PPAR𝛾 Gene and Atherosclerosis: Genetic Polymorphisms, Epigenetics and Therapeutic Implications.

Atherosclerosis is the leading cause of mortality and morbidity in the developed world. It is characterized by the formation of a plaque in the walls of middle and large arteries leading to macrovascular complications. Several risk factors are included, with diabetes being one of the most important for the onset and development of atherosclerosis. Due to an increase in the prevalence of diabetes in the world, the incidence of diabetic complications (microvascular and macrovascular) is increasing. Peroxisome proliferator-activated receptor γ (PPARγ) plays a important role in atherosclerotic processes. Peroxisome proliferator activated receptor γ belongs to the superfamily of nuclear receptors, has a great presence in fat tissue, macrophages, and regulates gene expression and most of the processes that lead to the onset and development of atherosclerosis. In this review, we discuss the basic patho-physiological mechanisms of atherosclerosis in type 2 diabetes mellitus (T2DM). Furthermore, we discuss the impact of PPARγ polymorphisms, and the epigenetic mechanisms affecting the onset of atherosclerosis, i.e, DNA methylation and demethylation, histone acetylation and deacetylation, and RNA-based mechanisms. Moreover, we add therapeutic possibilities for acting on epigenetic mechanisms in order to prevent the onset and progression of atherosclerosis.

ADIPONECTIN AS A POTENTIAL BIOMARKER OF LOW BONE MINERAL DENSITY IN POSTMENOPAUSAL WOMEN WITH METABOLIC SYNDROME.

CONTEXT: Adiponectin is an abundant adipokine, which has antiinflammatory, anti-atherosclerotic and vasoprotective actions, and potential antiresorptive effects on bone metabolism. It seems to be directly involved in the improvement and control of energy homeostasis, protecting bone health and predicting osteoporotic fracture risk. OBJECTIVE: To examine the relationship between adiponectin level and bone mineral density (BMD) in post-menopausal women with metabolic syndrome (MetS) and low BMD, and to estimate the prognostic significance of adiponectin in osteoporosis. DESIGN: Clinical-laboratory cross-sectional study including 120 middle-aged and elder women (average 69.18±7.56 years). SUBJECTS AND METHODS: The anthropometric parameters were measured for all examinees. Lumbar spine and hip BMD, as well as body fat percentage, were measured using a Hologic DEXA scanner. In all subjects serum adiponectin concentration was measured by ELISA method. RESULTS: The level of adiponectin was significantly positively correlated with BMD-total, BMD of the lumbar spine and BMD of the femoral neck (r=0.618, r=0.521, r=0.567; p<0.01). Levels of adiponectin and BMD are significantly lower in post-menopausal women with MetS and osteoporosis compared to patients with osteopenia (856.87±453.43 vs. 1287.32±405.21 pg/mL, p<0.01; BMD, p<0.05), and the highest values in healthy examinees. A cut-off value of adiponectin level for osteoporosis/osteopenia was 1076.22/1392.74 pg/mL. CONCLUSIONS: Post-menopausal women with MetS have significantly lower adiponectin level and low BMD compared to healthy examinees. Adiponectin may be an early, significant and independent predictor of developing osteoporosis in women with MetS, especially in post-menopausal period.

The impact of thyroid hormone replacement therapy on left ventricular diastolic function in patients with subclinical hypothyroidism.

OBJECTIVE: Subclinical hypothyroidism (SH) is associated with a moderately elevated risk of heart failure events among older adults. The objective of our prospective study was to assess the impact of thyroid hormone replacement therapy (HRT) with low doses of L-thyroxine (6.25-25 µg/day) on left ventricular diastolic function in patients with SH. MATERIALS AND METHODS: 33 patients with SH and 25 healthy controls were involved. All participants underwent standard echocardiography and Doppler imaging at baseline and, the patient group, also after a course of HRT. RESULTS: At baseline, patients with SH showed significantly lower E (0.79 ± 0.22 vs. 0.93 ± 0.19, p < 0.001), E/A ratio (1.19 ± 0.29 vs. 1.31 ± 0.25, p < 0.003), and higher intraventricular septum thickness (IVST) (0.99 ± 0.14 vs. 0.89 ± 0.18, p < 0.001) in comparison with healthy controls. After 6 months of therapy, the E/A ratio underwent significant increase (1.28 ± 0.21 vs. 1.19 ± 0.29, p < 0.001), while the IVS displayed a robust reduction (0.92 ± 0.16 vs. 0.99 ± 0.14, p < 0.001). CONCLUSIONS: HRT with low-dosed L-thyroxine may improve left ventricular diastolic function in patients with SH.

