Hormone therapy and the progression of coronary-artery atherosclerosis in postmenopausal women.

BACKGROUND: In postmenopausal women with coronary artery disease, conjugated equine estrogen with or without continuous administration of medroxyprogesterone acetate has failed to slow the progression of atherosclerosis. Whether 17beta-estradiol (the endogenous estrogen molecule) alone or administered sequentially with medroxyprogesterone acetate can slow the progression of atherosclerosis is unknown. METHODS: We conducted a double-blind, placebo-controlled trial in 226 postmenopausal women (mean age, 63.5 years) who had at least one coronary-artery lesion. Participants were randomly assigned to usual care (control group), estrogen therapy with micronized 17beta-estradiol alone (estrogen group), or 17beta-estradiol plus sequentially administered medroxyprogesterone acetate (estrogen-progestin group). In all patients the low-density lipoprotein (LDL) cholesterol level was reduced to a target of less than 130 mg per deciliter. The primary outcome was the average per-participant change between base-line and follow-up coronary angiograms in the percent stenosis measured by quantitative coronary angiography. RESULTS: After a median of 3.3 years of follow-up, the mean (+/-SE) change in the percent stenosis in the 169 participants who had a pair of matched angiograms was 1.89+/-0.78 percentage points in the control group, 2.18+/-0.76 in the estrogen group, and 1.24+/-0.80 in the estrogen-progestin group (P=0.66 for the comparison among the three groups). The mean difference in the percent stenosis between the estrogen group and the control group was 0.29 percentage point (95 percent confidence interval, -1.88 to 2.46), and the mean difference between the estrogen-progestin group and the control group was -0.65 (95 percent confidence interval, -2.87 to 1.57). CONCLUSIONS: In older postmenopausal women with established coronary-artery atherosclerosis, 17beta-estradiol either alone or with sequentially administered medroxyprogesterone acetate had no significant effect on the progression of atherosclerosis.

Direct detection of reactive nitrogen species in experimental autoimmune uveitis.

PURPOSE: Demonstrate unequivocally the generation of nitric oxide in experimental autoimmune uveoretinitis by electron spin resonance spectroscopy (ESR) using ferrous iron complex of N-methyl-D-glucamine dithiocarbamate, (MGD)(2)-Fe(2+), as a spin trap. METHODS: Experimental autoimmune uveitis was induced in Lewis rats, and at the peak of the intraocular inflammation, the animals received intravitreous injections of the spin trap. The retina and choroid dissected from the enucleated globes were subjected to ESR. Similarly, the retina and choroid obtained at the peak of experimental autoimmune uveo-retinitis (EAU) were placed in a vial containing luminal, and chemiluminescence was counted on a Packard liquid scintillation analyzer. RESULTS: The ESR three-line spectrum (g=2.04; a(N)=12.5 G) obtained was characteristic of the adduct [(MGD)(2)-Fe(2+)-NO]. The majority of this signal was eliminated by the inducible nitric oxide synthase (iNOS) specific inhibitor aminoguanidine injected inflamed retina was detected when compared with that of the non inflamed controls. The chemiluminescent activity was further increased two-fold by the addition of bicarbonate to the inflamed retina; the phenomenon is attributable only to the presence of a high steady-state concentration of peroxynitrite. CONCLUSIONS: The study shows an unequivocal presence of nitric oxide in EAU retina and choroid and the generation of peroxynitrite. High levels of these reactive nitrogen species generated in the inflamed retina and choroids are certain to cause irreversible tissue damage, especially at the susceptible sites such as photoreceptors.

Role of annexin II in estrogen-induced macrophage matrix metalloproteinase-9 activity: the modulating effect of statins.

