Asymmetric cell-matrix and biomechanics in aortopathy

Supplemental Figure 1. 3D reconstruction of regional aorta. A) Workflow for 3D reconstruction and image analysis. The aorta sections are histology processed, sectioned and stained (immunofluorescence) for the markers of interest. Images of serial sections of the aorta are aligned and tiled along z-axis using MATLAB, the area of reconstruction is defined, and 3D reconstruction is rendered using AMIRA software. The staining is analyzed using MetaMorph image analysis software. B) 3D models (long axis) generated for SM22 and SHH showing the anatomical planes. C) Integrated optical density (IOD) measures for SM22 and SHH along the directional progression of sectioning from the aortic annulus to ascending aorta.

Supplemental Figure 2. Regional VSMC disarray in Eln+/- aortas. Short axis view of the aorta and pulmonary artery (A, B; multiple images tiled) reveals heightened SM22 expression (C) specifically on the APS side (dotted circles, A, B) of WT aortas (E). High magnification image of the APS side of Eln+/- aorta demonstrate marked VSMC disarray (arrows, F), while WT APS aorta is smoothly stained for SM22 (E).

Supplemental Figure 3. Asymmetric regional SHH expression in the aortic root. Non-APS aortic root in WT mice is largely devoid of SHH staining demonstrating asymmetric CNC contribution (Supplemental Table 2) consistent with previous findings.41 However, Eln+/- mice show uniform and diffuse SHH staining throughout the circumference of the aortic root. Co - coronary sinuses (right and left), Non Co - non-coronary sinus. “Co” in lighter shade indicates out of plane. Hollow bold arrow in B shows the depth of aortic outflow tract along short axis with AAo at the top and AoV at the bottom.

Supplemental Video 1. Representative video showing the spatial distribution of SHH in the aged WT mouse aortic outflow tract, after performing 3D reconstruction.

Supplemental Video 2. Representative video showing the spatial distribution of SHH in the aged Eln+/- mouse aortic outflow tract, after performing 3D reconstruction.

Supplementary Table 1. SM22 expression in regional ascending aorta

APS
Ave IOD Ave Area Ave IOD/Area / Non-APS
Ave IOD Ave Area Ave IOD/Area
WT / 9.74 * 105 9.14 * 105 1.07 / 4.87 * 105 4.57 * 105 1.07
Eln+/- / 12.97 * 105 8.08 * 105 1.61 / 6.49 * 105 4.04 * 105 1.61

The APS side of ascending aorta demonstrates increased SM22 expression compared to non-APS side in both WT and Eln+/- mice (A). However, the area of APS aorta is significantly larger than that of non-APS aorta (consistent with Figure 2A-F). Therefore, when normalized to area, there is no relative change in SM22 expression between APS and non-APS sides (A). This system of normalization is employed for all markers.

Supplementary Table 2. SHH average IOD/Area

Root : Regional expression
APS non-APS
WT / 1.16 0.2*
Eln+/- / 0.74 # 0.8 #

* p<0.0001, APS vs. non-APS, WT. # p<0.0001, WT vs. Eln+/-.

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