Evidence for a specific influence of the nitrergic pathway on the peripheral pulse waveform in rabbits.

The height of the dicrotic notch between the systolic and diastolic peaks of the peripheral pulse wave, expressed as a fraction of the overall amplitude of the wave, is sensitive to nitric oxide (NO) bioactivity. This phenomenon might form the basis of a simple, non-invasive method for determining endothelial function in vivo. We assessed whether the phenomenon is specific to the NO pathway or whether other vasoactive agents have similar effects. The relative height of the dicrotic notch (RHDN) was determined by photoplethysmography in the rabbit ear. It was dose-dependently decreased by acetylcholine, a stimulator of endothelial NO synthesis, and increased by N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis. There was no effect on RHDN of the alpha-adrenergic blocker phentolamine or the beta-adrenergic blocker propranolol. The cyclo-oxygenase inhibitor indomethacin dose-dependently decreased RHDN but this effect was blocked by L-NAME, suggesting it was mediated by cross-talk with the NO pathway. Changes in RHDN appeared to be independent of heart rate and of the delay between the systolic peak and the notch, but were associated with changes in the slope of the dicrotic limb. Both L-NAME and phentolamine produced multiple diastolic peaks, indicative of wave reflections in the vasculature. These data support the view that changes in RHDN are specific to the NO pathway and provide additional information about the mechanisms involved.

Purinergic 2X1 receptors mediate endothelial dependent vasodilation to ATP.

ATP is an important endogenous mediator in the cardiovascular system. It induces endothelium dependent vasodilation, but the precise receptor pathway activated in this response is currently under debate. We have used traditional bioassay techniques to show that ATP-induced vasodilation in mesenteric vessels is endothelium-dependent. Furthermore, ATP-induced vasodilation was inhibited by both suramin and 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), consistent with a P2X(1)-, P2X(2)-, or P2X(3)-mediated event and was not potentiated by ivermectin, indicating that these responses were not P2X(4) receptor-mediated. ATP did not induce vasodilation in vessels from P2X (-/-)(1) mice, confirming an absolute requirement for this receptor. Finally, in pure cell populations of mouse mesenteric artery endothelial cells, we show that P2X(1) mRNA is specifically expressed. However, in line with observations in the brain, the P2X(1) present in endothelial cells does not seem to be recognized by conventional antibodies. Together, these results show that ATP-induced vasodilation is mediated by P2X(1) receptor activation on mesenteric arterial endothelial cells. These observations establish a critical role for P2X(1) receptors in the ATP vasodilator pathway.

Oenology: red wine procyanidins and vascular health.

Regular, moderate consumption of red wine is linked to a reduced risk of coronary heart disease and to lower overall mortality, but the relative contribution of wine's alcohol and polyphenol components to these effects is unclear. Here we identify procyanidins as the principal vasoactive polyphenols in red wine and show that they are present at higher concentrations in wines from areas of southwestern France and Sardinia, where traditional production methods ensure that these compounds are efficiently extracted during vinification. These regions also happen to be associated with increased longevity in the population.

Oxidative stress could precede endothelial dysfunction and insulin resistance in Indian Mauritians with impaired glucose metabolism.

AIMS/HYPOTHESIS: To measure oxidative stress, endothelial dysfunction and insulin resistance in Indian Mauritians at different stages of development of Type II (non-insulin-dependent) diabetes mellitus. METHODS: Plasma total 8-epi-PGF2alpha, an indicator of oxidative stress, was determined in age-matched subjects with normal glucose metabolism (n = 39), impaired glucose tolerance (n = 14), newly diagnosed diabetes (n = 8) and established diabetes (n = 14). Plasma glucose and insulin were measured at baseline and 2 h following an oral glucose tolerance test. Endothelial function was assessed by non-invasive digital pulse wave photoplethysmography. RESULTS: Plasma 8-epi-PGF2alpha increased in subjects with impaired glucose tolerance (p < 0.05) compared with control subjects, and was even higher in newly diagnosed diabetic patients (p < 0.01) and established (p < 0.01) diabetic patients. A tendency towards reduced endothelial function in subjects with impaired glucose tolerance became significant in patients with newly diagnosed and established diabetes (p < 0.01), and was correlated with 8-epi-PGF2alpha (r = 0.36, p < 0.01). Insulin resistance (homeostasis model assessment) did not change in subjects with impaired glucose tolerance compared with control subjects, but increased in newly diagnosed (p < 0.01) and established (p < 0.001) diabetic subjects. The 8-epi-PGF2alpha was correlated with fasting glucose (r = 0.50, p < 0.001), triglycerides (r = 0.40, p < 0.001) and insulin resistance (r = 0.35, p < 0.001). CONCLUSION/INTERPRETATION: Oxidant stress is an early event in the evolution of Type II diabetes and could precede the development of endothelial dysfunction and insulin resistance.

