Increased vascular smooth muscle cell stiffness: a novel mechanism for aortic stiffness in hypertension

Abstract

Increased vascular stiffness is fundamental to hypertension, and its complications, including atherosclerosis, suggest that therapy should also be directed at vascular stiffness, rather than just the regulation of peripheral vascular resistance. It is currently held that the underlying mechanisms of vascular stiffness in hypertension only involve the extracellular matrix and endothelium. We hypothesized that increased large-artery stiffness in hypertension is partly due to intrinsic mechanical properties of vascular smooth muscle cells. After confirming increased arterial pressure and aortic stiffness in spontaneously hypertensive rats, we found increased elastic stiffness of aortic smooth muscle cells of spontaneously hypertensive rats compared with Wistar-Kyoto normotensive controls using both an engineered aortic tissue model and atomic force microscopy nanoindentation. Additionally, we observed different temporal oscillations in the stiffness of vascular smooth muscle cells derived from hypertensive and control rats, suggesting that a dynamic component to cellular elastic stiffness is altered in hypertension. Treatment with inhibitors of vascular smooth muscle cell cytoskeletal proteins reduced vascular smooth muscle cell stiffness from hypertensive and control rats, suggesting their participation in the mechanism. This is the first study demonstrating that stiffness of individual vascular smooth muscle cells mediates vascular stiffness in hypertension, a novel concept, which may elucidate new therapies for hypertension and for vascular stiffness.

Keywords: atomic force microscopy; hypertension; pulse wave velocity; vascular smooth muscle; vascular stiffness.

Fig. 1.

Arterial pressure and aortic stiffness are increased in spontaneously hypertensive rats (SHRs) compared with Wistar-Kyoto (WKY) rats. A: arterial pressure in WKY and SHRs. SBP, systolic aortic blood pressure; DBP, diastolic aortic blood pressure; MBP, mean aortic blood pressure; PP, pulse pressure. B: in vivo aortic stiffness, determined by pulse wave velocity, in WKY and SHRs. C: ex vivo aortic stiffness, determined from stress and strain measurements during tensile testing of aortic ring segments. Tangential elastic stiffness is shown. *P < 0.05; **P < 0.01. Data are given as means ± SE; n = 4 per group.

Fig. 2.

Vascular smooth muscle cell (VSMC) stiffness is increased in SHRs compared with WKY rats. An example of the force recording generated from the reconstituted tissue model before and after cytochalasin D (CD) treatment using WKY (A) and SHR (B) VSMCs. C: average stiffness of reconstituted aortic tissues using VSMCs from WKY and SHRs (n = 7 for each group). D: cellular stiffness, determined after treatment with CD, for WKY and SHRs. E, distribution of force as a function of indentation in WKY and SHR VSMCs measured by atomic force microscopy. F: computed elastic stiffness of individual VSMCs, as determined by atomic force microscopy measurements, in WKY and SHRs (n = 30 from 4 rats for each group). Data are given as means ± SE. *P < 0.05.

Fig. 3.

Real-time oscillations in VSMC stiffness from SHRs and WKY rats. A: oscillations were detected after prolonged measurement of VSMC stiffness in both SHRs and WKY rats, as shown for representative cases. B: spectral analysis of these oscillations showed that amplitude was generally increased, whereas frequency was generally decreased in SHRs compared with WKY rats, as shown for representative cases. C: mean frequency was significantly decreased (first component). D: amplitude was significantly increased (all 3 components) in SHRs. *P < 0.05 and **P < 0.01, compared with WKY rats in post hoc comparisons. Column graph data are given as means ± SE.

Fig. 4.

Cytoskeletal protein inhibitors alter mean VSMC stiffness. Expression of cytoskeletal proteins actin (A), phosphorylated myosin light chain (p-MLC; B), myosin light chain kinase (MLCK; C), and MLC (D) in SHRs, compared with WKY rats. E: inhibition targeted at contractile response elements [actin with CD, and MLCK with 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7)] eliminated the baseline VSMC stiffness difference between SHRs and WKY rats. *P < 0.05 and **P < 0.01, compared with WKY rats. Data are from 4 rats per group and are given as means ± SE.

Fig. 5.

Contractile response to vasoactive stimulation. A: cell height was found to progressively increase in response to vasoactive stimulation by angiotensin II, as shown for representative cases. B: mean changes in height were increased in groups of WKY and SHR cells (n = 12 cells/group). C: mean VSMC stiffness in both WKY and SHRs (n = 12 cells/group) was also found to be progressively increase under this stimulation. *P < 0.05, ⁺P < 0.05, **P < 0.01, compared with untreated controls of the same groups in post hoc comparisons. Column data are represented as means ± SE.