In vivo phenotypic vascular dysfunction extends beyond the aorta in a mouse model for fibrillin-1 (Fbn1) mutation

Abstract

In individuals with Marfan Syndrome (MFS), fibrillin-1 gene (FBN1) mutations can lead to vascular wall weakening and dysfunction. The experimental mouse model of MFS (Fbn1^C1041G/+) has been advantageous in investigating MFS-associated life-threatening aortic aneurysms. It is well established that the MFS mouse model exhibits an accelerated-aging phenotype in elastic organs like the aorta, lung, and skin. However, the impact of Fbn1 mutations on the in vivo function and structure of various artery types with the consideration of sex and age, has not been adequately explored in real-time and a clinically relevant context. In this study, we investigate if Fbn1 mutation contributes to sex-dependent alterations in central and cerebral vascular function similar to phenotypic changes associated with normal aging in healthy control mice. In vivo ultrasound imaging of central and cerebral vasculature was performed in 6-month-old male and female MFS and C57BL/6 mice and sex-matched 12-month-old (middle-aged) healthy control mice. Our findings confirm aortic enlargement (aneurysm) and wall stiffness in MFS mice, but with exacerbation in male diameters. Coronary artery blood flow velocity (BFV) in diastole was not different but left pulmonary artery BFV was decreased in MFS and 12-month-old control mice regardless of sex. At 6 months of age, MFS male mice show decreased posterior cerebral artery BFV as compared to age-matched control males, with no difference observed between female cohorts. Reduced mitral valve early-filling velocities were indicated in MFS mice regardless of sex. Male MFS mice also demonstrated left ventricular hypertrophy. Overall, these results underscore the significance of biological sex in vascular function and structure in MFS mice, while highlighting a trend of pre-mature vascular aging phenotype in MFS mice that is comparable to phenotypes observed in older healthy controls. Furthermore, this research is a vital step in understanding MFS's broader implications and sets the stage for more in-depth future analyses, while providing data-driven preclinical justification for re-evaluating diagnostic approaches and therapeutic efficacy.

Figure 1

Age-dependent changes in aortic root diameters and wall stiffness in control and MFS mice. (A) Sinus of Valsalva diameter is increased in MFS male mice compared to age-matched CTRL male mice at 3M, 6M, and 9M of age. (B) Diameters in this region are increased in MFS female mice compared to age-matched CTRL female mice at 3M, 6M, 9M, and 12M of age. (C) 12M-CTRL female mice demonstrate increased diameter compared to 3M-CTRL mice, suggesting that age increased aneurysm growth. (D) In MFS mice, differences were not seen in aortic diameters between sexes over time, but 3M-MFS and 12M-MFS male mice demonstrated an age-dependent increase. (E) Aortic pulse wave velocity measures demonstrate increased aortic stiffness at 6M, 9M, and 12M of age in MFS male mice compared to age-matched CTRL male mice. (F) Aortic pulse wave velocity measures demonstrate increased aortic stiffness at 3M, 9M, and 12M of age in MFS female mice compared to age-matched CTRL female mice. (G) In CTRL mice, a sex-dependent effect was demonstrated at 3M where males were increased compared to female 3M-CTRL mice. (H) Sex did not contribute to any significant difference within the MFS mice over time.

Figure 2

Measurements of aortic root diameters in MFS mice. (A) Representative B-mode image of the aortic arch of a 6M-CTRL male evaluated for diameters of the aortic annulus, sinus of Valsalva, and sinotubular junction. (B) Representative B-mode image of the aortic arch of a 6M-MFS male with aortic diameters analyzed. (C) 6M-MFS male aortic annulus diameters were increased compared to 6M-CTRL male mice as well as 6M-MFS female mice. (D) 6M-MFS male and female mice demonstrate increased sinus of Valsalva diameters compared to sex- and age-matched CTRL mice. (E) 6M-MFS male sinotubular junction diameters were increased compared to 6M-CTRL male mice as well as 6M-MFS female mice. (F) In the Aortic Annulus, 6M-MFS and 12M-CTRL male mice have increased diameter compared to 6M-CTRL male mice. 6M-MFS male mice have increased (G) Sinus of Valsalva and (H) Sinotubular Junction diameters compared to 6M- and 12M-CTRL males. (I) 6M-MFS females and 12M-CTRL females have increased Aortic Annulus diameters compared to 6M-CTRL female mice. (J) 6M-MFS females have increased Sinus of Valsalva diameters compared to 6M- and 12M-CTRL female mice. (K) No differences were seen in the Sinotubular Junction diameter of our female cohorts.

