Zsuzsa Varga

Red Cells, Hemoglobin, Heme, Iron, and Atherogenesis

Objective—We investigated whether red cell infiltration of atheromatous lesions promotes the later stages of atherosclerosis. Methods and Results—We find that oxidation of ferro (FeII) hemoglobin in ruptured advanced lesions occurs generating ferri (FeIII) hemoglobin and via more extensive oxidation ferrylhemoglobin (FeIII/FeIV=O). The protein oxidation marker dityrosine accumulates in complicated lesions, accompanied by the formation of cross-linked hemoglobin, a hallmark of ferrylhemoglobin. Exposure of normal red cells to lipids derived from atheromatous lesions causes hemolysis and oxidation of liberated hemoglobin. In the interactions between hemoglobin and atheroma lipids, hemoglobin and heme promote further lipid oxidation and subsequently endothelial reactions such as upregulation of heme oxygenase-1 and cytotoxicity to endothelium. Oxidative scission of heme leads to release of iron and a feed-forward process of iron-driven plaque lipid oxidation. The inhibition of heme release from globin by haptoglobin and sequestration of heme by hemopexin suppress hemoglobin-mediated oxidation of lipids of atheromatous lesions and attenuate endothelial cytotoxicity. Conclusion—The interior of advanced atheromatous lesions is a prooxidant environment in which erythrocytes lyse, hemoglobin is oxidized to ferri- and ferrylhemoglobin, and released heme and iron promote further oxidation of lipids. These events amplify the endothelial cell cytotoxicity of plaque components.

The Serum Paraoxonase Activity in Patients with Chronic Renal Failure and Hyperlipidemia

Human serum paraoxonase is physically associated with an apolipoprotein (Apo-A1) and clusterin-containing high-density lipoprotein (HDL) and prevents low-density lipoprotein from lipid peroxidation. The aim of our study was to determine whether paraoxonase activity or phenotype is altered in patients with chronic renal failure and in hyperlipidemic subjects without renal insufficiency and to compare the values with those of healthy controls. We investigated the serum paraoxonase activity and polymorphism in 119 hemodialyzed uremic patients, 107 patients with primary hyperlipoproteinemia, and in 110 healthy control subjects. The serum paraoxonase activity was significantly decreased both in hyperlipidemic (p < 0.01) and uremic patients (p < 0.001) as compared with controls. On comparison, the serum paraoxonase activity was significantly lower (p < 0.001) in uremic than in hyperlipoproteinemic patients. The HDL and Apo-A1 levels were as follows: uremic < hyperlipidemic < control. To assess whether the observed reduction in paraoxonase activity was due to HDL and Apo-A1 level decreases, we standardized the enzyme activity for HDL and Apo-A1 concentrations. We found that the standardized paraoxonase activity (paraoxonase/HDL ratio) was also lower in the uremic patients (103.3 ± 69.5) as compared with hyperlipidemic patients (137.64 ± 81.0) and controls (194.45 ± 94.45). The standardized values for Apo-A1 showed a similar tendency: paraoxonase/Apo-A1 ratio in uremic patients 89.64 ± 47.8, in hyperlipidemic patients 128.12 ± 69.83, and in controls 161.40 ± 47.35. The phenotypic distribution of paraoxonase (AA, AB, BB) did not change significantly in the patient groups. These results suggest that HDL concentration and phenotypic distribution of paraoxonase may not be the only determining factors, but that other as yet undetermined factors could be involved in the enzyme activity changes.

Effect of elastin peptides and N-formyl-methionyl-leucyl phenylalanine on cytosolic free calcium in polymorphonuclear leukocytes of healthy middle-aged and elderly subjects

The effect of elastin peptides (kappa-elastin, KE) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) on cytosolic free calcium in polymorphonuclear leukocytes (PMNLs) of healthy middle-aged (35-45 years) and elderly (greater than 60 years) patients with normal and high serum cholesterol level was investigated. The cytosolic free calcium [( Ca2+]i) elevation after stimulation with these compounds was decreased in PMNLs of the aged groups compared to the healthy middle-aged group. The guanine nucleotide binding regulatory Gi protein inhibitor, pertussis toxin (PT) prevented the enhancing effect of KE and FMLP on PMNL free calcium of healthy middle-aged subjects, but could not completely abolish the [Ca2+]i elevation in PMNLs of aged subjects.

Hydrogen sulfide inhibits the calcification and osteoblastic differentiation of vascular smooth muscle cells

Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is involved in the pathogenesis of vascular calcification. Hydrogen sulfide (H2S) is a gas endogenously produced by cystathionine γ-lyase in VSMC. Here we determined whether H2S plays a role in phosphate-induced osteoblastic transformation and mineralization of VSMC. Hydrogen sulfide was found to inhibit calcium deposition in the extracellular matrix and to suppress the induction of the genes involved in osteoblastic transformation of VSMC: alkaline phosphatase, osteocalcin, and Cbfa1. Moreover, phosphate uptake and phosphate-triggered upregulation of the sodium-dependent phosphate cotransporter (Pit-1) were also prevented by H2S. Reduction of endogenous production of H2S by inhibition of cystathionine γ-lyase activity resulted in increased osteoblastic transformation and mineralization. Low plasma levels of H2S, associated with decreased cystathionine γ-lyase enzyme activity, were found in patients with chronic kidney disease receiving hemodialysis. Thus, H2S is a potent inhibitor of phosphate-induced calcification and osteoblastic differentiation of VSMC. This mechanism might contribute to accelerated vascular calcification in chronic kidney disease.