Mast Cells Might Have a Protective Role against the Development of Calcification and Hyalinisation in Severe Aortic Valve Stenosis.

Aortic valve stenosis is characterized by inflammation and extracellular matrix remodelling. The aim of this study was to analyse the impact of mast cells on the occurrence of histopathological changes of aortic valves in patients with severe grade, non-rheumatic degenerative aortic valve stenosis. Valve specimens were obtained from 38 patients undergoing valve replacement. The role of mast cells was analysed by dividing the specimens into two groups, characterized by the presence (group A, N = 13) or absence of mast cells (group B, N = 25). There were no significant differences in clinical data between the two groups. In group A, T cells and macrophages were present in all aortic valves, as compared to a significantly lower proportion of valves with T cells and macrophages in group B. Valves in group A were less often calcified and hyaline-degenerated than valves in group B. There were no changes in fibrosis between the two groups. We found a positive correlation between the presence of mast cells and macrophages/T cells, a negative correlation between the presence of mast cells and calcification/ hyaline degeneration, and no correlation between the presence of mast cells and fibrosis. There was also a negative correlation between the presence of macrophages/T cells and calcification. The linear regression model identified only the presence of mast cells as an independent negative prediction value for calcification. In conclusion, mast cells might have a protective role against the development of calcification and hyaline degeneration in severe grade, non-rheumatic aortic valve stenosis.

Polymorphism of angiotensin-converting enzyme (rs4340) and diabetic nephropathy in Caucasians with type 2 diabetes mellitus.

Diabetic nephropathy (DN) is the leading cause of endstage renal disease (ESRD) in developed countries. Several environmental and genetic factors predict the development and progression of DN. The renin-angiotensin system was demonstrated to be involved in the development of DN. We evaluated the association between rs4340 of the angiotensin-converting enzyme (ACE) gene and DN in Caucasians with type 2 diabetes mellitus (T2DM) in 276 Slovenian patients with T2DM who had DN, and 375 patients without clinical signs of DN. Genetic analysis was performed with either standard polymerase chain reaction (PCR) (for rs4340). Results were analyzed using the χ2 test and multivariate logistic regression analyses. We found no association between rs4340 and DN. Cystatin C was significantly higher in the DN+ group (p <0.001) than in the DN group. Cystatin C was a better marker for the estimation of renal function than estimated glomerular filtration rate (eGFR) according to the modification diet in renal disease (MDRD) equation mL/ min. We concluded that there was no association between the rs4340 of the ACE gene and DN in Caucasian patients who have T2DM.

PECAM-1 gene polymorphism (rs668) and subclinical markers of carotid atherosclerosis in patients with type 2 diabetes mellitus.

The platelet endothelial cell adhesion molecule 1 (PECAM-1) plays an important role in many inflammatory processes, including the development of atherosclerosis. Polymorphism rs668 of the PECAM-1 gene (373C/G) is functional, and it was reported to be associated with increased serum levels of PECAM-1. We investigated the association between the rs668 polymorphism of PECAM-1 and subclinical markers of carotid atherosclerosis in subjects with type 2 diabetes mellitus (T2DM). Five hundred and ninety-five T2DM subjects and 200 control subjects were enrolled. The carotid intima-media thickness (CIMT) and plaque characteristics (presence and structure) were assessed ultrasonographically. Biochemical analyses were performed using standard biochemical methods. Geno-typing of the PECAM-1 gene polymorphism (rs668) was performed using KASPar assays. The control examinations were performed 3.8 ± 0.5 years after the initial examination. Higher CIMT was found in patients with T2DM in comparison with subjects without T2DM. Statistically sig-nificantly faster progression of the atherosclerotic markers was shown in subjects with T2DM in comparison with the control group. When adjusted to other risk factors, the rs668 GG genotype was associated with an increased risk of carotid plaques in subjects with T2DM. We concluded that our study demonstrated a minor effect of the rs668 PECAM-1 on markers of carotid atherosclerosis in subjects with T2DM.

