Elevated uptake of plasma macromolecules by regions of arterial wall predisposed to plaque instability in a mouse model.

Atherosclerosis may be triggered by an elevated net transport of lipid-carrying macromolecules from plasma into the arterial wall. We hypothesised that whether lesions are of the thin-cap fibroatheroma (TCFA) type or are less fatty and more fibrous depends on the degree of elevation of transport, with greater uptake leading to the former. We further hypothesised that the degree of elevation can depend on haemodynamic wall shear stress characteristics and nitric oxide synthesis. Placing a tapered cuff around the carotid artery of apolipoprotein E -/- mice modifies patterns of shear stress and eNOS expression, and triggers lesion development at the upstream and downstream cuff margins; upstream but not downstream lesions resemble the TCFA. We measured wall uptake of a macromolecular tracer in the carotid artery of C57bl/6 mice after cuff placement. Uptake was elevated in the regions that develop lesions in hyperlipidaemic mice and was significantly more elevated where plaques of the TCFA type develop. Computational simulations and effects of reversing the cuff orientation indicated a role for solid as well as fluid mechanical stresses. Inhibiting NO synthesis abolished the difference in uptake between the upstream and downstream sites. The data support the hypothesis that excessively elevated wall uptake of plasma macromolecules initiates the development of the TCFA, suggest that such uptake can result from solid and fluid mechanical stresses, and are consistent with a role for NO synthesis. Modification of wall transport properties might form the basis of novel methods for reducing plaque rupture.

Endothelial microparticle release is stimulated in vitro by purified IgG from patients with the antiphospholipid syndrome.

IgG antiphospholipid antibodies (aPL) exert direct effects on various cell types, contributing to the pathogenesis of thrombosis and pregnancy morbidity in patients with the antiphospholipid syndrome (APS). Some IgG samples from these patients activate endothelial cells (EC) in vitro as judged by surface expression of adhesion molecules such as E-selectin, which can promote thrombosis. Endothelial microparticles (EMP), which themselves are potentially prothrombotic, are released by activated EC. Though elevated circulating EMP levels have been reported in patients with APS, it is not known whether these EMP are released due to a direct effect of aPL on the cells. We tested the effect of purified polyclonal IgG from patients with APS (APS-IgG) and healthy controls (HC-IgG) upon cultured human umbilical vein EC (HUVEC). HUVEC exposed to APS-IgG produced significantly more EMP than those exposed to HC-IgG (p=0.0036) and a greater proportion of these EMP carried surface E-selectin (6.2% ± 4.0 for APS-IgG vs. 3.4% ± 2.0 for HC IgG, p=0.0172). This study therefore demonstrates that purified polyclonal APS-IgG can drive EMP release. We propose that EMP generation may be a useful measure of aPL-mediated pathogenic effects upon EC.

High throughput en face mapping of arterial permeability using tile scanning confocal microscopy.

Elevated uptake of plasma macromolecules by the arterial wall has been implicated in the initiation of atherosclerosis. Here we describe a new method for mapping such uptake in laboratory animals. Albumin was labelled with a fluorescent dye and administered intravenously. After 10 min, the aorta was fixed in situ, excised and opened. En face confocal microscopy employing a computer-controlled stage was used to obtain contiguous tiles, each consisting of a stack of images of fluorescence emission at different depths in the wall. To obtain two-dimensional maps, intensities were summed in each column of voxels starting at the endothelial surface and extending 10 µm into the wall. Variation in the sensitivity of the system with time and in all three spatial directions was assessed and corrected using calibration standards and model specimens. In immature rabbits, uptake around aorto-intercostal branches was greatest in an arrowhead-shaped region around the downstream half of each ostium, and at its lateral margins. Uptake around branches in mature rabbits was more uniform; it was highest upstream of the ostium. Patches and streaks of high uptake were also seen at non-branch locations in the descending thoracic aorta. Transport was more uniform around branches in mice, except for small regions of high uptake at the ostial rim and at the leading edge of an intimal cushion upstream of the ostium, where lesions develop. The technique provides accurate quantification in three dimensions over large areas; it has high throughput, sensitivity and resolution and is suitable for widespread use.