Intimal cushions and endothelial nuclear elongation around mouse aortic branches and their spatial correspondence with patterns of lipid deposition

Abstract

Spatial variation in hemodynamic stresses acting on the arterial wall may explain the nonuniform distribution of atherosclerosis. In thoracic aortas of LDL receptor/apolipoprotein E double knockout mice, lesions develop preferentially around the entire circumference of intercostal branch ostia, regardless of age, with the highest prevalence occurring upstream. Additional chevron-shaped lesions occur further upstream of the ostia. This pattern differs from the age-related ones occurring in people and rabbits. In the present study, patterns of near-wall blood flow around intercostal ostia in wild-type mice were estimated from the morphology of endothelial nuclei, which were shown in vitro to elongate in response to elevated shear stress and to align with the flow, and wall structure was assessed from confocal and scanning electron microscopy. A triangular intimal cushion surrounded the upstream part of most ostia. Nuclear length-to-width ratios were lowest over this cushion and highest at the sides of branches, regardless of age. Nuclear orientations were consistent with flow diverging around the branch. The pattern of nuclear morphology differed from the age-related ones observed in rabbits. The intimal cushion and the distribution of shear stress inferred from these observations can partly account for the pattern of lesions observed in knockout mice. Nuclear elongation in nonbranch regions was approximately constant across animals of different size, demonstrating the existence of a mechanism by which endothelial cells compensate for the dependence of mean aortic wall shear stress on body mass.

Fig. 1.

En face montage of propidium iodide-stained endothelial nuclei around the origin of a mouse aortic branch. Arrow indicates direction of mean aortic blood flow. Bar = 100 μm. Inset: regions for which mean nuclear shape and orientation were calculated (U, upstream; D, downstream; L, anatomic left; R, anatomic right). Region U excludes the intimal cushion, which was analyzed separately.

Fig. 2.

Effect of applied shear stress on elongation (A) and deviation (B) from the most frequently occurring alignment of immortalized mouse aortic endothelial cell nuclei in vitro. Both effects were highly significant (P < 0.005 and P < 0.0005 by linear regression, respectively, n = 71 fields of view).

Fig. 3.

A: length-to-width ratios of endothelial nuclei from the 8 regions defined in Fig. 1, inset, over the intimal cushion, and in control regions away from the branch, in younger and older mice. B: angle between the long axis of nuclei and the average nuclear orientation around the branch for the regions defined in Fig. 1, inset, in the same mice. Negative angles indicate that the proximal end of the nucleus was displaced toward the anatomic right; positive angles, that it was displaced toward the left. Bars indicate means ± SE (n = 4 younger and 5 older mice, 99 branches in total).

Fig. 4.

Bars representing endothelial nuclear orientation, relative to the mean for the branch, in different regions around branch ostia in younger and older mice. Time-averaged blood flow is from top to bottom. The ostium is located in the central square in each image. Each square represents a region measuring 200 μm × 200 μm, defined in Fig. 1, inset.

Fig. 5.

A: projection of a confocal image stack showing en face view of tissue autofluorescence around the origin of a mouse aortic branch. A raised intimal cushion resembling the prow of a boat is present upstream of the ostium. Arrow indicates direction of mean aortic blood flow. B: scanning electron microscope image of a corrosion cast of the mouse aortic lumen at an intercostal branch. The intimal cushion, which protrudes into the lumen, is seen as a deep trench in the cast around the upstream (uppermost) side of the branch mouth. Raised endothelial nuclei leave pits in the cast. The downstream side of the branch is undercut where the flow divider projects into the lumen. C: chevron-shaped lesion, revealed by staining with oil red O, upstream of the mouth of an intercostal artery from an apolipoprotein E/low-density lipoprotein receptor double knockout mouse (detail from Fig. 2 of Ref. 31).

Fig. 6.

Changes with age in the pattern of endothelial nuclear length-to-width ratios (L/W) upstream and downstream of rabbit aortic branches. The upstream L/W has been divided by the downstream L/W, so values <1 indicate greater nuclear elongation downstream, and values >1 indicate greater elongation upstream. Each point indicates the data for a single rabbit (24 branches in total, 5–8 in each age group). Ages are the mean for each group.

Fig. 7.

Effect of age on the pattern of endothelial nuclear L/W around the branch. A: the mean L/W obtained in younger mice for each of the regions defined in Fig. 1, inset, plotted against the equivalent ratio in older mice. The positive correlation shows the broadly similar pattern with age, with the outlier (corresponding to the downstream region, D) revealing the only obvious change. The offset of the best-fit regression line shows the general increase in L/W with age. B: equivalent plot for the data obtained in regions upstream and downstream of rabbit ostia, showing a best-fit regression line of the opposite sign.

Fig. 8.

Mean endothelial nuclear L/W for control regions, away from branches, in mice and rabbits from different age groups, ranked according to body size. A decrease in mean aortic wall shear stress (∼10×) is expected with increasing size, but there is no corresponding decrease in L/W. Bars show means ± SE; n = 2–5 animals per group.