László Cervenak

Proinflammatory activation pattern of human umbilical vein endothelial cells induced by IL‐1β, TNF‐α, and LPS

Endothelial cells play a critical role in inflammation by responding to several endogenous and exogenous proinflammatory stimuli. The three most studied factors that provide inflammatory signals to endothelial cells are lipopolysaccharide (LPS), tumor necrosis factor (TNF)‐α, and interleukin (IL)‐1β; however, their effects on endothelial cells were thoroughly compared at the level of gene expression only. Therefore, our aim was to assess the differences in the signaling pathways, adhesion molecules, and cytokines induced by proinflammatory factors in human umbilical vein endothelial cells (HUVEC). In this study, we demonstrated that signaling of LPS was less effective than that of IL‐1β, and was significantly slower than that ofTNF‐α and IL‐1β, which can be partially explained by the special localization of Toll‐like receptor 4 (TLR4). We showed that TLR4 is mainly localized in Golgi apparatus in HUVEC. The proinflammatory capacity of TNF‐α was similar to that of IL‐1β in inducing NF‐κB nuclear translocation, while IL‐1β was the strongest activator of MAPK pathways. Moreover, expression of E‐selectin, IL‐6, and IL‐8 was induced most efficiently by IL‐1β, while LPS and TNF‐α had less effect, whereas we did not find such a difference in ICAM‐1 and MCP‐1 expression. Due to the higher induction of E‐selectin and IL‐8, IL‐1β might have more important role in neutrophil recruitment than LPS and TNF‐α. By above‐mentioned parameters we identified a signaling and expression pattern for the three proinflammatory molecules. This pattern illustrates how complex a proinflammatory process can be, and may enable us to predict and compare the pathomechanism of various inflammatory diseases.

Increased serum heat-shock protein 70 levels reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia

It has been previously reported that serum levels of 70-kDa heat-shock protein (Hsp70) are elevated in preeclampsia. The aim of the present study was to examine whether increased serum Hsp70 levels are related to clinical characteristics and standard laboratory parameters of preeclamptic patients, as well as to markers of inflammation (C-reactive protein), endothelial activation (von Willebrand factor antigen) or endothelial injury (fibronectin), trophoblast debris (cell-free fetal DNA) and oxidative stress (malondialdehyde). Sixty-seven preeclamptic patients and 70 normotensive, healthy pregnant women were involved in this case-control study. Serum Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Standard laboratory parameters (clinical chemistry) and C-reactive protein (CRP) levels were determined by an autoanalyzer using the manufacturer’s kits. Plasma von Willebrand factor antigen (VWF:Ag) levels were quantified by ELISA, and plasma fibronectin concentration by nephelometry. The amount of cell-free fetal DNA in maternal plasma was determined by quantitative real-time polymerase chain reaction analysis of the sex-determining region Y gene. Plasma malondialdehyde levels were measured by the thiobarbituric acid-based colorimetric assay. Serum Hsp70 levels were increased in preeclampsia. Furthermore, serum levels of blood urea nitrogen, creatinine, bilirubin and CRP, serum alanine aminotransferase and lactate dehydrogenase (LDH) activities, as well as plasma levels of VWF:Ag, fibronectin, cell-free fetal DNA and malondialdehyde were also significantly higher in preeclamptic patients than in normotensive, healthy pregnant women. In preeclamptic patients, serum Hsp70 levels showed significant correlations with serum CRP levels (Spearman R = 0.32, p = 0.010), serum aspartate aminotransferase (R = 0.32, p = 0.008) and LDH activities (R = 0.50, p < 0.001), as well as with plasma malondialdehyde levels (R = 0.25, p = 0.043). However, there was no other relationship between serum Hsp70 levels and clinical characteristics (age, parity, body mass index, blood pressure, gestational age, fetal birth weight) and laboratory parameters of preeclamptic patients, including markers of endothelial activation or injury and trophoblast debris. In conclusion, increased serum Hsp70 levels seem to reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia. Nevertheless, further studies are required to determine whether circulating Hsp70 plays a causative role in the pathogenesis of the disease.

