Bernhard M.K. Gmeiner

Copper- and magnesium protoporphyrin complexes inhibit oxidative modification of LDL induced by hemin, transition metal ions and tyrosyl radicals

The oxidative modification of LDL may play an important role in the early events of atherogenesis. Thus the identification of antioxidative compounds may be of therapeutic and prophylactic importance regarding cardiovascular disease. Copper-chlorophyllin (Cu-CHL), a Cu2+-protoporphyrin IX complex, has been reported to inhibit lipid oxidation in biological membranes and liposomes. Hemin (Fe3+-protoporphyrin IX) has been shown to bind to LDL thereby inducing lipid peroxidation. As Cu-CHL has a similar structure as hemin, one may assume that Cu-CHL may compete with the hemin action on LDL. Therefore, in the present study Cu-CHL and the related compound magnesium-chlorophyllin (Mg-CHL) were examined in their ability to inhibit LDL oxidation initiated by hemin and other LDL oxidizing systems. LDL oxidation by hemin in presence of H2O2 was strongly inhibited by both CHLs. Both chlorophyllins were also capable of effectively inhibiting LDL oxidation initiated by transition metal ions (Cu2+), human umbilical vein endothelial cells (HUVEC) and tyrosyl radicals generated by myeloperoxidase (MPO) in presence of H2O2 and tyrosine. Cu- and Mg-CHL showed radical scavenging ability as demonstrated by the diphenylpicrylhydracylradical (DPPH)-radical assay and estimation of phenoxyl radical generated diphenyl (dityrosine) formation. As assessed by ultracentrifugation the chlorophyllins were found to bind to LDL (and HDL) in serum. The present study shows that copper chlorophyllin (Cu-CHL) and its magnesium analog could act as potent antagonists of atherogenic LDL modification induced by various oxidative stimuli. As inhibitory effects of the CHLs were found at concentrations as low as 1 μmol/l, which can be achieved in humans, the results may be physiologically/therapeutically relevant.

Hydrogen sulphide induces HIF-1α and Nrf2 in THP-1 macrophages

The transcription factor HIF-1α regulates the adaptive response of cells to hypoxia and oxidative stress. In addition, an important regulatory role for HIF-1α in immune reactions and inflammation is suggested. The present study attempts to investigate the effect of the gaseous signalling molecule hydrogen sulphide (H2S) on HIF-1α in THP-1 macrophages using the slow H2S releasing donor GYY4137. We found that H2S induced HIF-1α protein accumulation in THP-1 macrophages in a concentration-dependent manner. Western blot analysis of cell fractions showed that HIF-1α protein translocates into the nucleus and leads to an increase of its target protein glucose transporter-1 (GLUT-1). Activation of nuclear factor-κB (NF-κB), as well as secretion of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were reduced in the presence of H2S. These findings indicate that HIF-1α accumulation due to H2S was not triggered by the NF-κB pathway. The antioxidant pathway Nrf2/HO-1 (nuclear factor erythroid 2-related factor 2/heme oxygenase-1) was activated by H2S. Inhibition of the p38 mitogen-activated protein kinase (MAPK) reversed H2S mediated effects, suggesting that the p38 MAPK pathway may be involved in H2S induced HIF-1α/Nrf2 signalling pathways.

S-carbamoylation impairs the oxidant scavenging activity of cysteine: its possible impact on increased LDL modification in uraemia.

Carbamoylation is the non-enzymatic reaction of cyanate with amino-, hydroxy- or thiol groups. In vivo, amino group modification (N-carbamoylation) resulting in altered function of proteins/amino acids has been observed in patients suffering from uraemia due to urea-derived cyanate. Uraemia has been linked to impaired antioxidant defense. As thiol-compounds like cysteine, N-acetyl cysteine and GSH have oxidant scavenging properties one may speculate that thiol-group carbamoylation (S-carbamoylation) may impair their protective activity. Here we report on the effect of S-carbamoylation on the ABTS free radical and HOCl scavenging property of cysteine as well on its ability to protect LDL from atherogenic modification induced by AAPH generated peroxylradicals or HOCl. The results show that S-carbamoylation impaired the ABTS free radical and HOCl scavenging property of the thiol-compounds tested. The ability of the thiols to protect LDL from lipid oxidation and apolipoprotein modification was strongly diminished by S-carbamoylation. The data indicate that S-carbamoylation could impair the free radical and HOCl scavenging of thiol-amino acids reducing their protective property against LDL atherogenic modification by these oxidant species. As S-carbamoylation is most effective at pH 7 to 5 in vivo thiol-carbamoylation may especially occur at sites of acidic extracellular pH as in hypoxic/inflammatory macrophage rich areas like the atherosclerotic plaque where increased LDL oxidation has been found and may contribute to the higher oxidative stress in uraemia.

