Tamara Djurić

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

OBJECTIVEnMatrix 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.nnnMETHODSnThe 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.nnnRESULTSnIn 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).nnnCONCLUSIONnOur 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.

The co-inertia approach in identification of specific microRNA in early and advanced atherosclerosis plaque

MicroRNAs (miRs) are short, non-coding RNAs that regulate gene expression by absolute or partial binding to mRNA, which results in transcript degradation and translation blocking. Atherosclerosis, as a complex and progressive disease, represents one of the main causes of cardiovascular clinical complications and even death. We applied co-inertia analysis (CIA) as a novel computation method, to determine which miRs are potentially associated with differences in gene expression levels originating from microarray data of early and advanced atherosclerotic plaque. As the CIA has not been applied in the field of atherosclerosis yet, we hypothesized that using CIA we can get novel information about the miRs that have significant role in early phase of disease or in severe phase of disease. The characteristic split in the data along the axes of performed CIA showed the difference in the gene expression pattern between early atherosclerosis and advanced atherosclerotic plaque. The advanced atherosclerotic plaques showed more homogenous gene expression pattern than early atherosclerosis samples. In early carotid lesions five out of five algorithms predicted miR-24, four out of five predicted miR-155, miR-145, and miR-100 as early active miRs. These miRs could be protective in plaque evolution context because they were not active in advanced plaques according to our results. They were reported previously as atheroprotective, which in a way represents confirmation of CIA application in atherosclerosis. We detected 13 new miRs which could be active in early plaque phenotype according to CIA prediction. In the advanced plaques we predicted miR-221, miR-222, miR-127 and miR-146 which were previously revealed to have atherogenic properties. In addition to miRs that have literature support, we also found new 8 miRs that, with described function so far, could present a novelty in research of atherosclerotic plaque evolution. All of these examples show that CIA results have a great potential to be of interest in future research in atherosclerotic plaque progression. We validated the applicability of CIA in the field of atherosclerosis, but we also found new interesting miR competitors that have strong potential to serve as markers and plaque development factors. These results should be experimentally confirmed in further research with ultimate goal to discover new mediators and blood markers, which could improve the prevention and therapy of this complex disease.

Matrix metalloproteinase-1 promoter genotypes and haplotypes are associated with carotid plaque presence.

OBJECTIVESnMatrix metalloproteinase (MMP)-1 degrades fibrillar collagens suggesting important role in vascular remodeling. Data about MMP-1 promoter polymorphisms and carotid atherosclerosis (CA) are scarce. The aim of this study was to evaluate association of MMP-1 genotypes/haplotypes with carotid plaque (CP) presence in Serbian population.nnnDESIGN AND METHODSnStudy enrolled a total of 702 participants: 274 controls and 428 consecutive patients with CA who underwent carotid endarterectomy. MMP-1 polymorphisms -1607 1G/2G, -519 A/G and -340 T/C were genotyped by PCR and RFLP methods.nnnRESULTSnIndividuals carrying MMP-1 -1607 2G allele had significantly increased allele dose-dependent risk for CP presence (1G1G vs. 1G2G vs. 2G2G; OR=1; OR=1.87 95% CI 1.29-2.07; OR=3.49 95% CI 1.67-7.30, p=0.0009, respectively). Compared to the referent haplotype 2G(-1607)-T(-340)-A(-519), the haplotypes 1G(-1607)-T(-340)-A(-519), 1G(-1607)-T(-340)-G(-519) and 2G(-1607)-C(-340)-A(-519) had statistically significant protective effect on CP presence (OR=0.41, 95% CI 0.29-0.81, p=0.01; OR=0.56, 95% CI 0.44-0.89, p=0.01; OR=0.43, 95% CI 0.27-0.86, p=0.02, respectively).nnnCONCLUSIONSnMMP-1 -1607 G/2G polymorphism solely and specific haplotypes of three analyzed promoter polymorphisms are significantly and independently associated with occurrence of CP. Replication studies in other populations are needed.

The association of V249I and T280M fractalkine receptor haplotypes with disease course of multiple sclerosis

We investigated the association of CX3CR1 genotypes/haplotypes with MS and performed the prediction analysis of protein sequence variants effects on CX3CL1/CX3CR1 interaction. We found no association of CX3CR1 with MS susceptibility. Frequency of I(249)T(280) haplotype was significantly lower in SP compared to RR patients (RR>10 years, OR=0.30, 95%CI=0.11-0.79, p=0.01; OR=0.53, 95%CI=0.18-1.56, p=0.2, in SP<10 years vs. RR>10 years). Prediction analysis showed that I249 T280 protein variant would significantly affect CX3CL1/CX3CR1 interaction. Our results suggest that CX3CR1 I₂₄₉T₂₈₀ haplotype could have protective effect for switch to SP MS. Further research is warranted to validate and replicate currently observed results.