Studies on the interactions between C-reactive protein and complement proteins

Several studies have investigated the interactions between C‐reactive protein (CRP) and various complement proteins but none of them took into consideration the different structural forms of CRP. The aim of our study was to investigate whether the different antigenic forms of CRP are able to bind C1q, to trigger activation of the C1 complex and to study the ability of the various CRP forms to bind complement factor H (FH) and C4b‐binding protein (C4BP). Interactions between various CRP forms and complement proteins were analysed in enzyme‐linked immunosorbent assay and surface plasmon resonance tests and activation of the C1 complex was followed in a reconstituted system using purified C1q, C1r and C1s in the presence of C1‐INH. Native, ligand‐unbound CRP activated the classical pathway weakly. After binding to phosphocholine, native CRP bound C1q and significantly activated C1. Native CRP complexed to phosphocholine did not bind the complement regulatory proteins FH and C4BP. After disruption of the pentameric structure of CRP, as achieved by urea‐treatment or by site‐directed mutagenesis, C1q binding and C1 activation further increased and the ability of CRP to bind complement regulatory proteins was revealed. C1q binds to CRP through its globular head domain. The binding sites on CRP for FH and C4BP seemed to be different from that of C1q. In conclusion, in parallel with the increase in the C1‐activating ability of different CRP structural variants, the affinity for complement regulatory proteins also increased, providing the biological basis for limitation of excess complement activation.

Relationship of circulating cell-free DNA levels to cell-free fetal DNA levels, clinical characteristics and laboratory parameters in preeclampsia

BackgroundThe aim of our study was to examine whether increased circulating total cell-free DNA levels are related to the clinical characteristics and standard laboratory parameters of preeclamptic patients, to markers of inflammation, endothelial activation or injury, oxidative stress and to cell-free fetal DNA levels.MethodsCirculating total cell-free DNA was measured by real-time quantitative PCR in plasma samples obtained from 67 preeclamptic and 70 normotensive pregnant women. Standard laboratory parameters, C-reactive protein, plasma von Willebrand factor antigen, plasma fibronectin, plasma malondialdehyde and cell-free fetal DNA levels were also determined.Results and ConclusionCirculating total cell-free and fetal deoxyribonucleic acid levels were significantly elevated in pregnancies complicated by preeclampsia (median: 11.395 vs. 32.460 and 0.001 vs. 0.086 pg/μl; P < .001). The quantity of plasma total cell-free DNA did not correlate with most of the laboratory parameters, except for serum aspartate aminotransferase and alanine aminotransferase activities (correlation coefficient: 0.31; P = 0.012 and 0.46; P < .001). There was no correlation with clinical characteristics, including body mass index. The releases of both free fetal and total cell-free deoxyribonucleic acid were found to be affected in preeclampsia. Hepatocellular necrosis seems to be responsible - at least partly - for increased circulating total DNA levels in preeclampsia, as suggested by the significant correlation with liver enzyme activities.

Adenosine Triphosphate Concentration of Exhaled Breath Condensate in Asthma

BACKGROUND Purinergic signaling is involved in asthma pathogenesis. Not only adenosine but also adenosine triphosphate (ATP) might play a role, but human evidence is scarce. ATP can be measured in exhaled breath condensate (EBC), a noninvasive airway sample suggested as being suitable for patient monitoring. We determined EBC ATP concentration in asthma, investigated its relation to disease parameters, and calculated airway ATP level. METHODS EBC was collected from 45 patients with persistent asthma (age 34.7 +/- 13.2 years; FEV(1), 87.0 +/- 15.5% predicted; mean +/- SD) and 32 healthy control subjects (age 36.9 +/- 12.6 years; FEV(1), 98.9 +/- 9.9% predicted). Exhaled nitric oxide concentration (FeNO) and lung function were measured, and Asthma Control Test (ACT) score was obtained. EBC ATP was measured in luciferin-luciferase assay. Airway ATP concentration was calculated using dilution estimated from conductivity of vacuum-treated EBC samples. Parametric tests were applied in the analyses. ATP concentrations and nitric oxide levels were logarithmically transformed. RESULTS EBC ATP and calculated airway ATP concentrations were not elevated in asthma, and none of them was related to FeNO or ACT score. EBC ATP concentration was influenced by airway droplet dilution (r = -0.32, P < .05), and there was a relation between calculated airway ATP level and FEV(1) (r = -0.35, P < .05). CONCLUSIONS EBC ATP concentration does not seem to be useful for asthma monitoring. The relation between EBC mediator concentration and EBC conductivity highlights the importance of further standardization of EBC methodology and the need for more studies to understand airway droplet formation.

Circulating anti-heat-shock-protein antibodies in normal pregnancy and preeclampsia

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.

