Induction of glutathione synthesis in human keratinocytes by Ginkgo biloba extract (EGb761).

The objective of the present study was to characterize the action of Ginkgo biloba extract (EGb761) and its sub-fractions on glutathione homeostasis in a human keratinocyte cell culture model. Cells were incubated with EGb761, its purified flavonoid (quercetin, kaempferol, rutin) or terpenoids (gingkolides A, B, C, J, bilobalide) constituents or the vehicle for up to 72 hours. Incubation of keratinocytes with the purified flavonoids or terpenoids did not affect cellular GSH levels. However, EGb761 treatment (up to 200 microg/ml) resulted in a dose-dependent increase of cellular GSH. Western blot analysis of extracts from cells treated with EGb761 revealed increased levels of the catalytic subunit of gamma-glutamylcysteinyl synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis. The abundance of mRNA for the catalytic subunit (assayed by RT-PCR) was also increased by the treatment with EGb761. Increased levels of cellular GSH by EGb761 were also observed in other cell lines including those from human bladder and liver as well as in murine macrophages indicating that the induction of gamma-GCS mRNA, protein and GSH may be an ubiquitous effect of EGb761 in mammalian cells.

Protein binding of procyanidins: studies using polyacrylamide gel electrophoresis and French maritime pine bark extract.

The application of PAGE to determine the interaction between procyanidins and proteins, as presented here, enables one to directly determine the binding of either a pure of a complex mixture of flavonoids to a particular protein. If the protein of interest is an enzyme, the combination of PAGE with quantitative activity measurements allows identifying whether a change in the enzyme activity is related to the binding. Data presented suggest that PBE and EGb 761 have protein-binding properties, which, in addition to their redox-based effects, could provide a biochemical basis for their action in biological systems.

Solar ultraviolet-induced erythema in human skin and nuclear factor-kappa-B-dependent gene expression in keratinocytes are modulated by a French maritime pine bark extract.

The procyanidin-rich French maritime pine bark extract Pycnogenol (PBE) has been investigated for its effect in protecting human skin against solar UV-simulated light-induced erythema. Twenty-one volunteers were given an oral supplementation of Pycnogenol: 1.10 mg/kg body weight (b. wt.)/d for the first 4 weeks and 1.66 mg/kg b. wt./d for the next 4 weeks. The minimal erythema dose (MED) was measured twice before supplementation (baseline MED), once after the first 4 weeks of supplementation, and a last time at the end of the study. The UVR dose necessary to achieve 1 MED was significantly increased during PBE supplementation. Since the activation of the pro-inflammatory and redox-regulated transcription factor NF-kappaB is thought to play a major role in UVR-induced erythema, the effect of PBE was also investigated in the human keratinocyte cell line HaCaT. PBE, added to the cell culture medium, inhibited UVR-induced NF-kappaB-dependent gene expression in a concentration-dependent manner. However, NF-kappaB-DNA-binding activity was not prevented, suggesting that PBE affects the transactivation capacity of NF-kappaB. These data indicate that oral supplementation of PBE reduces erythema in the skin. Inhibition of NF-kappaB-dependent gene expression by PBE possibly contributes to the observed increase in MED.

Enzyme inhibition and protein-binding action of the procyanidin-rich french maritime pine bark extract, pycnogenol: effect on xanthine oxidase.

Pycnogenol, an extract from French maritime pine bark (PBE), is a complex mixture of bioflavonoids with reported protective effects against disease. PBE is an effective scavenger of reactive oxygen species, and its main constituents are procyanidins of various chain lengths. To find out the biochemical basis of action of PBE on enzyme activity, involvement of its redox activity and direct binding to the enzyme in its subsequent action on enzyme activity have been investigated. PBE dose-dependently inhibited the activities of xanthine oxidase, xanthine dehydrogenase, horseradish peroxidase, and lipoxygenase, but it did not affect the activities of glucose oxidase, ascorbate oxidase, or elastase. To characterize the mechanism of PBE action, studies were focused on xanthine oxidase and glucose oxidase. Under non-denaturing conditions, PBE changed the electrophoretic mobility of xanthine oxidase but not of glucose oxidase. Gel filtration chromatography confirmed higher molecular weight complexes of xanthine oxidase and xanthine dehydrogenase in the presence of PBE. It was found that hydrophobic bonding might be the dominant mode of interaction between PBE and xanthine oxidase. The importance of the binding in the effect of PBE on enzyme activity was supported by the observation that PBE binds to and inhibits catalase, but not superoxide dismutase. However, no correlation was found between superoxide/hydroxyl radical scavenging activity and the inhibitory effect on xanthine oxidase activity of PBE, various purified flavonoids, or other complex mixtures of bioflavonoids. The results indicate that PBE selectively inhibits xanthine oxidase through binding to the enzyme rather than by the redox activity.

