Der Einfluss von Zellkulturbedingungen und Antioxidantien auf die Expression von Frataxin

Ziel dieser Diplomarbeit war, den Einfluss von Zellkulturbedingungen und Antioxidantien auf den Frataxingehalt in verschiedenen Zelllinien zu untersuchen. Frataxin ist ein mitochondriales Protein, welches bei Friedreich’s Ataxie vermindert exprimiert wird. Die genaue Funktion von Frataxin ist noch unklar, es wird aber vermutet, dass es für die Regulation der mitochondrialen Eisenhomöostase benötigt wird [BENCZE et al., 2006]. Oxidativer Stress spielt eine große Rolle im Krankheitsmechanismus von FRDA. Zusätzlich zum Risiko allgemeiner oxidativer Schädigung bei FRDA gibt es auch Anhaltspunkte, dass Frataxinmangel die Zellen anfälliger für oxidativen Stress macht [COOPER and SHAPIRA, 2003]. Aufgrund der Vermutung, dass eine Frataxinerhöhung durch Zusatz von Antioxidantien zu einer Reduktion von oxidativem Stress führen könnte, wurden verschiedene Antioxidantien in Bezug auf Frataxinexpression und Zellschädigung untersucht. Die Ergebnisse zeigen, dass die Antioxidantien Curcumin und Resveratrol eine Erhöhung der Frataxinexpression bei neuronalen P19 Zellen bewirken. Die Konzentration von 25 µM Resveratrol führte sogar zu einem signifikanten Anstieg der Frataxinexpression. Curcumin und Resveratrol führten hingegen bei den erythroleukämischen K562 Zellen zu einer nicht signifikanten Verminderung der Frataxinexpression. Die Antioxidantien α-Tocopherol Succinat und Kaffeesäure führten weder bei neuronalen P19, noch bei K562 Zellen zu einer Änderung der Frataxinexpression. Alle vier Antioxidantien führten bei T-Rex 293 Zellen zu kaum einer Veränderung bzw. zu einer leichten Verminderung der Zellschädigung. Diese war umso stärker, wenn die Zellen zusätzlich oxidativem Stress ausgesetzt waren, ist aber vermutlich darauf zurückzuführen, dass Tetracyclin und H2O2 einen Komplex bilden, der die Wirksamkeit des H2O2 verringert. Curcumin und Resveratrol zeigten bei T-Rex 293 Zellen eine konzentrationsabhängige Abnahme der Zytotoxizität. Im Rahmen dieser Arbeit wurde gezeigt, dass die verwendeten Antioxidantien nicht den erwarteten therapeutischen Effekt auf FRDA haben.The aim of this thesis was to determine the influence of cell culture conditions and antioxidants on the expression of frataxin in various cell lines. Frataxin is a mitochondrial protein, whose expression is decreased in Friedreich’s ataxia (FRDA). The exact function of frataxin remains unclear, but it is proposed that frataxin is required for the cellular regulation of mitochondrial iron homeostasis [BENCZE et al., 2006]. Oxidative stress plays a major role in the disease mechanism of FRDA. In addition to the increased risk of general oxidative damage in FRDA, there is evidence that frataxin deficiency makes cells more susceptible to oxidative damage [COOPER and SHAPIRA, 2003]. The experiments in this thesis were performed on the presumption that an increase in frataxin levels by supplementation of antioxidants could lead to a reduction of oxidative stress. Four antioxidants were analyzed with respect to frataxin expression and cell damage. The results show that the antioxidants curcumin and resveratrol induce an increase in the expression of frataxin in neuronal P19 cells. The concentration of 25 µM resveratrol lead to a significant increase in the frataxin expression. Curcumin and resveratrol had no significant increasing effect on the expression of frataxin in erythroleucemic K562 cells. The antioxidants α-tocopherol succinate and caffeic acid had no effect on frataxin expression in P19 or K562 cells. All four antioxidants induced either a decreasing effect or none at all on cell damage in T-Rex 293 cells. The decrease was higher when the cells were exposed to oxidative damage which is presumably a result of a complex formed between tetracycline and H2O2 which reduces the effect of H2O2. Curcumin and resveratrol showed a concentration-dependant decrease in cell cytotoxicity. It is shown in this thesis that the chosen antioxidants don’t have the expected therapeutic effect in FRDA