Annexin II (ANXII) is a receptor for tissue plasminogen activator and plasminogen for the conversion to plasmin, which, in turn, induces metalloproteinase-9 (MMP-9). 17beta-Estradiol (E(2)) is reported to decrease plasminogen activity inhibitor-1 and increase plasmin and matrix metalloproteinase activity. However, the combined effects of estrogen and statins on macrophage MMP-9 activity and ANXII expression remain unclear. Treatment of J774A.1 macrophages with 1.0-100 nM of E(2) for 24h increased both MMP-9 activity and ANXII expression in a dose-dependent manner (p<0.05). Preincubation with EGTA (10mM) released ANXII from the cell membrane and inhibited the E(2)-mediated MMP-9 activity as did incubation of macrophages with anti-annexin IgG. In the presence or absence of E(2) (5 nM), simvastatin treatment in the range of 0.1-5.0 microM significantly reduced macrophage MMP-9 enzymatic activity (p<0.005) in a dose-dependent manner. In the presence or absence of E(2), simvastatin also decreased ANXII expression (p<0.05). These findings indicate that ANXII plays a central role in modulating the enzymatic activity of MMP-9 in response to E(2) and that E(2)-mediated ANXII expression and MMP-9 activity can be prevented by simvastatin. Prevention of E(2)-mediated activation of MMP-9 by simvastatin suggests that concurrent statin use may account for early event risk of myocardial infarction seen with hormone therapy in recent clinical trials.

Pulsatile versus oscillatory shear stress regulates NADPH oxidase subunit expression: implication for native LDL oxidation.

Shear stress regulates endothelial nitric oxide and superoxide (O2-*) production, implicating the role of NADPH oxidase activity. It is unknown whether shear stress regulates the sources of reactive species production, consequent low-density lipoprotein (LDL) modification, and initiation of inflammatory events. Bovine aortic endothelial cells (BAECs) in the presence of 50 microg/mL of native LDL were exposed to (1) pulsatile flow with a mean shear stress (tau(ave)) of 25 dyne/cm2 and (2) oscillating flow at tau(ave) of 0. After 4 hours, aliquots of culture medium were collected for high-performance liquid chromatography analyses of electronegative LDL species, described as LDL- and LDL2-. In response to oscillatory shear stress, gp91phox mRNA expression was upregulated by 2.9+/-0.3-fold, and its homologue, Nox4, by 3.9+/-0.9-fold (P<0.05, n=4), with a corresponding increase in O2-* production rate. The proportion of LDL- and LDL2- relative to static conditions increased by 67+/-17% and 30+/-7%, respectively, with the concomitant upregulation of monocyte chemoattractant protein-1 expression and increase in monocyte/BAEC binding (P<0.05, n=5). In contrast, pulsatile flow downregulated both gp91phox and Nox4 mRNA expression (by 1.8+/-0.2-fold and 3.0+/-0.12-fold, respectively), with an accompanying reduction in O2-* production, reduction in the extent of LDL modification (51+/-12% for LDL- and 30+/-7% for LDL2-), and monocyte/BAEC binding. The flow-dependent LDL oxidation is determined in part by the NADPH oxidase activity. The formation of modified LDL via O2-* production may also affect the regulation of monocyte chemoattractant protein-1 expression and monocyte/BAEC binding.

Minimally modified electronegative LDL and its autoantibodies in acute and chronic coronary syndromes.

OBJECTIVES: To determine the minimally modified electronegative LDL (LDL-) and its autoantibodies in coronary syndromes. DESIGN AND METHODS: LDL(-) and its autoantibodies were determined by ELISA in patients with acute (ACS, unstable angina; AMI, acute myocardial infarction) and chronic coronary syndromes (stable angina, SA) and compared to subjects without coronary disease (controls). Results are expressed as median of LDL- (microg/mL) and anti-LDL(-) IgG (OD405 nm). RESULTS: The concentrations of LDL(-) were higher in patients with coronary disease (ACS: 40.7 microg/mL; SA: 35.0 microg/mL) as compared to controls (21.6 microg/mL). The highest LDL- concentrations were found in patients with AMI (41.8 microg/mL). Anti-LDL(-) IgG was elevated in ACS (1.143) in relation to CCS (0.527) and controls (0.467). A positive correlation was observed between anti-LDL- IgG and CRP levels (r = 0.34, p <0.01) in the studied groups. CONCLUSIONS: LDL(-) and anti-LDL(-) autoantibodies may be useful markers to follow patients with high risk for coronary events.

Detection of electronegative low density lipoprotein (LDL-) in plasma and atherosclerotic lesions by monoclonal antibody-based immunoassays.

