Fibrotic Aortic Valve Stenosis in Hypercholesterolemic/Hypertensive Mice

Abstract

Objective Hypercholesterolemia (HC) and hypertension (HT) are associated with aortic valve stenosis (AVS) in humans. We have examined aortic valve function, structure, and gene expression in HC/HT mice. Approach and Results Control, hypertensive (HT), hypercholesterolemic (Apoe−/−) (HC), and HC/HT mice were studied. Severe aortic stenosis (echocardiography) occurred only in HC/HT mice. There was minimal calcification of the aortic valve. Several structural changes were identified at the base of the valve. The intercusp raphe (or “seam” between leaflets) was longer in HC/HT mice than in other mice, and collagen fibers at the base of the leaflets were reoriented to form a mesh. In HC/HT mice, the cusps were asymmetrical, which may contribute to changes that produce AVS. RNA-Sequencing (RNA-Seq) was used to identify molecular targets during the developmental phase of stenosis. Genes related to structure of the valve were identified that differentially expressed before FAVS developed. Both RNA and protein of a profibrotic molecule, plasminogen activator inhibitor 1 (PAI-1), were increased greatly in HC/HT mice. Conclusions HC/HT mice are the first model of fibrotic AVS. HC/HT mice develop severe AVS in the absence of significant calcification, a feature which resembles AVS in children and some adults. Structural changes at the base of the valve leaflets include lengthening of the raphe, remodeling of collagen, and asymmetry of the leaflets. Genes were identified that may contribute to development of FAVS.

Discovery of an Experimental Model of Unicuspid Aortic Valve

“…In control aortic valves, sites of cusp attachment to aorta consisted of fibrillar collagen oriented predominantly parallel to or perpendicular to valve cusps (Figure 5A, C), as previously reported 12. In Velvet valves, histologic architecture was grossly distorted.…”

“…Adult mice that are subjected to acute severe pressure overload,18 and mice that acquire severe AS during adult life, develop LV systolic dysfunction or die shortly thereafter 11, 12. Humans who acquire severe AS during adult life usually develop symptoms within 2 years 26.…”

“…Severity of aortic valve stenosis (AS) was classified as follows: none, ACS ≥1.00 mm; mild, 0.80 mm≤ACS<1.00 mm; moderate, 0.66 mm≤ACS<0.80 mm; and severe, ACS <0.66 mm. Presence and severity of AS were subsequently confirmed visually, using 2‐dimensional short‐axis images, as previously described 12. For group analysis, AS was deemed “significant” if moderate or severe valve stenosis was present.…”

“…Some examples demonstrating either normal or anomalous valve anatomic features were then selected for scanning electron microscopy, as previously described 12. Briefly, trimmed hearts containing aortic valves were excised; fixed with 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer for 1 hour; postfixed with 1% osmium tetroxide for 1 hour; dehydrated through sequential incubation in 25%, 50%, 75%, 95%, and 100% ethanol; and dried using the CO 2 Sorvall Critical Point Drying System (DuPont, Wilmington, DE).…”

“…Sections, 10 μm thick, were obtained from proximal, mid, and distal aortic valve from each mouse. Slides were stained with Alizarin Red or Picrosirius Red to quantitate the amount of calcium or collagen, respectively, as described previously 12. Movat's Pentachrome stain was used to visualize proteoglycans and collagen in the aortic valve, as described previously 13.…”