Short communication: vascular smooth muscle cell stiffness as a mechanism for increased aortic stiffness with aging

Abstract

Rationale: Increased aortic stiffness, an important feature of many vascular diseases, eg, aging, hypertension, atherosclerosis, and aortic aneurysms, is assumed because of changes in extracellular matrix (ECM).

Objective: We tested the hypothesis that the mechanisms also involve intrinsic stiffening of vascular smooth muscle cells (VSMCs).

Methods and results: Stiffness was measured in vitro both by atomic force microscopy (AFM) and in a reconstituted tissue model, using VSMCs from aorta of young versus old male monkeys (Macaca fascicularis) (n=7/group), where aortic stiffness increases by 200% in vivo. The apparent elastic modulus was increased (P<0.05) in old (41.7+/-0.5 kPa) versus young (12.8+/-0.3 kPa) VSMCs but not after disassembly of the actin cytoskeleton with cytochalasin D. Stiffness of the VSMCs in the reconstituted tissue model was also higher (P<0.05) in old (23.3+/-3.0 kPa) than in young (13.7+/-2.4 kPa).

Conclusions: These data support the novel concept, not appreciated previously, that increased vascular stiffness with aging is attributable not only to changes in ECM but also to intrinsic changes in VSMCs.

Figure 1. Mechanical properties of VSMC from the reconstituted tissue model

Recording of the force generated by the aortic VSMCs from young (A) and old (B) monkeys in the reconstituted tissue. (C) Modulus of elasticity in reconstituted tissue. *, P<0.05 versus young monkeys (n=7/group).

Figure 2. Mechanical properties of single VSMC measured by AFM

(A) Distribution of force as a function of indentation in young (pink, n=40 cells) and old (blue, n=76 cells) monkeys. (B-C) Time course of elasticity for one single VSMC before and after CD treatment in young (B) versus old (C) monkeys. (D-E) Response of VSMC stiffness to CD (D) or ML7 (E) treatment in young and old monkeys. (F) Response of VSMC stiffness to colchicine treatment in young and old monkeys. *, P<0.05 versus young monkeys.

Figure 3. Cortical cytoskeletal density of VSMC by topographical AFM images

(A) Deflection Images of VSMC (left panel), gradient image (middle panel) and intermediate process image mask (right panel). (B) Example of deflection images in VSMCs from young versus old monkeys. (C) AFM image analysis. Old VSMC showed significantly greater surface gradients consistent with denser cortical cytoskeleton. (D) Fluorescent 3D image set (top panel) of young (left) and old (right) VSMC labeled for f-actin (red). AFM topographical image of same cell (middle panel) and overlay of AFM with fluorescent images (lower panel). *, P<0.05 versus young monkeys.

Figure 4. Alteration of cytoskeletal proteins in aging VSMCs

(A) Immunostaining for α-smooth muscle actin, α-tubulin, vimentin and β1-integrin in cultured VSMCs. (B) Western blotting of the corresponding proteins. *, P< 0.05 versus young monkeys. Identification of β1-integrin (C for young and D for old) and active integrin adhesion to FN measured by AFM on the surface of VSMCs from young (E) and old (F) monkeys. The binding probability and peak binding force (see supplementary file of the Methods) in VSMC was higher in old vs. young monkeys.