OBJECTIVES: To produce a monoclonal antibody (MAb) against electronegative LDL (LDL-) for detecting this modified lipoprotein in blood plasma and tissues. DESIGN AND METHODS: LDL- was isolated from human blood plasma and used as an antigen for immunization of Balb/c mice. Lymphocytes of immunized mice were fused with myeloma cells (SP2/0) to obtain the hybridomas. LDL- was detected in blood plasma and atherosclerotic lesions of humans and rabbits by MAb-based ELISA and immunohistochemistry, respectively. RESULTS: LDL- concentrations were higher (P < 0.05) in the blood plasma of hypercholesterolemic subjects (HC, 248 +/- 77 mg/dL of total cholesterol) than in normolipidemic subjects (NL, 173 +/- 82 mg/dL of total cholesterol) and rabbits (HC, 250 +/- 15 mg/dL of cholesterol versus NL, 81 +/- 12 mg/dL of cholesterol). Moreover, LDL- was detected in the atherosclerotic lesions of humans and rabbits. CONCLUSION: These MAb-based immunoassays are adequate to detect LDL- in biological samples and represent an important tool for investigating the role of LDL- in atherosclerosis.

Alpha-tocopherol supplementation in healthy individuals reduces low-density lipoprotein oxidation but not atherosclerosis: the Vitamin E Atherosclerosis Prevention Study (VEAPS).

BACKGROUND: Epidemiological studies have demonstrated an inverse relationship between vitamin E intake and cardiovascular disease (CVD) risk. In contrast, randomized controlled trials have reported conflicting results as to whether vitamin E supplementation reduces atherosclerosis progression and CVD events. METHODS AND RESULTS: The study population consisted of men and women > or =40 years old with an LDL cholesterol level > or =3.37 mmol/L (130 mg/dL) and no clinical signs or symptoms of CVD. Eligible participants were randomized to DL-alpha-tocopherol 400 IU per day or placebo and followed every 3 months for an average of 3 years. The primary trial end point was the rate of change in the common carotid artery far-wall intima-media thickness (IMT) assessed by computer image-processed B-mode ultrasonograms. A mixed effects model using all determinations of IMT was used to test the hypothesis of treatment differences in IMT change rates. Compared with placebo, alpha-tocopherol supplementation significantly raised plasma vitamin E levels (P<0.0001), reduced circulating oxidized LDL (P=0.03), and reduced LDL oxidative susceptibility (P<0.01). However, vitamin E supplementation did not reduce the progression of IMT over a 3-year period compared with subjects randomized to placebo. CONCLUSIONS: The results are consistent with previous randomized controlled trials and extend the null results of vitamin E supplementation to the progression of IMT in healthy men and women at low risk for CVD.

Signal transduction pathways leading to increased eIF4E phosphorylation caused by oxidative stress.

Phosphorylation of eIF4E is associated with increased activity of the translational machinery. Oxidative stress of resident vascular cells and macrophages potently enhances eIF4E phosphorylation. Oxidative stress activates numerous intracellular signaling pathways, including MAP-family kinase pathways and pathways leading to S6 kinase activation. The activation of MAP-family kinase pathways leads to the activation of Mnk and hence eIF4E phosphorylation, whereas the S6 kinase pathway is not involved, based on insensitivity to its inhibitors rapamycin and wortmannin. Ca-dependent pathways have been implicated in eIF4E phosphorylation, but the oxidative stress response pathway targeting eIF4E does not appear to require their participation. The results suggest that the potent activation of ERK and p38 protein kinases is sufficient to account for the enhanced eIF4E phosphorylation. Either is independently sufficient to effect the change, as neither PD098059 (Erk pathway inhibitor) nor SB202190 (p38 pathway inhibitor) alone can block the response, but when combined the response is almost completely abrogated. Mnk activation by oxidative stress leading to enhanced eIF4E phosphorylation may play a role in promoting stress-induced hyperproliferative diseases, such as smooth muscle cell proliferation and hypertrophy in cardiovascular disease, as the synthesis of several key regulators of cell growth has been shown to be held in check by moderation of eIF4E activity.

Oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine induces vascular endothelial superoxide production: implication of NADPH oxidase.

