Christian Seelos

Genistein, the Dietary-Derived Angiogenesis Inhibitor, Prevents LDL Oxidation and Protects Endothelial Cells From Damage by Atherogenic LDL

There is now growing evidence that the oxidative modification of LDL plays a potential role in atherosclerosis. In this study, genistein, a compound derived from a soy diet with a flavonoid chemical structure (4,5,7-trihydroxyisoflavone), which was found to inhibit angiogenesis, has been evaluated for its ability to act as an LDL antioxidant and a vascular cell protective agent against oxidized LDL. The results showed that genistein was able to inhibit the oxidation of LDL in the presence of copper ions or superoxide/nitric oxide radicals as measured by thiobarbituric acid-reactive substance formation, alteration in electrophoretic mobility, and lipid hydroperoxides. Bovine aortic endothelial cell- and human endothelial cell-mediated LDL oxidation was also inhibited in the presence of genistein. The 7-O-glucoside of genistein, genistin, was much less effective in inhibiting LDL oxidation in the cell-free and cell-mediated lipoprotein-oxidating systems. Incubating human endothelial cells in the absence or presence of genistein and challenging the cells with already oxidized lipoprotein revealed that in addition to its antioxidative potential during LDL oxidating processes, genistein effectively protected the vascular cells from damage by oxidized lipoproteins. The tyrosine kinase inhibitor genistein was found to block upregulation of two tyrosine-phosphorylated proteins of 132 and 69 kDa in endothelial cells induced by oxidized LDL. Parallel experiments with the inactive analogue daidzein, however, showed that the cytoprotective effect of the isoflavones seems not to be dependent on tyrosine phosphorylation. Our findings will support the suggested and documented beneficial action of a soy diet in preventing chronic vascular diseases and early atherogenic events.

Salicylate inhibits LDL oxidation initiated by superoxide/nitric oxide radicals

Simultaneously produced superoxide/nitric oxide radicals (O⋅− 2/NO⋅) could form peroxynitrite (OONO−) which has been found to cause atherogenic, i.e. oxidative modification of LDL. Aromatic hydroxylation and nitration of the aspirin metabolite salicylate by OONO− has been reported. Therefore we tested if salicylate may be able to protect LDL from oxidation by O⋅− 2/NO⋅ by scavenging the OONO− reactive decomposition products. When LDL was exposed to simultaneously produced O⋅− 2/NO⋅ using the sydnonimine SIN‐1, salicylate exerted an inhibitory effect on LDL oxidation as measured by TBARS and lipid hydroperoxide formation and alteration in electrophoretic mobility of LDL. The cytotoxic effect of SIN‐1 pre‐oxidised LDL to endothelial cells was also diminished when salicylate was present during SIN‐1 treatment of LDL. Spectrophotometric analysis revealed that salicylate was converted to dihydroxybenzoic acid (DHBA) derivatives in the presence of SIN‐1. 2,3‐ and 2,5‐DHBA were even more effective to protect LDL from oxidation by O⋅− 2/NO⋅. Because O⋅− 2/NO⋅ can occur in vivo, the results may indicate that salicylate could act as an efficacious inhibitor of O⋅− 2/NO⋅ initiated atherogenic LDL modification, thus further supporting the rationale of aspirin medication regarding cardiovascular diseases.

Paracetamol Catalyzes Myeloperoxidase-Initiated Lipid Oxidation in LDL

The oxidative modification of LDL may play a significant role in atherogenesis. Myeloperoxidase (MPO) expressed in human atherosclerotic plaques has been suggested to be operative in vivo, making LDL atherogenic. Tyrosyl radicals generated by MPO have been shown to act as physiological pro-oxidants of lipid peroxidation in LDL. Assuming that a variety of phenolic compounds are able to form phenoxyl radicals when exposed to peroxidases, we tested the ability of paracetamol, a known analgesic drug with a tyrosine-like monophenolic structure, to act as a pro-oxidant of lipid peroxidation in LDL. Spectroscopic analyses indicated that paracetamol, similar to tyrosine, could undergo peroxidase-induced phenoxyl radical formation, which was inhibited by the radical scavenger ascorbic acid as well as by heme poisons and catalase. Measurement of conjugated dienes and lipid hydroperoxides in LDL preparations exposed to MPO/H2O2 in the absence or presence of paracetamol revealed that the drug could act as a catalyst of lipid oxidation in LDL. Similar results were found when LDL oxidation was performed with activated human neutrophils, which use MPO to promote lipid peroxidation. In conclusion, the results suggest that paracetamol could act, via a phenoxyl radical, as a catalyst of LDL oxidative modification by MPO.