Association of the ACE rs4646994 and rs4341 polymorphisms with the progression of carotid atherosclerosis in slovenian patients with type 2 diabetes mellitus.

The current study was designed to reveal possible associations between the angiotensin-converting-enzyme (ACE) gene polymorphisms (rs4646994 and rs4341) with markers of carotid atherosclerosis in patients with type 2 diabetes mellitus (T2DM) in a 4-year-long follow-up study. Five hundred and ninety-five T2DM subjects and 200 control subjects were enrolled. Genotyping of ACE polymorphisms was performed using KASPar assays, and ultrasound examinations were performed twice (at the enrollment and at follow-up). With regard to the progression of atherosclerosis in subjects with T2DM, statistically significant differences were demonstrated in the change of the sum of carotid plaques thickness for the rs4646994 polymorphism. We did not demonstrate an association between the tested polymorphisms (rs4646994 and rs4341) and either carotid intima media thickness (CIMT) or CIMT progression in a 3.8-year period. In our study, we demonstrated that subjects with T2DM with the DD genotype of the rs4646994 [ACE insertion/deletion (I/D)] polymorphism had faster progression of atherosclerosis in comparison to subjects with other genotypes.

The semi-sewer river: hydraulic backwater effects and combined sewer overflow reverse flows in Central Brussels reduce deoxygenation impact further downstream.

In 2011 and 2012 the dissolved oxygen content in the low-discharge river Zenne was monitored continuously, every 5 minutes, downstream of Brussels city centre, making it possible to document the complex mechanisms by which combined sewer overflow (CSO) spills affect both the hydraulics and the oxygen balance of the hydrosystem. In addition to oxygen demand impacts, proportions of water volumes are such that the oxygen-devoid sewage water discharged from CSOs contributes significantly to the oxygen deficit observed in the river further downstream. It is shown that ensuing unexpected hydraulic behaviour, such as a full river-flow reversal, can explain the dual nature of oxygen sag following major CSO events. At times the semi-sewer river plays the role of an in-stream stormwater tank, effectively attenuating the environmental impacts of Brussels CSOs.

[Prosthetic rehabilitation in patient with forced class III malocclusion].

The paper presents clinical case of 63 years old edentulous patient with slight class III malocclusion. For 15 years he was using inadequately fabricated dentures causing forced severe class III malocclusion. Forced progeny was corrected by newly fabricated dentures which restored normal orofacial function and facial harmony.

Serum cystatin C levels in normal pregnancy.

AIM: The aim of this study was to determine the levels of cystatin C, creatinine and creatinine clearance in different trimesters of uncomplicated pregnancy in women with normal kidney function. SUBJECTS AND METHODS: A total of 109 pregnant women were included: group 1 - 38 women (average age 29.63 ± 4.3 y) in the first trimester, Group 2 - 32 women (average age 33.56 ± 5.95 y) in the second trimester and Group 3 - 39 pregnant women (average age 30.1 ± 6.95 y) in the third trimester. Serum cystatin C was determined by the PENIA method (Particle-Enhanced Nephelometric Immuno-Assay), using Behring tests (Behring Diagnostics GmbH, Marburg, Germany). Results were statistically analyzed using the ANOVA. RESULTS: A statistically significant increase in serum cystatin C level was found in the third trimester of pregnancy (0.69 ± 0.16 mg/l vs. 0.78 ± 0.26 mg/l vs. 1.21 ± 0.30 mg/l). CONCLUSION: It appears that cystatin C is not a reliable marker of kidney function in pregnancy and that its increase is connected with a combination of several factors, including endotheliasis, hormonal influence and glomerular filtration rate (GFR) alterations.