Modified low-density lipoprotein (LDL) induces reactive oxygen species (ROS) production by vascular cells. It is unknown if specific oxidized components in these LDL particles such as oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) can stimulate ROS production. Bovine aortic endothelial cells (BAEC) were incubated with ox-PAPC (50 microg/ml). At 4 h, ox-PAPC significantly enhanced the rate of O2- production. Pretreatment of BAEC in glucose-free Dulbecco's modified Eagle's medium plus 10 mM 2-deoxyglucose (2-DOG), the latter being an antimetabolite that blocks NADPH production by the pentose shunt, significantly reduced the rate of O2- production. The intensity of NAD(P)H autofluorescence decreased by 28 +/- 12% in BAEC incubated with ox-PAPC compared to untreated cells, with a further decrease in the presence of 2-DOG. Ox-PAPC also increased Nox4 mRNA expression by 2.4-fold +/- 0.1 while pretreatment of BAEC with the small interfering RNA (siNox4) attenuated Nox4 RNA expression. Ox-PAPC further reduced the level of glutathione while pretreatment with apocynin (100 microM) restored the GSH level (control = 22.54 +/- 0.23, GSH = 18.06 +/- 0.98, apocynin = 22.55 +/- 0.60, ox-PAPC + apocynin = 21.17 +/- 0.36 nmol/10(6) cells). Treatment with ox-PAPC also increased MMP-2 mRNA expression accompanied by a 1.5-fold increase in MMP-2 activity. Ox-PAPC induced vascular endothelial OO2-(.) production that appears to be mediated largely by NADPH oxidase activity.

Oxysterol-induced up-regulation of MCP-1 expression and synthesis in macrophage cells.

To investigate the proinflammatory potential of cholesterol and cholesterol oxidation products (oxysterols), which are present in oxidized low-density lipoproteins, foam cells, and fibrotic plaque, we used an in vitro model mimicking the challenge of macrophage cells by the cholesterol accumulating within the central core of atheroma. A biologically representative oxysterol mixture was shown to be potentially able to sustain a chronic inflammatory process within the vascular wall by up-regulating the expression of defined proinflammatory genes. In particular, expression and synthesis of the major chemokine for monocytes/macrophages, namely monocyte chemotactic protein-1 (MCP-1), were consistently increased when cells of the macrophage lineage (U937 cell line) were incubated with this mixture. On the contrary, an identical concentration of unoxidized cholesterol in no case modified expression or synthesis of the chemokine. Up-regulated expression and synthesis of MCP-1 by the oxysterol mixture was clearly dependent on a net increment of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor kappaB (NF-kappaB) nuclear binding. The results indicate that cholesterol may contribute to the progression of atherosclerotic lesions by strongly up-regulating crucial proinflammatory factors like MCP-1, but only after having been oxidized to oxysterols.

Role of lipid peroxidation in the epidemiology and prevention of breast cancer.

We have recently proposed a common mechanistic pathway by which obesity and hypertension lead to increased renal cell cancer risk. Our hypothesis posits lipid peroxidation, which is a principal mechanism in rodent renal carcinogenesis, as an intermediate step that leads to a final common pathway shared by numerous observed risks (including obesity, hypertension, smoking, oophorectomy/hysterectomy, parity, preeclampsia, diabetes, and analgesics) or protective factors (including oral contraceptive use and alcohol) for renal cell cancer [Cancer Causes Control 2002;13:287-93]. During this exercise, we have noticed how certain risk factors for renal cell carcinoma are protective for breast cancer and how certain protective factors for renal cell carcinoma increase risk for breast cancer. Parity and oophorectomy, for example, are positively associated with renal cell carcinoma but are negatively associated with breast cancer. Similarly, obesity and hypertension are positively associated with renal cell carcinoma, but obesity is negatively associated with breast cancer in premenopausal women and hypertension during pregnancy is negatively associated with breast cancer. Furthermore, alcohol intake, negatively associated with renal cell carcinoma, is also positively associated with breast cancer. We propose here the possibility that lipid peroxidation may represent a protective mechanism in breast cancer. Although this runs counter to the conventional view that lipid peroxidation is a process that is harmful and carcinogenic, we present here the chemical and biological rationale, based on epidemiologic and biochemical data, which may deserve further consideration and investigation.

Long-term effect of estrogen replacement on plasma nitric oxide levels: results from the estrogen in the prevention of atherosclerosis trial (EPAT).