Distribution of lipid deposits around aortic branches of mice lacking LDL receptors and apolipoprotein E.

Mice with inactivated genes are increasingly used as models of human atherosclerosis. The aim of the present study was to determine whether the characteristic age-related distributions of lipid deposition seen around human arterial branches are replicated in such mice. Lesions occur downstream of branch ostia in immature human aortas, but these regions are spared in adult vessels, with lesions occurring more frequently at the sides or upstream of the branches. We determined the pattern of lipid staining around 102 intercostal branch ostia from apolipoprotein E/low density lipoprotein receptor double-knockout mice aged 9 to 20 weeks by using en face microscopy and a frequency-mapping technique. Lesion prevalence was high in the ostium and the region immediately surrounding it. Frequencies were 2.12+/-0.30 (mean+/-SEM, n=11) times higher upstream than downstream (P<0.01), but the pattern did not resemble the adult human pattern: there were no peaks in frequency at the sides or upstream of the branch, and there was no sparing downstream. Furthermore, a patch of sparing upstream of the branch was seen, which has not been reported for human vessels, and there was no trend toward a more upstream pattern with age. We conclude that knockout mice may not be a suitable model in which to investigate localizing factors.

Carnosine, the anti-ageing, anti-oxidant dipeptide, may react with protein carbonyl groups.

Carnosine (beta-alanyl-L-histidine) is a physiological dipeptide which can delay ageing and rejuvenate senescent cultured human fibroblasts. Carnosine's anti-oxidant, free radical- and metal ion-scavenging activities cannot adequately explain these effects. Previous studies showed that carnosine reacts with small carbonyl compounds (aldehydes and ketones) and protects macromolecules against their cross-linking actions. Ageing is associated with accumulation of carbonyl groups on proteins. We consider here whether carnosine reacts with protein carbonyl groups. Our evidence indicates that carnosine can react non-enzymically with protein carbonyl groups, a process termed 'carnosinylation'. We propose that similar reactions could occur in cultured fibroblasts and in vivo. A preliminary experiment suggesting that carnosine is effective in vivo is presented; it suppressed diabetes-associated increase in blood pressure in fructose-fed rats, an observation consistent with carnosine's anti-glycating actions. We speculate that: (i) carnosine's apparent anti-ageing actions result, partly, from its ability to react with carbonyl groups on glycated/oxidised proteins and other molecules; (ii) this reaction, termed 'carnosinylation,' inhibits cross-linking of glycoxidised proteins to normal macromolecules; and (iii) carnosinylation could affect the fate of glycoxidised polypeptides.

Characteristics of the pulse waveform during altered nitric oxide synthesis in the rabbit.

Nitrovasodilators produce characteristic changes in the shape of the peripheral pulse wave. Similar changes might also be caused by alteration of endogenous NO activity, which would allow such activity to be assessed in vivo. We investigated whether manipulation of the NO pathway influences the pulse waveform, and the mechanisms involved. The pulse wave in the ear of normal rabbits was examined by reflectance photoplethysmography before and during infusion of vasoactive agents. Pulse wave velocity was assessed by using an additional sensor on the rear foot. A diastolic peak was observed in the ear pulse; its timing was consistent with it being a reflection of the systolic peak from the lower body. The height of the dicrotic notch marking the start of this diastolic wave was decreased by acetylcholine or an NO donor, and further decreased by a phosphodiesterase type V inhibitor. The acetylcholine-induced decreases were blocked by inhibiting NO synthesis with N(G)-nitro-L-arginine methyl ester (L-NAME) but were unaffected by the inactive enantiomer D-NAME. These data demonstrate that NO influences the height of the notch in the pulse wave. Heart rate and blood pressure were altered during acetylcholine or L-NAME infusion, but there were no changes in pulse wave amplitude or velocity, or in the timing of the diastolic peak or dicrotic notch. The slope of the pulse wave between the systolic peak and notch changed substantially. These effects are most convincingly explained by changes in wave reflection, not only from the lower body but also from more proximal sites.