Interleukin-18 Promoter Gene Polymorphisms are not Associated with Myocardial Infarction in Type 2 Diabetes in Slovenia.

Type 2 diabetes is a major risk factor for myocardial infarction (MI) and chronic inflammation may play a central role in both diseases. Interleukin (IL)-18 is a potent proinflammatory cytokine, which is considered important in acute coronary syndromes and type 2 diabetes. We investigated the association of the -137 (G>C), polymorphism (rs187238) and the -607 (C>A) polymorphism (rs1946518) of the IL-18 gene promoter region in 495 Caucasians with type 2 diabetes, of whom 169 had MI and 326 subjects had no clinically evident coronary artery disease (controls). We also investigated the impact of these polymorphisms on the serum IL-18 level in subsets of both groups and in a normal group. Genotype distributions of the polymorphisms showed no significant difference between cases and controls. However, IL-18 serum levels were significantly lower in diabetics with the 137 CC genotype than in those with other genotypes (241.5 ± 132.7 ng/L vs. 340.2 ± 167.4 ng/L; p <0.05). High sensitivity C-reactive protein and IL-18 serum levels were higher in diabetics in the MI group than in the control group. We conclude that these IL-18 promoter gene polymorphisms are not risk factors for MI in Caucasians with type 2 diabetes.

Cardiotoxic injury caused by chronic administration of microcystin-YR.

Microcystins are cyclic peptide toxins. Chronic intoxication with well-known members of the microcystin family--microcystins-LR--induces liver tumour formation, injury of kidney and heart. Despite worldwide distribution in the environment, the effects of microcystins-YR have not been studied extensively. The aim of the study was to evaluate whether microcystins-YR, in relatively low doses, have a toxic effect on cardiomyocytes of chronically treated rats. Male adult Wistar rats were treated every second day for 8 months with microcystins-YR (10 microg/kg i.p., N = 5). Control groups were treated either with vehicle (ethanol and methanol 4 : 1 v/v; N = 5) or with physiologic saline (N = 4). The heart sections of microcystin-YR-treated rats revealed decreased volume density of cardiac muscle tissue (microcystins- YR = 0.485 mm3/mm3 +/- 0.003; vehicle = 0.493 mm3/mm3 +/- 0.002; saline = 0.492 mm3/mm3 +/- 0.002) due to fibrous proliferation. A few lymphocyte infiltrates were observed. Most of cardiomyocytes were enlarged (microcystins-YR = 20.19 microm +/- 1.34, vehicle = 17.45 microm +/- 0.52, saline = 16.00 microm +/- 1.43), with enlarged and often bizarre-shaped nuclei and decreased myofibril volume fraction (microcystins- YR = 0.416 mm3/mm3 +/- 0.009; vehicle = 0.472 mm3/ mm3 +/- 0.009; saline = 0.479 mm3/mm3 +/- 0.010). No TUNEL-positive cells were found in the heart sections of rats in all groups. The results allow the conclusion that chronic exposure to low doses of microcystins-YR may cause atrophy and fibrosis of the heart muscle.

Oral status in patients receiving 5-fluorouracil for colorectal cancer.

PURPOSE: Oral complications are frequent and troublesome symptoms for those undergoing chemotherapy for cancer. Several antineoplastic agents are proved to have stomatotoxic potential, among them 5-fluorouracil (5-FU). The aim of the present study was to evaluate the oral status and patient experiences during chemotherapy with 5-FU for colorectal cancer. METHODS: Twenty-eight patients treated with 5-day 5-FU plus leucovorin entered this study. Positive data about oral symptoms were taken by anamnesis. Mucositis severity index, gingival index, plaque index, probing pocket depth and bleeding on probing have been used to assess oral mucosa and periodontal status of the patients. Patients were examined prior to chemotherapy and 14 days after the start of the chemotherapy cycle. RESULTS: Mild to moderate subjective complaints concerning oral cavity were reported by 17.9% of patients before and 39.2% of patients after chemotherapy. Clinical examination revealed oral mucosa damage in 10.7% and 35.7% of patients, with mean mucositis score of 0.14 and 0.54 before and after chemotherapy, respectively. Although mean values of all periodontal indices were elevated after chemotherapy, only increase in gingival index was statistically significant (p=0.035). Mucositis was significantly correlated with oral pain (p=0.00), xerostomia (p=0.00), and plaque index (p=0.077), while the correlation between mucositis and the rest of the examined parameters was not significant. CONCLUSION: Oral complications were not highly expressed in this study. Although 5-FU is considered to exert significant stomatotoxic effect, severe mucositis was far less common in this study compared to studies reported elsewhere.