Serum amyloid A: high-density lipoproteins interaction and cardiovascular risk

Aims High-density lipoproteins (HDLs) are considered as anti-atherogenic. Recent experimental findings suggest that their biological properties can be modified in certain clinical conditions by accumulation of serum amyloid A (SAA). The effect of SAA on the association between HDL-cholesterol (HDL-C) and cardiovascular outcome remains unknown. Methods and results We examined the association of SAA and HDL-C with mortality in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study, which included 3310 patients undergoing coronary angiography. To validate our findings, we analysed 1255 participants of the German Diabetes and Dialysis study (4D) and 4027 participants of the Cooperative Health Research in the Region of Augsburg (KORA) S4 study. In LURIC, SAA concentrations predicted all-cause and cardiovascular mortality. In patients with low SAA, higher HDL-C was associated with lower all-cause and cardiovascular mortality. In contrast, in patients with high SAA, higher HDL-C was associated with increased all-cause and cardiovascular mortality, indicating that SAA indeed modifies the beneficial properties of HDL. We complemented these clinical observations by in vitro experiments, in which SAA impaired vascular functions of HDL. We further derived a formula for the simple calculation of the amount of biologically ‘effective' HDL-C based on measured HDL-C and SAA from the LURIC study. In 4D and KORA S4 studies, we found that measured HDL-C was not associated with clinical outcomes, whereas calculated ‘effective' HDL-C significantly predicted better outcome. Conclusion The acute-phase protein SAA modifies the biological effects of HDL-C in several clinical conditions. The concomitant measurement of SAA is a simple, useful, and clinically applicable surrogate for the vascular functionality of HD

High-Density Lipoprotein from Chronic Kidney Disease Patients Modulates Polymorphonuclear Leukocytes

The anti-inflammatory properties of high-density lipoproteins (HDL) are lost in uremia. These HDL may show pro-inflammatory features partially as a result of changed protein composition. Alterations of polymorphonuclear leukocytes (PMNLs) in chronic kidney disease (CKD) may contribute to chronic inflammation and high vascular risk. We investigated if HDL from uremic patients is related to systemic inflammation by interfering with PMNL function. PMNL apoptosis was investigated by assessing morphological features and DNA content. CD11b surface expression was quantified by flow cytometry. Oxidative burst was measured via cytochrome c reduction assay. Chemotaxis was assessed by using an under-agarose migration assay. We found that HDL from CKD and hemodialysis (HD) patients significantly attenuated PMNL apoptosis, whereas HDL isolated from healthy subjects had no effect on PMNL apoptosis. The use of signal transduction inhibitors indicated that uremic HDL exerts anti-apoptotic effects by activating pathways involving phosphoinositide 3-kinase and extracellular-signal regulated kinase. Healthy HDL attenuated the surface expression of CD11b, whereas HDL from CKD and HD patients had no effect. All tested isolates increased the stimulation of oxidative burst, but did not affect PMNL chemotactic movement. In conclusion, HDL may contribute to the systemic inflammation in uremic patients by modulating PMNL functions

Molecular remodeling of the renin-angiotensin system after kidney transplantation

