Experimental evidence of the genetic hypothesis on the etiology of bicuspid aortic valve aortopathy in the hamster model

Abstract

Bicuspid aortopathy occurs in approximately 50% of patients with bicuspid aortic valve (BAV), the most prevalent congenital cardiac malformation. Although different molecular players and etiological factors (genetic and hemodynamic) have been suggested to be involved in aortopathy predisposition and progression, clear etiophysiopathological mechanisms of disease are still missing. The isogenic (genetically uniform) hamster (T) strain shows 40% incidence of BAV, but aortic dilatations have not been detected in this model. We have performed comparative anatomical, histological and molecular analyses of the ascending aorta of animals with tricuspid aortic valve (TAV) and BAV from the T strain (TTAV and TBAV, respectively) and with TAV from a control strain (HTAV). Aortic diameter, smooth muscle apoptosis, elastic waviness, and Tgf-β and Fbn-2 expression were significantly increased in T strain animals, regardless of the valve morphology. Strain and aortic valve morphology did not affect Mmp-9 expression, whereas Mmp-2 transcripts were reduced in BAV animals. eNOS protein amount decreased in both TBAV and TTAV compared to HTAV animals. Thus, histomorphological and molecular alterations of the ascending aorta appear in a genetically uniform spontaneous hamster model irrespective of the aortic valve morphology. This is a direct experimental evidence supporting the genetic association between BAV and aortic dilatation. This model may represent a population of patients with predisposition to BAV aortopathy, in which increased expression of Tgf-β and Fbn-2 alters elastic lamellae structure and induces cell apoptosis mediated by eNOS. Patients either with TAV or BAV with the same genetic defect may show the same risk to develop bicuspid aortopathy.

Keywords: animal model; aortic dilatation; bicuspid aortic valve (BAV); etiology; hamster; pathophysiology.

FIGURE 1

Scanning electron micrographies of TAVs (A,C) and a BAV (B) of hamsters from the affected (A,B) and the control (C) strains. Cranial views. D, dorsal (non-coronary) leaflet; L, left leaflet; R, right leaflet; V, ventral leaflet. Arrowhead: raphe. Scale bar: 500 μm. (D) Analysis of the relative diameter of the ascending aorta of hamsters grouped according to the strain and the valve morphology. H, control strain; TBAV, bicuspid aortic valve from the affected strain; TTAV, tricuspid aortic valve from the affected strain. Only significant p-values are detailed.

FIGURE 2

Transverse sections of the ascending aorta convexity (Cx; A,B) and concavity (Cn; C,D) of hamsters from the affected (A,C) and the control (B,D) strains, stained with hematoxylin and eosin. Scale bar: 100 μm. (E) Quantification of the waviness index in the aortic convexity and concavity of animals from the control strain (H) and from the affected strain with TAV (TTAV) and BAV (TBAV). Only significant p-values are detailed.

FIGURE 3

Confocal images of the convexity (Cx; A,C,E) and concavity (Cn; B,D,F) of the ascending aorta of hamsters from the control (A,B) and affected (C–F) strains. TUNEL fluorescence (green) demonstrated abundant apoptotic cells (arrowheads) in the media of the aortic convexity of animals of the affected strain both with TAV (C) and BAV (E). DAPI (blue) stained nuclei. No TUNEL signal was detected in aortas from control animals (A,B) and in the negative control (not shown), and only a few signals were found in the concavity of animals of the affected strain (D,F). Note that the elastic sheets show some autofluorescence. A, adventitia; L, lumen. Scale bar: 50 μm. (G) Quantification of the percentage of TUNEL⁺ cells revealed significant differences between convexity and concavity, and between the affected and control strains, irrespective of aortic valve morphology. H, control strain; TBAV, bicuspid aortic valve from the affected strain; TTAV, tricuspid aortic valve from the affected strain. Only significant p-values are detailed.

FIGURE 4

Endothelial nitric oxide synthase immunoperoxidase in the convexity (Cx; A,C,E) and concavity (Cn; B,D,F) of the ascending aorta of hamsters from the control (A,B) and the affected (C–F) strains. All specimens showed strong immunoreactivity in the endothelium, close to the lumen (L), and the adventitia (A). Medial eNOS⁺ smooth muscle cells (arrowheads) were abundant and homogeneously distributed in animals from the control strain (A,B), but they were scarce and scattered in animals of the T strain (C–F), particularly at the aortic concavity (D,F). No signal was detected in the negative control (not shown). Scale bar: 50 μm. (G) Quantification of the aortic media area occupied by eNOS⁺ cells. Expression was reduced almost by halve in both the convexity and the concavity of animals of the T strain. Convexity and concavity showed similar eNOS expression except in animals with BAV, in which the reduction in the concavity was even more pronounced. H, control strain; TBAV, bicuspid aortic valve from the affected strain; TTAV, tricuspid aortic valve from the affected strain. Only significant p-values are detailed.

FIGURE 5

Analysis of Tgf-β, Mmp-2, Mmp-9, Fbn-1, and Fbn-2 mRNA expression in the ascending aorta of hamsters grouped according to the strain and the valve morphology. Tgf-β expression (A) was significantly higher in animals of the affected strain irrespective of aortic valve morphology. Mmp-2 expression (B) was significantly reduced in animals with BAV from the affected strain. Mmp-9 expression (C) increased with age, although significantly only for T-TAV animals, but did not change significantly with the strain and the valve morphology. Fbn-1 expression (D) did not significantly change in animals of the affected strain, both with TAV and BAV. Fbn-2 expression (E) was higher in animals of the affected strain, although differences were significant only for animals with BAV. Fbn-1/Fbn-2 expression ratio (F) was over fivefold reduced in animals from the T strain, both with TAV and BAV. The expressions were normalized against the reference gene Cdkn1β. H, control strain; TBAV, bicuspid aortic valve from the affected strain; TTAV, tricuspid aortic valve from the affected strain. Only significant p-values are detailed.