Oxidative stress impairs insulin internalization in endothelial cells in vitro.

AIMS/HYPOTHESIS: Because oxidative stress has been suggested to be a significant contributing factor in the development of endothelial dysfunction and insulin resistance, we investigated whether reactive oxygen species contribute to insulin resistance by impairing insulin uptake through an effect on endothelial insulin receptor function. METHODS: Following a 2-h pro-oxidant challenge with xanthine oxidase, we examined the temporal pattern of insulin processing in the human umbilical endothelial cell line Ea.Hy926 and bovine aortic endothelial cells equilibrated with [125I]-insulin. Insulin receptor mRNA concentrations were analysed by RT-PCR and insulin receptor tyrosine phosphorylation and protein concentrations were estimated by western blotting. RESULTS: Xanthine oxidase exposure resulted in a major reduction in total insulin receptor-mediated [125I]-insulin internalization over a 1-h period in both Ea.Hy926 and bovine aortic endothelial cells. After 15 min, untreated bovine aortic endothelial cells internalized fivefold more cell-bound [125I]-insulin than pro-oxidant treated cells. The [125I]-insulin disappeared from the cell surface at a similar rate in both pro-oxidant and untreated cells, with relatively more [125I]-insulin being released into the medium in pro-oxidant treated cells. Although xanthine oxidase reduced insulin receptor mRNA and protein concentrations, cell surface insulin binding capacity was not affected. Following 5 min insulin exposure, insulin receptor auto-phosphorylation was considerably reduced in cells challenged with xanthine oxidase for 2 h, which could be important for insulin receptor activation and internalization. CONCLUSION/INTERPRETATION: Oxidative stress impairs insulin endocytosis in both arterial and venous endothelial cell lines. This was not a consequence of modified insulin binding capacity but could involve insufficient insulin receptor activation.

Oxidized LDL-mediated monocyte adhesion to endothelial cells does not involve NFkappaB.

Oxidised LDL (oxLDL) is a key pathogenic mediator of atherogenesis, exhibiting many proatherogenic properties. We have examined the effect of oxLDL on monocyte adhesion in the endothelial cell line, EA.hy 926. This has included the role of endothelial cell adhesion molecule expression (ICAM-1 and VCAM-1), monocyte chemoattractant protein-1 (MCP-1), and the transcription factor NFkappaB in this interaction. In response to oxLDL (10-100 microg/ml), monocyte adhesion to cells increased dose-dependently. Adhesion of oxLDL at 100 microg/ml was equivalent to that seen with TNFalpha (10 ng/ml). Unmodified LDL (nLDL, 100 microg/ml) had no effect. Both oxLDL and nLDL increased MCP-1 mRNA levels. Interestingly, oxLDL had no effect on the expression of ICAM-1 and VCAM-1. In addition NFkappaB was not activated as shown by western blots of IkappaB-alpha degradation and electrophoretic mobility shift assay. In summary these data show that increased monocyte adhesion to EA.hy 926 cells occurs independently of ICAM-1, VCAM-1, and NFkappaB activation and may involve novel adhesive mechanisms.

Endothelin-1 synthesis reduced by red wine.

Statistical evidence of reduced coronary heart disease in areas of high wine consumption has led to the widespread belief that wine affords a protective effect. Although moderate drinking of any alcohol helps to reduce the incidence of coronary heart disease, there is no clear evidence that red wine confers an additional benefit. Here we show that red wines strongly inhibit the synthesis of endothelin-1, a vasoactive peptide that is crucial in the development of coronary atherosclerosis. Our findings indicate that components specific to red wine may help to prevent coronary heart disease.

