Maja Živković

Angiotensin II type 1 receptor gene polymorphism and essential hypertension in Serbian population

Essential hypertension is considered to be a multifactorial trait resulting from the combined influence of environmental and genetic determinants. Due to the controversial results about the role of the ATR1 gene locus in hypertension and understanding that ethnic origin should be carefully considered in studying the association between gene polymorphism and disease etiology, we investigated the role of A1166C polymorphism in Serbian hypertensives. A total of 298 subjects, 100 hypertensive and 198 normotensive, age- and sex-matched controls, were included in this study. All subjects were genotyped for the A1166C polymorphism in ATR1 gene using allele-specific PCR-based technique. There were significant differences in both allele and genotype frequencies between hypertensive and normotensive male subjects (p<0.05). There is significant association between hypertension and CC genotype (CC vs. AC+AA OR=2.56, p=0.04) in the males only. These results suggest that a genetic variant of the ATR1 gene locus influences the risk of essential hypertension in the sex-specific manner in the Serbian population.

Association of MMP-3 5A/6A gene polymorphism with susceptibility to carotid atherosclerosis

OBJECTIVES Stromelysin-1 (MMP-3) as a key member of metalloproteinase family could have an important role in atherogenesis. The 5A/6A polymorphism in the promoter of MMP-3 gene affects the level of MMP-3 gene expression. We assessed whether the MMP-3 promoter low- and high-activity genotypes are related to susceptibility for carotid atherosclerosis (CA) in Serbian population. DESIGN AND METHODS The study group of case-control design consisted of 515 participants. The 265 patients with ultrasonographic evidence of carotid plaque presence were recruited for the study. The 5A/6A polymorphism was typed by RFLP-PCR. RESULTS There was significantly higher prevalence of genotypes containing 6A allele in the patients with CA compared to controls (p<0.05). The model of inheritance with the dominant effect of 6A allele gave elevated and significant OR for carotid atherosclerosis (adjusted OR 2.35, CI=1.0-5.5, p=0.048). CONCLUSIONS Subjects carrying genotypes with 6A allele had significantly higher susceptibility to carotid atherosclerosis.

Association of polymorphisms in CTLA-4, IL-1ra and IL-1β 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.

Association of MMP-8 promoter gene polymorphisms with carotid atherosclerosis: Preliminary study

OBJECTIVE Matrix metalloproteinases (MMPs) are involved in the remodeling of the extracellular matrix in the arterial wall. Collagen I is associated with vascular smooth muscle cell (VSMC) migration and monocyte differentiation. MMP-8 is expressed in atherosclerotic plaque and preferentially cleaves collagen type I. The aim of this study was to investigate the associations of two MMP-8 promoter polymorphisms, rs11225395 (-799C/T) and rs1320632 (-381 A/G), with carotid plaque occurrence, and the influence of these polymorphisms on MMP-8 mRNA expression in plaque tissue. METHODS The study included a total of 766 participants: 277 controls and 489 patients with carotid atherosclerosis undergoing endarterectomy. The two investigated polymorphisms were genotyped by PCR-RFLP. The gene expression analysis was performed by real-time PCR. RESULTS In females only, a significantly higher frequency of the -381G allele was found in patients with carotid atherosclerosis compared to controls (OR, 1.7; 95% CI 1.1-2.9; p = 0.001). Significant up-regulation of MMP-8 gene expression was observed in patients carrying the -381G allele compared to those with the AA genotype (mean factor, 3.54; S.E. range, 0.643-19.551; p = 0.007). Carotid plaque tissue of the haplotype G(-381)T(-799) showed a significantly higher mRNA level compared with the reference A(-381)C(-799) haplotype (p = 0.003). CONCLUSION Our preliminary results indicate that MMP-8 -381A/G and -799C/T gene polymorphisms could be risk factors for carotid atherosclerosis. Further validation and functional studies are needed to establish the potential regulatory role of these polymorphisms and their impact on susceptibility to carotid atherosclerosis.

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. J. Clin. Lab. Anal. 24:246–251, 2010.