CXCL16 haplotypes in patients with human carotid atherosclerosis: preliminary results.

AIMnChemokine CXC ligand 16 (CXCL16) has chemoattractive, adhesive and scavenging properties and may play a role in the formation of atherosclerotic lesions. However, studies of CXCL16 polymorphisms in patients with atherosclerosis are scarce. The missense polymorphisms I123T and A181V are potentially important factors in the regulation of presentation and shedding of the CXCL16 chemokine domain. The aim of this study was to analyze the association between I123T and A181V polymorphism haplotypes and the accumulation of carotid plaque as well as the effect of the haplotype on the CXCL16 mRNA expression in carotid plaques in patients with advanced atherosclerosis. Additionally, we performed a bioinformatic prediction analysis of the impact of CXCL16 protein sequence variation on CXCL16-CXCR6 interactions and analyzed the soluble CXCL16 plasma levels according to the CXCL16 haplotype.nnnMETHODSnThis study evaluated a total of 733 participants, including 283 controls and 450 patients with carotid atherosclerosis (CA) undergoing endarterectomy. Analyses of the polymorphisms and the gene expression were performed using real-time PCR. The soluble CXCL16 levels were measured with ELISA.nnnRESULTSnThe missense allele haplotype, T123V181, was found to be significantly and independently associated with the occurrence of CA plaque (OR=1.27;1.02-1.57, p=0.03). This haplotype was predicted to significantly change the CXCL16-CXCR6 interaction, compared to I123A181. Neither the CXCL16 mRNA expression in the human plaques nor the soluble CXCL16 plasma levels differed according to the haplotype.nnnCONCLUSIONSnThese results indicate that the CXCL16 T123V181 haplotype is a moderate genetic risk factor for the development of carotid plaque. Further functional and replication studies are needed to clarify the mechanisms by which this combination of alleles promotes advanced CA and validate its impact on disease progression.

The gender-specific association of CXCL16 A181V gene polymorphism with susceptibility to multiple sclerosis, and its effects on PBMC mRNA and plasma soluble CXCL16 levels: preliminary findings

CXC ligand 16 (CXCL16) is a multifunctional chemokine involved in cell adhesion and chemoattraction as well as in the scavenging of oxidized lipoproteins. Experimental data suggest the roles of CXCL16 in pathogenesis of multiple sclerosis (MS). A181V polymorphism in the human CXCL16 gene has been associated with the clinical course of certain chronic inflammatory diseases. The aim of this study was to analyze the effects of CXCL16 A181V polymorphism on: (1) susceptibility to MS and disease course, (2) peripheral blood mononuclear cells (PBMC) CXCL16 mRNA levels and plasma soluble CXCL16 levels of patients with MS and healthy controls. In this study, 459 MS patients and 303 controls were included. Real-time PCR-based methods were applied for genotyping of CXCL16 A181V and for CXCL16 gene expression analysis. Quantitative sandwich enzyme immunoassay was performed for quantification of plasma soluble CXCL16. CXCL16 AA genotype had a significant protective effect on MS susceptibility in women (ORxa0=xa00.53, ±95xa0% CIxa0=xa00.35–0.82, pxa0=xa00.004). The V allele-containing genotypes were associated with significantly higher CXCL16 mRNA levels in PBMC of both female (mean factorxa0=xa01.81, S.E.xa0=xa01.14–2.77, pxa0<xa00.01) and male (mean factorxa0=xa01.58, S.E.xa0=xa01.35–1.73, pxa0<xa00.01) controls. No significant association of the CXCL16 polymorphism was established either with soluble CXCL16 plasma levels or with clinical parameters and course of MS. The main finding of this study is gender-specific association of CXCL16 A181V polymorphism with susceptibility to MS in females. The current results should be replicated and validated in the larger sample group.

Fructose-rich diet induces gender-specific changes in expression of the renin-angiotensin system in rat heart and upregulates the ACE/AT1R axis in the male rat aorta.