The estrogen in the prevention of atherosclerosis trial (EPAT) was a 2-year randomized controlled trial in which unopposed 17beta-estradiol reduced subclinical atherosclerosis progression, measured as change in carotid intima-media thickness (CIMT). This study was conducted to determine whether long-term 17beta-estradiol 1mg daily increased plasma nitric oxide (NO) levels and whether this accounted for atheroprotection in EPAT. Although the on-trial serum estradiol level was significantly higher in the estradiol-treated group (n = 91 subjects) than the placebo group (n = 89 subjects) (mean (S.D.) = 59.0 (31.7) pg/ml versus 14.3 (10.4) pg/ml, p < 0.0001), there was no significant difference in the on-trial plasma NO levels, 18.5 (8.2) microM versus 20.1 (9.3) microM. Correlation between on-trial estradiol level and NO change was -0.22 (p = 0.003) in the total sample (placebo- and estradiol-treated subjects) and -0.21 (p = 0.049) in the estradiol-treated group. Change in NO levels was inversely correlated to change in LDL-cholesterol in the estradiol group (r = -0.23, p = 0.03). An NO response to 17beta-estradiol according to age, time since menopause and baseline CIMT was not found arguing against a possible NO effect in healthy versus diseased endothelium. NO levels were not related to CIMT progression. In this study, we found no evidence for an estrogen-induced effect on plasma total NO levels which unlikely accounted for the mechanism underlying the 17beta-estradiol atheroprotective effect on subclinical atherosclerosis progression.

Atherosclerosis: another protein misfolding disease?

The secondary structure and conformation of apo-B 100 in low-density lipoproteins (LDL) are imposed by lipid-protein interactions and dynamics, and affected by the introduction or removal of lipids during the course of lipoprotein metabolism. Following an alteration of the water-lipid interface as a result of, for example, oxidation of lipids, the supramolecular structure becomes destabilized and apoB can misfold. These events have been observed in LDL(-), a fraction of oxidatively modified LDL isolated in vivo. This modified lipoprotein possesses several atherogenic properties and represents an in vivo counterpart of in vitro modified LDL that is implicated in atherosclerosis. The misfolding of apoB, its aggregation, resistance to proteolysis, and cytotoxicity are common motifs shared by LDL(-) and amyloidogenic proteins. Based on these analogies, we propose that atherogenesis could be considered as a disease produced by the accumulation of cytotoxic and pro-inflammatory misfolded lipoproteins.

Determinants of the effect of estrogen on the progression of subclinical atherosclerosis: Estrogen in the Prevention of Atherosclerosis Trial.

OBJECTIVE: To determine the extent to which the estrogen-induced changes in lipids and markers of carbohydrate metabolism explain the beneficial effect of estrogen therapy on the progression of carotid artery intima-media thickness (IMT) in postmenopausal women. DESIGN: A randomized, double-blind, placebo-controlled, single-center trial enrolling 222 postmenopausal women 45 years and older without cardiovascular disease and with low-density lipoprotein (LDL) cholesterol levels of 3.37 mmol/L or greater (> or = 130 mg/dL). Intervention was unopposed micronized 17beta-estradiol versus placebo. Measurements were made using high-resolution B-mode ultrasonography to measure carotid artery IMT at baseline and every 6 months on-trial. RESULTS: Progression of carotid IMT was inversely related to on-trial high-density lipoprotein (HDL) cholesterol (P = 0.04) and was directly related to on-trial LDL-cholesterol (P = 0.005). Compared with placebo, women randomized to estradiol showed a higher mean on-trial HDL-cholesterol level and a lower mean on-trial LDL-cholesterol level. In contrast, fasting glucose, insulin, and hemoglobin A1C were lowered and insulin sensitivity increased with estradiol therapy, but the changes were not related to carotid IMT progression. On-trial HDL-cholesterol and LDL-cholesterol were significant independent determinants of carotid IMT progression, jointly explaining 30% of the treatment effect of unopposed estrogen on the progression of carotid IMT. CONCLUSION: Unopposed 17beta-estradiol reduced carotid IMT progression in postmenopausal women in part by increasing HDL-cholesterol and decreasing LDL-cholesterol. Although women randomized to estradiol showed improvement in all the markers of carbohydrate metabolism, these factors did not play a significant role in carotid IMT progression.