HLA-association of serum levels of natural antibodies

Natural antibodies of IgM or IgG types are present in sera of most healthy individuals and are important participants of the immune response. Little is known, however, about the genetic regulation of their plasma levels in humans. We determined the concentrations of three IgM type natural autoantibodies (NAAbs) reactive to certain conserved self-antigens (citrate synthase (A-CIT), chondroitin sulphate C (A-COS) and 60 kDa heat shock proteins (A-HSP) in the sera of 78 healthy individuals and in their 86 children. In case of all the 164 individuals alleles of several polymorphisms were determined in class II (HLA-DQ, -DR), class III (AGER-429T>C, HSP70-2 1267A>G, TNF-308G>A, CFB S/F, copy number of the C4A and C4B genes), and class I (HLA-A, -B) regions of the major histocompatibility complex (MHC). Since the samples originated from a family study, extended MHC haplotypes were also determined for each study participant. Our results show that children of parents with low NAAb concentration have significantly lower serum concentrations of all the three NAAbs, as compared to offsprings of parents without reduced serum concentration. This indicates that the serum levels of these NAAbs were partly regulated by factors which are inherited from the parents to offsprings. In further studies performed only in genetically independent parents, we found significant differences in the serum levels of the IgM type A-CIT and A-COS antibodies (Abs) between carriers and non-carriers of the HLA-DR2 (15 and 16) antigens. In both cases the Ab concentrations were higher in the HLA-DR15 carriers (p=0.002 and p=0.008, respectively) and lower in DR16 carriers (p=0.029 and p=0.049, respectively) than in the non-carriers. Even more significant differences were found when the levels of two Abs were evaluated together. Frequency of the DR15 carriers was significantly lower among subjects with one or two low (in the lowest quartile) titers of A-CIT/A-COS Abs (p=0.014), A-CIT/A-HSP Abs (p=0.016) and A-COS/A-HSP Abs (p=0.013) as compared to those with normal Ab titers for both antigens. By contrast, frequency of the DR16 carriers was significantly higher among subjects with one or two low A-CIT/A-COS Abs (p=0.001), A-CIT/A-HSP Abs (p=0.002) and A-COS/A-HSP Abs (p=0.021) as compared to those with normal Ab titers for both antigens. Similar differences were found for both IgM type antibodies when carriers and non-carriers of the HLA-DR15-DQ6 and HLA-DR16-DQ5 haplotypes were considered. These novel observations indicate that not only adaptive immune response but also natural autoantibody pattern, as a part of innate immune response, is influenced by the MHC allele composition.

Doxorubicin selectively suppresses mRNA expression and production of endothelin-1 in endothelial cells.

Doxorubicin (DXR) is a widely used cytostatic agent, but its administration is limited by its cardiovascular side effects. The endothelium is one of the largest organs in the human body and due to its direct contact with blood; it is exposed to the toxic effects of DXR. The aim of this study was to investigate in endothelial cells the effects of DXR on the expression of genes involved in cardiovascular diseases. We used in vitro cultured human umbilical vein endothelial cells (HUVEC) as a model; gene expression was assessed by SuperArray and qPCR. Out of the 96 representative genes of cardiovascular importance, the expression of only the ET-1 gene changed significantly. ET-1 mRNA expression was 10.9% of the untreated control (p=0.0049). This result was confirmed by qPCR (2.41% of control, p=0.0022). DXR also suppressed ET-1 production at protein level (p=0.0116). Both the early decrease in endothelial ET-1 production in the presence of DXR and the high plasma level of DXR during chemotherapy may influence the toxic effects of the drug.

Protein kinase inhibitor-induced endothelial cell cytotoxicity and its prediction based on calculated molecular descriptors

Protein kinase inhibitors (PKIs) as potent signal transduction therapeutic compounds represent a very rapidly expanding group of anticancer drugs. These agents may be toxic for endothelial cells, however, very few experimental data exist on the cytotoxicity of PKIs. The aim of this study was to set up an appropriate test system for endothelial cells and to assess the structure-related cytotoxic effects of a selected library of PKIs. The inhibitor library contains several lead molecules with different basic structures and a set of modified derivatives of the lead compounds. The toxicity of PKIs did not correlate directly with the structural features of the molecules. However, we successfully built up a model based on 15 calculated molecular descriptors, which is capable of predicting cytotoxicity with acceptable probability. Our results show that the cytotoxic effects of PKIs should be taken into account for optimal drug development to overcome endothelial cell-related side effects.