Ferric nitrilotriacetate induced DNA and protein damage: inhibitory effect of a fermented papaya preparation.

The carcinogen Fe-NTA catalyzes the hydrogen peroxide-derived production of free radicals and possibly acts through a mechanism involving oxidative stress. Fermented papaya preparation (FPP) has been reported as a natural antioxidant able to prevent lipid peroxidation in vitro and in vivo. However, little is known about the antioxidant properties of FPP regarding iron-mediated oxidative damage to DNA and proteins. In the present study FPP protected supercoiled plasmid DNA against Fe-NTA plus H2O2 induced single and double strand breaks. Similar protective effects of FPP were evident when human T-lymphocytes were challenged with Fe-NTA/H2O2 and DNA damage was determined using the Comet assay. Fe-NTA/H2O2 also induced fragmentation of bovine serum albumin (BSA) in vitro and depleted cellular GSH levels in lymphocytes. BSA fragmentation and GSH depletion were dose-dependently counteracted by FPP. EPR spin trapping studies demonstrated that antioxidant properties of FPP are related to both hydroxyl scavenging as well as iron chelating properties.

Protective effect of defibrotide on perfusion induced endothelial damage.

In the present study, in vitro effects of Defibrotide (D) on perfusion-induced changes in the morphology of endothelium were investigated by scanning (SEM) and transmission (TEM) electron microscope. Human umbilical cord veins were incubated or perfused with platelet-rich plasma alone (PRP) or platelet-rich plasma with Defibrotide (PRP+D) at 3ml/min or 14ml/min and the changes observed were compared. SEM examination of luminal surfaces demonstrated that perfusion with high flow rates may damage endothelial cells and lead to morphological changes which may be prevented by the presence of Defibrotide in the perfusate. Also, the marked reduction in the number of adhered platelets on luminal surface of veins incubated or perfused with Defibrotide compared to veins treated with platelet-rich plasma only revealed that Defibrotide has anti-thrombotic effects. TEM examination of ruthenium red (RR) stained thin sections of veins demonstrated that perfusion disrupts the glycosaminoglcan (GAG) coat on endothelial cells. But the presence of D in the perfusate preserves the integrity of GAG, indicating further cytoprotective effects of the drug on endothelium.

Long-term effect of 17beta-estradiol and thrombin on tissue factor pathway inhibitor release from HUVEC.

In spite of the increasing evidence that estrogens have protective effects on the vascular system, the evidence that estrogens may contribute to the risk of thrombosis is still being debated. We investigated the effect of 17beta-estradiol (E2) on tissue factor pathway inhibitor (TFPI) release from of cultured human umbilical vein endothelial cells (HUVEC). In this study HUVEC were harvested by collegenase treatment and cultured in multiwelled plates with medium 199 supplemented with 10% fetal calf serum and antibiotics. The cells were incubated in the presence or absence of E2 (1 and 100 nM) with/without thrombin (4 U/mL) for 6 or 24 hours. After the incubations TFPI level of media were measured by IMUBIND Total Eliza kit. Our results demonstrates that E2 at physiological concentrations decreases the release of TFPI from HUVEC significantly. Thrombin also decreases TFPI antigen levels detected in culture media. When combined with thrombin the effect of estrogen is not visible due the much higher effectivity of thrombin in diminishing TFPI levels. These results show that E2 shifts the hemostatic balance towards the procoagulant phase through lowering the TFPI levels secreted by the endothelium.

17Beta-estradiol modulates endothelin-1 expression and release in human endothelial cells.