Figure 3

Measurements of aortic root wall stiffness in MFS mice. (A) Representative image of a 6M-CTRL female traced aortic arch in B-mode. (B) PW Doppler Mode waveform of the ascending aortic arch (Time 1, T1) with representative times analyzed. (C) PW Doppler Mode waveform of the descending aortic arch (Time 2, T2) with representative times analyzed. (D) 6M-MFS male and female mice have increased aortic stiffness, compared to sex- & age-matched CTRL mice. (E) 6M-MFS male mice have increased aortic stiffness, measured as aortic pulse wave velocity, compared to 6M- and 12M-CTRL male mice. (F) 6M-MFS female and 12M-CTRL female mice have increase aortic pulse wave velocity compared to 6M-CTRL female mice.

Figure 4

Measurements of coronary artery peak blood flow velocity during diastole in MFS mice. (A) Representative image of coronary artery outflow visualized in the parasternal long axis view with pulse wave doppler mode waveform analyzed with five consecutive velocity measurements in a 6M-CTRL female mouse. (B) Genotype and sex did not induce differences in coronary peak blood flow velocity during diastole at 6M. No differences were seen in coronary peak blood flow velocity during diastole as a function of age in (C) male cohorts or (D) female cohorts.

Figure 5

Assessments of left pulmonary artery peak blood flow velocity in MFS mice. (A) Representative image of left pulmonary artery outflow visualized in the parasternal long axis view with pulse wave doppler mode waveform analyzed with five consecutive velocity measurements in a 6M-CTRL male mouse. (B) 6M-MFS male and females have decreased left pulmonary artery peak blood flow velocity compared to sex- and age-matched 6M-CTRL mice. 6M-MFS and 12M-CTRL mice have decreased left pulmonary peak blood flow velocity compared to 6M-CTRL in (C) male and (D) female cohorts.

Figure 6

Measurements of posterior cerebral artery peak blood flow velocity in MFS mice. (A) Representative image of color doppler mode of the PCA in a 6M-CTRL female. (B) Representative image of Pulse Wave Doppler Mode waveform of the PCA analyzed with five consecutive velocity measurements. (C) 6M-MFS male mice and 6M-CTRL female mice demonstrated decreased PCA blood flow velocity compared to 6M-CTRL male mice. (D) 6M-MFS and 12M-CTRL male mice have decreased PCA blood flow velocity compared to 6M-CTRL mice. (E) Female mice showed no differences amongst cohorts.

Figure 7

Representative M-mode images of parasternal long axis view to visualize the left ventricle in a 6M-CTRL male mouse. (A) The left ventricle tracing of the anterior and posterior walls is visualized. (B) Parasternal long axis left ventricle trace (PSLAX LV trace) analysis allows for tracing of the ventricle over three consecutive contractions to measure cardiac parameters and (C) intraventricular wall septum in systole and diastole.

Figure 8

Comprehensive evaluation of mitral valve function in MFS mice. (A) Representative Image of Pulse wave Doppler Mode of the mitral valve inflow while in the apical four chamber view with measurements for peak early filling (MV E), peak atrial filling (MV A), and E/A ratio in a 6M-CTRL male mouse. (B) Male and female 6M-MFS mice have decreased MV E filling velocity compared to sex-and age-matched CTRL mice but without differences in (C) MV A filling velocity or (D) E/A ratio. (E) MV E filling velocity is decreased in 6M-MFS males compared to both 6M- and 12M-CTRL male mice. No differences were demonstrated in (F) MV A filling velocity or (G) E/A ratio in the male cohorts. (E) MV E filling velocity was decreased in 6M-MFS and 12M-CTRL female mice compared to 6M-CTRL female mice. (F) MV A filling velocity and (G) the E/A ratio is unchanged in female mice.

Figure 9

Summary of results. Depiction of the measured in vivo parameters, presenting the ultrasound measurements for both male and female outcomes. These include the Aortic Annulus (AA), Sinus of Valsalva (SV), and Sinotubular Junction (SJ) diameters of the aortic root, as well as the aortic Pulse Wave Velocity (PWV). Additionally, we have provided summaries for blood flow velocity measurements in the posterior cerebral artery, pulmonary artery, and coronary artery, along with cardiac parameters and mitral valve measures. The results are compared to 6M-CTRL (6-month-old control group), 6M-MFS (6-month-old Marfan syndrome group), and 12M-CTRL (12-month-old control group) for reference.