Dysfunction of endothelial and smooth muscle cells in small arteries of a mouse model of Marfan syndrome

Abstract

Background and purpose: Marfan syndrome, a connective tissue disorder caused by mutations in FBN1 encoding fibrillin-1, results in life-threatening complications in the aorta, but little is known about its effects in resistance vasculature.

Experimental approach: Second-order mesenteric arteries from mice at 3, 6 and 10 months of age (n= 30) heterozygous for the Fbn1 allele encoding a cysteine substitution (Fbn1(C1039G/+)) were compared with those from age-matched control littermates.

Key results: Stress-strain curves indicated that arterial stiffness was increased at 6 and 10 months of age in Marfan vessels. Isometric force measurement revealed that contraction in response to potassium (60 mM)-induced membrane depolarization was decreased by at least 28% in Marfan vessels at all ages, while phenylephrine (3 microM)-induced contraction was reduced by at least 40% from 6 months. Acetylcholine-induced relaxation in Marfan vessels was reduced to 70% and 45% of control values, respectively, at 6 and 10 months. Sensitivity to sodium nitroprusside was reduced at 6 months (pEC(50)= 5.64 +/- 0.11, control pEC(50)= 7.34 +/- 0.04) and 10 months (pEC(50)= 5.99 +/- 0.07, control pEC(50)= 6.99 +/- 0.14). Pretreatment with N(omega)-Nitro-L-arginine methyl ester (200 microM) had no effect on acetylcholine-induced relaxation in Marfan vessels, but reduced vasorelaxation in control vessels to 57% of control values. Addition of indomethacin (10 microM) and catalase (1000 U.mL(-1)) further inhibited vasorelaxation in Marfan vessels to a greater degree compared with control vessels.

Conclusions and implications: Pathogenesis of Marfan syndrome in resistance-sized arteries increases stiffness and impairs vasomotor function.

Figure 1

Vessel elasticity during ageing in mesenteric arteries from Marfan and control mice from (A) 3, (B) 6 and (C) 10 months of age (*P < 0.05 vs. control, n= 8–12).

Figure 3

Reversibility of contractile function of mesenteric artery from (A) 6 and (B) 10 months of age after stretching at ΔL/L₀= 2.5, 3.0, 3.5 and 4.0. After being stretched for 3 min and restored to optimal tension, vessels were stimulated with 60 mM KCl (*P < 0.05 vs. control, n= 6–8). Values (%) are changes of force generation normalized to that at the optimal tension.

Figure 2

Reversibility of mesenteric artery elasticity at 10 months of age in (A) control and (B) Marfan mice. Reversibility of elasticity was tested by performing two consecutive stress–strain measurements. Vessel elasticity from the first and second measurement was compared in each group. Representative results are shown from three independent experiments.

Figure 4

Isometric force measurement. Maximal force generated in response to (A) 60 mM KCl and (B) phenylephrine (3 µM) was compared between control and Marfan vessels (*P < 0.05 vs. control, n= 8–12).

Figure 5

Active force, the difference between total and passive force, was compared between control and Marfan vessels from (A) 3, (B) 6 and (C) 10 months of age (*P < 0.05 vs. control, n= 8–12).

Figure 6

Concentration–response curve of acetylcholine (ACh)-induced relaxation in phenylephrine-precontracted mesenteric arteries from control and Marfan mice at (A) 3, (B) 6 and (C) 10 months of age (*P < 0.05 vs. control, n= 8–12).

Figure 7

Concentration–response curve of sodium nitroprusside (SNP)-induced relaxation in phenylephrine-precontracted mesenteric arteries from control and Marfan mice at (A) 6 and (B) 10 months of age (*P < 0.05 vs. control, n= 8–12).

Figure 8

Effects of N_ω-nitro-L-arginine methyl ester (L-NAME), indomethacin (Indo) and catalase on acetylcholine (ACh)-induced relaxation, from 10-month-old control and Marfan mice. The concentration–response curves of ACh are shown. (A) Control and (B) Marfan vessels were preincubated with L-NAME (200 µM) or Indo (10 µM) for 30 min and then contracted with phenylephrine (3 µM). (C) Control and (D) Marfan vessels were preincubated with catalase (1000 U·mL⁻¹) or carbenoxolone (100 µM) in the presence of L-NAME (200 µM) and Indo (10 µM) for 30 min and then contracted with phenylephrine (3 µM) (n= 8–12).

Figure 9

Effect of superoxide dismutase (SOD, 150 U·mL⁻¹) on phenylephrine (3 µM)-stimulated contraction and acetylcholine (ACh) (10 µM)-mediated relaxation in mesenteric arteries from control and Marfan mice at 10 months of age. Bar graphs show (A) E_max and (B) pEC₅₀ in response to phenylephrine in the presence and absence of SOD, while (C) E_max and (D) pEC₅₀ show responses to ACh-mediated relaxation (*P < 0.05 vs. control, n= 5–8).

Figure 10

Acetylcholine (ACh, 10 µM)-induced production of H₂O₂ by the endothelium, detected as an increase in fluorescence intensity in dichlorodihydrofluorescein diacetate-loaded endothelial cells in small mesenteric arteries of mice. The ACh-induced increase in fluorescence intensity was abolished when the artery was preincubated with catalase (1000 U·mL⁻¹). All experiments were performed in the presence of N_ω-nitro-L-arginine methyl ester (200 µM) and indomethacin (10 µM). Representative traces shown are typical of the responses obtained in 23 cells from four mice.