Peter D. Weinberg

Regulation of cell signalling by vitamin E.

Vitamin E, the most important lipid-soluble antioxidant, was discovered at the University of California at Berkeley in 1922. Since its discovery, studies of the constituent tocopherols and tocotrienols have focused mainly on their antioxidant properties. In 1991 Angelo Azzis group (Boscoboinik et al. 1991a,b) first described non-antioxidant cell signalling functions for alpha-tocopherol, demonstrating that vitamin E regulates protein kinase C activity in smooth muscle cells. At the transcriptional level, alpha-tocopherol modulates the expression of the hepatic alpha-tocopherol transfer protein, as well as the expression of liver collagen alphal gene, collagenase gene and alpha-tropomyosin gene. Recently, a tocopherol-dependent transcription factor (tocopherol-associated protein) has been discovered. In cultured cells it has been demonstrated that vitamin E inhibits inflammation, cell adhesion, platelet aggregation and smooth muscle cell proliferation. Recent advances in molecular biology and genomic techniques have led to the discovery of novel vitamin E-sensitive genes and signal transduction pathways.

Effect of genistein and daidzein on platelet aggregation and monocyte and endothelial function.

There has been much recent interest in the cardiovascular benefits of dietary isoflavones. The aim of the present in vitro studies was to investigate potential anti-thrombogenic and anti-atherogenic effects of the isoflavones genistein and daidzein in platelets, macrophages and endothelial cells. Pre-treatment with either isoflavone inhibited collagen-induced platelet aggregation in a dose-dependent manner. In a macrophage cell line (RAW 264.7) activated with interferon gamma plus lipopolysaccharide, both isoflavones were found to inhibit NO production and tumour necrosis factor alpha (TNF-alpha) secretion dose-dependently, but they did not affect mRNA levels for inducible nitric oxide synthase and cyclo-oxygenase-2. Both isoflavones also dose-dependently decreased monocyte chemoattractant protein-1 secretion induced by TNF-alpha in human umbilical vein endothelial cells. Compared with daidzein, genistein exerted greater inhibitory effects for all parameters studied. The present data contributes to our knowledge on the molecular mechanisms by which isoflavones may protect against coronary artery disease. Further studies are required to determine whether the effects of isoflavones observed in the current in vitro studies are relevant to the aetiology of coronary artery disease in vivo.

Effect of circulating forms of soy isoflavones on the oxidation of low density lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.

Contrasting Patterns of Spontaneous Aortic Disease in Young and Old Rabbits

The pattern of spontaneous lipid deposition around aortic branch ostia was mapped in New Zealand White rabbits aged 1 month or 2 to 6 years. The young rabbits were studied within 1 day of weaning, and the older rabbits had been maintained on a low-protein, low-fat diet. Plasma concentrations of total cholesterol for the two groups averaged 75 and 18 mg/dL, respectively. Aortas were fixed in situ at a pressure of 90 to 100 cm H2O, stained with oil red O, and photographed en face under an epifluorescence microscope. Areas of staining contrasted in color with the fixative-stimulated autofluorescence of nondiseased tissue and were mapped by placing grids over the photomicrographs. Disease around intercostal ostia was rare, but two distributions were established by combining data from many branches. In weanlings, staining was seen within a triangular area downstream of the branch. In old animals, this area had the lowest frequency of disease; lesions tended to occur downstream of the spared region, along axes lying to either side of it, and at the lateral and upstream margins of the ostium. Disease was less rare at celiac branches. It occurred mainly downstream of the ostium in weanlings, whereas upstream sites were most affected in old animals, although significant disease remained at the juvenile locations. Earlier reports have described similar age-related distributions of disease in human aortas, consistent with a common underlying mechanism. The distributions also correlate with the spatial variations in arterial transport properties established in previous studies, and may be determined by them.

Rate-limiting steps in the development of atherosclerosis: the response-to-influx theory.

A large number of processes are involved in the pathogenesis of atherosclerosis but it is unclear which of them play a rate-limiting role. One way of resolving this problem is to investigate the highly non-uniform distribution of disease within the arterial system; critical steps in lesion development should be revealed by identifying arterial properties that differ between susceptible and protected sites. Although the localisation of atherosclerotic lesions has been investigated intensively over much of the 20th century, this review argues that the factor determining the distribution of human disease has only recently been identified. Recognition that the distribution changes with age has, for the first time, allowed it to be explained by variation in transport properties of the arterial wall; hitherto, this view could only be applied to experimental atherosclerosis in animals. The newly discovered transport variations which appear to play a critical role in the development of adult disease have underlying mechanisms that differ from those elucidated for the transport variations relevant to experimental atherosclerosis: they depend on endogenous NO synthesis and on blood flow. Manipulation of transport properties might have therapeutic potential.