Endothelial dysfunction accompanies a pro-oxidant, pro-diabetic challenge in the insulin resistant, obese Zucker rat in vivo.

We have recently made the novel observation that a pro-oxidant challenge with hydroquinone in combination with buthionine sulfoximine (each at 50 mg/kg i.p. daily for 7 days) provokes the onset of type II diabetes mellitus in a model of insulin resistance, the obese Zucker rat. Since endothelial dysfunction in oxidant stress may aggravate in vivo insulin resistance, we have now investigated endothelium-dependent and nitric oxide (NO)-mediated vascular responses in the obese Zucker rat in vivo following this pro-oxidant insult. Pro-oxidant-treated animals exhibited defective vasodepression to the endothelium-dependent agent acetylcholine and to a lesser extent, the NO donor glyceryl trinitrate, together with a reduction in circulating levels of cGMP. Our data therefore suggest that the progression to type II diabetes mellitus in the obese Zucker rat mediated by a pro-oxidant insult is associated with impairments in agonist-stimulated, endothelium-dependent vasodilation and vascular NO signalling.

Antioxidants, diabetes and endothelial dysfunction.

While a damaged endothelium is recognised to be a key accessory to diabetic macroangiopathy, awareness is developing that impairments concerning endothelium- and nitric oxide (NO)-dependent microvascular function, may contribute to several other corollaries of diabetes, such as hypertension, dyslipidaemia and in vivo insulin resistance. There are now several reports describing elevations in specific oxidant stress markers in both insulin resistance syndrome (IRS) and diabetes, together with determinations of reduced total antioxidant defence and depletions in individual antioxidants. Such a pro-oxidant environment in diabetes may disrupt endothelial function through the inactivation of NO, resulting in the attenuation of a fundamental anti-atherogenic and euglycaemic vascular influence. Indeed, experimental and clinical data suggest that the supplementation of insulin resistant or diabetic states with antioxidants such as vitamin E, normalises oxidant stress and improves both endothelium-dependent vasodilation and insulin sensitivity. However, the promising potential efficacy of antioxidant therapy in cardiovascular disease and diabetes, in either a primary or secondary preventative role, awaits definitive clinical demonstration.

Investigation of endothelial hyperreactivity in the obese Zucker rat in-situ: reversal by vitamin E.

The obese Zucker rat, a popular model of insulin resistance allied with oxidant stress, is associated with either normal or paradoxically enhanced endothelial vasodilator function compared with its lean litter mate. We have investigated hindquarter endothelium-dependent vasodilation in the obese Zucker rat in-situ and have examined its relationship with oxidant stress. In perfused hindquarter preparations equivalently preconstricted with phenylephrine, vasodilator responses to the endothelium-dependent agent acetylcholine (0.03-1000 pmol) were greater in obese (pD2 = 11.03+/-0.19) compared with lean (pD2 = 10.53+/-0.13) animals (P < 0.01, two-way analysis of variance). In contrast, maximal vasodilation to the nitric oxide (NO) donor sodium nitroprusside (100 nmol) was similar in obese (59.6+/-19.8%) and lean (51.9+/-2.6%) preparations (P > 0.05). However, this exaggerated vasodilator reactivity to acetylcholine in obese animals was abolished following four-week dietary supplementation with the lipophilic antioxidant vitamin E (obese pD2 = 10.74+/-0.18; lean pD2 = 10.74+/-0.08). This antioxidant-mediated effect was associated with a reduction (P < 0.02, two-way analysis of variance) and an enhancement (P < 0.01, two-way analysis of variance) in endothelium-dependent vasodilator responses in obese and lean hindlimb preparations, respectively. Our data therefore now point to a differential modulation of hindquarter endothelium-dependent vasodilation in the obese and lean Zucker rat by the prevailing oxidant tone, resulting in an agonist-stimulated endothelial vasodilator hyperreactivity in obese animals.

Investigation of basal endothelial function in the obese Zucker rat in vitro.

