No description available.

It is becoming increasingly evident that the development of atherosclerotic coronary heart disease (CHD) can largely be regulated by lifestyle and dietary choices. The type of fatty acids regularly consumed may promote or prevent atherogenesis. Flaxseed, the richest plant source of the omega-3 fatty acid alpha-linolenic acid (ALA) is thought to protect against atherosclerotic disease. However, the mechanism(s) by which flaxseed exerts these anti-atherogenic effects requires further investigation. Alternatively, there are dietary fatty acids that are thought to induce significant deleterious effects upon our cardiovascular health. Epidemiological evidence associates dietary trans fatty acids (TFAs) with atherosclerotic CHD. This evidence has largely focused on the main source of TFAs in the North American diet, industrially hydrogenated vegetable shortening (iTFAs). It is assumed that TFAs stimulate atherosclerosis but the only studies to date have shown no effect of TFAs on atherosclerosis. Even less is known of the impact of naturally occurring TFAs from dairy and meat products of ruminant animals (rTFAs) on atherosclerotic disease. We investigated the effects of flaxseed supplementation on atherosclerosis and vascular function in two animal models, the hypercholesterolemic rabbit and the cholesterol fed, low density lipoprotein receptor (LDLr-/-) deficient mouse. New Zealand White rabbits and LDLr-/- mice were fed a diet containing flaxseed in the absence or presence of dietary cholesterol for a period of 6 to 24 weeks. We found that dietary flaxseed inhibits the atherogenic effects of a high cholesterol diet in both animal models. The anti-atherogenic effect was achieved in the mouse model through a capacity to lower circulating cholesterol levels and at a cellular level by inhibiting cell proliferation and inflammation. This reduction is also associated with an improved vascular relaxation response as demonstrated in the rabbit model. We also investigated the effects of consuming TFAs from two sources, industrially hydrogenated iTFAs rich in elaidic TFA (C18:1t-9) or naturally-occurring ruminant rTFAs rich in vaccenic TFA (C18:1t-11), on atherosclerotic development in the LDLr-/- mouse in the presence or absence of elevated dietary cholesterol. Our results demonstrate that consuming iTFAs dose dependently initiates atherosclerotic development but not beyond the effects of dietary cholesterol alone. However, consuming rTFAs rich in vaccenic acid protects against hyperlipidemia and atherosclerosis in the presence or absence of dietary cholesterol. The effects of combining dietary flaxseed and iTFAs in the diet were also examined in this model. Adding whole ground flaxseed or flaxseed oil (ALA) to diets containing low and high doses of iTFAs completely prevented atherosclerotic development in the absence of dietary cholesterol. Flaxseed was also able to partially prevent atherosclerosis caused by iTFAs and cholesterol. Our results suggest that the omega-3 ALA fatty acid rich content of flaxseed is mainly responsible for the anti-atherogenic effects of flaxseed. Our results highlight potential mechanisms for the beneficial effects of dietary flaxseed and the mixed effects of TFAs on cardiovascular health and underscore the need for further basic and clinical investigations.

No description available.

It is becoming increasingly evident that the development of atherosclerotic coronary heart disease (CHD) can largely be regulated by lifestyle and dietary choices. The type of fatty acids regularly consumed may promote or prevent atherogenesis. Flaxseed, the richest plant source of the omega-3 fatty acid alpha-linolenic acid (ALA) is thought to protect against atherosclerotic disease. However, the mechanism(s) by which flaxseed exerts these anti-atherogenic effects requires further investigation. Alternatively, there are dietary fatty acids that are thought to induce significant deleterious effects upon our cardiovascular health. Epidemiological evidence associates dietary trans fatty acids (TFAs) with atherosclerotic CHD. This evidence has largely focused on the main source of TFAs in the North American diet, industrially hydrogenated vegetable shortening (iTFAs). It is assumed that TFAs stimulate atherosclerosis but the only studies to date have shown no effect of TFAs on atherosclerosis. Even less is known of the impact of naturally occurring TFAs from dairy and meat products of ruminant animals (rTFAs) on atherosclerotic disease. We investigated the effects of flaxseed supplementation on atherosclerosis and vascular function in two animal models, the hypercholesterolemic rabbit and the cholesterol fed, low density lipoprotein receptor (LDLr-/-) deficient mouse. New Zealand White rabbits and LDLr-/- mice were fed a diet containing flaxseed in the absence or presence of dietary cholesterol for a period of 6 to 24 weeks. We found that dietary flaxseed inhibits the atherogenic effects of a high cholesterol diet in both animal models. The anti-atherogenic effect was achieved in the mouse model through a capacity to lower circulating cholesterol levels and at a cellular level by inhibiting cell proliferation and inflammation. This reduction is also associated with an improved vascular relaxation response as demonstrated in the rabbit model. We also investigated the effects of consuming TFAs from two sources, industrially hydrogenated iTFAs rich in elaidic TFA (C18:1t-9) or naturally-occurring ruminant rTFAs rich in vaccenic TFA (C18:1t-11), on atherosclerotic development in the LDLr-/- mouse in the presence or absence of elevated dietary cholesterol. Our results demonstrate that consuming iTFAs dose dependently initiates atherosclerotic development but not beyond the effects of dietary cholesterol alone. However, consuming rTFAs rich in vaccenic acid protects against hyperlipidemia and atherosclerosis in the presence or absence of dietary cholesterol. The effects of combining dietary flaxseed and iTFAs in the diet were also examined in this model. Adding whole ground flaxseed or flaxseed oil (ALA) to diets containing low and high doses of iTFAs completely prevented atherosclerotic development in the absence of dietary cholesterol. Flaxseed was also able to partially prevent atherosclerosis caused by iTFAs and cholesterol. Our results suggest that the omega-3 ALA fatty acid rich content of flaxseed is mainly responsible for the anti-atherogenic effects of flaxseed. Our results highlight potential mechanisms for the beneficial effects of dietary flaxseed and the mixed effects of TFAs on cardiovascular health and underscore the need for further basic and clinical investigations.

