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The role of oxidative stress (OXS) due to myocardial nitric oxide synthase (NOS) uncoupling related to oxidative depletion of its cofactor tetrahydrobiopterin (BH4) emerged in the pathogenesis of heart failure with preserved ejection fraction. We determined the prevalence of six single nucleotide polymorphisms (SNPs) of genes encoding enzymes related to OXS, BH4 metabolism, and NOS function in ≥60-year-old 94 patients with hypertension and 18 age-matched controls with normal ejection fraction. Using echocardiography, 56/94 (60%) patients with hypertension had left ventricular (LV) diastolic dysfunction (HTDD+ group) and 38/94 (40%) patients had normal LV diastolic function (HTDD- group). Four SNPs (rs841, rs3783641, rs10483639, and rs807267) of guanosine triphosphate cyclohydrolase-1, the rate-limiting enzyme in BH4 synthesis, one (rs4880) of manganese superoxide dismutase, and one (rs1799983) of endothelial NOS genes were genotyped using real-time polymerase chain reaction method and Taqman probes. Protein carbonylation, BH4, and total biopterin levels were measured from plasma samples. No between-groups difference in minor allele frequency of SNPs was found. We calculated a genetic score indicating risk for OXS based on the minor allele frequencies of the SNPs. A high genetic risk for OXS was significantly associated with HTDD+ even after adjustment for confounding variables (odds ratio [95% confidence interval]:4.79 [1.12-20.54]; P = .035). In both patient groups protein carbonylation (P < .05 for both), plasma BH4 (P < .01 for both) and in the HTDD+ group total biopterin (P < .05) increased versus controls. In conclusion, in patients with hypertension and normal ejection fraction, a potential precursor of heart failure with preserved ejection fraction, a partly genetically determined increased OXS, seems to be associated with the presence of LV diastolic dysfunction., (Copyright © 2016 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.)

Predicting the survival of a patient with heart failure (HF) is a complex problem in clinical practice. Our previous study reported that extracellular HSP70 (HSPA1A) correlates with markers of heart function and disease severity in HF, but the predictive value of HSP70 is unclear. The goal of this study was to analyze extracellular HSP70 as predictive marker of mortality in HF. One hundred ninety-five patients with systolic heart failure were enrolled and followed up for 60 months. By the end of follow-up, 85 patients were alive (survivors) and 110 died (nonsurvivors). HSP70 (measured by ELISA in the serum) was elevated in nonsurvivors, compared with survivors (0.39 [0.27-0.59] vs. 0.30 [0.24-0.43] ng/ml, respectively, p = 0.0101). In Kaplan-Meier survival analysis higher HSP70 levels above median were associated with a significantly increased mortality. In multivariable survival models, we show that HSP70 level above the median is an age-, sex-, body mass index-, creatinine-, and NT-proBNP-independent predictor of 5-year mortality in HF. Extracellular HSP70 could prove useful for estimating survival in patients with HF.

Predicting the prognosis of comatose, post-cardiac-arrest patients is a complex problem in clinical practice. There are several established methods to foretell neurological outcome; however, further prognostic markers are needed. HSP70 (HSPA1A), which increases rapidly in response to severe stress (among others after ischemic or hypoxic events), is a biomarker of cell damage in the ischemic brain and spinal cord. We hypothesized that HSP70 might be a reliable predictor of mortality in post-cardiac-arrest patients. The aim of this study was to analyze the role of extracellular HSP70 in the systemic inflammatory response over time, as well as the predictive value in cardiac arrest patients. Here, we show that the elevation of HSP70 levels in resuscitated patients and their persistence is an independent predictor of 30-day mortality after a cardiac arrest. Forty-six cardiac arrest patients were successfully cooled to 32-34 °C for 24 h, and followed up for 30 days. Twenty-four patients (52.2 %) were alive by the end of follow-up, and 22 patients (47.8 %) died. Forty-six patients with stable cardiovascular disease served as controls. Extracellular HSP70 (measured by ELISA in blood samples) was elevated in all resuscitated patients (1.31 [0.76-2.73] and 1.70 [1.20-2.37] ng/ml for survivors and non-survivors, respectively), compared with the controls (0.59 [0.44-0.83] ng/ml). HSP70 level decreased significantly in survivors, but persisted in non-survivors, and predicted 30-day mortality regardless of age, sex, complications, and the APACHE II score. Extracellular HSP70 could prove useful for estimating prognosis in comatose post-cardiac-arrest patients.