Human cytomegalovirus increases HUVEC sensitivity to thrombin and modulates expression of thrombin receptors

Human cytomegalovirus (HCMV) establishes a life-long persistent infection. HCMV infection could be associated with chronic inflammatory diseases, such as cardiovascular disease and atherosclerosis. Here we observed that in HCMV (AD-169) pre-exposed human umbilical vein endothelial cells (HUVEC), thrombin-induced expression of IL-1α and M-CSF is markedly enhanced compared to the un-exposed cells. Study of the expression of thrombin receptor genes in HUVEC showed that HCMV triggered a time- and concentration-dependent expression of the thrombin receptors PAR1, PAR3 and PAR4 at the mRNA level. Induction of PAR1 and PAR3 mRNA expression is due to transcriptional activation of their promoters as shown by gene reporter assay. Furthermore, the virus induced expression of PAR1 and PAR3 but not PAR4 proteins, as analyzed by Western immunoblotting. However, flow cytometric analysis revealed that only PAR3, expressed at very low level in control HUVEC, is induced at the surface during the exposure to the virus. Our data suggest that although exposure to HCMV induces a minor increase of cell-surface receptors expression, it does make endothelial cells more responsive to additional thrombin stimulation.

Expression profiling of the AT2R mRNA in affected tissue from children with CAKUT

OBJECTIVES Congenital anomalies of the kidney and urinary tract (CAKUT) are common causes of chronic renal failure in children. The angiotensin II receptor type 2 (AT2R) is one of proposed candidate genes for CAKUT, but the expression was never explored in humans. The aim was to establish the AT2R gene expression in human CAKUT concerning -1332A/G polymorphism, which might affect alternative splicing. DESIGN AND METHODS Forty-eight patients with CAKUT constitute the basis of this study. Genotyping for -1332A/G, RT-PCR for AT2R gene expression and confirmation sequencing were performed. RESULTS The expression of Ex 1/2/3 and Ex 1/3 transcript splice variants of the AT2R mRNA were detected in human CAKUT tissue. The pattern was observed independently of A to G transition. CONCLUSIONS The expression of AT2R mRNA in human CAKUT was established for the first time and was not affected by -1332A/G polymorphism in children with CAKUT.

Estradiol enhances effects of fructose rich diet on cardiac fatty acid transporter CD36 and triglycerides accumulation

Fructose rich diet increases hepatic triglycerides production and has deleterious cardiac effects. Estrogens are involved in regulation of lipid metabolism as well, but their effects are cardio beneficial. In order to study effects of fructose rich diet on the main heart fatty acid transporter CD36 and the role of estrogens, we subjected ovariectomized female rats to the standard diet or fructose rich diet, with or without estradiol (E2) replacement. The following parameters were analyzed: feeding behavior, visceral adipose tissue mass, plasma lipids, cardiac CD36 expression, localization and insulin regulation, as well as the profile of cardiac lipids. Results show that fructose rich diet significantly increased plasma triglycerides and decreased plasma free fatty acid (FFA) concentration, while E2 additionally emphasized FFA decrease. The fructose diet increased cardiac plasma membrane content of CD36 in the basal and insulin-stimulated states, and decreased its low density microsomes content. The E2 in fructose-fed rats raised the total cardiac protein content of CD36, its presence in plasma membranes and low density microsomes, and cardiac deposition of triglycerides, as well. Although E2 counteracts fructose in some aspects of lipid metabolism, and separately they have opposite cardiac effects, in combination with fructose rich diet, E2 additionally enhances CD36 presence in plasma membranes of cardiac cells and triglycerides accumulation, which paradoxically might promote deleterious effects of fructose diet on cardiac lipid metabolism. Taken together, the results presented in this work are of high importance for clinical administration of estrogens in females with a history of type 2 diabetes.

The glutathione S-transferase T1 deletion is associated with susceptibility to multiple sclerosis

BACKGROUND Multiple sclerosis (MS) occurs as a result of interaction between genetic and environmental factors. Recent data support the view that oxidative damage is one of an early event in MS tissue injury. The safe elimination of reactive oxygen species and toxins via glutathione S-transferase (GST) pathways is required in order to protect cells against reactive oxygen-induced damage. The aim of our study was to analyze the possible association of GSTM1 and GSTT1 gene polymorphisms with the susceptibility and clinical parameters of MS, in 455 consecutive patients and 366 controls. METHODS A multiplex polymerase chain reaction (PCR) was used to detect the deletions in GSTM1 and GSTT1 genes. RESULTS Patients with MS had significantly higher frequency of GSTT1 null genotype compared to controls (37.36% vs. 21.86%, respectively, p<0.0001, adjusted OR 2.13 (1.56-2.90)), as well as double deletions (15.38% vs. 10.38%, respectively, p<0.05). The carriers of GSTM1 deletion had significantly earlier onset of MS compared to the wild-type carriers (28.31 ± 8.45 vs. 30.64 ± 9.30 years, respectively, p = 0.03). CONCLUSION This study suggests the potential pathogenic role of GSTT1 deletion on MS susceptibility. There are no similar data published so far, yet this study should be replicated in other populations.

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.