Erik E. Änggård

Plasma 8‐epi‐PGF2α levels are elevated in individuals with non‐insulin dependent diabetes mellitus

This study reports plasma levels of a specific nonenzymatic peroxidation product of arachidonic acid, esterified 8‐epi‐PGF2α, from healthy‐ and NIDDM individuals as an index of oxidative stress in vivo. Plasma 8‐epi‐PGF2α was isolated by solid‐phase extraction on a C,8 followed by an NH2 cartridge and analyzed by GC‐MS/NICI as PFB‐ester/TMS‐ether derivative. We found that the average concentration of esterified 8‐epi‐PGF2α among NIDDM subjects (0.93 ± 0.07 nM, n = 39) was higher (P < 0.0001, Mann‐Whitney test) than in healthy individuals (0.28 ± 0.04 nM, n = 15). These data indicate that NIDDM is associated with increased plasma lipid peroxidation.

Photoplethysmographic assessment of pulse wave reflection: Blunted response to endothelium-dependent beta2-adrenergic vasodilation in type II diabetes mellitus☆

OBJECTIVES We sought to determine whether a simple index of pressure wave reflection may be derived from the digital volume pulse (DVP) and used to examine endothelium-dependent vasodilation in patients with type II diabetes mellitus. BACKGROUND The DVP exhibits a characteristic notch or inflection point that can be expressed as percent maximal DVP amplitude (IP(DVP)). Nitrates lower IP(DVP), possibly by reducing pressure wave reflection. Response of IP(DVP) to endothelium-dependent vasodilators may provide a measure of endothelial function. METHODS The DVP was recorded by photoplethysmography. Albuterol (salbutamol) and glyceryl trinitrate (GTN) were administered locally by brachial artery infusion or systemically. Aortic pulse wave transit time from the root of the subclavian artery to aortic bifurcation (T(Ao)) was measured by simultaneous Doppler velocimetry. RESULTS Brachial artery infusion of drugs producing a greater than threefold increase in forearm blood flow within the infused limb was without effect on IP(DVP), whereas systemic administration of albuterol and GTN produced dose-dependent reductions in IP(DVP). The time between the first and second peak of the DVP correlated with T(Ao) (r = 0.75, n = 20, p < 0.0001). The effects of albuterol but not GTN on IP(DVP) were attenuated by N(G)-monomethyl-L-arginine. The IP(DVP) response to albuterol (400 microg by inhalation) was blunted in patients with type II diabetes mellitus as compared with control subjects (fall 5.9 +/- 1.8% vs. 11.8 +/- 1.8%, n = 20, p < 0.02), but that to GTN (500 microg sublingually) was preserved (fall 18.3 +/- 1.2% vs. 18.6 +/- 1.9%, p = 0.88). CONCLUSIONS The IP(DVP) is influenced by pressure wave reflection. The effects of albuterol on IP(DVP) are mediated in part through the nitric oxide pathway and are impaired in patients with type II diabetes.

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.

Analysis of F2-isoprostanes as indicators of non-enzymatic lipid peroxidation in vivo by gas chromatography-mass spectrometry: development of a solid-phase extraction procedure

Recently, it has been reported that a series of prostaglandin F2-like compounds (F2-isoprostanes) are produced in vivo during peroxidation of arachidonic acid by a mechanism independent of the cyclooxygenase pathway. Of these, 8-epi-PGF2 alpha is shown to be a potent vasoconstrictor. We describe an improved method for analysing F2-isoprostanes in biological fluids. The method involves solid-phase extraction on an octadecylsilane (C18) and an aminopropyl (NH2) cartridge. After conversion to pentafluorobenzyl ester and trimethylsilyl ether derivatives, F2-isoprostanes are analysed by negative-ion chemical ionization mass spectrometry using tetradeuterated PGF2 alpha as the internal standard. The limit of detection of the assay was 10 pg/ml, with a coefficient of variation ranging from 9.4 to 15.1%. Analysis of plasma samples from healthy volunteers (n = 7) revealed no quantifiable levels of free (unesterified) 8-epi-PGF 2 alpha. However, the plasma samples contained 58 to 166 pg/ml of 8-epi-PGF2 alpha when analyzed for the total (sum of free and esterified) F2-isoprostanes. The main advantages of the method lie in the improved recovery, gas chromatographic separation and speed compared to existing techniques.