Previously, we reported on the presence of antibodies to linear epitopes of human and mycobacterial 60 kD heat shock proteins (HSP) in the sera of healthy blood donors. Since many recent findings indicate that the levels of these antibodies may be altered in coronary heart disease (CHD) and also inflammatory bowel diseases (IBD), it seemed worthwhile to compare the epitope specificity of the anti-HSP60 and anti-HSP65 antibodies in the sera of patients with these diseases to those in healthy subjects. The multipin enzyme-linked immunosorbent assay method was applied with a large overlapping set of synthetic 10-mer peptides covering selected regions of human HSP60 and Mycobacterium bovis HSP65. Sera of 12 healthy persons (HP), 14 CHD, and 14 IBD patients with the same concentration of total anti-HSP60 and HSP65 IgG antibodies were tested. We have identified CHD-specific epitopes in the equatorial domain of the HSP60 protein but in neither region of the HSP65 molecule, indicating that the formation of anti-HSP60 antibodies is not or only partially due to the cross-reaction between human HSP60 and bacterial HSP65. IBD-specific epitopes were found in many regions of the HSP60 and in even more regions of the HSP65 molecule including an IBD-specific T cell epitope in region X as well. These findings indicate that the epitope specificity of the anti-human and anti-mycobacterial HSP60 antibodies associated with various diseases is different.

Background: We have previously reported that serum levels of 70 kDa heat shock protein (Hsp70, HSPA1A) are increased and reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia. The purpose of this study was to determine whether increased serum Hsp70 concentrations in women with preeclampsia are related to circulating levels of cytokines, chemokines, adhesion molecules and angiogenic factors, the key players in the pathogenesis of the disease., Methods: Sixty preeclamptic patients and 60 normotensive, healthy pregnant women were involved in this case-control study. Levels of Hsp70 (HSPA1A) and transforming growth factor (TGF)-beta1 in maternal sera were assessed by ELISA. Serum levels of interleukin (IL)-1 beta, IL-1 receptor antagonist, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-18, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interferon-gamma-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were determined by multiplex suspension array. Serum total soluble fms-like tyrosine kinase-1 (sFlt-1) and biologically active placental growth factor (PlGF) levels were measured by electrochemiluminescence immunoassay. For statistical analyses, the Mann-Whitney U-test, the Fisher exact and Pearson chi-square tests, the Spearman rank order correlation, multiple linear regression and logistic regression were applied., Results: Serum levels of Hsp70 were significantly higher in preeclamptic patients than in healthy pregnant women. Additionally, most of the measured inflammatory variables differed significantly between the two study groups except for serum IL-1 beta and TGF-beta1 levels and IL-18/IL-12p70 and IL-12p70/IL-12p40 ratios, indicating a bias toward a pro-inflammatory status in preeclampsia. Preeclamptic patients had significantly higher sFlt-1 levels and sFlt-1/PlGF ratio and significantly lower PlGF concentrations as compared to healthy pregnant women. In the preeclamptic group, serum Hsp70 concentrations showed significant correlations with serum levels of IL-12p40 (R=0.59, p<0.001), MCP-1 (R=0.43, p<0.001), ICAM-1 (R=0.39, p=0.0020) and VCAM-1 (R=0.46, p<0.001). Furthermore, elevated serum Hsp70 level and sFlt-1/PlGF ratio had a synergistic (joint) effect in the risk of preeclampsia, as shown by the substantially higher odds ratios of their combination than of either alone., Conclusions: Increased serum Hsp70 concentrations in women with preeclampsia were associated with pro-inflammatory changes in circulating cytokine profile, suggesting that circulating Hsp70 might contribute to the development of the excessive systemic inflammatory response characteristic of the maternal syndrome of the disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