The salicylate metabolite gentisic acid, but not the parent drug, inhibits glucose autoxidation-mediated atherogenic modification of low density lipoprotein

Oxidation of low density lipoprotein (LDL) by glucose‐derived radicals may play a role in the aetiology of atherosclerosis in diabetes. Salicylate was shown to scavenge certain radicals. In the present study, aspirin, salicylate and its metabolites 2,5‐ and 2,3‐dihydroxybenzoic acid (DHBA) were tested for their ability to impair LDL oxidation by glucose. Only the DHBA derivatives, when present during LDL modification, inhibited LDL oxidation and the increase in endothelial tissue factor synthesis induced by glucose oxidised LDL. The LDL glycation reaction was not affected by DHBA. The antioxidative action of DHBA may be attributed to free radical scavenging and/or chelation of transition metal ions catalysing glucose autoxidation.

Salicylate promotes myeloperoxidase-initiated LDL oxidation: antagonization by its metabolite gentisic acid

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. Tyrosyl radicals generated by myeloperoxidase (MPO) can act as prooxidants of LDL oxidation. Taking into consideration, that monophenolic compounds are able to form phenoxyl radicals in presence of peroxidases, we have tested salicylate, in its ability to act as a prooxidant in the MPO system. Measurement of conjugated dienes and lipid hydroperoxides were taken as indicators of lipid oxidation. Exposure of LDL preparations to MPO in presence of salicylate revealed that the drug could act as a catalyst of lipid oxidation in LDL. The radical scavenger ascorbic acid as well as heme poisons (cyanide, azide) and catalase were inhibitory. The main metabolite of salicylic acid, gentisic acid, showed inhibitory action in the MPO system. Even when lipid oxidation was maximally stimulated by salicylate the LDL oxidation was efficaciously counteracted in presence of gentisic acid at salicylate/gentisic acid ratios that could be reached in plasma of patients receiving aspirin medication. Gentisic acid was also able to impair the tyrosyl radical catalyzed LDL peroxidation. The results suggest that salicylate could act like tyrosine via a phenoxyl radical as a catalyst of LDL oxidative modification by MPO. But the prooxidant activity of this radical species is effectively counteracted by the salicylate metabolite gentisic acid.

Preoperative TNM-classification is a better prognostic indicator for recurrence nof hepatocellular carcinoma after liver transplantation than albumin mRNA in peripheral blood

Abstract Background/Aims: Survival after orthotopic liver transplantation for hepatocellular carcinoma is limited by a high rate of tumor recurrence. A polymerase chain reaction assay based on the detection of albumin mRNA expression in peripheral blood for detection of hematogenous micrometastasis of hepatocellular carcinoma has been described, which may help to select candidates for orthotopic liver transplantation. Methods: The prognostic value of a highly sensitive nested reverse transcription-polymerase chain reaction assay was evaluated in comparison with the TNM-classification of the Internal Union against Cancer in a population of liver transplant candidates. Results: Eighty patients with liver disease and 42 control patients were evaluated. Six of 21 patients with hepatocellular carcinoma and 11 of 59 patients with other diseases of the liver were positive for albumin reverse transcription-polymerase chain reaction, making this assay an indicator of ongoing liver damage without absolute specificity for hepatocellular carcinoma. Twelve patients with hepatoma were followed after liver transplantation and seven of those patients had a tumor recurrence within 12 months. Six of these patients with recurrence had International Union against Cancer stage IV A tumors preoperatively, while only one of them was positive for albumin reverse transcription-polymerase chain reaction before transplantation. Only one patient with a stage I to III tumor had a recurrence within 12 months. Conclusions: Detection of albumin mRNA in peripheral blood by reverse transcription-polymerase chain reaction seems to be an unreliable marker for assessing hematogenous spread of hepatocellular carcinoma. With International Union against Cancer stage IV A being a much better predictor of tumor recurrence, the practical value of albumin mRNA reverse transcription-polymerase chain reaction for patient selection in liver transplant candidates seems to be very limited.

