Differences in the Thoracic Aorta by Region and Sex in a Murine Model of Marfan Syndrome

Abstract

Marfan syndrome (MFS) is a hereditary disorder of the connective tissue that causes life-threatening aortic aneurysm, which initiates at the aortic root and can progress into the ascending portion. However, analysis of ascending aorta reactivity in animal models of MFS has remained elusive. Epidemiologic evidence suggests that although MFS is equally prevalent in men and women, men are at a higher risk of aortic complications than non-pregnant women. Nevertheless, there is no experimental evidence to support this hypothesis. The aim of this study was to explore whether there are regional and sex differences in the thoracic aorta function of mice heterozygous for the fibrillin 1 (Fbn1) allele encoding a missense mutation (Fbn1^C1039G/+), the most common class of mutation in MFS. Ascending and descending thoracic aorta reactivity was evaluated by wire myography. Ascending aorta mRNA and protein levels, and elastic fiber integrity were assessed by qRT-PCR, Western blotting, and Verhoeff-Van Gieson histological staining, respectively. MFS differently altered reactivity in the ascending and descending thoracic aorta by either increasing or decreasing phenylephrine contractions, respectively. When mice were separated by sex, contractions to phenylephrine increased progressively from 3 to 6 months of age in MFS ascending aortas of males, whereas contractions in females were unchanged. Endothelium-dependent relaxation was unaltered in the MFS ascending aorta of either sex; an effect related to augmented endothelium-dependent hyperpolarization-type dilations. In MFS males, the non-selective cyclooxygenase (COX) inhibitor indomethacin prevented the MFS-induced enhancement of phenylephrine contractions linked to increased COX-2 expression. In MFS mice of both sexes, the non-selective nitric oxide synthase inhibitor L-NAME revealed negative feedback of nitric oxide on phenylephrine contractions, which was associated with upregulation of eNOS in females. Finally, MFS ascending aortas showed a greater number of elastic fiber breaks than the wild-types, and males exhibited more breaks than females. These results show regional and sex differences in Fbn1^C1039G/+ mice thoracic aorta contractility and aortic media injuries. The presence of more pronounced aortic alterations in male mice provides experimental evidence to support that male MFS patients are at increased risk of suffering aortic complications.

Keywords: Marfan syndrome; aortic aneurysm; ascending thoracic aorta contraction; cyclooxygenase; elastin fragmentation; gender medicine; nitric oxide synthase; sex differences.

Figure 1

(A) Diagram illustrating the descending and ascending thoracic aorta segments used in the present study. Concentration-response curves to phenylephrine in descending (B) and ascending (C,D) aorta from wild-type and Marfan syndrome mice. Results are the mean ± SE from wild-type (descending n = 10; ascending n = 10) and Marfan syndrome (descending n = 10; ascending n = 10–13) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA.

Figure 2

Concentration-response curves to phenylephrine in descending (A) and ascending (B,C) aorta from wild-type and Marfan syndrome male (left) and female (right) mice. Results are the mean ± SE from wild-type (male descending n = 5; male ascending n = 5; female descending n = 5; female ascending n = 5) and Marfan syndrome (male descending n = 5; male ascending n = 5–7; female descending n = 5; female ascending n = 5–6) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA.

Figure 3

Concentration-response curves to phenylephrine (A) and analysis of area under the curve (B) in the presence of the non-selective cyclooxygenase (COX) inhibitor indomethacin (Indo; 10⁻⁵ M) in ascending aorta from wild-type and Marfan syndrome male (left) and female (right) mice. Results are the mean ± SE from wild-type (male n = 5; female n = 6) and Marfan syndrome (male n = 5; female n = 5) mice. (C) Comparative analysis of ascending aorta mRNA levels of COX isoforms, COX-1 and COX-2. mRNA levels are expressed as 2^−ΔΔCt using Ribosomal Protein S28 as internal control. Results are the mean ± SE from wild-type (male n = 13-15; female n = 14) and Marfan syndrome (male n = 13; female n = 12) mice. (D) Western blot analysis for COX-1 (left) and COX-2 (right) protein expression. Bar graphs (bottom) show the results of densitometric analyses from pooled data. The molecular weight (kDa) of the protein is shown on the right side of the blot. Results are the mean ± SE from wild-type (male n = 6; female n = 5–6) and Marfan syndrome (male n = 6; female n = 6) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA.

Figure 4

Concentration-response curves to phenylephrine (A) and analysis of area under the curve (B) in the presence of the nonselective nitric oxide synthase (NOS) inhibitor L-NAME (3 × 10⁻⁴ M) in ascending aorta from wild-type and Marfan syndrome male (left) and female (right) mice. Results are the mean ± SE from wild-type (male n = 4; female n = 5) and Marfan syndrome (male n = 5; female n = 6) mice. (C) Comparative analysis of ascending aorta mRNA levels of NOS isoforms, endothelial NOS (eNOS) and inducible NOS (iNOS). mRNA levels are expressed as 2^−ΔΔCt using Ribosomal Protein S28 as internal control. Results are the mean ± SE from wild-type (male n = 15; female n = 13–14) and Marfan syndrome (male n = 14–15; female n = 12–13) mice. (D) Western blot analysis for eNOS protein expression and phosphorylation at Ser-1177 (left), and iNOS protein expression (right). Bar graphs (bottom) show the results of densitometric analyses from pooled data. The molecular weight (kDa) of the protein is shown on the right side of the blot Results are the mean ± SE from wild-type (male n = 3–6; female n = 3–6) and Marfan syndrome (male n = 3–6; female n = 3–6) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA.

Figure 5

Concentration-response curves to acetylcholine in the absence (A) or presence of L-NAME (3 × 10⁻⁴ M) plus Indo (10⁻⁵ M) (B) or L-NAME plus Indo plus the IK_Ca and BK_Ca channel blocker charybdotoxin (Charybd; 100 nM) plus the specific SK_Ca channel blocker apamin (Apa; 100 nM) (C) in ascending aorta from wild-type and Marfan syndrome male (left) and female (right) mice. Results are the mean ± SE from wild-type (male n = 4–6; female n = 5–6) and Marfan syndrome (male n = 5–6; female n = 5–6) mice. *P < 0.05, **P < 0.01, ***P < 0.001 by two-way ANOVA.

Figure 6

Representative images (A) and analysis of elastic fiber breaks (B) in the tunica media of the ascending aorta from wild-type and Marfan syndrome male and female mice. Scale bar represents 100 μm. Representative examples of elastin breaks are indicated with arrows. Results are the mean ± SE from wild-type (male n = 8; female n = 8) and Marfan syndrome (male n = 7; female n = 8) mice. *P < 0.05, ***P < 0.001 by two-way ANOVA.