It has been previously reported that circulating anti-heat-shock-protein (Hsp) antibody levels are elevated in cardiovascular disorders. The aim of the present study was to determine circulating antihuman Hsp60, antimycobacterial Hsp65, and antihuman Hsp70 antibody levels in healthy pregnant women and preeclamptic patients and to investigate their relationship to the clinical characteristics of the study subjects, as well as to the markers of inflammation (C-reactive protein (CRP)), endothelial activation (von Willebrand factor antigen), or endothelial injury (fibronectin), oxidative stress (malondialdehyde) and to serum Hsp70 levels. Ninety-three preeclamptic patients and 127 normotensive healthy pregnant women were involved in this case control study. Serum anti-Hsp60, anti-Hsp65, anti-Hsp70, and Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Serum CRP levels were determined by an autoanalyzer using the manufacturer's kit. Plasma von Willebrand factor antigen levels were quantified by ELISA, while plasma fibronectin concentration by nephelometry. Plasma malondialdehyde levels were measured by the thiobarbituric-acid-based colorimetric assay. For statistical analyses, nonparametric methods were applied. Anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibodies were detected in all of our serum samples. There were no significant differences in serum anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibody levels between the control and preeclamptic groups. Serum levels of Hsp70 and CRP, as well as plasma levels of VWF antigen, fibronectin, and malondialdehyde, were significantly higher in preeclamptic patients than in normotensive healthy pregnant women. Serum anti-Hsp60 antibody levels showed significant correlations with serum anti-Hsp65 antibody levels both in the control and the preeclamptic groups (Spearman R = 0.55 and 0.59; p < 0.001, respectively). However, no other relationship was found between clinical features (maternal age, smoking status, parity, body mass index, gestational age at blood draw, systolic and diastolic blood pressure, gestational age at delivery, and fetal birth weight) and measured laboratory parameters of the study subjects and serum anti-Hsp antibody levels in either study group. In conclusion, anti-Hsp60 and anti-Hsp70 antibodies as naturally occurring autoantibodies are present in the peripheral circulation of healthy pregnant women. Nevertheless, humoral immunity against heat shock proteins was not associated with preeclampsia. Further studies are warranted to explore the role of heat shock proteins and immune reactivity to them in the immunobiology of normal pregnancy and preeclampsia.

Objective: According to data from animal models, complement activation plays a major role in the brain injury after acute ischemic stroke. Scarce findings are, however, available on the detection of complement activation products in stroke patients., Methods: We have measured plasma levels of the five complement activation products (C1rC1sC1inh, C4d, C3a, C5a and SC5b-9) in samples of 26 patients with ischemic stroke upon admission. Twenty-six patients with severe carotid atherosclerosis served as patient controls., Results: Levels of two activation products (SC5b-9 and C4d)) were significantly elevated in the plasma of stroke patients, SC5b-9 levels, exhibited significant positive correlation with the clinical severity of stroke, the severity of neurological deficit, as well as with the level of functional disability., Conclusion: These findings suggest that complement activation plays an active role in the development of brain infarct. The measurement of complement activation products might help to determine the clinical prognosis after acute ischemic stroke. Furthermore, there is potential usefulness of complement modulating therapy in ischemic stroke.