Oxysterol mixtures prevent proapoptotic effects of 7-ketocholesterol in macrophages: implications for proatherogenic gene modulation.

Oxysterols are common components of oxidized low-density lipoprotein and accumulate in the core of fibrotic plaques as a mixture of cholesterol and cholesteryl ester oxidation products. The proapoptotic effects of a biologically representative mixture of oxysterols was compared with equimolar amounts of 7-ketocholesterol and unoxidized cholesterol. The oxysterol mixture in a concentration range actually detectable in hypercholesterolemic patients did not stimulate programmed cell death in cultivated murine macrophages. Unoxidized cholesterol also produced no effect. By contrast, when given alone, 7-ketocholesterol strongly stimulated the mitochondrial pathway of apoptosis with cytochrome c release, caspase-9 activation, and eventually caspase-3 activation. Subsequent experiments showed that when 7-ketocholesterol was administered to cells together with another oxysterol, namely 7betaOH-cholesterol, the strong proapoptotic effect of 7-ketocholesterol was markedly attenuated. As regards the mechanism underlying this quenching, we found that the combined oxysterol treatment counteracted the ability of 7-ketocholesterol, when administered alone, to strongly up-regulate the steady-state levels of reactive oxygen species (ROS) without interfering with sterol uptake. Furthermore, this increase in intracellular ROS appeared to be responsible for the up-regulation of proapoptotic factor, p21, after treatment with 7-ketocholesterol but not in cells challenged with the oxysterol mixture. Competition among oxysterols, apparently at the level of NADPH oxidase, diminishes the ROS induction and direct toxicity that is evoked by specific oxysterols. As a consequence, a more subtle gene modulation by oxysterols becomes facilitated in vascular cells.

Monocyte recruitment to endothelial cells in response to oscillatory shear stress.

Leukocyte recruitment to endothelial cells is a critical event in inflammatory responses. The spatial, temporal gradients of shear stress, topology, and outcome of cellular interactions that underlie these responses have so far been inferred from static imaging of tissue sections or studies of statically cultured cells. In this report, we developed micro-electromechanical systems (MEMS) sensors, comparable to a single endothelial cell (EC) in size, to link real-time shear stress with monocyte/EC binding kinetics in a complex flow environment, simulating the moving and unsteady separation point at the arterial bifurcation with high spatial and temporal resolution. In response to oscillatory shear stress (tau) at +/- 2.6 dyn/cm2 at a time-averaged shear stress (tau(ave))=0 and 0.5 Hz, individual monocytes displayed unique to-and-fro trajectories undergoing rolling, binding, and dissociation with other monocyte, followed by solid adhesion on EC. Our study quantified individual monocyte/EC binding kinetics in terms of displacement and velocity profiles. Oscillatory flow induces up-regulation of adhesion molecules and cytokines to mediate monocyte/EC interactions over a dynamic range of shear stress +/- 2.6 dyn/cm2 (P=0.50, n=10).

The phytoestrogen equol increases nitric oxide availability by inhibiting superoxide production: an antioxidant mechanism for cell-mediated LDL modification.

Estrogen replacement therapy (ERT) is reported to lower the incidence of cardiovascular disease in postmenopausal women. ERT also lowers the levels of oxidatively modified low-density lipoprotein (LDL). Because modified LDL can mediate the development of atherosclerosis by inflammatory processes, ERT may exert its LDL protective effect through enhanced antioxidant activity in vascular tissues. Plant sources of estrogenic compounds have been used as alternatives for ERT because they avoid a number of negative health effects produced by estrogen. In this study, the antioxidant properties of the soy isoflavone metabolite, equol (an estrogenic metabolite of daidzein) were studied. Equol has a greater antioxidant activity than the parent isoflavone compounds genistein and daidzein, found in high concentration in soy. Equol inhibits LDL oxidation in vitro and LDL oxidative modification by J774 monocyte/macrophages to LDL(-), an electronegative modified LDL found in human plasma. An antioxidant effect of equol was found to be mediated by inhibition of superoxide radical (O(2)(-*)) production and manifested through enhanced levels of free nitric oxide (NO) that prevents LDL modification. Thus, when NO levels were increased by donor agents, generators, or compounds that facilitate nitric oxide synthase activity, LDL(-) formation by J774 cells was strongly inhibited. Conversely, inhibition of NO production enhanced LDL(-) formation, and the combination of reduced NO and increased O(2)(-*) production yielded maximum LDL(-) formation. Pretreatment of cells with equol inhibited production of O(2)(-*) by J774 cells apparently via the inactivation of the reduced nicotinamide adenine dinucleotide phosphate oxidase complex. Decreased O(2)(-*) production resulted in increased free NO levels (but not total NO production) indicating that decreased reactions between O(2)(-*) and NO are an outcome of equol's antioxidant activity in cell culture.