Carbamoylation abrogates the antioxidant potential of hydrogen sulfide

Hydrogen sulfide (H2S) has been identified as the third gasotransmitter. Beside its role as signaling molecule in the cardiovascular and nervous system the antioxidant and cyto-protective properties of H2S have gained much attention. In the present study we show that cyanate, an uremic toxin which is found in abundant concentration in sera of patients suffering from chronic kidney disease (CKD), can abrogate the antioxidant and cytoprotective activity of H2S via S-carbamoylation reaction, a reaction that previously has only been shown to have a physiological effect on cysteine groups, but not on H2S. Carbamoylation strongly inhibited the free radical scavenging (ABTS(+·) and alkylperoxyl ROO(·)) properties of H2S. The extent of intracellular ROS formation induced by ROO(·) was diminished by H2S whereas carbamoylation counteracted the protective effect. Reagent HOCl was rapidly inactivated by H2S in contrast to the carbamoylated compound. Protein modification by HOCl was inhibited by H2S but carbamoylation significantly reduced the effect. Thus, S-carbamoylation of low molecular weight thiols by abrogating their antioxidant potential may contribute to the higher oxidative stress observed in CKD.

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: Implication in hemodialysis mediated atherogenic LDL modification

Objective: Al3+ stimulates Fe2+ induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al3+ and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al3+ on LDL oxidation. Materials and methods: Using different LDL modifying systems (Fe2+, Cu2+, free radical generating compounds, human endothelial cells, hemin/H2O2 and HOCl), the influence of Al3+ on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation. Results: Al3+ could stimulate the oxidizability of LDL by Fe2+, but not in the other systems tested. Al3+ and Fe2+ were found to bind to LDL and Al3+could compete with Fe2+ binding to the lipoprotein. Fluorescence polarization data indicated that Al3+ does not affect the phospholipid compartment of LDL. Conclusions:The results indicate that increased LDL oxidation by Fe2+ in presence of Al3+ might be due to blockage of Fe2+ binding sites on LDL making more free Fe2+ available for lipid oxidation.

Detection of Survival Motor Neuron Protein in Buccal Cells Through Electrochemiluminescence-Based Assay

Spinal muscular atrophy (SMA) is a severe autosomal recessive disorder affecting one in every 10,000 live births. The disease is characterized by loss of alpha-motor neurons in the spinal cord that leads to progressive atrophy and weakness of limb and trunk muscles. This neuromuscular disorder results from deletions and/or mutations within the survival motor neuron 1 (SMN1) gene, leading to a pathologically decreased expression of functional full-length SMN protein. Here we report on the investigation to measure SMN protein levels through electrochemiluminescence immunoassay (ECLIA). This simple assay is a highly quantitative method able to measure SMN protein levels in human, mouse, and rat samples throughout a wide working range with low intra- and interassay error. The sensitivity for human SMN is 30u2009pg/mL and provides a new tool for the set up of high-throughput screening for basic research. Moreover, we describe a novel tool for a noninvasive assessment of SMN in buccal cells derived from healthy donors, SMA carriers, and SMA patients. The availability of a validated quantitative ECLIA should improve the investigation of novel compounds for the treatment of SMA.

Carbamoylated free amino acids in uremia: HOCl generates volatile protein modifying and cytotoxic oxidant species from N-carbamoyl-threonine but not threonine.

N-carbamoylation is the non-enzymatic reaction of cyanate with amino groups. Due to urea-formed cyanate in uremic patients beside carbamoylated proteins also free amino acid carbamoylation has been detected, a modification which has been linked to disturbed protein synthesis as NH(2)-derivatisation interferes with peptide bond formation. HOCl the product of the activated MPO/H(2)O(2)/Cl(-) system is known to react with the NH(2)-group of free amino acids to form chloramines which could exert some protective effect against protein modification and cytotoxicity induced by HOCl. As N-carbamoylation may inhibit formation of chloramines we have used N-carbamoyl-threonine as a model amino acid to study its ability to limit the reactivity of HOCl with proteins (LDL and human serum albumin) and cells (THP-1 monocytes and coronary artery endothelial cells). The data indicate that N-carbamoylation completely abolished the protein- and cell-protective effect of threonine against HOCl attack. In contrast to threonine the reaction of HOCl with carbamoyl-threonine resulted in the formation of volatile oxidant species with protein modifying and cytotoxic potential. The volatile lipophilic inorganic monochloramine (NH(2)Cl) was identified as a breakdown product of this reaction.