Background: Hereditary angioedema (HAE) is characterized by episodic edematous attacks due to the deficiency of the C1-inhibitor (C1-INH). Recently, we have described that the long-term use of danazol affects lipid metabolism, resulting in decreased high-density lipoprotein (HDL) and increased low-density lipoprotein (LDL) cholesterol levels, which might lead to accelerated, early atherosclerosis. Our aim in the present study was to investigate the impact of danazol treatment on the risk of atherosclerosis in HAE patients., Methods: The prevalence of vascular disease, as well as carotid intima-media thickness (IMT)--an objective marker of atherosclerosis--was determined in 32 HAE patients undergoing danazol prophylaxis, and compared to 25 HAE patients without danazol treatment, as well as to 20 healthy controls. Distinct atherosclerosis risk profiles were determined in addition., Results: HAE patients with danazol prophylaxis had higher body mass index (p=0.0055 and 0.0020), creatinine (p=0.0001 and 0.0130), alanine aminotransferase (p=0.0298 and 0.0457), LDL (p=0.0060 and <0.0001) and decreased HDL (p<0.0001 and <0.0001) levels compared to both control groups. The prevalence of vascular diseases did not differ in the two patient groups. No significant differences were observed in mean (0.43 (0.37-0.50)mm vs. 0.40 (0.35-0.49)mm, p=0.5465) carotid IMT values, when comparing patients with or without long-term danazol prophylaxis., Conclusions: Thickening of IMT due to danazol use was not observed in HAE patients. We hypothesize that the functional deficiency of C1-INH might confer protection against atherosclerosis in these patients.

Background: Ischemic stroke is a multifactorial disorder with genetic and environmental components. The aim of our study was to investigate whether two polymorphisms of the estrogen receptor alpha (ESR1) gene (ESR1 c.454-397T>C and c.454-351A>G) are associated with ischemic stroke in a Caucasian population from Hungary., Methods: One hundred and ninety-eight patients with ischemic stroke and 180 control subjects were enrolled in this case-control study. Ischemic stroke subtypes were categorized according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification as large-artery atherosclerosis, small-artery occlusion, cardioembolism or stroke of other determined etiology. The ESR1 PvuII and XbaI genotypes were determined using the PCR-RFLP method., Results: There were no significant differences in the genotype, allele and haplotype frequencies of PvuII and XbaI polymorphisms between the group of patients with ischemic stroke and the control group. Furthermore, ESR1 PvuII and XbaI genotypes, alleles and haplotypes were not associated with any subtype of ischemic stroke., Conclusions: We did not observe an association between ESR1 PvuII and XbaI gene polymorphisms and ischemic stroke or any subtype of ischemic stroke. However, further studies are needed to explore the complex interaction between environmental factors and ESR1 gene polymorphisms in the risk of ischemic stroke, particularly in ethnically different populations.

Background: Estrogen is known to affect lipoprotein metabolism, the haemostatic system and inflammatory markers. Our aim was to determine whether estrogen receptor alpha (ESR1) PvuII and XbaI gene polymorphisms can influence lipid, haemostatic and inflammatory variables in healthy Caucasian women and men of reproductive age., Methods: 58 healthy women (aged between 18 and 45 years) and 55 healthy men (aged between 21 and 45 years) of reproductive age were enrolled in our study. FSH levels, lipid (total cholesterol, triglyceride, HDL cholesterol, lipoprotein(a), apo A-I, apo B), haemostatic (prothrombin time, activated partial thromboplastin time (aPTT), thrombin time, fibrinogen, factor V, VII, VIII, protein C, protein S, antithrombin III) and inflammatory (CRP) variables were measured on autoanalyzers using commercially available kits. Serum VLDL and LDL cholesterol concentrations were calculated with the equation of Friedewald. The ESR1 PvuII and XbaI genotypes were determined with PCR-RFLP method., Results: In the total group, the ESR1 XbaI GG genotype carriers had significantly higher serum lipoprotein(a) concentrations than the AA or AG genotype carriers. Serum total cholesterol concentrations were significantly higher in healthy women with the PvuII CC genotype than in those with the TT or TC genotypes, whereas healthy women with the GG genotype of the ESR1 XbaI polymorphism had significantly higher serum total cholesterol and LDL cholesterol levels compared to those with the AA or AG genotypes. No other effects of the ESR1 PvuII and XbaI polymorphisms were found on the investigated lipid, haemostatic and inflammatory variables either in the total group or in women and men separately., Conclusions: The ESR1 PvuII and XbaI gene polymorphisms seem to affect lipoprotein metabolism in healthy subjects of peak reproductive age. However, further studies are needed to determine the molecular mechanisms by which the two polymorphisms could influence serum lipid levels.