Introduction: The cardiovascular renin–angiotensin system (RAS) could be affected by gender and dietary regime. We hypothesized that male rats will be more susceptible to activation of RAS in the heart and aorta, as a response to a fructose-rich diet (FRD). Materials and methods: Both male and female Wistar rats were given a 10% (w/v) fructose solution for 9 weeks. We measured the biochemical parameters, blood pressure (BP) and heart rate. We used Western blot and real-time polymerase chain reaction (PCR) to quantify protein and gene expression. Results: In the male rats, the FRD elevated BP and expression of cardiac angiotensin-converting enzyme (ACE), while the expression of angiotensin-converting enzyme 2 (ACE2) and angiotensin II Type 2 receptor (AT2R) were significantly decreased. In female rats, there were no changes in cardiac RAS expression due to FRD. Furthermore, the ACE/AT1R axis was overexpressed in the FRD male rats’ aortae, while only AT1R was upregulated in the FRD female rats’ aortae. ACE2 expression remained unchanged in the aortae of both genders receiving the FRD. Conclusions: The FRD induced gender-specific changes in the expression of the RAS in the heart and aortae of male rats. Further investigations are required in order to get a comprehensive understanding of the underlying mechanisms of gender-specific fructose-induced cardiovascular pathologies.

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 AIMSnThe 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 AT1Rs 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.nnnMETHODSnOur 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.nnnRESULTSnGenotypes containing the C allele bore a significant association with the hypoechoic plaque phenotype (adjusted OR 1.87, 95% CI 1.16-3.00, pxa0=xa00.01). 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 (pxa0=xa00.032, pxa0=xa00.015, respectively). AT1R protein expression was also significantly higher for CC genotypes (pxa0<xa00.01).nnnCONCLUSIONnOur 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.

CXCL16 in Vascular Pathology Research: from Macro Effects to microRNAs

Chemokines and their receptors have become significant factors in atherosclerosis research. CXCL16 is a multifunctional agent located on a separate locus to all other known chemokines and binds only to its unique receptor named CXCR6. As a scavenger receptor, adhesion molecule, and chemokine, it quickly became an interesting target in atherosclerosis research as all its functions have a role in vascular pathology. The investigation of the role of CXCL16 in atherosclerosis, although shown in in vitro studies, animal knockout models, and CXCL16 gene polymorphisms, haplotypes, and circulating levels, still shows puzzling results. Genetic and epigenetic studies have just scratched the surface of research necessary for a better assessment of the significance and perspective of this marker in plaque development and progression. In this review, we will summarize current knowledge about CXCL16 in atherosclerosis. Additionally, we will point out the importance of bioinformatics tools for the detection of potentially new CXCL16 regulatory networks through microRNA activity. This review aims to provide a better understanding of the underlying mechanisms, define more specific biomarkers, and discover new therapeutic targets.

MMP-1 and -3 haplotype is associated with congenital anomalies of the kidney and urinary tract

BackgroundCongenital anomalies of the kidney and urinary tract (CAKUT) are a common cause of progressive chronic kidney disease that may lead to end-stage renal disease and renal replacement therapy in childhood. Altered expression or activity of matrix metalloproteinases (MMPs) have been found in CAKUT. The MMP-1, -3, and -8 polymorphisms studied here are located in the gene promoters and alter expression. Our aim was to investigate associations of MMP polymorphisms, solely and in haplotypes, with CAKUT in children.MethodsA case–control study with 101 pediatric patients and 281 controls was performed. The MMP-1 (-1607 1G/2G), -3 (5A/6A), and -8 (-799 C/T) genotypes were determined by PCR–restriction fragment length polymorphism.ResultsWe found statistically significant associations of MMP-3 5A/6A polymorphism (pu2009<u20090.0001) and 1G−1607-6A haplotype, with no preferences for MMP-8 -799C or T alleles, with CAKUT (ORu2009=u20092.93, 95xa0%xa0CIxa01.43–5.98, adjusted for gender, pu2009=u20090.003) and with obstructive uropathies in a subgroup of patients (ORu2009=u20094.57, 95xa0%xa0CI 2.74–7.61, adjusted for gender, pu2009<u20090.0001).ConclusionsMMP-3 genotypes and MMP-3 and -1 haplotypes encompassing either MMP-8 -799C or T alleles were associated with CAKUT and obstructive uropathies in pediatric patients. Still, functional and association studies are needed to elucidate evident roles of MMPs in CAKUT.