Objective: We aimed at assessing the molecular adaptation of the renin-angiotensin system (RAS) after successful kidney transplantation (KTX). Materials and methods: In this prospective, exploratory study we analyzed 12 hemodialysis (HD) patients, who received a KTX and had excellent graft function six to 12 months thereafter. The concentrations of plasma Angiotensin (Ang) peptides (Ang I, Ang II, Ang-(17), Ang-(15), Ang-(28), Ang-(38)) were simultaneously quantified with a novel mass spectrometry-based method. Further, renin and aldosterone concentrations were determined by standard immunoassays. Results: Ang values showed a strong inter-individual variability among HD patients. Yet, despite a continued broad dispersion of Ang values after KTX, a substantial improvement of the renin/Ang II correlation was observed in patients without RAS blockade or on angiotensin receptor blocker (HD: renin/Ang II R2 = 0.660, KTX: renin/Ang II R2 = 0.918). Ang-(17) representing the alternative RAS axis was only marginally detectable both on HD and after KTX. Conclusions: Following KTX, renin-dependent Ang II formation adapts in non-ACE inhibitor-treated patients. Thus, a largely normal RAS regulation is reconstituted after successful KTX. However, individual Ang concentration variations and a lack of potentially beneficial alternative peptides after KTX call for individualized treatment. The long-term post-transplant RAS regulation remains to be determined.(VLID)456375

Heart Failure with Preserved and Reduced Ejection Fraction in Hemodialysis Patients: Prevalence, Disease Prediction and Prognosis

Background/Aims: Heart failure (HF) is a main cause of mortality of hemodialysis (HD) patients. While HF with reduced ejection fraction (HFrEF) is known to only affect a minority of patients, little is known about the prevalence, associations with clinical characteristics and prognosis of HF with preserved ejection fraction (HFpEF). Methods: We included 105 maintenance HD patients from the Medical University of Vienna into this prospective single-center cohort study and determined the prevalence of HFpEF (per the 2013 criteria of the European Society of Cardiology) and HFrEF (EF <45%), using standardized post-HD transthoracic echocardiography. We also assessed clinical, laboratory and volume status parameters (by bioimpedance spectroscopy). These parameters served to calculate prediction models for both disease entities, while clinical outcomes (frequency of cardiovascular hospitalizations and/or cardiac death) were assessed prospectively over 274 months of follow-up. Results: All but 4 patients (96%) had evidence of diastolic dysfunction. 70% of the entire cohort fulfilled HF criteria (81% HFpEF, 19% HFrEF). Age, female sex, body mass index, blood pressure and dialysis vintage were predictive of HFpEF (sensitivity 86%, specificity 63%; AUC 0.87), while age, female sex, NT pro-BNP, history of coronary artery disease and atrial fibrillation were predictive of HFrEF (sensitivity 85%, specificity 90%; AUC 0.95). Compared to patients without HF, those with HFpEF and HFrEF had a higher risk of hospitalization for cardiovascular reason and/or cardiac death (adjusted HR 4.31, 95% CI 0.46-40.03; adjusted HR 3.24, 95% CI 1.08-9.75, respectively). Conclusion: Diastolic dysfunction and HFpEF are highly prevalent in HD patients while HFrEF only affects a minority. Distinct patient-specific characteristics predict diagnosis of either entity with good accuracy.(VLID)486423

International Journal of Medical Sciences / Sports and HDL-Quality Reflected By Serum Amyloid A and Surfactant Protein B

Background: The aim of this prospective study was to investigate the influence of long-term physical activity on HDL quality, reflected by serum amyloid A (SAA) and surfactant protein B (SPB). Methods and results: 109 healthy subjects were recruited, 98 completed the study. Participants perform within the calculated training pulse for 8 months. The performance gain was measured/quantified by bicycle stress tests at the beginning and end of the observation period. SAA and SPB were measured at baseline and after 4 and 8 months by ELISA. In contrary to HDL-quantity, there was no sports-induced change in SAA or SPB observable. However, significant predictors for SPB-levels were smoking status, BMI and weekly alcohol consumption and for SAA weekly alcohol consumption together with sex and hsCRP-levels. Conclusions: Long-term physical activity increases HDL-quantity but has no impact on HDL-quality reflected by SAA and SPB. Smoking is associated with higher SPB-levels and the weekly alcohol intake is associated with both higher SAA and SPB-levels suggesting a damaging effect of smoking and drinking alcohol on the HDL-quality. We assume that HDL-quality is at least as important as HDL-quantity when investigating the role of HDL in (cardiovascular) disease and should receive attention in further studies dealing with HDL.(VLID)486411