Uptake and endocytic pathway of transferrin and iron in perfused rat liver.

Uptake and distribution of transferrin and iron in perfused rat liver are dependent on perfusion temperature, time and uptake affinity. Transferrin passes at least two different compartments on its receptor-mediated recycling pathway, which are separable by centrifugation in a shallow Nycodenz gradient. Perfusion at lowered temperature (16 degrees C) is sufficient for internalization of transferrin and iron. Passage of radiolabelled iron to other than endosomal compartments as well as recycling of labelled transferrin are largely suppressed at this perfusion temperature, as much less is released by further perfusion with unlabelled transferrin than at 4 degrees C where the ligand is largely washed off the surface, or 37 degrees C, where the recycling pathway is operating. But also at lowered temperature only a part of the iron in endosomal fractions can be assigned to transferrin. A considerable part of the total uptake of transferrin and iron can be attributed to low-affinity mechanisms even at very low transferrin concentrations. Transferrin receptors are concentrated in endosomal fractions in comparison to fractions representing different plasma membrane domains of the liver. Endosomal fractions specifically display detergent-activated NADH-acceptor oxidoreductase which may be part of the iron uptake system.

Phosphorylation of tyrosine prevents dityrosine formation in vitro

Treatment of L‐tyrosine in a peroxidase/H2O2, system results in the formation of dityrosine. However, the phosphoester derivative of tyrosine, O‐phospho‐L‐tyrosine, was unable to form dityrosine even in mixtures with free L‐tyrosine. Dephosphorylation of O‐phospho‐L‐tyrosine by alkaline phosphatase followed by horseradish peroxidase/H2O2, treatment resulted in the formation of dityrosine. Our in vitro results indicate that phosphorylation/dephosphorylation of L‐tyrosine may regulate dityrosine formation, and is supposed to play an important role in protein‐protein interactions, i.e. cross‐linking.

Tyrosine phosphorylation blocks tyrosine free radical formation and, hence, the hormonogenic iodination reaction.

It is known that a tyrosine free radical is produced during the hormonogenic iodination reaction of tyrosine residues on thyroglobulin. In the hormonogenic region of thyroglobulin, phosphorylated tyrosine residues have been detected. Using an vitro tyrosine iodinating system we report that the hormonogenic reaction cannot go off if tyrosine becomes phosphorylated. Enzymatic dephosphorylation of the modified amino acid restored the ability of the molecule to become iodinated. Considering the mechanism of the tyrosine free radical formation, these observations are due to the inability of the phosphorylated amino acid to form a free radical. Our data may suggest a putative regulatory mechanism in thyroid hormone synthesis by phosphorylation of hormonogenic tyrosine residues on thyroglobulin.

OXIDIZED LOW-DENSITY-LIPOPROTEIN TRIGGERS PROGRAMMED DEATH OF ENDOTHELIAL CELLS

Incubation of bovine aortic as well as human umbilical vein endothelial cells with either oxidized or native low‐density‐lipoprotein in the presence of trace amounts of copper induced morphological changes of the cells and chromatin fragmentation characteristic for programmed cell death. Shrinkage of cells was evident after 6 to 8 hours of incubation and clearly preceded release of lactate dehydrogenase as a marker of cell permeability. Condensation of nuclear chromatin and internucleosomal cleavage was demonstrated by Hoechst staining and gel electrophoresis, respectively. Thus, by inducing active death of endothelial cells oxidized low‐density‐lipoprotein might negatively influence tissue homeostasis of the endothelium and thereby promote the development of atherosclerotic plaques.