There is little doubt that disturbed hemodynamic forces play a role in the development of focal atherosclerotic lesions; however, these forces are difficult to measure directly. Instead, it has been proposed that artery geometry, as the primary determinant of local hemodynamics, could be a clinically feasible surrogate “local” risk factor for atherosclerosis. To date this hypothesis has not been satisfactorily tested, owing to superficial geometric surrogates of disturbed flow, small sample sizes (effect of systemic factors) and/or confounding effects of disease on geometry. The primary objective of this thesis was to test this “geometric risk hypothesis” via direct association of definitive geometric factors and an early atherosclerosis marker (e.g. wall thickness), made possible through our access to magnetic resonance imaging and risk factor data from the Atherosclerosis Risk in Communities’ Carotid MRI sub-study. First, it was shown that the 3D geometry of the carotid bifurcation could be characterized rapidly and reliably, even for routine clinical acquisitions. Second, two novel individual geometric variables were proposed, inspired by the influence of flare and tortuosity on flow separation, which were shown to improve the prediction of disturbed flow burden compared to “conventional” shape-based geometric variables. Third, these redefined geometric factors, but not their shape-based counterparts, were shown by multiple regression to be independent predictors of wall thickness, but only after thoroughly accounting for the secondary effects of wall thickening on geometry. These findings provide strong evidence for the geometric risk hypothesis of atherosclerosis in humans group study, and provide important guidance for future investigations of geometric risk; however, the incremental value of optimized geometric risk factors is questionable relative to conventional cardiovascular risk factors, which challenges their future clinical usage as additional non-modifiable local risk factors.

There is little doubt that disturbed hemodynamic forces play a role in the development of focal atherosclerotic lesions; however, these forces are difficult to measure directly. Instead, it has been proposed that artery geometry, as the primary determinant of local hemodynamics, could be a clinically feasible surrogate “local” risk factor for atherosclerosis. To date this hypothesis has not been satisfactorily tested, owing to superficial geometric surrogates of disturbed flow, small sample sizes (effect of systemic factors) and/or confounding effects of disease on geometry. The primary objective of this thesis was to test this “geometric risk hypothesis” via direct association of definitive geometric factors and an early atherosclerosis marker (e.g. wall thickness), made possible through our access to magnetic resonance imaging and risk factor data from the Atherosclerosis Risk in Communities’ Carotid MRI sub-study. First, it was shown that the 3D geometry of the carotid bifurcation could be characterized rapidly and reliably, even for routine clinical acquisitions. Second, two novel individual geometric variables were proposed, inspired by the influence of flare and tortuosity on flow separation, which were shown to improve the prediction of disturbed flow burden compared to “conventional” shape-based geometric variables. Third, these redefined geometric factors, but not their shape-based counterparts, were shown by multiple regression to be independent predictors of wall thickness, but only after thoroughly accounting for the secondary effects of wall thickening on geometry. These findings provide strong evidence for the geometric risk hypothesis of atherosclerosis in humans group study, and provide important guidance for future investigations of geometric risk; however, the incremental value of optimized geometric risk factors is questionable relative to conventional cardiovascular risk factors, which challenges their future clinical usage as additional non-modifiable local risk factors.

