Calcific Aortic Valve Disease Is Associated with Layer-Specific Alterations in Collagen Architecture.

Disorganization of the valve extracellular matrix (ECM) is a hallmark of calcific aortic valve disease (CAVD). However, while microarchitectural features of the ECM can strongly influence the biological and mechanical behavior of tissues, little is known about the ECM microarchitecture in CAVD. In this work, we apply advanced imaging techniques to quantify spatially heterogeneous changes in collagen microarchitecture in CAVD. Human aortic valves were obtained from individuals between 50 and 75 years old with no evidence of valvular disease (healthy) and individuals who underwent valve replacement surgery due to severe stenosis (diseased). Second Harmonic Generation microscopy and subsequent image quantification revealed layer-specific changes in fiber characteristics in healthy and diseased valves. Specifically, the majority of collagen fiber changes in CAVD were found to occur in the spongiosa, where collagen fiber number increased by over 2-fold, and fiber width and density also significantly increased. Relatively few fibrillar changes occurred in the fibrosa in CAVD, where fibers became significantly shorter, but did not otherwise change in terms of number, width, density, or alignment. Immunohistochemical staining for lysyl oxidase showed localized increased expression in the diseased fibrosa. These findings reveal a more complex picture of valvular collagen enrichment and arrangement in CAVD than has previously been described using traditional analysis methods. Changes in fiber architecture may play a role in regulating the pathobiological events and mechanical properties of valves during CAVD. Additionally, characterization of the ECM microarchitecture can inform the design of fibrous scaffolds for heart valve tissue engineering

Visualization of collagen fiber microarchitecture using SHG microscopy.

<p>Representative images from <b>(A)</b> healthy and <b>(B)</b> diseased leaflets show microarchitectural changes in collagen in the fibrosa and spongiosa. Scale bar = 50 μm.</p

Quantification of collagen fiber characteristics in the fibrosa and spongiosa of healthy vs. diseased aortic valve leaflets.

<p>Quantification of collagen fiber characteristics in the fibrosa and spongiosa of healthy vs. diseased aortic valve leaflets.</p

Human valve specimen information.

<p>Human valve specimen information.</p

Visualization of collagen fibers via picrosirius red staining and quantification of collagen content.

<p>Picrosirius red staining of <b>(A)</b> healthy and <b>(B)</b> diseased leaflets was visualized using brightfield microscopy and polarized light. Birefringence hue and amount were quantified as a percent of total tissue area (N = 5, n = 3) in the <b>(C)</b> fibrosa and <b>(D)</b> spongiosa. Collagen production was also quantified via <b>(E)</b> qRT-PCR analysis of <i>COL1A1</i> gene expression (N = 4, n = 3) and <b>(F)</b> measurement of total collagen protein via dot blot (N = 5 healthy; N = 4 diseased, n = 3).</p

Immunohistochemical detection and quantification of lysyl oxidase (LOX) expression.

<p>Immunohistochemical staining of <b>(A)</b> healthy and <b>(B)</b> diseased leaflets shows distribution of LOX production throughout leaflet, with some areas of localized high-intensity staining (indicated by arrows). F = fibrosa, S = spongiosa. N = 5. Scale bar = 0.50 mm. <b>(C)</b> Layer-specific quantification of LOX staining, indicating significantly greater amounts of LOX in the diseased fibrosa relative to the healthy condition (p = 0.001728). N = 5, n = 3. Total LOX expression across the entire leaflet was also quantified via <b>(D)</b> qRT-PCR for LOX (N = 5, n = 3) and <b>(E)</b> measurement of LOX protein via dot blot (N = 5, n = 3).</p