Determination of DNA damage induced by oxidative stress in hyperlipidemic patients

In the present paper, we report data on the genotoxic properties of hydrogen peroxide in polymorphonuclear neutrophils (PMNLs) separated from normolipidemic and type II/a hyperlipidemic patients. In all, 15 hyperlipidemic patients (11 female, 4 male, mean age 54.6+/-10.25 years) were involved in the study, and 7 normolipidemic patients (5 female, 2 male, mean age 53.4+/-8.07 years) served as controls. Using the comet assay, there was a significant difference in the degree of DNA damage between the two groups. The visual score characteristic of the degree of DNA damage was 350.97+/-31.31 in the hyperlipidemic group, while it was 289.5+/-29.49 in the control group (P<0.001). In the hyperlipidemic patients, a positive correlation was found between the degree of DNA damage and the basic oxidation of PMNLs (r=0.517), and the superoxide anion production of the cells stimulated with phorbolmiristate acetate (PMA) (r=0.326) and formyl-Met-Leu-Phe (FMLP) (r=0.525) as well. There was a negative correlation between DNA damage and HDL-associated antioxidant paraoxonase (PON) activity (r=-0.469), and the PON/HDL ratio (r=-0.631). No correlation was found between the degree of DNA damage and the plasma concentration of nitric oxide (NO) (r=0.098) and thiobarbituric acid-reactive substances (TBARS) (r=0.061) in hyperlipidemic patients. Our results show that in hyperlipidemic patients there is an increase in lymphocyte DNA damage caused by oxidative stress when compared to normolipidemic individuals as demonstrated by comet assay. Decreased antioxidant capacity in hyperlipidemic patients may play a significant role in this process.

Increased levels of platelet activation markers are positively associated with carotid wall thickness and other atherosclerotic risk factors in obese patients

The role of platelets in the development of atherosclerosis and obesity-related prothrombotic state is still under investigation. In this cross-sectional cohort study, we measured the levels of different platelet activation markers and evaluated their relationship with carotid intima-media thickness (IMT) along with other atherosclerotic risk factors in obese patients with or without atherosclerotic co-morbidities. We enrolled 154 obese patients, including 98 with either hypertension, type 2 diabetes mellitus or dyslipidaemia, 56 without these co-morbidities and 62 age- and sex-matched healthy controls. Platelet P-selectin expression and the number of platelet-derived microparticles (PMPs) were measured by flow cytometry; soluble P-selectin levels were analysed by ELISA and Thr715Pro P-selectin polymorphism was determined by PCR-RFLP. Carotid IMT was examined by ultrasonography. The levels of platelet activation parameters were significantly elevated in all obese subjects with increased carotid IMT compared to healthy controls. There was no effect of Thr715Pro genotype on soluble P-selectin levels in obese individuals contrary to normal subjects. Significant and positive association was revealed between carotid IMT and platelet P-selectin (p<0.0001), soluble P-selectin (p=0.039) and PMP (p=0.0001) levels. After adjusting for multiple variables, independent association was found between soluble P-selectin and fibrinogen (p=0.007), PMP levels and body mass index (p<0.0001) as well as platelet P-selectin and carotid IMT (p=0.012) plus plasminogen activator inhibitor-1 (p=0.009). In conclusion, P-selectin and PMP levels showed positive associations with abnormal carotid IMT and other risk factors in obesity suggesting a critical role of enhanced platelet reactivity in atherosclerotic wall alteration.

Altered phosphatidylinositol breakdown after K-elastin stimulation in PMNLs of elderly.

The degradation of elastic fibres during atherosclerotic plaque formation in arterial wall is a well known process. The liberated elastin peptides such as K-elastin possess various biological activities: They are chemotactic for monocytes and fibroblasts, stimulate the oxidative burst and the intracellular free Ca2+ mobilisation through the phosphatidylinositol (PIP2) breakdown in PMNLs. It was found that the PIP2 breakdown induced by K-elastin is a pertussis toxin sensitive process in PMNLs of young subjects. In the case of the elderly, the K-elastin-induced oxidative burst, intracellular free Ca2+ elevation was less than in young, and could not be inhibited by pertussis toxin. Studying the K-elastin-induced inositol phosphate (IP) formation in PMNLs of elderly a disturbed PIP2 breakdown was found. K-elastin stimulated the IP formation at a very low level in PMNLs of elderly. This alteration of the second messenger formation (e.g. IP3 and Ca2+) after KE stimulation, might be the consequence of their originally elevated levels in resting PMNLs of elderly.