OBJECTIVE: In this study the role of 17beta-estradiol (E2) in the regulation of endothelin-1 (ET-1) mRNA expression and secretion was investigated in cultured human umbilical vein endothelial cells (HUVECs). METHODS: Endothelial cells were either deprived of or treated with 17beta-estradiol (10(-9), 10(-7) M) for 48 h. After the incubation, the effect of E2 on ET-1 gene expression was evaluated by Northern blot analysis. ET-1 release into the media was measured by radioimmunoassay after 6 h of incubation under basal conditions and upon stimulation with thrombin (4 U/ml). In addition, the cyclic guanosine 5'-monophosphate (cGMP) content of cells was assayed by immunoassay. In order to exclude the role of nitric oxide (NO) in E2-induced effects on endothelin-1 gene expression and secretion, nitric oxide synthase (NOS) inhibitor, N-nitro L-arginine methyl ester (1 mM) (L-NAME) was added to the media of some cultures. RESULTS: Incubation of HUVECs with 10(-9) and 10(-7) M E2 for 48 h resulted in a 30 and 47% inhibition of ET-1 mRNA expression, respectively. Incubation with E2 also decreased the basal and thrombin-stimulated ET-1 release while increasing the cGMP content of cells significantly. NOS inhibitor L-NAME increased the release of ET-1 from E2-incubated cells but did not alter the ET-1 release from hormone-deprived cells. However, ET-1 secretion of E2-treated cells were significantly less than the deprived ones. Northern blot analyses also demonstrated that inhibition of NOS only partly attenuated the effect of E2 on ET-1 gene expression. In the presence of L-NAME, treatment with 10(-7) M E2 caused a 12% decrease in ET-1 gene expression. CONCLUSION: The results demonstrate that E2 may play both direct and indirect role in regulation of ET-1 gene expression and production in human endothelial cells. E2-induced increase in NO but decrease in ET-1 production may partly explain the mechanism of the protective effects of the hormone on the cardiovascular system.

Molecular aspects of procyanidin biological activity: disease preventative and therapeutic potentials.

There is a growing interest in the utilization of procyanidins for their dietary and pharmacological properties. A wide spectrum of beneficial activity for human health has been advocated for procyanidins due, in part, to their strong antioxidant activity. More recently the ability of procyanidins to affect gene expression and cell response in vitro has been reported, providing a novel mechanistic perspective on the biological activity of these phytochemicals. This article reviews recent cellular and molecular aspects of the biological activity of procyandins and discusses their disease preventative and therapeutic potentials.

Nitric oxide synthesis and TNF-alpha secretion in RAW 264.7 macrophages: mode of action of a fermented papaya preparation.

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.

Role of endothelin in obstructive jaundice.

Mediators responsible for renal changes in obstructive jaundice are not specified. This study is designed to study the role of endothelin-1 (ET-1) in obstructive jaundice in rats. Animals were randomly placed into five experimental groups. Group 1 (N = 3) was the sham-operated group. Group 2 (N = 8) after common bile duct (CBD) ligation, received bosentan, which is a nonselective endothelin receptor blocker, 50 mg/kg/day for seven days. Group 3 (N = 7) received 1 microg/kg/day captopril. Group 4 (N = 7) was given both drugs orally for seven days. Group 5 (N = 6) after CBD ligation, received Arabic gum as the vehicle. Blood was drawn from the infrahepatic vena cava for the determination of ET-1, bilirubin, creatinine, protein oxidation products, hyaluronic acid, and beta-N-acetyl-hexosaminase. Liver tissue samples were obtained to determine glutathione levels. ET-1, protein oxidation products, hyaluronic acid, bilirubin, and creatinine levels increased significantly in the control group when compared with sham. Bosentan effectively prevented ET-1 elevation but could not reverse creatinine or bilirubin elevation. Captopril with or without bosentan was cytoprotective but did not reverse increased creatinine levels. It is concluded that increased ET-1 in obstructive jaundice may be one of the contributing factors of renal damage.

Bioflavonoid effects on the mitochondrial respiratory electron transport chain and cytochrome c redox state.

The polyphenolic structure common to flavonoids enables them to donate electrons and exert antioxidant activity. Since the mitochondrial electron transport chain consists of a series of redox intermediates, the effect of flavonoids in a complex mixture of polyphenols, as well as related pure flavonoids, was evaluated on the rat liver mitochondrial electron transport chain. A French maritime pine bark extract (PBE), a complex mixture of polyphenols and related pure flavonoids, was able to reduce cytochrome c reversibly, possibly by donation of electrons to the iron of the heme group; the donated electrons can be utilized by cytochrome c oxidase. Among single flavonoids tested, (-)-epicatechin gallate had the greatest ability to reduce cytochrome c. In addition, PBE competitively inhibited electron chain activity in both whole mitochondria and submitochondrial particles. A 3.5-fold increase in the apparent Km value for succinate was calculated from reciprocal plots. Among the flavonoids tested, taxifolin and (-)-epicatechin gallate showed minor inhibitory effects, while (+/-)-catechin and (+)-epicatechin were ineffective. Activities of NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases were inhibited by low concentrations of PBE to a similar extent. However, inhibition of cytochrome c oxidase activity required 4-fold higher PBE concentrations. These results suggest that flavonoids reduce cytochrome c and that PBE inhibits electron transport chain activity mainly through NADH-ubiquinone, succinate-ubiquinone, and ubiquinol-cytochrome c reductases.

