaque presence and affect PHACTR1 mRNA expression in carotid plaque tissue.

BACKGROUND: Carotid plaque is a hallmark of advanced carotid atherosclerosis and there is evidence of phosphatase and actin regulator 1 (PHACTR1) involvement in the processes that lead to atherosclerosis. PHACTR1 intronic variants have been associated with coronary artery disease and carotid dissection. Up to date the PHACTR1 haplotypes were not investigated in association with carotid plaque presence (CPP). So, the aims of this study were to investigate possible association of PHACTR1 haplotypes inferred from the intronic variants rs9349379, rs2026458 and rs2876300 with CPP in patients with advanced carotid atherosclerosis and to analyze their possible effect on PHACTR1 relative mRNA expression in carotid plaque tissue specimens. METHODS: The study group consisted of 501 patients with evidence of carotid plaque presence admitted for carotid endarterectomy and 310 healthy controls. PHACTR1 rs9349379, rs2026458, rs2876300 and relative mRNA expression were detected by TaqMan® technology. RESULTS: We have found significant and independent association of haplotype ACA with the CPP, compared to the referent haplotype GTA (adjusted OR = 1.54 95% CI = 1.07-2.21, p = 0.02). The OR was adjusted for gender, age, BMI, hypertension and total cholesterol. The relative expression of PHACTR1 mRNA in carotid plaque tissue proved to be significantly higher in carriers of the ACG haplotype compared to the referent haplotype GTA (p = 0.03). CONCLUSION: Our results suggest that PHACTR1 haplotypes inferred from the variants rs9349379, rs2026458 and rs2876300 affect PHACTR1 mRNA and bear the risk for CPP in patients with advanced carotid atherosclerosis. Further replication and validation studies are inevitable.

CDKN2B gene expression is affected by 9p21.3 rs10757278 in CAD patients, six months after the MI.

BACKGROUND: Chromosomal region 9p21.3 is most robustly associated with coronary artery disease (CAD) in western European populations. However, heterogeneity in CAD phenotypes leads to uncertainty whether 9p21.3 is associated with stable and/or acute clinical presentations of CAD. 9p21.3 is rich in regulatory elements, but the underlying mechanisms of its actions in CAD remain unclear. We investigate the association of 9p21.3 two haplotype blocks lead variants (rs10757278 and rs518394) with first-ever non-fatal myocardial infarction (MI) in CAD patients and their association with CDKN2B mRNA expression in peripheral blood mononuclear cells 6 months after the event. METHODS: We included CAD patients with sustained first MI (n = 523) and controls (n = 583). Gene expression was assessed in 72 patients 6 months after MI and 43 healthy controls. TaqMan® technology was used for the gene expression and genotyping analysis. RESULTS: CDKN2B mRNA was significantly lower in MI patients compared with the controls (p = 0.002) and in patients carrying the rs10757278 G risk allele versus AA homozygotes (p = 0.012) 6 months after the event. While we confirmed the association of rs10757278 with CDKN2B expression in MI patients, we failed to find an association between the investigated variants and MI or disease burden. CONCLUSIONS: We suggest a dysregulation of gene expression in the 9p21.3 region six months after acute MI, which is affected by a genetic variant in patients. The rs10757278 rare allele is one factor that might lead to prolonged risk for proatherogenic complications.

Lipoprotein lipase: A bioinformatics criterion for assessment of mutations as a risk factor for cardiovascular disease.

Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism. Decrease of the LPL enzymatic activity leads to elevated triglycerides (TG) and reduced high-density lipoprotein (HDL-C levels), both risk factors for cardiovascular disease (CVD). Therefore, mutations, which decrease the LPL activity, may confer susceptibility to CVD. Here, the informational spectrum method (ISM), a virtual spectroscopy method for structure/function analysis of nucleotide and protein sequences, is applied for identification of evolutionary highly conserved information encoded by the primary structure of LPL. It was demonstrated that mutations, which alter the LPL enzymatic activity also alter this information. On the basis of this finding, an efficient and simple bioinformatics criterion for assessment of the pathogenic effect of LPL nonsynonymous single nucleotide substitution as a risk factor of CVD has been proposed.

Association of the MMP-3 5A/6A gene polymorphism with multiple sclerosis in patients from Serbia.

Matrix metalloproteinases (MMPs) are proteolytic enzymes involved in remodeling of the extracellular matrix. MMPs are suggested to play a role in the influx of inflammatory cells into the CNS, disruption of the blood brain barrier, and to degrade myelin in vitro. In this study, we have investigated the possible association of MMP-3 5A/6A gene polymorphism with MS susceptibility and/or severity in patients from Serbia. A total of 184 MS patients (150 RR, 34 SP) and 236 controls have been studied. Results show that the distribution of MMP-3 5A/6A genotype frequencies between MS patients and controls were not significantly different. In bout onset patients, carriers of MMP-3 6A/6A genotype had significantly higher mean MSSS values compared to the carriers of 5A allele (6.29+/-1.89 vs. 5.29+/-2.62, respectively, ANCOVA, p=0.01 Scheffe post-hoc test). In conclusion, our results indicate association of MMP-3 6A/6A genotype with significantly higher mean MSSS values. Thus, the obtained results suggest that it should be carefully considered during follow up of patients with MS. Further genetic and functional studies are needed to resolve the complex role of MMPs and their tissue inhibitors in MS pathology and/or regeneration.

Transcriptome-driven integrative exploration of functional state of ureter tissue affected by CAKUT.