Adiponectin resistance parameter as a marker for high normal blood pres-sure and hypertension in patients with metabolic syndrome.

INTRODUCTION: The different degrees of adiponectin/insulin sensitivity and dysfunctional adipose tissue lead to the development of hypertension (HT). This study aimed to determine adiponectin (AD) concentration in patients with metabolic syndrome (MetS) and high-normal blood pressure or hypertension and to investigate the importance of Homeostatic Model Assessment-AD (HOMA-AD) index in assessing adiponectin/insulin resistance in hypertension. METHODS: This cross-sectional study involved 150 subjects divided into two groups: with MetS (and high-normal blood pressure, n =50; and HT, n =50), and controls without MetS (n =50). In all subjects, serum adiponectin concentration was measured by enzyme-linked immunosorbent assay method. Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) and HOMA-AD index were calculated. RESULTS: The results showed that, compared to the control group, serum AD concentrations were significantly lower in patients with MetS and high-normal blood pressure (p =0.008), and the lowest in group MetS and HT (p =0.001). High AD levels and low HOMA-AD were significantly associated with decreased blood pressure values. In patients with MetS, the value of HOMA-AD≥1.13 was associated with a higher risk of developing high-normal blood pressure. Furthermore, the value of HOMA-AD≥2.63 was associated with a higher risk of developing hypertension. CONCLUSIONS: Hypoadiponectinemia is associated with hypertension, especially in the early stages of the disease. The serum AD levels and HOMA-AD index may be useful markers for identifying patients at risk for high-normal blood pressure and hypertension. HIPPOKRATIA 2020, 24(1): 3-7.

Allergenicity and immunogenicity of the major mugwort pollen allergen Art v 1 chemically modified by acetylation.

BACKGROUND: Treating allergies with modified allergens is an approach to make the treatment safer and more efficient. Art v 1 is the most prominent allergen of mugwort pollen and a significant cause of hayfever around Europe. The aim of this study was to reduce the allergenicity of Art v 1 by acetylation, and to investigate the capacity of the modified protein to generate blocking antibodies. METHODS: The reduction of allergenicity of Art v 1 following acetylation was monitored by immunoblot, ELISA inhibition using a pool of sera from mugwort pollen allergic patients, basophil activation assay and by skin prick testing of mugwort-allergic patients. Rabbits were immunized against Art v 1 and acetylated Art v 1 (acArt v 1) and the rabbit antisera were tested for their capacity to block human IgE binding in ELISA. Human T cell proliferation against Art v 1 and acArt v 1 was examined in peripheral blood mononuclear cells (PBMCs) of mugwort pollen allergic patients and cytokine release in PBMC cultures was monitored. RESULTS: Acetylation of Art v 1 gave a derivative of reduced allergenicity in the in vitro and ex vivo tests applied. The skin test reactivity to acArt v 1 was significantly reduced in 19 patients when compared with the reactivity to Art v 1. Rabbit antibodies to acArt v 1 and Art v 1 showed similar capacity to block human IgE binding to Art v 1 in inhibition ELISA. Both proteins were able to induce proliferation of PBMCs and CD3/CD4(+) cells of mugwort-allergic patients. Release of IL-5 was significantly reduced in cultures stimulated with acArt v 1. CONCLUSIONS: Art v 1 modified by acetylation had a significantly reduced allergenicity in vitro and in vivo, while its immunogenicity was retained. Modification of allergens by acetylation could be a new strategy for allergen-specific immunotherapy.

Detection of genetically modified organisms in processed meat products on the Serbian food market.