17 beta-Estradiol increases intracellular free calcium concentrations of human vascular endothelial cells and modulates its responses to acetylcholine.

In this study, we have investigated the effect of 17 beta-estradiol (E2) on intracellular free calcium concentrations ([Ca2+]i) in human umbilical vein endothelial cells (HUVEC) using fura-2 fluorescence. E2 at concentrations of 1nM -1 microM was added subsequently to HUVEC cultures which were either deprived of estrogens or preincubated with E2 (100 nM) for 24 hours. In both groups of cultures, E2 stimulated significant increases in [Ca2+]i in a dose-dependent manner. The effects were more prominent in E2-deprived cells. Preincubation of cells with tamoxifen or the presence of it in the buffer during the experiments did not inhibit the response of the cells to E2. Experiments performed in Ca2+ free/EGTA buffer yielded transient increases in [Ca2+]i suggesting release of Ca2+ from intracellular stores was responsible for the initial peak, while sustained elevations were supported by Ca2+ influx from the extracellular space. Addition of La3+ abolished the sustained [Ca2+]i elevations. Carbachol (CCh) (1nM, 100 nM) did not induce changes in [Ca2+]i of estrogen-deprived cells but produced significant increases in [Ca2+]i of the same cells after incubation with E2 for 30 minutes. The cultures which were preincubated with E2 for 24 hours responded to carbachol directly. The results of our study indicate that E2 may modulate the functions of endothelial cells after only a brief exposure and also may be necessary for the response to acetylcholine especially at low concentrations.

Involvement of free radicals in the cardioprotective effect of defibrotide.

Ischemia followed by reperfusion has deleterious effects on myocardial tissue and a wide range of drugs have been investigated to modulate these changes. Defibrotide (polydeoxyribonucleotides from bovine lung), a drug with antithrombotic and fibrinolytic activities, has also proven to be cardioprotective against myocardial ischemia/reperfusion damage. However, the mechanism of this protective effect has not been clarified yet. The aim of this study was to determine whether this effect is due to protection against free radical induced changes. The experimental model in rabbits includes coronary artery ligation for 60 min followed by a reperfusion period of 45 min. In this model, free radical damage was estimated by different parameters of lipid peroxidation such as diene conjugation, carbonyl content, and thiobarbituric acid reactive substances, together with protein oxidation determinations. The results demonstrate that defibrotide prevents free radical induced changes after myocardial ischemia/reperfusion.

Protective effects of cilazapril against free radical injury in myocardial ischaemia-reperfusion.

Cilazapril is a prodrug which is rapidly hydrolysed to the pharmacologically active cilazaprilat following absorption to the bloodstream. In clinical pharmacological studies, administration of cilazapril resulted in potent, reversible, selective and competitive angiotensin converting enzyme inhibition. In this study, we have examined the protective effect of cilazapril on a myocardial ischaemia-reperfusion model by using different parameters of lipid peroxidation and protein oxidation. We have observed increased levels of diene conjugates, carbonyls and malondialdehyde as well as protein carbonyls after ischaemia-reperfusion, whereas protein sulphydryl groups were decreased. Our results clearly demonstrate that cilazapril, a non-sulphydryl, long-acting angiotensin converting enzyme inhibitor, has free-radical-scavenging potential in a model comparable to the clinical situation observed in humans.

Morphological changes in carotid arteries of rabbits induced by defibrotide infusion.

Defibrotide is an antithrombotic and profibrinolytic drug which modulates endothelial function. The drug increases prostacyclin and tissue plasminogen activator while it decreases plasminogen activator inhibitor synthesis by endothelial cells. In this study, in vivo effects of defibrotide on the morphology of endothelial cells and vessel wall of the healthy rabbits were investigated by light and electron microscopy. The examination of the carotid arteries of healthy rabbits after infusion of saline or defibrotide (10 mg/kg/hr) in saline solution for three hours revealed that the drug had induced dramatic morphological changes in all the test animals while no change was observed in control group. The changes observed after defibrotide administration, such as the decrease in hill and valley-like appearance of endothelial surface, and thinning of the intimal layer provides evidence for the vasorelaxant effect of the drug, while the decrease in the number of blood cells adhering to the endothelial surface confirms the antithrombotic effect of defibrotide. Finally the decrease in the number of crater-like structures may be due to the cytoprotective effect of the drug.