Flow mediated dilation (FMD) is a non-invasive test that assesses endothelial health and nitric oxide bioavailability; it is commonly used to examine changes in vascular health due to disease or de-conditioning. Currently, a wide variety of protocols are being used to assess upper and lower extremity conduit artery health. The current project was embarked upon to gain a better understanding of the FMD protocols currently being used to asses conduit artery FMD and how these results impact our understanding of a participant’s vascular health. More specifically occlusion duration, cuff placement and artery location were examined in three commonly examined conduit arteries. The FMD responses in the brachial artery (BA), superficial femoral artery (SFA), and popliteal artery (PA) of ten healthy men, mean age of 27, after five and/or two-minutes of distal occlusion were examined. When the two-minute protocol was performed on the SFA and PA, low-resistance static calf exercise was added to augment the shear stimulus. It was hypothesized that percent FMD and shear stress responses of the SFA and PA would not be significantly different after five-minutes of occlusion, thereby allowing leg conduit artery FMD to be performed on either artery. It was further hypothesized that there would be no significant differences between the shear stress and percent FMD responses of the leg conduit arteries after five or two-minutes of occlusion; inferring that shorter occlusion durations when combines with ischemic muscle contractions can be used to assess SFA or PA FMD. With regards to comparisons between arm and leg conduit arteries, it was hypothesized that there would be significant between limb differences in baseline diameter, FMD and shear stress post five-minutes of distal occlusion. These differences will be used to better understand the effects of artery location and size on conduit artery FMD IV responses. Limitations with the traditional edge-detection method of determining arterial diameter prompted the creation of a new method of measuring artery diameter, the center-based method. It was hypothesized that there would be no significant differences in the percent FMD and time to FMD after five-minutes of BA occlusion (n=7). The results of the current study demonstrated that five-minutes of calf occlusion elicited a significant PA FMD but not a significant SFA FMD. FMD post two-minutes of PA occlusion with exercise was not significantly different than that produced by five-minutes of occlusion. Conversely, two-minutes of calf occlusion with exercise was unable to elicit a SFA FMD response. Significant differences in shear stress and FMD were reported between arm and leg conduit arteries, demonstrating different responses to five-minutes of distal occlusion due to artery size and location. Finally, no significant differences were noted between FMD and time to FMD when the center-based or edge-detection method was used. This study has demonstrated that the calf occlusion protocol was unable to elicit a FMD response in the SFA FMD; this occlusion location is only able to elicit a PA FMD response. Furthermore, two-minutes of occlusion with one-minute of exercise can be used in place of the five-minute protocol to examine PA FMD but not SFA FMD. Differences between arm and leg conduit arteries are noted and it has been suggested that this is likely due to leg conduit artery adaptations to gravity. Lastly, preliminary data suggest that the center-based method is an appropriate method of measuring conduit artery diameter.

Atherosclerosis 'is a key pathological process underlying the development and progression of three major diseases of the vascular system- coronary artery disease, cerebro-vascular and peripheral vascular disease. Chronic vascular wall inflammation is considered as a principal cause in the initiation and progression of atherosclerosis. Intimal thickening that develops in arteries and veins as an adaptive response to an injury has many similarities with atherosclerosis, but at the same time represents a unique pathological entity. This Thesis explores the utility of applying a novel DNAzyme based approach that targets "master-regulator" transcription factors c-jun and Egr-1 to in vivo and in vitro models of acute inflammation and intimal thickening. Studies included in this Thesis reveal that transcription factor c-jun plays a, key regulatory role in controlling leucocyte movement during an acute inflammation induced by IL-1 f3 through regulation of the expression of adhesion molecules ICAM, VCAM-1, E-selectin and VE-cadherin. Similarly, by applying ED5, a DNAzyme that targets transcription factor Egr-1 to the rat model of mesenteric microcirculation I demonstrate that Egr-1 controls leucocyte movement during an acute inflammation as evidenced by almost complete inhibition of leucocyte flux, adhesion and extravasation by ED5. The rabbit model of bypass grafting shows that Dz13 (a DNAzyme targeting transcription factor c-jun) significantly reduces intimal thickening in bypassed vein grafts of chow-fed animals at 28 days in vivo and in culture-grown human saphenous veins in vitro. Taken together these findings suggest that a DNAzyme based approach of targeting transcription factor c-jun has the potential to be used as a modulator of the acute inflammatory response and of intimal thickening formation. Further work needs to be done before this technology is ready for clinical use in humans.