The addition of soybean proteins to processed meat products has significantly increased in recent years due to the interesting functional and nutritional properties of these vegetable proteins. Since the Roundup Ready (RR) soybean is the only transgenic soybean line approved for market in EU this work was aimed at monitoring its presence in meat products on the Serbian food market. The extracted DNA was analyzed using duplex polymerase chain reaction (PCR) with primer pairs aimed at the lectin gene and 35S promoter. Samples positive for the presence of GM soybean were subjected to a real-time quantification of the percentage of RR soya. The results indicated that out of fifty processed meat products examined, twelve gave positive results with 35S promoter and all contained RR soya below 0.1%.

Peroxisome proliferator-activated receptor gamma/Pro12Ala polymorphism and peroxisome proliferator-activated receptor gamma coactivator-1 alpha/Gly482Ser polymorphism in patients with sarcoidosis.

BACKGROUND AND AIM OF THE WORK: Reduced expression and activity of the peroxisome proliferator-activated receptor gamma (PPARG) have been measured in cells of bronchoalveolar lavage fluid in sarcoidosis patients. PPARG, together with its transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARGC1A), has important modulating effects on immune response and apoptosis. In the present study, we investigated whether the polymorphisms Pro12Ala (rs1805192) in the PPARG gene and Gly482Ser (rs8192678) in the PPARGC1A gene, which affect transcriptional activities, are associated with sarcoidosis. METHODS: We performed an integrative "omic" approach and identified the PPARG gene as a suitable candidate. Polymerase chain reaction was performed followed by restriction fragment length polymorphism to determine PPARG/Pro12Ala and PPARGC1A/Gly482Ser genotypes of 104 sarcoidosis patients and 112 healthy control subjects. RESULTS: A higher frequency of the Ala allele (p=0.0101, OR=1.84, CI 1.18-2.88), as well as a significantly higher frequency of Pro/Ala heterozygotes and Ala/Ala homozygotes at the Pro12Ala/PPARG polymorphism (p=0.0020, OR=2.45, CI 1.42-4.25) were found in patients with sarcoidosis. In addition, a higher frequency of the Ser allele (p=0.013, OR=1.69, CI 1.13-2.53) and Gly/Ser heterozygotes and Ser/Ser homozygotes (p=0.0470, OR=1.80, CI 1.04-3.10) at the Gly482Ser/PPARGC1A polymorphism were found in patients with sarcoidosis as compared to healthy control subjects. CONCLUSION: Our results indicate that the presence of the Ala allele at the PPARG/Pro12Ala polymorphism and the Ser allele at the PPARGC1A/Gly482Ser polymorphism may be a predisposing factor for sarcoidosis.

Inflammatory changes in small blood vessels in the endomyocardium of cardiac syndrome X in female patients with increased C-reactive protein.

The pathogenesis and pathohistological changes of CSX, a syndrome characterized by anginal chest pain and normal coronary arteries on coronary angiography, is poorly understood. The purpose of this study was to analyse morphological changes in small blood vessels of the CSX patients with increased CRP levels (above 5 mg/l). EMB was performed for diagnostic purposes in 31 female patients with CSX, and EMB specimens were histologically and immunohistochemically analysed. Increased CRP was found in 18 (58.1%) female patients with CSX. Signs of inflammation in the walls of small blood vessels were demonstrated in 13 (76%) and TUNEL-positive endothelial cells in 3 (17%) women with increased CRP. Morphological analysis of small blood vessels in EMB in CSX female patients with increased CRP levels revealed signs of inflammation and apoptosis of endothelial cells, indicating the role of inflammation in the pathogenesis of CSX.

Penile self-mutilation - two cases in one family.

Male genital self-mutilation is an infrequently reported event in the general medical literature. The majority of cases deal with a single episode of self-mutilation. These cases are usually focused on surgical repair. We present an extremely rare situation of two cases of male genital self-mutilation occurring in the same family. Both patients were treated for schizophrenia. Both patients had religious psychotic experiences and guilt feelings associated with sexual conflicts. Both patients had an experience of setting fire. Although they were close relatives, the younger did not imitate the older one when he mutilated his genitals. The possible causes of such behavior have been analyzed in the light of the recent literature.