Hilde Laggner

Hydrogen sulphide: A novel physiological inhibitor of LDL atherogenic modification by HOCl

Hypochlorite (HOCl), the product of the activated myeloperoxidase/H2O2/chloride (MPO/H2O2/Cl− ) system is favored as a trigger of LDL modifications, which may play a pivotal role in early atherogenesis. As HOCl has been shown to react with thiol-containing compounds like glutathione and N-acetylcysteine protecting LDL from HOCl modification, we have tested the ability of hydrogen sulfide (H2S)—which has recently been identified as an endogenous vasorelaxant—to counteract the action of HOCl on LDL. The results show that H2S could inhibit the atherogenic modification of LDL induced by HOCl, as measured by apolipoprotein alterations. Beside its HOCl scavenging potential, H2S was found to inhibit MPO (one may speculate that this occurs via H2S/heme interaction) and destroy H2O2. Thus, H2S may interfere with the reactants and reaction products of the activated MPO/H2O2/Cl− system. Our data add to the evidence of an anti-atherosclerotic action of this gasotransmitter taking the role of HOCl in the atherogenic modification of LDL into account.

The novel gaseous vasorelaxant hydrogen sulfide inhibits angiotensin-converting enzyme activity of endothelial cells

Objective Beside NO (nitric monoxide) and CO (carbon monoxide), H2S (hydrogen sulfide) has been identified recently as the third gasotransmitter. By acting directly on KATP-channels on smooth muscle cells (SMC) H2S possesses vasorelaxing properties. It has the potential to react with metal ions (i.e. Cu, Fe, Zn) in metalloproteins. Angiotensin-converting enzyme (ACE), responsible for vasoconstriction, is a zinc (Zn2+) containing enzyme. We therefore hypothesized that H2S may interact with the Zn2+ in the active center of ACE, modulating (inhibiting) enzyme activity. Methods ACE activity was measured on the surface of human endothelial cells (HUVECs) monolayers in culture, ex-vivo in umbilical veins and in HUVEC protein extracts. Quantitative real-time polymerase chain reaction (PCR) was used to study the effect of H2S on ACE mRNA expression in HUVECs. Results H2S inhibited the activity of ACE in HUVEC protein extracts in a dose-dependent manner, and only Zn2+ but not Cd2+, Ca2+ or Mg2+ could counteract the inhibitory effect. Cell-surface ACE activity was inhibited by H2S on HUVEC monolayers and in ex-vivo umbilical veins. No influence of H2S on ACE mRNA expression was observed. Conclusion H2S exhibits direct inhibitory action on ACE activity in HUVECs, obviously by interfering with the Zn2+ in the active center of the enzyme. Thus, beside the known influence of H2S on SMC KATP-channels, the observed direct ACE inhibitory effect may add to the vasorelaxant effect of H2S in the vasculature by reducing angiotensin II production and inhibiting bradykinin degradation.

Hydrogen sulfide destroys lipid hydroperoxides in oxidized LDL.

LOOHs (lipid hydroperoxides) in oxLDL [oxidized LDL (low-density lipoprotein)] are potentially atherogenic compounds. Recently, H2S was identified as the third endogenous gasotransmitter in the vasculature. H2O2 is known to be destroyed by H2S. Assuming that H2S may also react with LOOHs, the results show that H2S can destroy LOOHs in oxLDL. The ability of LOOH-enriched LDL to induce HO-1 (haem oxygenase 1) in endothelial cells was abolished by H2S pretreatment. HPLC analysis showed that 9-HPODE [(9S)-hydroperoxy-(10E,12Z)-octadecadienoic acid], a compound found in oxLDL, was reduced to 9-HODE [(9S)-hydroxy-(10E,12Z)-octadecadienoic acid] in the presence of H2S. Thus H2S may act as an antiatherogenic agent by reducing LOOHs to the less reactive LOHs and could abrogate the pathobiological activity of oxLDL.

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.

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.

The main components of St John's Wort inhibit low-density lipoprotein atherogenic modification: A beneficial “side effect” of an OTC antidepressant drug?

Hypericin and pseudohypericin are polycyclic–phenolic structurally related compounds found in Hypericum perforatum L. (St Johns wort). As hypericin has been found to bind to LDL one may assume that it can act as antioxidant of LDL lipid oxidation, a property which is of prophylactic/therapeutic interest regarding atherogenesis as LDL oxidation may play a pivotal role in the onset of atherosclerosis. Therefore, in the present paper hypericin, pseudohypericin and hyperforin, an other structurally unrelated constituent in St Johns wort were tested in their ability to inhibit LDL oxidation. LDL was isolated by ultracentrifugation and oxidation was initiated either by transition metal ions (copper), tyrosyl radical (myeloperoxidase/hydrogen peroxide/tyrosine) or by endothelial cells (HUVEC). LDL modification was monitored by conjugated diene and malondialdehyde formation. The data show that all compounds (hypericin, pseudohypericin and hyperforin) at doses as low as 2.5 μmol/l are potent antioxidants in the LDL oxidation systems used. The results indicate that the derivatives found in Hypericum perforatum have possible antiatherogenic potential.

Vitamin C inhibits NO-induced stabilization of HIF-1α in HUVECs

Abstract HIF-1α represents the oxygen-regulated sub-unit of the transcription factor HIF-1, which regulates the transcription of numerous genes involved in cellular response to hypoxia and oxidative stress. It is shown here that nitric oxide (NO) induces HIF-1α stabilization in human endothelial cells from umbilical cords (HUVECs) under normoxic conditions. HIF-1α protein was increased ∼ 36-fold after incubation with 500 μM DETA-NO, which releases a steady state NO concentration of roughly one thousandth of the initial concentration of the donor. Loading of the cells with vitamin C counteracted NO-induced HIF-1α accumulation. Based on the observations that oxidative and nitrosative stress can influence the activity of the proteasomal system, which is responsible for the non-lysosomal degradation of proteins, among them HIF-1α, it was investigated whether NO-induced stabilization of HIF-1α might be due to reduced 20S proteasomal activity. This hypothesis could not be proved, because NO concentrations to inhibit 20S proteasomal activity were about one order of magnitude higher than that to inhibit HIF-1α degradation.