Known players, new interplay in atherogenesis: Chronic shear stress and carbamylated-LDL induce and modulate expression of atherogenic LR11 in human coronary artery endothelium

In this study we examined whether low-density lipoprotein (LDL) receptor family members represent a link between blood flow characteristics and modified low-density lipoproteins involved in endothelial injury, a pivotal factor in atherogenesis. We demonstrated the expression of pro-atherogenic LDL receptor relative (LR11) for the first time in human coronary artery endothelial cells (HCAEC) in vitro and in vivo. Next, LR11 expression and regulation were explored in HCAEC cultured conventionally or on the inner surface of hollow fiber capillaries under exposure to shear stress for 10 days in the presence or absence of LDL. There was no LR11 expression under static conditions. When exposed to chronic low shear stress (2.5 dynes/cm²) transmembrane and soluble endothelial-LR11 were detected in high levels irrespective of the type of LDL added (carbamylated or native). In contrast, chronic high shear stress (25 dynes/cm²) inhibited the LR11-inducing effect of LDL such that transmembrane and soluble LR11 expression became non-detectable with native LDL. Carbamylated LDL significantly counteracted this atheroprotective effect of high shear stress as shown by lower, yet sustained expression of soluble and transmembrane LR11. Oxidised LDL showed similar effects compared to carbamylated LDL but caused significantly lower LR11 expression under chronic high shear stress. Medium from HCAEC under LR11-inducing conditions enhanced vascular smooth muscle cell migration, which was abrogated by the anti-LR11 antibody. Expression of LR11 depended entirely on p38MAPK phosphorylation. We conclude that coronary endothelial LR11 expression modulated by LDL and chronic shear stress contributes to atherogenesis. LR11 and p38MAPK are potential targets for prevention of atherosclerosis.

The uremic toxin indoxyl sulfate acts as a pro- or antioxidant on LDL oxidation

Uremic toxins have been shown to play a role in chronic kidney disease (CKD) associated oxidative stress. Oxidative stress and inflammation have been associated with increased risk of cardiovascular disease in uraemia. The oxidative modification of LDL may play a role in early atherogenesis. Enhanced LDL oxidation has been found in uremic patients which may account for accelerated atherosclerosis observed in CKD. The uremic toxin indoxyl sulfate (IS) has been reported to exert oxidative and antioxidative activity. Thus, in the present study we have investigated the influence of IS on the atherogenic modifications of LDL exposed in vitro to different oxidising systems. The transition metal ion (Cu(2) ) and hemin/H2O2 induced lipid oxidation reactions monitored by conjugated diene formation, were inhibited by the presence of IS, which points to possible antioxidant effects by this uremic toxin. A protective effect of IS on LDL apoprotein modification by the exposure to the product of the myeloperoxidase/H2O2/Cl(-) system HOCl, was also observed as estimated by protein carbonyl formation. In contrast, a marked increase in conjugated dienes and lipid hydroperoxides was observed when lipid oxidation was initiated by the free radical generator AAPH in presence of IS. The GC-MS analysis revealed the formation of indole-2,3-dione and 6,12-dihydro-6,12-dioxo-indolo[2,1-b]quinazoline (tryptanthrine) in IS/AAPH reaction. A scheme for the generation of tryptanthrine from IS via indoxyl radicals is proposed, which may facilitate LDL lipid oxidation. Our observations add further insight in the Janus-faced properties of this important uremic toxin.

Investigating the Role of H2S in 4-HNE Scavenging

4-HNE (4-hydroxy-2-nonenal) is a highly reactive α,β-unsaturated aldehyde generated from oxidation of polyunsaturated fatty acids and has been suggested to play a role in the pathogenesis of several diseases. 4-HNE can bind to amino acids, proteins, polynucleotides, and lipids and exert cytotoxicity. 4-HNE forms adducts (Michael adducts) with cysteine, lysine, as well as histidine on proteins with the thiol function as the most reactive nucleophilic moiety. Thus, detoxification strategies by 4-HNE scavenging compounds might be of interest. Recently, hydrogen sulfide (H2S) has been identified as an endogenous vascular gasotransmitter and neuromodulator. Assuming that the low-molecular thiol H2S may react with 4-HNE, methods to monitor the ability of H2S to counteract the protein-modifying and cytotoxic activity of 4-HNE are described in this chapter.