Age-dependent changes in transmembrane signalling: Identification of G proteins in human lymphocytes and polymorphonuclear leukocytes

In human neutrophils (PMNLs) we found that in the elderly IP3 formation was significantly decreased compared to that of young subjects. For FMLP receptor binding affinity and number no measurable differences occurred upon ageing, studying both the low or the high affinity receptors. The amount of ADP-ribosylated G proteins, catalysed by pertussis toxin (PT) or cholera toxin (CT), was significantly increased in PMNLs of the elderly. In lymphocytes, the PT-catalysed ADP ribosylation of G proteins was also increased with ageing, while the CT-catalysed ribosylation was decreased. The autoradiogram of [32P]ADP-ribosylated proteins by CT in lymphocytes of young individuals showed a major polypeptide of 40,000 M(r). In contrast, in lymphocytes of the elderly, the major polypeptide was 45,000 M(r). In PMNLs, CT labelled quite strongly the 45,000 M(r) band, mainly in the elderly. When PT was used, no age-related pattern changes could be demonstrated, while differences could be observed between the two types of cells. The use of antiserum P680 (G alpha common) showed no age-related pattern changes, while the intensity of the labelled proteins varies with age and cell type. The antiserum U46 (Go alpha) could identify in lymphocytes of young subjects two polypeptides 68,000 and 41,000 M(r). The prominent polypeptide in lymphocytes of the elderly was the 70,000 M(r) and no other polypeptides could be recognized. In PMNLs of young subjects the U46 and serum identified a range of species. In PMNLs of the elderly all these bands were weakly labelled. The present data indicate changes in the pattern and the quantity of G proteins in lymphocytes and PMNLs of elderly subjects.

Relationship of Serum Resistin Level to Traits of Metabolic Syndrome and Serum Paraoxonase 1 Activity in a Population with a Broad Range of Body Mass Index

UNLABELLED The relationship between resistin, one of the adipokines, and metabolic syndrome is not fully elucidated. Altered activity of the HDL-associated antioxidant enzyme paraoxonase 1 (PON1) that participates in the antioxidant defense mechanisms of HDL may have an important role in the obesity-related accelerated atherosclerosis. Inverse associations of PON1 with obesity and serum levels of leptin have been demonstrated. Our aim was to investigate the association of serum levels of resistin with (i) PON1 activity, and (ii) parameters of metabolic syndrome, including some that are additional for research. A total of 74 Caucasian subjects were recruited into the study and divided into 3 age and sex-matched groups. Group 1, 25 non-diabetic overweight/obese subjects with BMI of 28-39.9 kg/m (2); group 2, 25 non-diabetic obese patients with BMI >or=40 kg/m (2); and the control group 3, 24 healthy, normal-weight control subjects. Serum levels of resistin were correlated negatively with BMI (r=-0.27, P<0.05), waist circumference (r=-0.28, P<0.05), serum levels of leptin (r=-0.28, P<0.05), non-esterified fatty acids (NEFA) (r=-0.23, P<0.05), and HbA (1C) (r=-0.26, P<0.05), systolic BP (r=-0.28, P<0.05), and lipid peroxidation (measured by TBARS) (r=-0.40, P<0.01), and correlated positively with PON1 (r=0.24, P<0.05). No association was detected between the serum concentrations of resistin and the following investigated parameters: diastolic BP, levels of uric acid, glucose, insulin, or insulin resistance (measured by homeostasis model assessment, HOMA-IR), triglyceride, total cholesterol, LDL-C, and HDL-C. During multiple regression analyses BMI and TBARS were independent predictors of PON1, while age, gender, blood pressure, HOMA-IR, LDL-C, HDL-C, and resistin were not. CONCLUSIONS Among the study subjects, serum levels of resistin showed a positive, although not independent correlation with serum PON1, and a negative correlation with numerous parameters of the metabolic syndrome (i.e. adiposity, blood pressure, levels of leptin, free fatty acid, glycosylated hemoglobin, and lipid peroxidation). BMI and TBARS are independent predictors of PON1 activity.

Relative Abundance of Some Free Fatty Acids in Plasma of Uremic Patients: Relationship between Fatty Acids, Lipid Parameters, and Diseases

The unesterified fatty acid patterns in plasma of predialytic (PHD) and hemodialysis (HD) patients were determined. The HD patients were divided into three groups: (1) HD without cardiovascular disease (HD-norm); (2) HD with cardiomyopathy (HD-CAD), and (3) HD with hyperlipidemia (HD-hyp). The relative abundance of saturated fatty acids (SFAs) was greater in the plasma of HD-norm and HD-CAD patients (73.3 and 70.0%, respectively) than that in controls (62.6%), and the relative abundance of monounsaturated fatty acids (MUFAs) was significantly greater in the plasma of HD-hyp patients than that in controls (38.9 vs. 21.6%, p < 0.01). In all HD patients the relative concentration of polyunsaturated fatty acids (PUFAs) was lower than in controls. There were no significant differences in the fatty acid patterns of PHD patients. In conclusion, the relative abundance of SFAs and MUFAs in plasma of HD patients is associated with their concomitant lipid disorders and cardiomyopathy, while the low relative abundance of PUFAs was common in all HD patients.