Bicuspid valve aortopathy is associated with distinct patterns of matrix degradation

BAV-A is stiffer relative to DA, at a >5-cm aorta diameter.

Figure 1. Workflow for the study. Aortic tissue from control, BAV-A, and tricuspid aortic valve idiopathic DA patients were used for micromechanical characterization. Using oscillatory nanoindentation, we measured the elastic modulus (E) at the medial layer of the tissue. Subsequently, all tissues were fully digested for biochemistry. Levels of GAG, collagen, and elastin were quantified. Using the same tissue cohorts, Verhoeff Van Gieson–stained cross-sections were used to characterize elastin microstructure. From the acquired images, we measured elastin lamellar units, lamellar spacing, and elastic lamellae thickness, over the media. Furthermore, tissue thickness, elastin content, number of elastin segments, and the length of the segments were also measured over the entire tissue cross-section. BAV-A, Bicuspid aortic valve aneurysm; DA, degenerative aneurysm; GAG, glycosaminoglycan.

Figure 2. Image processing procedure to obtain microstructural properties of elastin. A, Representative stitched Verhoeff Van Gieson–stained aortic wall image. B, Middle media segment of the tissue highlighted was used in pixel classification. Pixel classifier was trained using 6 images before (C) simple segmented mask image was exported. Typically to quantify elastic lamellae properties in the media, a line was drawn at the area of interest like the red central line drawn in (C) to produce (D) a plot profile, and subsequently the number of lamellar units, lamellar spacing, and elastic lamellae thickness were measured. To quantify elastin segments over the entire tissue cross-section, in E, a downscaled and skeletonized image of the mask image was required. Our in-house macro script identifies F, the number of elastin segments (red lines separated out with purple ends) and G, the length of elastin segments (between the white ends). All images were taken at 40× magnification. Scale bar: 20 μm.

Figure 3. Micromechanical and biochemical data for control (n = 8), BAV-A (n = 15), and tricuspid aortic valve idiopathic DA (n = 15). All data was represented as box plots and data overlaid with upper and lower borders of the box to represent the upper and lower quartiles, and the middle horizontal line to represent the median. The upper and lower whiskers represent 5th and 95th percentile of the data. Variables acquired from the specimens included A, elastic modulus (E) from oscillatory nanoindentation, and levels of B, GAG; C, collagen; and D, elastin from biochemical assays. BAV-A was significantly more elastic in comparison with the control and DA. However, it was DA that had elevated collagen levels relative to the control. Only E was statistically different between the aneurysmal groups. Therefore, we can note that changes in micromechanical behavior between BAV-A and DA cannot be explained by only the levels of GAG and structural proteins within the tissue. Noticeably, BAV-A was overall more homogeneous than control and DA for majority of the in vitro measurements. BAV-A, Bicuspid aortic valve aneurysm; DA, degenerative aneurysm; GAG, glycosaminoglycan.

Figure 4. Quantitative analysis of tissue thickness and elastin content for control (n = 8), BAV-A (n = 15) and tricuspid aortic valve idiopathic DA (n = 15), expressed as mean ± standard error. A, Representative stitched original and mask image of the Verhoeff Van Gieson–stained aortic section for the 3 tissue groups (40× magnification). Tissue cross-sections were divided into 10 sections that covered innermost media (In), inner media (M1), middle media (M2), outer media (M3), and adventitia (Ad) layers. Over the tissue cross-section, B, tissue thickness, and C, elastin content of layers were quantified. Elastin content was quantified by the fraction area occupied by elastin relative to the entire area of the mask image. DA had a thicker innermost media and adventitia layer relative to BAV-A. More elastin was present in control and BAV-A compared with DA. Both BAV-A and DA had an extensive reduction in content between middle media and adventitia. Although there are thickness differences in the tissue layers between the aneurysmal groups, it was found that only these groups had a similar percentage reduction in elastin content toward the outer portion of the aortic wall. Scale bar: 20 μm.…

Figure 5. Quantitative analysis of elastin microstructure within control (n = 8), BAV-A (n = 15) and tricuspid aortic valve idiopathic DA (n = 15) aortic tissues. Quantitative analysis of elastin microstructure within bicuspid aortic valve aneurysm aortic tissues with normal valve function (Norm; n = 1), AI (n = 2), AS (n = 7), and a mixture of AI and AS (Mix; n = 5) are also shown F-J. Data in A-C and F-H were represented as box plots and data overlaid with upper and lower borders of the box to represent the upper and lower quartiles, and the middle horizontal line to represent the median. The upper and lower whiskers represent 5th and 95th percentile of the data. A, The number of elastin lamellar units; B, the lamellar spacing; and C, the thickness of the elastic lamellae were measured within the media. D, The number of elastin segments; and E, the length of the segments were measured over the entire tissue cross-section, expressed as mean ± standard error. Tissue cross-section was divided into 10 sections that covered innermost media (In), inner media (M1), middle media (M2), outer media (M3), and adventitia (Ad) layers. BAV-A had significantly more lamellar units with narrow lamellar…

Figure 6. Summary schematic highlighting the phenotypic differences between the control, BAV-A, and tricuspid aortic valve idiopathic DA. Over the tissue cross-sections, there was more elastin content in the control and BAV-A tissues relative to DA tissues. Although control and BAV-A tissues appear to be similar microstructurally, subtle differences appear in the media and adventitia layers. BAV-A tissues had more elastin content, lamellar units, and elastin segments, thicker lamellae, and narrower lamellar spacing compared with DA tissues. These microstructural differences along with differences in collagen level could explain for the elevated micromechanical response. BAV-A, Bicuspid aortic valve aneurysm; DA, degenerative aneurysm.

Video 1. Elastin microstructural organisation has a unique pattern in bicuspid aortic valve aneurysm relative to idiopathic aortic aneurysm. Video available at: https://www.jtcvs.org/article/S0022-5223(19)31891-4/fulltext.

Figure E1. Microstructural differences of the aorta amongst BAV-A patients with different valve function. Elastin content within bicuspid aortic valve aneurysm aortic tissues with normal valve function (Norm; n = 1), AI (n = 2), AS (n = 7), and a mixture of AI and AS (Mix; n = 5), expressed as mean ± standard error of mean. AI, Aortic insufficiency; AS, aortic stenosis.

Figure E2. Micromechanical and biochemical data for control, BAV-A and DA, according to age <55 years old, and age >55 years old. Variables acquired included A, elastic modulus; B, GAG level; C, collagen level; and D, elastin level. BAV-A, Bicuspid aortic valve aneurysm; DA, degenerative aneurysm; GAG, glycosaminoglycan.