Antioxidant effect of simvastatin is not enhanced by its association with alpha-tocopherol in hypercholesterolemic patients.

Among the pleiotropic effects of statins, their antioxidant action may be involved in their protective effects. Thus, we investigated the antioxidant effect of simvastatin, associated or not with alpha-tocopherol, on levels of electronegative low-density lipoprotein (LDL-), nitrotyrosine, thiols (homocysteine, glutathione, cysteine, methionine), and lipid-soluble antioxidants in blood plasma of hypercholesterolemic subjects. In this study, 25 hypercholesterolemic subjects were treated for 2 months with simvastatin (20 mg/day) and with simvastatin (20 mg/day) + alpha-tocopherol (400 IU/day). Concentrations of thiols were determined by high-performance capillary electrophoresis-laser-induced fluorescene. Lipid-soluble antioxidants were determined by HPLC, and LDL-, and nitrotyrosine by ELISA. Simvastatin, independent of its association with alpha-tocopherol, reduced plasma concentrations of LDL-, nitrotyrosine, total cholesterol, and LDL cholesterol and the LDL cholesterol/HDL cholesterol ratio. Neither simvastatin nor simvastatin plus alpha-tocopherol altered plasma levels of the thiols analyzed. alpha-Tocopherol did not change the antioxidant effect of simvastatin on the levels of LDL- and nitrotyrosine in hypercholesterolemic subjects. The reduction of LDL- and nitrotyrosine by simvastatin seems to be related to the pleiotropic effects of this statin, and it may have an important protective effect against endothelial dysfunction and atherosclerosis.

Oxidative stress during ex vivo hemodialysis of blood is decreased by a novel hemolipodialysis procedure utilizing antioxidants.

The high cardiovascular mortality in patients receiving hemodialysis (HD) has been attributed, in part, to oxidative stress. Here we examined the effectiveness of antioxidants introduced by means of a novel hemolipodialysis (HLD) procedure in terms of reducing oxidative stress during ex vivo blood circulation. Oxidative stress was studied in a model HD system resembling the extracorporeal circulation of blood during clinical HD. Blood circulation produced an increase of up to 280% in free hemoglobin levels and an increase of 320% in electronegative LDL (LDL(-)) subfraction. A significant correlation between LDL(-) and free hemoglobin levels confirmed previous findings that LDL(-) formation during ex vivo circulation of blood can be mediated by the oxidative activity of free hemoglobin. These effects were significantly attenuated during HLD using a dialysis circuit containing vitamin E with or without vitamin C. By contrast, HLD with vitamin C alone had a marked pro-oxidant effect. TBARS, lipid hydroperoxides, vitamin E and beta-carotene content in LDL were not significantly altered by the HD procedure. These findings demonstrate the occurrence of oxidative stress in human plasma where lipoproteins are a target and indicate antioxidant-HLD treatment as a specific new approach to decreasing the adverse oxidative stress frequently associated with cardiovascular complications in high-risk populations of uremic patients.

Evolution of oxidative stress and inflammation during hemodialysis and their contribution to cardiovascular disease.

End-stage renal disease patients have increased cardiovascular morbidity and mortality. These patients have many unique risk factors, such as an accumulation of uremic toxins, electrolyte imbalances, metabolic disturbances, anemia, chronic inflammation, and thrombogenic disturbances. Oxidative stress has been implicated in many of these disturbances. This review will focus on some of the factors that may accelerate cardiovascular disease in uremic patients, with an emphasis on mechanisms and interactions of various components of oxidative stress and inflammation. Understanding the mechanisms of these pathways may be useful in developing effective prevention and treatment strategies.