People with diabetes suffer from an increased incidence of atherosclerosis, possibly due to the hyperglycaemia associated with this disease. Glucose may covalently modify proteins via glycation and glycoxidation reactions. Reactive aldehydes (e.g. methylglyoxal and glycolaldehyde) generated from these glycation and glycoxidation reactions, lipid peroxidation and other metabolic pathways may also modify proteins in glycation and glycoxidation reactions. These reactions can result in the formation of advanced glycation end-products, which are increased in diabetes and associated complications such as atherosclerosis. Low-density lipoproteins (LDLs) are the main source of lipid in atherosclerotic plaques, and the lipid-laden foam cells contained within. Modification of the single protein in LDL, apolipoprotein B-100 (apo B) by glucose and aldehydes may result in recognition of these altered LDL particles by macrophage scavenger receptors and cellular accumulation of cholesteryl esters; such accumulation is characteristic of atherosclerotic foam cells. The extent and nature of the modifications of LDLs that give rise to this behaviour have been poorly characterised, especially in regards to modification/oxidation of protein versus lipid components induced by glucose and low-molecular-mass aldehydes. Therefore the aims of this project were to: 1) characterise LDL modification by glucose, methylglyoxal and glycolaldehyde; 2) examine the effect of these modified LDLs on arterial cells by monitoring cellular viability, proliferation and cholesterol and cholesteryl ester levels; and 3) examine macrophage handling of apo B from these modified LDLs. Glycolaldehyde induced more rapid and more extensive changes to LDL than methylglyoxal, which was significantly more modified than LDL exposed to glucose, in the presence or absence of Cu2+. LDL was modified by glycolaldehyde and methylglyoxal in a time- and concentration-dependent manner. These aldehyde-modified LDLs were significantly more negatively charged relative (determined by changes in relative electrophoretic mobility), more aggregated (by SDS-PAGE) and lost more Arg, Lys and Trp residues (assessed by fluorescence-based assays) than glucose-modified and control LDLs. Glucose-modified LDL had more modest increases in net negative charge, aggregation and only significantly lost Arg residues. Under the conditions examined none of the modified LDLs contained significant levels of the protein oxidation products DOPA and o-tyrosine, the lipid oxidation products 7-ketocholesterol and cholesteryl ester hydro(pero)oxides, nor marked depletion of the major antioxidant α-tocopherol or significant radical formation (EPR spectroscopy). Therefore these LDLs were glycated, but not (glyc)oxidised, and so allowed the cellular uptake of glycated LDL, rather than glycoxidised LDL, to be examined. These glycated LDLs had no effect on the cellular viability (assessed by LDH release), cell protein (BCA assay), and cholesterol and cholesteryl ester levels (quantified by reverse-phase HPLC) of endothelial and smooth muscle cells. The glycated LDLs also had no effects on human and mouse macrophage viability, protein and free cholesterol levels. However, exposure of macrophages to some of the glycated LDLs resulted in significant accumulation of cholesteryl esters and apo B. The greatest cellular accumulation of cholesteryl esters was in cells exposed to glycolaldehyde-modified LDL, which occurred in a time- and concentration-dependent manner. Less cholesteryl ester accumulation was observed in cells exposed to methylglyoxal-modified LDL, but some conditions resulted in significantly more cellular cholesteryl esters as compared to control LDLs, unlike glucose-modified LDL. Macrophages endocytosed significantly more apo B from glycolaldehyde-modified LDL labelled with 125I on the apo B, than methylglyoxal-modified 125I-LDL. Apo B from methylglyoxal-modified 125I-LDL was also endocytosed and degraded in greater amounts than control 125I-LDLs, unlike glucose-modified 125I-LDLs. The glycation of LDL by some low-molecular-mass aldehydes have been shown to result in model foam cell formation as characterised by cholesteryl ester and apo B accumulation. This accumulation correlated with increases in net negative charge, aggregation and loss of Lys and Trp residues of the apo B in glycated LDL particles. However, the differences in cellular uptake of glycolaldehyde- versus methylglyoxal-modified LDL were not completely resolved and it is postulated that this may arise from the extent or type of products formed on key amino acid residues, resulting in differential uptake by macrophage scavenger receptors, rather than loss of particular amino acids per se. Therefore these studies provide a potential mechanism to explain the increased atherosclerosis in people with diabetes, and a suitable model to examine the potential inhibition of the effects of glycated LDLs. This could provide potential therapeutic interventions to reduce diabetes-induced atherosclerosis.

Thesis Erasmus University Rotterdam. / ook verschenen in gedrukte versie. With bibliogr., with a summary in Dutch.