(a) We studied basal endothelial function in the insulin-resistant, obese Zucker rat, including the influence of superoxide anion on the regulation of contractile reactivity by nitric oxide (NO), following treatment in vivo with the antioxidant tiron or the pro-oxidant combination hydroquinone+buthionine sulfoximine. (b) The leftward shift in the contractile potency of phenylephrine due to NO synthase inhibition with N(G)-nitro-L-arginine methyl ester (L-NAME) was greater in the isolated aorta of the obese Zucker rat relative to its insulin-sensitive littermate, the lean Zucker rat. (c) Pretreatment with tiron depressed vasoconstriction to phenylephrine and comparably enhanced the L-NAME-mediated leftward shift in contractile reactivity in the obese and lean Zucker rats in hydroquinone+buthionine sulfoximine-sensitive manner. (d) Our data therefore indicate superior endothelial function in the obese relative to the lean Zucker rat, reflected by a greater regulation of vasoconstrictor reactivity by basal NO, while the regulation of NO bioavailability by superoxide anion is similar.

Pro-oxidant challenge in vivo provokes the onset of NIDDM in the insulin resistant obese Zucker rat.

We investigated the ability of an acute pro-oxidant challenge in vivo to deteriorate glycaemic control and insulin action in the obese Zucker rat, a model of insulin resistance associated with oxidant stress. In obese animals, the daily administration for 1 week of hydroquinone (HQ) in combination with L-buthionine sulphoximine (BSO), elevated fasting plasma glycaemia and insulinaemia and markedly aggravated i.v. glucose-stimulated hyperinsulinaemia without significantly affecting i.v. glucose tolerance, suggesting exacerbated insulin resistance. Intermediate effects on hyperinsulinaemia in obese animals were determined with HQ treatment alone while BSO treatment alone had no effect. In contrast, none of the pro-oxidant treatments affected age-matched, insulin sensitive, lean Zucker rats. Our data therefore demonstrate for the first time, a vulnerability to deterioration in insulin action in an established insulin resistant state following an environmental pro-oxidative insult. This may have relevance in the conversion of insulin resistance syndrome (IRS) to non-insulin dependent diabetes mellitus (NIDDM).

Endothelial cell dysfunction and the pathogenesis of diabetic macroangiopathy.

Type 2 diabetes mellitus (DM) represents a high risk condition for the development of atherosclerotic and thromboembolic macroangiopathy, which make major contributions to diabetic mortality and morbidity. While many cardiovascular risk factors are common to both atherosclerosis and Type 2 DM, the enhanced risk of diabetic macroangiopathy may be attributable to additional pro-atherogenic mediators associated with insulin resistance syndrome. Given the central pathogenic role of endotheliopathy in atherosclerosis, it is likely that this vascular monolayer is the ultimate target of injury in response to such mediators. Furthermore, a pro-oxidative, dysfunctional endothelium may actively contribute to the pro-atherogenic environment through an inappropriate regulation of vascular tone, permeability, coagulation, fibrinolysis, cell adhesion and proliferation. Such dysfunction may mediate hypertension, dyslipidaemia and altered haemostasis, in addition to aggravating in vivo insulin resistance.

F2-isoprostane evidence of oxidant stress in the insulin resistant, obese Zucker rat: effects of vitamin E.

We have concurrently investigated oxidant stress, glucose tolerance and glucose-stimulated insulin responses in the obese Zucker rat, a widely used model of insulin resistance. The plasma level of the lipid peroxidation product 8-epi-prostaglandin F2alpha, a sensitive in vivo marker of oxidant stress, was elevated approximately 5-fold in 13-week old obese relative to age-matched, insulin-sensitive lean Zucker rats. Supplementation of the diet with vitamin E (as (+)-alpha-tocopherol acetate, 0.5% w/w) for 4 weeks, reduced plasma 8-epi-prostaglandin F2alpha and concomitantly reversed glucose-stimulated hyperinsulinaemia in the obese Zucker rat without worsening glucose tolerance. We therefore provide evidence of oxidant stress, measured as elevated plasma 8-epi-prostaglandin F2alpha, for the first time in the obese Zucker rat which now provides a rationale for the beneficial effects of antioxidants on insulin action previously reported in this model of insulin resistance.

