Mechanics of ascending aortas from TGFβ-1, -2, -3 haploinsufficient mice and elastase-induced aortopathy

Abstract

Transforming growth factor-beta (TGFβ-1, -2, -3) ligands act through a common receptor complex yet each is expressed in a unique and overlapping fashion throughout development. TGFβ plays a role in extra-cellular matrix composition with mutations to genes encoding TGFβ and TGFβ signaling molecules contributing to diverse and deadly thoracic aortopathies common in Loeys-Dietz syndrome (LDS). In this investigation, we studied the TGFβ ligand-specific mechanical phenotype of ascending thoracic aortas (ATA) taken from 4-to-6 months-old Tgfb1^+/-, Tgfb2^+/-, and Tgfb3^+/- mice, their wild-type (WT) controls, and an elastase infusion model representative of severe elastolysis. Heterozygous mice were studied at an age without dilation to elucidate potential pre-aortopathic mechanical cues. Our findings indicate that ATAs from Tgfb2^+/- mice demonstrated significant wall thickening, a corresponding decrease in biaxial stress, decreased biaxial stiffness, and a decrease in stored energy. These results were unlike the pathological elastase model where decreases in biaxial stretch were found along with increases in diameter, biaxial stress, and biaxial stiffness. ATAs from Tgfb1^+/- and Tgfb3^+/-, on the other hand, had few mechanical differences when compared to wild-type controls. Although aortopathy generally occurs later in development, our findings reveal that in 4-to-6 month-old animals, only Tgfb2^+/- mice demonstrate a significant phenotype that fails to model ubiquitous elastolysis.

Keywords: Connective tissue disorders; Loeys-Dietz syndrome (LDS); Transforming growth factor-beta.

Figure 1.

Tgfb1, Tgfb2, and Tgfb3 mRNA expression in ascending thoracic aortas taken from Tgfb1^+/− (n=3), Tgfb2^+/− (n=4), and Tgfb3^+/− (n=4), mice and their wild-type (WT) controls (n=3 each). All expression levels are relative to the B2m housekeeping gene. * Denotes statistical significance between heterozygotes and their WT controls at p<0.05.

Figure 2.

Representative images of the ascending thoracic aortas from Tgfb1^+/−, Tgfb2^+/−, Tgfb3^+/−, wild-type, and Elastase infused wild-type mice prior to, (top) and following cannulation, axial extension, and pressurization to 100 mmHg (bottom). (top) Ruler with mm units and (bottom) 1 mm scale bars. Elastase infusion is performed in the loaded configuration.

Figure 3.

Biaxial mechanical data from the ascending aortas of Tgfb1^+/− (red diamonds), Tgfb2^+/− (yellow squares), Tgfb3^+/− (blue triangles), wild-type (WT; black circles), and elastase infused wild-type (Elastase; green ☓) mice. As a subset of the total biaxial mechanical data, (a-c,e) are reported at a single axial stretch ratio and (d,f) at a single pressure of 100 mmHg. Experimental data are shown as mean values ± the standard error of the mean.

Figure 4.

Passive structural and mechanical properties from the ascending aortas of Tgfb1^+/− (red), Tgfb2^+/− (yellow), Tgfb3^+/− (blue), wild-type (WT; black), and elastase infused wild-type (Elastase; green) mice. All values are mean ± standard deviation. * Denotes statistical significance between groups at p<0.05 while ** simplifies statistical significance between the elastase and all other groups. # Elastase samples were tested at a single axial stretch ratio.

Figure 5.

Averaged and fitted pressure-force data taken from the ascending aortas of Tgfb1^+/−, Tgfb2^+/−, Tgfb3^+/−, wild-type (WT), and elastase infused wild-type (Elastase) mice. Experimental data are shown as discrete values ± the standard error of the mean at low (blue circle), medium (red square), and high (yellow triangle) axial stretch ratios. The medium condition represents the force-invariant pressurization axial stretch ratio. Fitted results using the 4-fiber-family Holzapfel-Gasser-Ogden (HGO) model are represented by curves (black). The elastase sample (green diamond) was tested at a single axial stretch ratio.

Figure 6.

Stored elastic energy of ascending aortas shown as (top) contour plots with open circles at in vivo conditions and (bottom) discrete values of stored energy taken at 20, 60, 100, and 140 mmHg taken from Tgfb1^+/− (red), Tgfb2^+/− (yellow), Tgfb3^+/− (blue) and (d) wild-type (WT; black) mice. * Denotes statistical significance between groups at p<0.05.

Figure 7.

Ascending aortic (left) circumferential and (right) axial stiffness moduli from Tgfb1^+/− (red), Tgfb2^+/− (yellow), Tgfb3^+/− (blue), and wild-type (WT; black) mice. Linearizations were performed around conditions corresponding to force-invariant axial stretch and (a) 20, (b) 60, (c) 100, and (d) 140 mmHg. * Denotes statistical significance between individual groups at p<0.05. conditions.

Figure 8.

Histological features using (top) Hematoxylin and Eosin staining and (bottom) elastin autofluorescence of the ascending aortas taken from Tgfb1^+/−, Tgfb2^+/−, Tgfb3^+/−, wild-type (WT), and elastase infused wild-type (Elastase) mice. All images at 40x.