Arterial response to mechanical injury: balloon catheter de-endothelialization

Coronary angioplasty has been used clinically for over a decade. Its initial promise as an alternative to coronary bypass surgery has only partially been fulfilled because of the high rate of post-operative restenosis. A number of animal models have been devised to study this phenomenon and although none is entirely satisfactory, they have, together with recent advances in molecular biology provided an insight into the cellular mechanisms that may contribute to this complication. This knowledge may ultimately lead to a means of therapeutic intervention. This review summarises our present understanding of the pathology of post-angioplasty re-stenosis as revealed by studies using the balloon catheter de-endothelialization model, and discusses some of the intervention strategies that have been attempted.

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.

Nitric oxide from vascular smooth muscle cells: regulation of platelet reactivity and smooth muscle cell guanylate cyclase.

1 Incubation of smooth muscle cells (SMC) from bovine aorta for 3 min with human washed platelets treated with indomethacin (10 μm) promoted a cell number‐related inhibition of platelet aggregation induced by thrombin (40 mu ml−1). This inhibition was not attributable to products of the cyclooxygenase pathway for the SMC were also treated with indomethacin (10 μm). 2 The inhibitory activity of the SMC on platelet aggregation was enhanced by incubating the SMC with E. coli lipopolysaccharide (LPS, 0.5 μg ml−1) for a period of 9 to 24 h. This effect was attenuated when cycloheximide (10 μg ml−1) was incubated together with LPS. Cycloheximide did not prevent the inhibitory activity of the non‐treated cells. 3 The inhibition of platelet aggregation obtained with non‐treated or LPS‐treated SMC was potentiated by superoxide dismutase (SOD, 60 u ml−1) and ablated by oxyhaemoglobin (OxyHb, 10 μm). Preincubation of the SMC with NG‐monomethyl‐l‐arginine (l‐NMMA, 30–300 μm) for 60 min prevented their antiaggregatory activity. This effect was reversed by concurrent incubation with l‐arginine (l‐Arg, 100 μm) but not with d‐arginine (d‐Arg, 100 μm). 4 Exposure of the non‐treated SMC (5 × 105 cells) to stirring (1000 r.p.m., 37°C) for 10 min led to a significant increase in their levels of guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) but not adenosine 3′:5′‐cyclic monophosphate (cyclic AMP). l‐NMMA (300 μm) attenuated the increase in cyclic GMP induced by stirring but did not affect the basal levels of cyclic GMP in the cells. The inhibitory activity of l‐NMMA was reversed by co‐incubation with l‐Arg (100 μm) but not d‐Arg (100 μm). l‐Arg alone had no effect on the levels of cyclic GMP. In the absence of stirring, a 10 min stimulation of the non‐treated SMC with glyceryl trinitrate (GTN, 200 μm) or atrial natriuretic factor (ANF, 10−7 m) led to an increase in the levels of cyclic GMP but not cyclic AMP. The increase in cyclic GMP promoted by GTN or ANF was not affected by l‐NMMA. The levels of cyclic GMP were higher in the LPS (0.5 μg ml−1, 18 h)‐treated cells (5 × 10−5) and stirring was more effective in increasing the levels of cyclic GMP in these cells. 5 These findings support the idea that non‐treated or LPS‐treated cultured SMC can produce an NO‐like factor. Production by the latter requires protein synthesis as evidenced by blockade with cycloheximide. This NO‐like factor may play a role in the auto‐regulation of smooth muscle cell reactivity through a cyclic GMP‐dependent mechanism.

Cultured endothelial cells maintain their L-arginine level despite the continuous release of EDRF

Endothelial cells cultured from bovine aorta and grown on microcarrier beads contain 107 +/- 9 microM L-arginine (Arg; n = 11). When packed into a jacketed chromatography column and perfused with Krebs solution, the cells showed a substantial and sustained release of endothelium-derived relaxing factor (EDRF) for up to 2 h, which was further enhanced by infusions of adenosine diphosphate (4 microM). In contrast to other amino acids, such as L-alanine, L-aspartate, L-glutamine, L-glutamate or L-serine, which showed a time-dependent decrease to less than 30% of their original level within 2 h, Arg remained at control levels for 30 min and decreased only by 25% after 2 h. Thus endothelial cells can generate Arg from an intracellular source to maintain their Arg level despite the continuous formation of EDRF.