AIMS: (1) to identify the most dysregulated genes in ureter tissue affected by congenital anomalies of the kidney and urinary tract (CAKUT) and to extract the biological meaning of these markers; (2) to describe the key molecular networks in CAKUT and to provide expression validation of the genes selected from these networks. MAIN METHODS: Transcriptome data was obtained from ureter samples of CAKUT patients and controls by Illumina iScan microarray. Identification of differentially expressed genes was coupled with subsequent bioinformatics analyses. Expression of candidate genes was validated by qRT-PCR. KEY FINDINGS: Analysis of the transcriptome led to the identification of 78 commonly dysregulated genes in CAKUT tissue compared to controls. Integrative bioinformatic analyses of differentially expressed genes identified 7 major networks. The targets for qRT-PCR validation were selected as members of the major molecular networks in CAKUT, which had both, the significant high fold change and biological relevance for CAKUT. By qRT-PCR the substantial increase of LCN2, PROM1, SOSTDC1, and decrease of INA, RASD1 and TAC3 mRNA levels was confirmed. SIGNIFICANCE: Since CAKUT is a leading cause of end-stage renal disease in children, the search for molecular targets for postnatal therapy is of particular interest. Data described in this study represents the gene expression profile and significant molecular networks specific to human ureter affected by CAKUT. The discovery of impaired molecular factors and processes is the step towards the uncovering of the key mechanisms that reflect CAKUT postnatally and could lead to the affected tissue deterioration and end organ damage.

The HACD4 haplotype as a risk factor for atherosclerosis in males.

The 9p21.3 region is rich in regulatory elements and the variants in this region had been robustly associated with carotid plaque (CP) and coronary artery disease (CAD). Recently, the HACD4 was detected as one of the six 9p21.3 differentially expressed genes associated with accelerated atherosclerosis and greater mean lesion area in the Athsq1 congenic mice. We aimed to investigate association of two potentially regulatory HACD4 variants (rs36212560 I/D, rs2275888 T/C) and their haplotypes with CP occurrence and the level of HACD4 and FOCAD mRNA in human CP tissue. Association study was replicated in CAD patients who suffered the first myocardial infarction. Study included 477 CP patients, 303 healthy controls and replication sample of 224 CAD males from the population of Serbia. Genotypes were determined by polymerase chain reaction (PCR) and real-time PCR using TaqMan® technology. The gene expression was detected with TaqMan® technology. We have found significant and independent association of DT haplotype with CP presence in men (adjusted OR=1.64 CI=1.12-2.42, p=0.011). The result was replicated in CAD males (adjusted OR=1.84 CI=1.21-2.80, p=0.004). We have found significant effect of the HACD4 rs2275888 on FOCAD mRNA level in human CP tissue. Correction for multiple testing was performed. Independent association of HACD4 haplotypes with atherosclerotic phenotypes connotes a further validation and replication in larger cohorts as well as functional studies to enlighten the potential mechanism of its action in pathophysiology of atherosclerosis.

Matrix metalloproteinase-9 -1562 C/T gene polymorphism in Serbian patients with multiple sclerosis.

Matrix metalloproteinase-9 (MMP-9) is suggested to play a role in MS by mediating T cell migration across subendothelial basement membrane and by contribution to myelin breakdown. We studied the association of MMP-9 -1562 C/T gene polymorphisms with MS susceptibility and severity in 187 patients from Serbia. The significant decrease in T allele carriership (p = 0.01), was found in female MS patients. In addition, a trend toward lower MSSS in T allele carriers was noticed (CC, mean 5.7 +/- 2.5 vs. CT+TT, mean 4.9 +/- 2.5). Further studies in different populations are needed to resolve the potential influence of MMP-9 gene polymorphism on MS.

X-linked angiotensin II type 2 receptor gene polymorphism -1332A/G in male patients with essential hypertension.

BACKGROUND: The role of AT2R in regulation of blood pressure (BP) was mainly investigated in animal models. It is proposed to be a negative regulator of BP. X-linked AT2R -1332 A/G polymorphism has been denoted as functional. This polymorphism was associated with certain cardiovascular phenotypes in hypertensive patients, but it was poorly investigated in essential hypertension. The aim of our study was to evaluate possible association of -1332 A/G gene polymorphism with essential hypertension in males from Serbian population. METHODS: The study group included 304 men of Caucasian origin, 190 normotensive (NT) and 114 hypertensive (HT), free of cardiovascular disorders. Genotyping was done by PCR RFLP method. RESULTS: G/- genotype was in association with HT (OR 1.6, CI=1.0-2.6, p=0.04). Stratification by age (<40 years, mean 31.65+/-5.29 and >40 years, mean 51.36+/-8.32) pronounced significance only in older males (OR 2.4, CI=1.2-5.0, p=0.02). After adjustment for confounding factors the OR for hypertension remained unchanged and significant (adjusted OR 2.3, CI=1.0-5.4, p=0.04). CONCLUSION: Hemizygosity for the G allele was found to be susceptibility factor for hypertension in males. Still, clarifying the role of AT2R in development of human hypertension requires further replication studies in larger and different populations.

Association of polymorphisms in CTLA-4, IL-1ra and IL-1beta genes with multiple sclerosis in Serbian population.

We have investigated separate as well as combined influence of IL-1beta TaqI, IL-1ra VNTR and CTLA-4 + 49 A/G polymorphisms on susceptibility, clinical course and progression of MS in 162 Serbian patients. We found significant independent relative risk for MS susceptibility in noncarriers of IL-1ra allele 2 (OR = 2.2, CI = 1.3-3.7, p = 0.003) and CTLA-4 + 49 AA genotype (OR = 2.0, CI = 1.2-3.5, p = 0.01) as well as their combined effect (OR = 4.4, CI = 2.0-9.7, p = 0.0003). Our result supports the significant and combined effect of IL-1ra VNTR and CTLA-4 polymorphisms on MS justifying the need for further haplotype analysis in different populations.