Background: The role of factor V (Leiden) mutation, thrombophilia, and apolipoprotein E (apoE) alleles in the pathogenesis of accelerated atherosclerosis and restenosis was studied in patients requiring reoperation within five years after femoropopliteal angioplasty with artificial grafts., Methods: One hundred ninety-eight consecutive patients with femoropopliteal atherosclerotic disease, reoperated for restenosis were contacted by phone and 100 of them returned for laboratory and clinical work-up. In addition to clinical evaluation and routine laboratory investigations, parameters of lipoprotein metabolism, factor V (Leiden) mutation and apolipoprotein E (apoE) allele were studied by PCR amplification of DNA and endonuclease digestion techniques., Results: A significantly higher incidence of factor V (Leiden) mutation was found in patients with atherosclerosis and restenosis, compared to 445 healthy blood donors (13/200, 6.5% vs. 34/890, 3.8%, p=0.0379). Distribution of the alleles of the apolipoprotein E (apoE) gene was different, when the patients were compared to 372 controls; however, the difference only approached the level of statistical significance (25/200, 12.5% vs. 56/744, 7.5%, p=0.0515). Comparing the two groups, the number of epsilon4 allele carriers was significantly higher among patients with restenosis (25/100, 25% vs. 53/272, 14%, p=0.0147)., Conclusion: Factor V (Leiden) mutation may influence the progression of atherosclerosis and the development of restenosis after revascularization in patients with accelerated femoropopliteal atherosclerosis. Further investigation is needed whether long-term anticoagulation has an impact or not on the course of disease in such cases. ApoE epsilon4 allele should be screened in patients with femoropopliteal atherosclerosis, because it indicates a faster progression of atherosclerosis and may predict restenosis after revascularization procedure.

Long-term dietary fatty acid intake alters the development of left ventricular hypertrophy, but the linking signaling pathways are unclear. We studied the role and underlying signaling mechanisms of dietary fat intake in the early phase of the hypertrophic process. Rats assigned for 4 weeks of high-oil, high-fat, or standard diet were subjected to angiotensin II (Ang II; 33 microg/kg/h, subcutaneous) or vehicle infusion for 24 h. The Ang II-induced increase in left ventricular mRNA levels of hypertrophy-associated genes was higher in rats fed the high-oil diet compared with the standard diet. Western blotting revealed that, in parallel with changes in gene expression, the high-oil diet increased c-Jun N-terminal kinase phosphorylation (P < 0.001). Ang II increased p38 mitogen-activated protein kinase (MAPK) phosphorylation in rats fed the high-fat diet (3-fold; P < 0.01). The increase in transcription factor activator protein-1 (AP-1) DNA binding activity in response to Ang II was higher in rats fed the high-oil diet compared with those fed the standard diet (P < 0.001). Ang II downregulated inducible nitric oxide synthase mRNA levels in fatty acid-supplemented groups compared with the standard diet group. These results show that dietary fat type modulates the early activation of hypertrophic genes in pressure-overloaded myocardium involving the distinct activation of AP-1 and MAPK signal transduction pathways.