Modulation of nitric oxide-dependent vascular and platelet function in-vitro by the novel phosphodiesterase type-V inhibitor, ONO-1505.

We have characterized the in-vitro modulation of both nitric oxide (NO)-dependent vasodilator activity and anti-platelet function by the novel type-V phosphodiesterase inhibitor, ONO-1505 (4-[2-(2-hydroxyethoxy)ethylamino]-2-(1H-imidazol-1-yl)-6-methoxyquin azoline methanesulphonate). ONO-1505 elicited vasorelaxation in the rat isolated aorta. If the concentration of ONO-1505 was < or = 10 microM the vasorelaxation was abolished by N(G)-nitro-L-arginine methyl ester (L-NAME), by methylene blue, and by endothelial denudation. Furthermore, pretreatment of the rat isolated aorta for 10 min with ONO-1505 in the presence of L-NAME potentiated vasorelaxation to the NO-donor, sodium nitroprusside. Similarly, ONO-1505, although having no effect on adenosine diphosphate (ADP)-induced rat platelet aggregation in-vitro, augmented established anti-aggregatory effects of sodium nitroprusside. The data therefore show that the novel phosphodiesterase V inhibitor ONO-1505 augments endogenous and exogenous nitrovasodilator activity in-vitro; they also imply modulation of the NO pathway in the haemodynamic actions of this compound, previously reported in-vivo.

Physiological microassay of plasma total antioxidant status in a model of endothelial dysfunction in the rat following experimental oxidant stress in vivo.

We have developed a photometric microassay for the assessment of total antioxidant status in plasma at physiological pH and temperature and applied it to evaluate experimental oxidant stress in vivo associated with endothelial dysfunction in vitro. Rat plasma or l-ascorbic acid inhibited the peroxidase-mediated accumulation after 6 min at pH 7.4 and 37°C of ABTS(+) (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical), measured at 405 nm, in a concentration-dependent manner. Plasma total antioxidant status, expressed as the ascorbate equivalent antioxidant concentration, was subsequently found to be significantly reduced in rats treated daily for 7 days in vivo with the oxidant compounds hydroquinone (50 mg/kg i.p.) and triethylenetetramine (100 mg/kg i.p.), either alone or in combination with the glutathione-depleting agent l-buthionine sulfoximine (50 mg/kg i.p). Furthermore, basal endothelial function in isolated aorta was impaired after hydroquinone or triethylenetetramine in a manner aggravated by l-buthionine sulfoximine.

Investigation of oxidant stress and vasodepression to glyceryl trinitrate in the obese Zucker rat in vivo.

1. We examined the relationship between oxidant stress and the vasodepressor activity of glyceryl trinitrate (GTN) in vivo, including rapid GTN tolerance development, in 13-week old obese and age-matched lean Zucker rats which had been maintained for 4 weeks on either control diet or diets enriched with the lipophilic, chain-breaking antioxidants vitamin E (0.5% w w(-1)) or probucol (0.5% w w(-1)) or the superoxide anion scavenger tiron (1% w v(-1) in drinking water). 2. The basal plasma level of the isoprostane 8-epi-PGF2alpha, an in vivo marker of lipid peroxidation, was elevated by approximately 5 fold in the obese Zucker rat and markedly reduced by dietary lipophilic antioxidants and depressed by dietary tiron. 3. Vasodepression to bolus does GTN (0.1-100 microg kg(-1) i.v.), but not endothelium-dependent vasodepression to bolus dose acetylcholine (ACh, 0.02-2.0 microg kg(-1) i.v.), was impaired in obese animals and completely restored by dietary antioxidants. 4. Nitrate tolerance developed in vivo during a I h infusion of GTN (40 microg kg(-1) min(-1) i.v.) appeared more severe in obese animals. However, rapid nitrate tolerance was not affected by dietary antioxidants in either the obese or lean Zucker rat. 5. We therefore provide evidence that elevated oxidant stress in the obese Zucker rat is associated with an impairment in nitrate vasodepressor activity. However, our data are not consistent with either a role for oxidant stress in rapid nitrate tolerance development in the anaesthetized Zucker rat or the aggravation of this tolerance by pre-existing oxidant stress.