Endothelial dysfunction and compromised eNOS/Akt signaling in the thoracic aorta during the progression of Marfan syndrome

Abstract

Background and purpose: Aortic complications account for the major mortality in Marfan syndrome (MFS), a connective tissue disorder caused by mutations in FBN1 encoding fibrillin-1. We hypothesized that MFS impaired endothelial function and nitric oxide (NO) production in the aorta.

Experimental approach: Mice (at 3, 6, 9 and 12 months of age) heterozygous for the Fbn1 allele encoding a cysteine substitution (Fbn1 (C1039G/+), Marfan mice, n=75), the most common class of mutation in MFS, were compared with age-matched control littermates (n=75). Thoracic and abdominal aortas from the two groups were studied.

Key results: Isometric force measurements revealed that relaxation to ACh (but not to sodium nitroprusside) was diminished in the phenylephrine-precontracted Marfan thoracic aorta at 6 months of age (pEC(50)=6.12+/-0.22; maximal response, E(max)=52.7+/-6.8%; control: pEC(50)=7.34+/-0.19; E(max)=84.8+/-2.2%). At one year, both inhibition of NO production with N(omega)-nitro-L-arginine methyl ester, or denudation of endothelium increased the phenylephrine-stimulated contraction in the control thoracic aorta by 35%, but had no effect in the Marfan aorta, indicating a loss of basal NO production in the Marfan vessel. From 6 months, a reduced phosphorylation of endothelial NOS (eNOS)(Ser1177) and Akt(Thr308) detected by Western blotting was observed in the Marfan thoracic aorta, which was accompanied by decreased levels of cGMP. Expressions of Akt and eNOS in the abdominal aorta were not different between the two groups.

Conclusions and implications: MFS impairs endothelial function and signaling of NO production in the thoracic aorta, suggesting the importance of NO in the age-related progression of thoracic aortic manifestations.

Figure 1

Concentration–response curves of ACh-induced relaxation in phenylephrine (3 μM)-precontracted (a) TA and (b) AA from control (open bars) and Marfan (closed bars) mice at 6 months of age. In the TA, the ACh-induced maximal relaxation response (E_max) is significantly higher than that in the control (n=8–14, ^*P<0.05). The concentration-response curve of Marfan TA was right-shifted compared with the control, indicating a decrease in the sensitivity to ACh-induced relaxation. Similar observations were not made in the AA. Values of E_max and pEC₅₀ at different age groups are presented in the bar graphs (c–f).

Figure 2

Effect of L-NAME (200 μM) on phenylephrine-induced contraction in aortas from Marfan and control mice. Bar graphs showing E_max (mN) in response to phenylephrine stimulation (3 μM) plus and minus L-NAME at the age of (a) 3, (b) 6, (c) 9 and (d) 12 months (n=8–16, ^*P<0.05).

Figure 3

Concentration–response curves of SNP-induced relaxation in phenylephrine precontracted (a) TA and (b) AA from control and Marfan mice at 6 months of age. The endothelium-independent relaxation stimulated by SNP was not different between two animal groups, suggesting that the sensitivity of smooth muscle to NO was not altered in MFS.

Figure 4

Bar graphs presenting the levels of cGMP in the (a) TA and (b) AA homogenate from control (open bars) and Marfan (closed bars) mice. Values shown, expressed as %, are normalized to the control level at 3 months of age (n=4, ^*P<0.05, vs age-matched control groups).

Figure 5

Mechanisms of impaired NO production in TA: Western immunoblotting showing the protein expression of eNOS, phospho-eNOS^Ser1177, Akt, phospho-Akt^Thr308 and β-actin in the (a) TA and (b) AA from control and Marfan mice at different ages. Expression of β-actin served as loading control. Because of the limited aortic samples from each mouse, TA and AA were pooled from each group of animals at different ages.