Mechanism of aortic medial matrix remodeling is distinct in patients with bicuspid aortic valve

Abstract

Objectives: Patients with bicuspid aortic valves (BAV) are predisposed to developing ascending thoracic aortic aneurysms (TAA) at an earlier age than patients who develop degenerative TAAs and have a tricuspid aortic valve (TAV). The hypothesis tested is that BAV-associated aortopathy is mediated by a mechanism of matrix remodeling that is distinct from that seen in TAAs of patients with tricuspid aortic valves.

Methods: Aortic specimens were collected during ascending aortic replacement, aortic valve replacement, and heart transplants from nonaneurysmal (NA) donors and recipients. Matrix architecture of the aortic media was assessed qualitatively using multiphoton microscopy followed by quantification of collagen and elastin fiber orientation. α-Elastin was determined and matrix maturity was assessed by quantifying immature and mature collagen and lysyl oxidase (Lox) expression and activity in aortic specimens. Matrix metalloproteinase-2/9 activity was quantified in aortic smooth muscle cells.

Results: Elastin and collagen fibers were more highly aligned in BAV-NA and BAV-TAA cases than in TAV-TAA cases, whereas TAV-TAA cases were more disorganized than TAV-NA cases. α-Elastin content was unchanged. Immature collagen was reduced in BAV-NA and BAV-TAA cases when compared with TAV-NA and TAV-TAA cases. Mature collagen was elevated in TAV-TAA cases compared with TAV-NA and BAV-TAA cases. There was a trend toward elevated Lox gene expression and activity and matrix metalloproteinase-2/9 activity for TAV-TAA, BAV-NA, and BAV-TAA specimens.

Conclusions: The highly aligned matrix architecture in patients with BAVs indicates that wall remodeling is distinct from TAV-TAA. Altered matrix architecture and reduced collagen maturity suggest that the effector molecules mediating the remodeling of TAAs are different in BAV and TAV cases.

Figure 1

Matrix architecture of human ascending thoracic aorta. Representative images of collagen (green) and elastin (red) fiber orientation acquired using multi-photon microscopy (left panel). Magnification is 25X. Histograms of collagen and elastin fiber angle counts (right panels).

Figure 2

Determination of matrix fiber orientation. A) Representative monochromatic image of collagen fiber matrix of a BAV-TAA patient using multi-photon microscopy. Magnification is 25X. B) Vector map (blue arrows) of fiber alignment for image in (A). C) Increased magnification of box indicated in (B) to illustrate fiber alignment vectors. D) Representative histogram of collagen fiber angle counts for BAV-TAA generated from multi-photon image in (A) to calculate the orientation index (Δθ). E–F) Normalized orientation indices (NOIs) for elastin and collagen respectively. Bars represent mean ± SEM, n=5 (TAV-NA), 8,9 (TAV-TAA), 6 (BAV-NA) and 10 (BAV-TAA) *Significant from TAV-NA, ** Significant from TAV-TAA, p<0.05.

Figure 3

Assessment of matrix composition and maturity. A) Verhoeff’s van Giemsa (top panel) and Masson’s trichrome (bottom panel) for representative ascending thoracic aortic specimens. Images were oriented such that the adventitial side is located at the top and intimal side on the bottom. Images were captured at 20X magnification. Scale bar= 50 μm. B) Elastin content, n=10 (TAV-NA), 9 (TAV-TAA), 5 (BAV-NA) and 11 (BAV-TAA). C) Immature collagen n=8 (TAV-NA), 9 (TAV-TAA), 4 (BAV-NA) and 9 (BAV-TAA) and D) Mature collagen n=8 (TAV-NA), 7 (TAV-TAA), 3 (BAV-NA) and 8 (BAV-TAA) and bars represent mean ± SEM. *Significant from TAV-NA, **Significant from TAV-TAA, p<0.05.

Figure 4

Picrosirius red staining for collagen fibers in ascending aorta. A) Representative micrographs of aortic specimens imaged using linear polarized light showing thinner (green) and thicker collagen fibers (red). Images were oriented such that the adventitial side is located at the top and intimal side on the bottom. Scale bar=100 μm. B) Quantification of the area fraction of green (open bars) and red (solid bars). Bars represent mean ± SEM, n=3 (TAV-NA), 4 (TAV-TAA) and 5 (BAV-NA, BAV-TAA).

Figure 5

Lox and MMP analysis. A) Lox mRNA expression in aortic tissue specimens n=17 (TAV-NA), 8 (TAV-TAA), 8 (BAV-NA) and 15 (BAV-TAA). B) Lox n=5 (TAV-NA), 4 (TAV-TAA), 3 (BAV-NA) and 15 (BAV-TAA) and C) MMP-2/9 activity n=4 (TAV-NA), 5 (TAV-TAA), 3 (BAV-NA) and 6 (BAV-TAA)