Changes With Age in the Influence of Endogenous Nitric Oxide on Transport Properties of the Rabbit Aortic Wall Near Branches

Uptake of circulating albumin by the aortic wall is greater downstream than upstream of branches in immature rabbits, but the opposite pattern occurs in mature animals. We investigated the role of NO in determining these variations. Descending thoracic aortas of rabbits were cannulated using techniques that avoid depressurization, overstretching, and excessive fluid dynamic stresses at the endothelial surface. They were perfused in situ at a constant pressure and flow rate with oxygenated, protein-containing physiological buffer, with or without N omega-monomethyl-L-arginine, an inhibitor of NO synthesis. Aortas were fixed 7 to 8 minutes after the addition of rhodamine-labeled albumin to this perfusate, and uptake of the tracer near intercostal ostia was measured by digital imaging fluorescence microscopy of sections through the wall. Despite the absence of pulsatile flow, blood cells, and many plasma components, patterns of transport in control experiments were the same as those occurring in vivo; uptake was greatest downstream of ostia in immature vessels and upstream in mature ones, although mean uptake was higher than previously reported. In the presence of the inhibitor, mean uptake in immature arteries was elevated threefold and the maximum tracer concentration occurred deeper in the wall, but there was no change in the fractional difference between regions. Conversely, the reverse of the control pattern of transport was observed in mature arteries exposed to the inhibitor, but there was no change in mean uptake. The reversal was almost entirely prevented by adding excess L-arginine to the perfusate and was largely stereospecific. Endogenous NO thus appears to determine the mature pattern of transport near branches and helps to maintain the barrier function of the immature wall.

Effect of Age on the Pattern of Short-term Albumin Uptake by the Rabbit Aortic Wall Near Intercostal Branch Ostia

Lipid deposition occurs more frequently downstream than upstream of branches in immature human aorta but the opposite pattern is seen in mature vessels. These distributions may reflect variation in the uptake of plasma macromolecules by the aortic wall. We have recently shown that the quasi-steady state uptake of albumin is greater downstream than upstream of branches in immature rabbit aortas and that the opposite pattern occurs in mature animals. Additionally, there is a sharp drop in the mean uptake shortly after weaning. In the present study, the mechanisms underlying these phenomena were investigated by examining the short-term uptake of albumin and its distribution across the wall. Albumin was labeled with a fluorescent dye and introduced into the circulation of conscious New Zealand White rabbits. Thoracic aortas were fixed in situ 10 minutes later and were sectioned through the center of intercostal ostia. Fluorescence from sections was measured by using digital imaging fluorescence microscopy and was converted to tracer concentrations after appropriate autofluorescence levels had been subtracted. In animals aged 45 days, more tracer was detected in the wall downstream than upstream of branches; the difference between regions was > 100% of the mean value. This percentage halved and the mean uptake decreased almost threefold by 75 days. In mature animals, the mean value remained at the 75-day level but the converse distribution was seen; 22% more tracer was detected upstream than downstream. These trends were insensitive to the depth of the intimal-medial layer examined. In each region, the maximum tracer concentration occurred close to the luminal surface but not always within the first 2.9-microns-thick layer of the wall. Maxima were similar in magnitude to those observed at quasi-steady state, but the fall with increasing distance into the wall was much sharper. In many cases concentrations remained constant over most of the media, and rises toward the adventitial boundary were rarely seen. Uptake after 10 minutes predominantly reflects the rate at which tracer enters the wall. The concentration profiles were consistent with most of the tracer having entered from the luminal surface and with the involvement of convective transport. The trends observed with age closely paralleled those occurring at quasi-steady state. Consequently, the latter are also likely to be determined by changes in the resistance of the wall to macromolecule influx.

Age-related variations in transport properties of the rabbit arterial wall near branches

Lipid accumulation in the human aorta occurs predominantly downstream of branches in foetuses, neonates and infants but upstream at later ages. The lipid in these deposits may derive from plasma lipoproteins. We have examined uptake of plasma proteins by the rabbit aortic wall near branches as a function of age. Albumin was labelled with a fluorescent dye and introduced into the circulation of animals fed a normal diet. The aorta was fixed in situ 3 h later and the distribution of tracer in sections through the wall was measured by using digital imaging fluorescence microscopy. Net uptake by the intima-media was higher downstream of intercostal ostia than upstream in young animals but this difference decreased and then reversed with age. Furthermore, the average of uptake by both regions was higher shortly after weaning than at later ages. These age-related variations in transport properties may explain discrepancies between previous studies of uptake, resolve apparent inconsistencies between the properties of rabbit and human arteries and, if applicable to man, might account for the non-uniform and changing pattern of lipid accumulation around arterial branches.

Techniques for reducing the interfering effects of autofluorescence in fluorescence microscopy: improved detection of sulphorhodamine B-labelled albumin in arterial tissue.

Measurements of the transport of circulating sulphorhodamine B‐labelled albumin into the arterial wall, made by applying digital imaging fluorescence microscopy to sections of arteries fixed in situ, are limited in sensitivity by the low levels of tracer fluorescence and high levels of autofluorescence emitted from the tissue. Three attempts to improve these ratios are described. In the first, spectra of the tracer in solution and of arterial autofluorescence were used to design novel microscope filters for rhodamine‐like dyes. By exciting with the rarely used yellow lines of the mercury arc lamp and detecting a narrow band of emission with Stokes shifts as small as 15u2003nm, the ratio of tracer fluorescence to autofluorescence was tripled. In the second, effects of different fixatives were investigated. Using a model system, it was confirmed that Karnovskys fixative gives good tracer immobilization but elevates autofluorescence, whereas fixative‐free buffer solutions give low autofluorescence but do not retain the tracer. It was further found that simple formaldehyde‐based fixatives, hitherto considered to be poor fixatives of albumin, immobilized the tracer as well as the glutaraldehyde‐based fixative, whilst giving autofluorescence levels comparable to those seen with buffer alone; they therefore give excellent tracer fluorescence to autofluorescence ratios. In the third, lowering specimen temperature by 50u2003°C was found to increase the intensity of tracer fluorescence by 30% whilst autofluorescence was unaffected. These data may have relevance to microscopical studies using other tissues and fluorescent tracers.

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 NG‐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.