Abstract
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.
Highlights
The aortic heart valve is comprised of three distinct extracellular matrix (ECM) layers: the fibrosa, spongiosa, and ventricularis
Healthy aortic heart valves were obtained within 24 hours post-mortem from individuals with no previous diagnosis of calcific aortic valve disease (CAVD); this protocol was reviewed by the UW-Madison IRB and granted an exemption (#2012–0721) due to not qualifying as human subjects research as defined under 45 CFR 46.102(f)
Collagen was prevalent in the fibrosa and ventricularis, while glycosaminoglycans comprised the spongiosa, and elastin was localized to the ventricularis (Fig 1B). This layered ECM structure was clearly disrupted in diseased leaflets (Fig 1A)
Summary
Methods and MaterialsReagents and MaterialsAll reagents used were purchased from Sigma Aldrich Aortic valve leaflets from individuals 50–75 years of age were collected from the University of Wisconsin Hospital and the William S. Healthy aortic heart valves were obtained within 24 hours post-mortem from individuals with no previous diagnosis of CAVD; this protocol was reviewed by the UW-Madison IRB and granted an exemption (#2012–0721) due to not qualifying as human subjects research as defined under 45 CFR 46.102(f). Written consent was obtained from patients by the cardiothoracic surgery team, in accordance with protocol #2009–1094 approved by the UW-Madison IRB. These valves were confirmed as diseased by the cardiothoracic surgery team and a pathologist, and all had macroscopically visible and palpable calcification (example pictured in S1 Fig).
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