The study of the influence of flow on vascular endothelial biology.

It is now recognized that the mechanical environment of a cell has an influence on its structure and function. For the vascular endothelial cell that resides at the interface of the flowing blood and the underlying vessel wall, there is mounting evidence of the importance of flow and the associated wall shear stress in the regulation of endothelial biology. Not only is it a sensitive regulator of endothelial structure and function, but different flow environments will influence endothelial cell biology differently. Furthermore, there may be an interaction between the chemical environment of a cell and its mechanical environment. This is illustrated by the inhibition by steady laminar shear stress of the cytokine induction of VCAM-1. Results also are presented in which flow studies have been conducted using a co-culture model of the vessel wall. These experiments provide evidence of a quiescent endothelium; however, much more needs to be done to engineer the cell culture environment to make it more physiologic.

Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: role of a superoxide-producing NADH oxidase.

Atherosclerotic lesions are found opposite vascular flow dividers at sites of low shear stress and oscillatory flow. Since endothelial proinflammatory genes prominent in lesions are regulated by oxidation-sensitive transcriptional control mechanisms, we examined the redox state of cultured human umbilical vein endothelial cells after either oscillatory or steady laminar fluid shear stress. Endothelial oxidative stress was assessed by measuring activity of the superoxide (O2.- )-producing NADH oxidase (a major source of reactive oxygen species in vascular cells), intracellular O2.- levels, induction of the redox-sensitive gene heme oxygenase-1 (HO-1), and abundance of Cu/Zn superoxide dismutase (Cu/Zn SOD), an antioxidant defense enzyme whose level of expression adapts to changes in oxidative stress. When cells were exposed to oscillatory shear (+/-5 dyne/cm2, 1 Hz) for 1, 5, and 24 hours, NADH oxidase activity and the amount of HO-1 progressively increased up to 174+/-16% (P<0.05) and 505+/-111% (P<0.05) versus static conditions, respectively, whereas levels of Cu/Zn SOD remained unchanged. This upregulation of HO-1 was completely blocked by the antioxidant N-acetylcysteine (NAC, 20 mmol/L). In contrast, steady laminar shear (5 dyne/cm2) induced NADH oxidase activity and NAC-sensitive HO-1 mRNA expression only at 1 and 5 hours, a transient response that returned toward baseline at 24 hours. Levels of Cu/Zn SOD mRNA and protein were increased after 24 hours of steady laminar shear. Furthermore, intracellular O2.-, as measured by dihydroethidium fluorescence, was higher in cells exposed to oscillatory than to laminar shear. These data are consistent with the hypothesis that continuous oscillatory shear causes a sustained activation of pro-oxidant processes resulting in redox-sensitive gene expression in human endothelial cells. Steady laminar shear stress initially activates these processes but appears to induce compensatory antioxidant defenses. We speculate that differences in endothelial redox state, orchestrated by different regimens of shear stress, may contribute to the focal nature of atherosclerosis.

Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium.

Low and oscillatory shear stresses are major features of the hemodynamic environment of sites opposite arterial flow dividers that are predisposed to atherosclerosis. Atherosclerosis is a focal inflammatory disease characterized initially by the recruitment of mononuclear cells into the arterial wall. The specific characteristics of the hemodynamic environment that facilitate the generation of arterial inflammatory responses in the presence of, for example, hyperlipidemia are unknown. We show here that prolonged oscillatory shear stress induces expression of endothelial cell leukocyte adhesion molecules, which are centrally important in mediating leukocyte localization into the arterial wall. Vascular cell adhesion molecule-1 was upregulated an average 9-fold relative to endothelial monolayers in static culture. Intercellular adhesion molecule-1 and E-selectin exhibited 11-fold and 7.5-fold increases, respectively. Upregulation of these adhesion molecules was associated with enhanced monocyte adherence. Cytokine stimulation of surface vascular cell adhesion molecule-1 was maximally induced after 6 and 8 hours of cytokine incubation. Oscillatory shear stress for these time periods elicited respective vascular cell adhesion molecule-1 levels of 16% and 30% relative to those observed for cytokine stimulation. Surface intercellular adhesion molecule-1 induction by cytokine stimulation for 24 hours was found to be approximately five times the level detected after 24 hours of oscillatory shear stress. Experiments performed in the presence of the antioxidant N-acetylcysteine demonstrated that the expression of vascular cell adhesion molecule-1 could be almost totally abolished, whereas that of intercellular adhesion molecule-1 was typically reduced by approximately 70%. These results imply that oscillatory shear stress per se is sufficient to stimulate mononuclear leukocyte adhesion and, presumptively, migration into the arterial wall. These results further indicate that atherosclerotic lesion initiation is likely related, at least in part, to unique signals generated by oscillatory shear stress and that the mechanism of upregulation is, to some extent, redox sensitive.

Focal and regional variations in the composition of the glycocalyx of large vessel endothelium.

The glycocalyx of the endothelium of the systemic arteries and vena cava of the rabbit was visualised by in situ perfusion fixation with glutaraldehyde containing Alcian blue. The thickness of the layer ranged from 45 +/- 1 nm in the coronary artery to 81 +/- 2 nm in the carotid. The glycocalyx was 20 +/- 1.5 nm thicker on the downstream side of intercostal ostia than on the upstream side. Changes in the staining pattern with increasing concentrations of MgCl2 indicated that carboxyl groups made the major contribution to the surface charge, though sulphate groups were also present, particularly in the aortic arch and carotid artery. Segments of the thoracic aorta and carotid artery were also stained in vitro with fluorescence labelled wheat germ agglutinin, and fluorescence intensity in histological sections was quantified using a video microscope equipped with a microcomputer-based image analysis system. The fluorescence intensity in the carotid was 1.65 +/- 0.15 times that in the aorta. Pretreatment with neuraminidase reduced fluorescence intensity by 60 +/- 4% in the carotid and 53 +/- 2% on the upstream side of intercostal ostia, but only by 37 +/- 3% on the downstream side. Chondroitinase and heparanase both reduced binding and when used together their effect was additive, reducing fluorescence by 27 +/- 3, 51 +/- 4, and 32 +/- 3% at the three sites, respectively. Though the interpretation of the lectin binding experiments is complicated by a number of factors, these results support previous reports that sialyl groups are abundant in the endothelial glycocalyx. Glycosaminoglycans are also present, however, in significant amounts.

Mechanisms of interaction of albumin with arterial elastin.

Binding of proteins, lipoproteins and calcium salts to arterial elastin occurs with age and in disease. In order to clarify the mechanisms of interaction we investigated the binding of native and charge-modified albumins to elastin isolated by alkali extraction of the porcine aorta. Fluorescence microscopy was used to quantify the distribution of lissamine-rhodamine labelled albumins in native and charge-modified elastins under different solution conditions. For native albumin uptake was approximately twice as high on the intimal and adventitial sides as in the mid media. The distribution of anionic albumin was very similar, but methylation either of the albumin or the elastin greatly reduced the transmural variation. The uptake of methylated albumin was approximately three-fold greater than the native albumin. The uptake of the native albumin was reduced preferentially in the intimal region in the presence of calcium and only the charge-modified proteins were able to penetrate the intralamellar space. These observations demonstrated the importance of hydrophobic interactions in albumin binding.