Substantial inhibition of neo-intimal response to balloon injury in the rat carotid artery using a combination of antibodies to platelet-derived growth factor-BB and basic fibroblast growth factor

Thirty percent of patients undergoing percutaneous transluminal coronary angioplasty develop recurrent disease within a year. This is usually due to the rapid accumulation of intimal smooth muscle cells and extracellular matrix, which causes luminal narrowing, and is probably orchestrated by several mitogenic and chemotactic factors, of which platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) appear to be particularly important. We have investigated the effects of administering a combination of neutralizing antibodies directed against PDGF-BB and bFGF on neo-intima development following balloon catheter injury in the rat carotid artery. Purified sheep anti-PDGF-BB and anti-bFGF immunoglobulins (IgGs) were administered singly and in combination prior to mechanical injury and daily until sacrifice, 8 days later. Plasma titres of exogenous anti-PDGF-BB and anti-bFGF were maintained at levels 10-20-fold higher than those required to neutralise the mitogenic and chemotactic effects of 20 ng/ml of PDGF-BB, or 10 ng/ml bFGF in vitro. Used singly, anti-PDGF IgG treatment was associated with a 47% reduction in intimal thickness and a 59% reduction in intimal:medial area ratio; anti-bFGF IgG administration caused a 53% reduction in intimal thickness, and a 50% reduction in intimal:medial area ratio. Treatment with a combination of these antibodies resulted in a 83.8% reduction in intimal thickness (P < 0.05), and a 91% reduction in intimal:medial area ratio (P < 0.01). The latter treatment was also associated with a significantly higher intimal cell density (14.2 +/- 1.6 x 10(3) nuclei/mm2) compared to animals receiving non-immune IgG (7.8 +/- 0.8 x 10(3) nuclei/mm2; P < 0.025), although intimal and medial cell proliferation indices were not significantly different between the groups (P > 0.05). Our results suggest that in this particular model, PDGF-BB and bFGF are the major factors controlling neointimal hyperplasia, and that these growth factors are operating principally via an effect on smooth muscle cell migration and extracellular matrix protein accumulation.

Oral treatment with an antioxidant (raxofelast) reduces oxidative stress and improves endothelial function in men with Type II diabetes

Aims/hypothesis. To determine whether raxofelast, a new water soluble antioxidant decreases oxidative stress and improves endothelial function in men with Type II (non-insulin dependent) diabetes mellitus. Methods. We treated ten normotensive, normocholesterolaemic men with Type II diabetes and as controls ten healthy men matched with them for age with raxofelast (600 mg twice daily) for 1 week. Plasma 8-epi-PGF2α, a non-enzymic oxidation product of arachidonic acid was measured by gas chromatography/mass spectrometry as an index of oxidative stress. Forearm vasodilator responses to brachial artery infusion of acetylcholine (7.5, 15 and 30 μg min–1) and of the nitric oxide donor nitroprusside (1, 3 and 10 μg min–1) were measured by strain gauge plethysmography. Results. Plasma concentrations of 8-epi-PGF2α were greater in diabetic than in control men (0.99 ± 0.20 vs 0.18 ± 0.01 nmol l–1, means ± SEM, p < 0.001) and fell after raxofelast (from 0.99 ± 0.20 to 0.47 ± 0.07 nmol l–1, p < 0.05) in diabetic men but not in control men. Blood flow responses to acetylcholine were lower (p < 0.05) in diabetic than in control men (7.4 ± 1.0 vs 12.9 ± 2.3 ml · min–1· 100 ml–1 for the highest dose). In diabetic men, but not in control men, raxofelast increased (p < 0.05) blood flow responses to acetylcholine (from 7.4 ± 1.0 ml · min–1· 100 ml–1 to 11.3 ± 2.3 ml · min–1· 100 ml–1 at highest dose). Blood flow responses to nitroprusside were similar in control and diabetic men and in both groups were similar before and after raxofelast. Conclusion/interpretation. Oral treatment with raxofelast for 1 week reduces oxidative stress and improves endothelial function in men with Type II diabetes. [Diabetologia (2000) 43: 974–977]