Vascular smooth muscle cell-specific progerin expression in a mouse model of Hutchinson-Gilford progeria syndrome promotes arterial stiffness: Therapeutic effect of dietary nitrite

Abstract

Vascular stiffness is a major cause of cardiovascular disease during normal aging and in Hutchinson-Gilford progeria syndrome (HGPS), a rare genetic disorder caused by ubiquitous progerin expression. This mutant form of lamin A causes premature aging associated with cardiovascular alterations that lead to death at an average age of 14.6 years. We investigated the mechanisms underlying vessel stiffness in Lmna^G609G/G609G mice with ubiquitous progerin expression, and tested the effect of treatment with nitrites. We also bred Lmna^LCS/LCS Tie2Cre^+/tg and Lmna^LCS/LCS SM22αCre^+/tg mice, which express progerin specifically in endothelial cells (ECs) and in vascular smooth muscle cells (VSMCs), respectively, to determine the specific contribution of each cell type to vascular pathology. We found vessel stiffness and inward remodeling in arteries of Lmna^G609G/G609G and Lmna^LCS/LCS SM22αCre^+/tg , but not in those from Lmna^LCS/LCS Tie2Cre^+/tg mice. Structural alterations in aortas of progeroid mice were associated with decreased smooth muscle tissue content, increased collagen deposition, and decreased transverse waving of elastin layers in the media. Functional studies identified collagen (unlike elastin and the cytoskeleton) as an underlying cause of aortic stiffness in progeroid mice. Consistent with this, we found increased deposition of collagens III, IV, V, and XII in the media of progeroid aortas. Vessel stiffness and inward remodeling in progeroid mice were prevented by adding sodium nitrite in drinking water. In conclusion, Lmna^G609G/G609G arteries exhibit stiffness and inward remodeling, mainly due to progerin-induced damage to VSMCs, which causes increased deposition of medial collagen and a secondary alteration in elastin structure. Treatment with nitrites prevents vascular stiffness in progeria.

Keywords: aging; dietary nitrite; progeria; smooth muscle cells; vascular stiffness.

Figure 1

The aortas of progeroid mice exhibit arterial stiffness and inward remodeling. (a) Wire myography analysis of diameter–tension relationships, linear regression slope, and diameter estimated at 100 mmHg for aortic rings (n = 11 Lmna^G609G/G609G mice and n = 13 Lmna^+/+ littermate controls). (b) Magnetic resonance imaging (MRI) of the thoracic aorta in Lmna^+/+mice (n = 19) and Lmna^G609G/G609G mice (n = 17) and quantification of aortic size in area units (mm²) over a complete cardiac cycle. Distensibility is expressed as the slope of the ascending part of the aortic size–time curve

Figure 2

Mice with VSMC‐specific progerin expression display arterial stiffness and inward remodeling, whereas mice with EC‐specific progerin expression do not. (a, b) Wire myography analysis of diameter–tension relationships, linear regression slope, and diameter estimated at 100 mmHg for each vessel segment in aortic rings from Lmna^LCS/LCSSM22αCre^tg/+mice (n = 11) (a) and Lmna^LCS/LCSTie2Cre^tg/+mice (n = 8) (b). Mice of both genotypes are compared with Lmna^LCS/LCSlittermate controls (n = 13 and 8, respectively)

Figure 3

Small mesenteric vessels from ubiquitous and VSMC‐specific progeroid mice exhibit arterial stiffness and inward remodeling. Pressure–diameter curves for the vessel (outer) and lumen (inner) diameters, corresponding stress–strain curves, and representative images of the pressurized arteries at 60 mmHg. (a) Arteries from Lmna^G609G/G609G mice (n = 10), compared with Lmna^+/+littermate controls (n = 9). (b) Arterioles from Lmna^LCS/LCSSM22αCre^tg/+mice (n = 6), compared with Lmna^LCS/LCS littermate controls (n = 6)

Figure 4

Collagen disruption prevents stiffness in aortas of Lmna^G609G/G609G mice. Diameter–tension relationships and corresponding linear regression slopes in aortic rings from Lmna^+/+mice (n = 5–10) and Lmna^G609G/G609G mice (n = 5–10) after incubation with drugs affecting vessel structure: collagenase (collagen degradation), elastase (elastin–fiber degradation), and mycalolide B (depolymerization of F‐actin to G‐actin). Vehicle was used as control

Figure 5

Aortic media of Lmna^G609G/G609G mice shows increased collagen deposition, a decreased amount of smooth muscle tissue, and altered elastin waving. (a) Histological analysis of aortic sections from Lmna^G609G/G609G mice (n = 13) and Lmna^+/+mice (n = 11) stained with H&E and Masson's trichrome, showing increased collagen deposition and decreased smooth muscle area in the aortic medial layer of progeroid mice. (b) Confocal microscopy images of elastin autofluorescence and DAPI nuclear staining (n = 13–14), showing increased elastin wave linearity in aortic sections from Lmna^G609G/G609G mice unaccompanied by significant changes in nuclear number. (c) Morphological analysis of whole aortic sections (n = 9–12), showing no change in medial layer thickness and a decreased lumen perimeter relative to controls, indicating inward remodeling in the Lmna^G609G/G609G aorta

Figure 6

Increased deposition of collagens III, IV, V and XII in aortic media of Lmna^G609G/G609G mice. Representative images of immunofluorescence staining of collagens I, III, IV, V, and XII in Lmna^+/+ and Lmna^G609G/G609G aortic sections (n = 7‐10) and quantification of the normalized mean signal intensities. Media (M) layer is denoted in collagen I images between dashed lines. Scale bar 100 µm

Figure 7

Dietary nitrite supplementation protects Lmna^G609G/G609G mice against vascular stiffness and inward remodeling. (a) Study design. (b) Pressure–diameter curves for vessel (outer) and lumen (inner) diameters, corresponding stress–strain curves, and representative images of the pressurized arteries at 60 mmHg in nitrite‐treated Lmna^{G609G/G609G+/+}(n = 7) and Lmna^+/+ (n = 6) mice. (c) Effect of dietary nitrite supplementation on diameter–tension relationships, slope, and physiological diameter in aortic rings from Lmna^+/+mice (n = 10–14) and Lmna^G609G/G609G mice (n = 9–13). Nitrites attenuate arterial stiffness, as evidenced by increases in the force–diameter slope and the physiological diameter in aortic rings of treated Lmna^G609G/G609G mice