Discrete contributions of elastic fiber components to arterial development and mechanical compliance

Abstract

Objective: Even though elastin and fibrillin-1 are the major structural components of elastic fibers, mutations in elastin and fibrillin-1 lead to narrowing of large arteries in supravalvular aortic stenosis and dilation of the ascending aorta in Marfan syndrome, respectively. A genetic approach was therefore used here to distinguish the differential contributions of elastin and fibrillin-1 to arterial development and compliance.

Methods and results: Key parameters of cardiovascular function were compared among adult mice haploinsufficient for elastin (Eln(+/-)), fibrillin-1 (Fbn1(+/-)), or both proteins (dHet). Physiological and morphological comparisons correlate elastin haploinsufficiency with increased blood pressure and vessel length and tortuosity in dHet mice, and fibrillin-1 haploinsufficiency with increased aortic diameter in the same mutant animals. Mechanical tests confirm that elastin and fibrillin-1 impart elastic recoil and tensile strength to the aortic wall, respectively. Additional ex vivo analyses demonstrate additive and overlapping contributions of elastin and fibrillin-1 to the material properties of vascular tissues. Lastly, light and electron microscopy evidence implicates fibrillin-1 in the hypertension-promoted remodeling of the elastin-deficient aorta.

Conclusions: These results demonstrate that elastin and fibrillin-1 have both differential and complementary roles in arterial wall formation and function, and advance our knowledge of the structural determinants of vascular physiology and disease.

Figure 1. Morphology of ascending aortae and left common carotid arteries

Panel A: Representative left common carotid artery of WT, Fbn1^+/−, Eln^+/−, and dHet mice with the tracing of vessels shown on the right side of each image. Arrow points to a vessel twist. Panel B: Representative carotid arteries of the indicated genotypes upon excision with the arrows highlighting arterial tortuosity. Scale bar = 1 mm.

Figure 2. Mechanical testing of wild-type and mutant arteries

Panels A and B: Pressure-diameter curves of ascending aorta (A) and left common carotid artery (B) of the indicated genotypes. Vertical solid-line arrows indicate the outer diameter of WT (black) and Eln^+/− (grey) aorta, and dotted lines indicate the outer diameter of Fbn1^+/− (black) and dHet (grey) aorta at their respective physiological pressures. Compared with WT aorta, line graphs in Panel A show significant reduction of Eln^+/− outer diameter at 0, 25, 125, 150 and 175 mmHg (p≤ 0.05); significant increase of Fbn1^+/− outer diameter between 50 and 150 mmHg (p≤ 0.05); and reduced or increased outer diameter of dHet aorta at 0, 125, 150 and 175 mmHg, and at 75 and 100 mm Hg, respectively (p≤ 0.05). Line graphs also show that outer diameter of dHet aorta is larger than Eln^+/− at pressure ≥ 50 mmHg (p≤ 0.05) and smaller than Fbn1^+/− at 25, 75, 125, 150, and 175 mmHg (p≤ 0.05), whereas the outer diameter of Eln +/- aorta is smaller than Fbn1^+/− at all pressures (p≤ 0.05). Line graphs in Panel B show significant reductions in outer diameters of Eln^+/− left common carotids at 0 and 100 mmHg and above compared with WT (p≤ 0.05), and above 0 mmHg compared to Fbn1^+/− (p≤ 0.05). Line graphs also show no differences between Fbn1^+/− and WT carotids (p> 0.05), overlapping values between dHet and Eln^+/− carotids (p> 0.05), smaller values than Fbn1^+/− at all pressures (p≤ 0.05), and smaller values than WT at all pressures except 50 mm Hg (p≤ 0.05). Arteries were held at the in vivo longitudinal stretch ratio during testing. Values are means ± SD; n = 4–6 for each artery type and genotype. Panel C: Line graphs of the percent diameter change plotted against pressure show higher than WT values at 50 and 75 mmHg, and lower than WT values between 125 and 175 mmHg (p≤ 0.05) of the Eln^+/− and Fbn1^+/− aorta; and higher values between 25 and 75 mmHg and lower values at 100 mmHg and above (p≤ 0.05) of the dHet aorta compared to WT. The dHet aorta also has less percent diameter change than Fbn1^+/− at 100 and 125 mmHg (p≤ 0.05). Panel D: Line graphs of E_inc plotted against pressure show no differences between the dHet and Eln^+/− aorta, and between the Eln^+/− and Fbn1^+/− aorta at any pressure (p > 0.05); lower dHet than WT values at pressures below 75mmHg and higher dHet than WT values above 100mmHg, and higher dHet than Fbn1^+/− values at 125 mmHg (p≤ 0.05); lower Eln^+/− than WT values at 50 mmHg and higher Eln^+/− than WT values at 125 mmHg (p≤ 0.05); and higher Fbn1^+/− than WT values at 125 mmHg (p≤ 0.05). Values are means ± SD; n = 4–6 for each genotype.

Figure 3. Circumferential stress and stretch ratio in wild-type and mutant aortae

Average circumferential stress (Panel A) and stretch ratio (Panel B) vs. pressure, and circumferential stress vs. circumferential stretch ratio (Panel C) in the ascending aorta of the indicated genotypes. Compared with the WT aorta, line graphs in Panel A show increased circumferential stress of the dHet or Eln^+/− aorta at 75 and 100 mmHg, and of the Fbn1^+/− aorta at 75, 100 and 125 mmHg (p≤0.05). Compared with the WT aorta, line graphs in Panel B show greater circumferential stretch ratio of the Eln^+/− aorta at 50–125 mmHg, and of the Fbn1^+/− aorta at 75–100 mmHg (p≤0.05). Line graphs also show that the stretch ratio of the dHet aorta is greater than the WT, Eln^+/−, or Fbn1^+/− aorta between 25 and 175 mmHg, 50 and 125 mmHg, or 25 and 125 mmHg, respectively (p≤0.05). There are no statistical differences between the singly haploinsufficient vessels (p>0.05). Line graphs in Panel C represent circumferential stress and stretch ratio plotted against each other for all genotypes; Fbn1^+/− and Eln^+/− profiles are very close to each other, Fbn1^+/− and WT profiles overlap at both ends, and the dHet profile is appreciably shifted to the right. Values are means ± SD; n = 4–6 per genotype.

Figure 4. Aortic wall morphology

Panel A: Elastic fiber staining in the ascending aortae of the indicated genotypes. Bar graphs at the bottom summarize the average number of lamellar units in the WT (black), Fbn1^+/− (white), Eln^+/− (dark grey) or dHet (light grey) aorta. Asterisks indicate statistically significant differences in the number of elastic lamellae (p≤0.05). Values are means ± SD; n = 4 for each genotype. Scale bar = 100 µm. Panel B: EM images of the central medial layer of the ascending aortae in mice of the indicated genotypes. Scale bar = 5µm.