Atherosclerosis is an inflammatory disease. The complement system plays an important role in the atherosclerotic process. However, lesser data is available on the possible role of C3 as a risk factor for atherosclerosis. Therefore, in a follow up study we determined C3 levels in 266 patients with pre-existing severe coronary artery disease (CAD) and compared their serum C3 concentrations with the cause of the disease. We investigated whether C3 levels predict the major complications of severe CAD during a 5-year long follow up period in patients, who have received an aorto-coronary bypass graft surgery. C3 concentrations were elevated in the patients with severe CAD compared to 182 healthy controls, and women had higher C3 concentrations than men. Pathological C3 levels (C3> or =1.8 g/L) were able to predict major complications of atherosclerosis (death by cardiac events, new acute myocardial infarction, stroke, carotid surgery and peripheral arterial disease) that developed during the follow up period only in women (OR: 4.1, 95% C.I. 1.23-13.61, p = 0.0249) independent of other risk factors for atherosclerosis. Our data supports the assumption that high C3 indicates the progression of atherosclerosis as a special marker of chronic inflammation.

Previously we detected more than 3 times higher anti-cholesterol antibody (ACHA) levels in HIV positive patients compared to healthy individuals, however, this level significantly decreased during highly active anti-retroviral therapy (HAART). In our present study we examined whether these findings could also be detected in patients with chronic hepatitis C (CHC). We calculated the correlation between the ACHA levels and the C5b-9 complement activation product. 39 patients with CHC were treated with IFN-alpha-2b (Schering-Plough) 5 MU daily for 6 weeks, followed by 5 MU TIW. Serum levels of ACHA and complement activation products were measured with ELISA. Serum HCV RNA was measured by a highly sensitive branched DNA technique before and 3, 6 and 12 months after the beginning of IFN-alpha-2b therapy. 52 healthy persons served as controls. At the onset of treatment ACHA level was significantly (p = 0.0062) higher in patients (40 (24-69) AU/ml) (median (interquartile range)) than in control sera (26 (20-35) AU/ml). In the 26 responder patients ACHA levels decreased to the normal level during the therapy, but no change was observed in the 13 non-responders. In patients with a sustained response ACHA levels remained low till the end of the 12 months IFN treatment. ACHA levels were significantly (p = 0.0422) higher in the patients with low (< 4.0 mmol/l) than in those with normal (> or = 4.0 mmol/l) cholesterol concentrations. The ACHA level before the therapy strongly correlated (r = 0.5499, p = 0.0014) with C5b-9 serum levels. ACHA levels are elevated in CHC, but this elevation is not as high as in HIV. Decrease of viral load by IFN-alpha-2b treatment in the responders results in normalization of ACHA concentration. High ACHA levels in patients with low serum cholesterol concentration suggest that high ACHA levels may contribute to the decrease in cholesterol levels. The correlation between the ACHA and C5b-9 levels indicate, that the ACHA may play a role in the complement activation in CHC.

Background: Clinical and experimental studies suggest that increased activity of semicarbazide-sensitive amine oxidase (SSAO) and the production of cytotoxic metabolites (e.g., formaldehyde and hydrogen peroxide) may play an important role in the pathogenesis of atherosclerosis. The present study was designed to assess the relationship between the increased activity of the enzyme and the severity of atherosclerosis in diabetic and control subjects., Methods: The study included 29 patients with type 2 diabetes mellitus and 25 control subjects. Human serum SSAO activity was determined by using 14C-benzylamine as substrate. Mean common carotid intima-media thickness (IMT), Crouse score and Bogousslawsky score was evaluated by color-coded, high-resolution duplex carotid sonography., Results: Serum SSAO activity was significantly increased in patients with type 2 diabetes compared to controls. Carotid plaque score (Crouse score), total cholesterol level and age-corrected intima-media thickness showed positive correlation with enzyme activity in control subjects. In patients with diabetes, serum SSAO activity correlated with the severity of carotid stenosis (Bogousslawsky score) as well as the carotid plaque score., Conclusions: Determination of serum SSAO activity might be a candidate biochemical marker of early atherosclerosis and diabetic macrovascular complications.