Augmented vascular smooth muscle cell stiffness and adhesion when hypertension is superimposed on aging

Abstract

Hypertension and aging are both recognized to increase aortic stiffness, but their interactions are not completely understood. Most previous studies have attributed increased aortic stiffness to changes in extracellular matrix proteins that alter the mechanical properties of the vascular wall. Alternatively, we hypothesized that a significant component of increased vascular stiffness in hypertension is due to changes in the mechanical and adhesive properties of vascular smooth muscle cells, and that aging would augment the contribution from vascular smooth muscle cells when compared with the extracellular matrix. Accordingly, we studied aortic stiffness in young (16-week-old) and old (64-week-old) spontaneously hypertensive rats and Wistar-Kyoto wild-type controls. Systolic and pulse pressures were significantly increased in young spontaneously hypertensive rats when compared with young Wistar-Kyoto rats, and these continued to rise in old spontaneously hypertensive rats when compared with age-matched controls. Excised aortic ring segments exhibited significantly greater elastic moduli in both young and old spontaneously hypertensive rats versus Wistar-Kyoto rats. were isolated from the thoracic aorta, and stiffness and adhesion to fibronectin were measured by atomic force microscopy. Hypertension increased both vascular smooth muscle cell stiffness and vascular smooth muscle cell adhesion, and these increases were both augmented with aging. By contrast, hypertension did not affect histological measures of aortic collagen and elastin, which were predominantly changed by aging. These findings support the concept that stiffness and adhesive properties of vascular smooth muscle cells are novel mechanisms contributing to the increased aortic stiffness occurring with hypertension superimposed on aging.

Keywords: aorta; collagen; elastin; fibronectins; focal adhesions; microscopy, atomic force; vascular stiffness.

Figure 1

Hypertension advances from young to old SHR. Thoracic aortic pressure was measured in the descending thoracic aorta in young (16 weeks old) and old (64 weeks old) WKY and SHR. In both age groups, (A) systolic pressure, (B) diastolic pressure, (C) mean aortic pressure, and (D) pulse pressure is increased in the SHR. Systolic and pulse pressures were significantly increased with age in the SHR, but not WKY. Data are shown as mean ± SEM, with n = 8 animals per group. Post-hoc comparisons, * p < 0.05, *** p < 0.001, compared to WKY; # p < 0.05, compared to young.

Figure 2

Stiffness of the thoracic aorta and individual VSMCs increase with hypertension and aging. Excised aortic rings from young (16 weeks old) and old (64 weeks old) WKY and SHR were mechanically stretched, and stiffness was determined from the stress-strain relationship, as shown for representative rings (A) (SHR = dashed; WKY = solid; Old = filled symbols: Young = open symbols). Stiffness was increased in the SHR at the young age, and further augmented with age (B). The stiffness of VSMCs derived from the thoracic aorta was determined by AFM nanoindentation. Each cell was individually probed with a microcantilever, and the force response was determined from the laser deflection of the microcantilever as it touched the cell surface, as shown for representative approach (blue) and retraction (red) curves (C). The stiffness was increased in SHR at both young and old ages (D). Data are shown as mean ± SEM. Post-hoc comparisons, parisons among the four groups were determined * p < 0.05, ** p < 0.01, compared to WKY; # p < 0.05, ## p < 0.01, compared to young.

Figure 3

Adhesion properties of VSMCs are increased in SHR. In separate atomic force microscopy experiments, the microcantilever probe was coated with fibronectin, to promote the formation of VSMC surface attachments to the probe. This resulted in several stepwise adhesion breaks (arrows), as the cantilever was pulled away from the cell surface, as shown for representative approach (blue) and retraction (red) curves (A). SHR cells formed a greater number of adhesion attachments per approach-retraction cycle (B). The total force generated from these adhesion attachments was also greater in the SHR (C). Data are shown as mean ± SEM, with n = 60 cells per group. Post-hoc comparisons, * p < 0.05, ** p < 0.01, **** p < 0.0001 compared to WKY; # p < 0.05, ## p < 0.01, ### p < 0.001 compared to young.

Figure 4

Aortic structural remodeling in hypertension differs from that of age. Overall morphology was compared from Mason's Trichrome staining of the thoracic aorta (scale bar = 1.0 mm), shown with high-magnification insert (scale bar = 0.1 mm) (A). At both ages, the SHR had increased lumen diameter (B), medial layer thickness (C), and medial thickness-diameter ratio (D), and medial cross-sectional area (E), and these changes further increased with age. Data are shown as mean ± SEM, with n = 8 animals per group. Post-hoc comparisons, * p < 0.05, ** p < 0.01, compared to WKY; # p < 0.05, ## p < 0.01, ### p < 0.001, compared to young.

Figure 5

Collagen and elastin composition in hypertension differs from that of age. Collagen fibers were visualized by under circularly-polarized light (A), and neither the content within individual image fields (B) nor that computed for the total tissue (C) was not significantly different in hypertension. The collagen content was normalized to the tissue area within that field to determine a tissue density, which was decreased in the SHR (D). Elastin was quantified from Van Gieson's staining (E). Older age decreased elastin content within individual fields (F), but overall this was increased for the total tissue ring(G). Elastin density was decreased by age, and not hypertension (H). Data are shown as mean ± SEM, with n = 8 animals per group. Post-hoc comparisons, * p < 0.05, ** p < 0.01, compared to WKY; # p < 0.05, ## p < 0.01, ### p < 0.001, compared to young.