Dysregulation of Endothelial Nitric Oxide Synthase Does Not Depend on Hemodynamic Alterations in Bicuspid Aortic Valve Aortopathy

Abstract

Background Bicuspid aortic valves (BAVs) predispose to ascending aortic aneurysm. Turbulent blood flow and genetic factors have been proposed as underlying mechanisms. Endothelial nitric oxide synthase (eNOS) has been implicated in BAV aortopathy, and its expression is regulated by wall shear stress. We hypothesized that if turbulent flow induces aneurysm formation in patients with a BAV, regional differences in eNOS expression would be observed in BAVs. Methods and Results Ascending aortic specimens were harvested intraoperatively from 48 patients with tricuspid aortic valve (19 dilated, 29 nondilated) and 38 with BAV (28 dilated, 10 nondilated) undergoing cardiac surgery. eNOS mRNA and protein concentration were analyzed at the convex and concave aortic wall. In nondilated aortas, eNOS mRNA and protein concentration were decreased in BAV compared with tricuspid aortic valve (all P<0.05). eNOS expression was increased in association with dilation in BAV aortas (P=0.03), but not in tricuspid aortic valve aortas (P=0.63). There were no regional differences in eNOS mRNA or protein concentration in BAV aortas (all P>0.05). However, eNOS expression was increased at the concave wall (versus convexity) in tricuspid aortic valve dilated aortas (all P<0.05). Conclusions Dysregulated eNOS occurs independent of dilation in BAV aortas, suggesting a potential role for aberrantly regulated eNOS expression in the development of BAV-associated aneurysms. The absence of regional variations of eNOS expression suggests that eNOS dysregulation in BAV aortas is the result of underlying genetic factors associated with BAV disease, rather than changes stimulated by hemodynamic alterations. These findings provide insight into the underlying mechanisms of aortic dilation in patients with a BAV.

Keywords: aortic valve; ascending aortic aneurysm; bicuspid aortic valve; endothelial nitric oxide synthase; hemodynamics.

Figure 1. Schematic representation of the human aorta, with tissue sampling sites indicated.

To assess for endothelial nitric oxide synthase (eNOS) mRNA expression and protein concentration in the ascending aorta of patients with a bicuspid aortic valve, tissue samples were harvested from the convex and concave walls of the aorta, adjacent to the sinotubular junction (STJ) and at the level of the mid‐ascending aorta. Samples from patients with a tricuspid aortic valve were used as comparison. Sample sites are indicated by rectangular boxes: samples of convex wall (A1) and concave wall (A2) adjacent to the STJ and samples of convex (B1) and concave wall (B2) from the mid‐ascending aorta were harvested. This schematic representation indicates a bicuspid aortic valve, with a partially fused commissure (gray dotted line).

Figure 2. Schematic summary of endothelial nitric oxide synthase (eNOS) expression in normal and dilated aortas associated with tricuspid and bicuspid aortic valves.

A, Schematic representation of a normal (nondilated) aorta associated with a tricuspid aortic valve (TAV). eNOS expression is relatively high (green shading) in both the convex and concave walls of the aorta. B, Schematic representation of a dilated aorta associated with a TAV. eNOS expression is relatively high in the concave wall, with decreased expression (darker green) in the convex wall of the aorta. C, Schematic representation of a nondilated aorta associated with a bicuspid aortic valve (BAV). eNOS expression is relatively low (red shading) in both the convex and concave walls. D, Schematic representation of a dilated aorta associated with a BAV. eNOS expression is relatively lower than in nondilated BAV aortas (darker red shading) and similar to that of dilated TAV aortas in the convex wall (dark shading), Within the BAV dilated aorta, eNOS is equally expressed between concave and convex walls.

Figure 3. Endothelial nitric oxide synthase (eNOS) expression is altered in bicuspid aortic valve (BAV) nondilated aortas compared with tricuspid aortic valve (TAV) nondilated aortas.

A, Graph depicting the eNOS mRNA expression in nondilated (normal) aortas from patients with a TAV or a BAV. B, Graph and corresponding western blot image depicting the eNOS protein expression in normal aortas from patients with a TAV and patients with a BAV. eNOS protein expression values are normalized to beta‐actin protein expression. C, Graph depicting the eNOS mRNA expression in dilated aortas from patients with a TAV or BAV. D, Graph and corresponding western blot image depicting the eNOS protein expression in dilated aortas from patients with a TAV and patients with a BAV. eNOS protein expression values are normalized to beta‐actin protein expression. *=significance at P<0.05; AU indicates arbitrary units. Data are presented as mean (bars)±SEM (error bars).

Figure 4. Endothelial nitric oxide synthase (eNOS) expression in the aorta is altered in response to dilation in patients with a bicuspid aortic valve (BAV) but not patients with a tricuspid aortic valve (TAV).

A, Graph depicting the eNOS mRNA expression in patients with a TAV with nondilated (normal) and dilated aortas. B, Graph and corresponding western blot image depicting the eNOS protein expression in normal and dilated aortas from patients with a TAV. eNOS protein expression values are normalized to beta‐actin protein expression. C, Graph depicting the eNOS mRNA expression in normal and dilated aortas from patients with a BAV. D, Graph and corresponding western blot image depicting the eNOS protein expression in normal and dilated aortas from patients with a BAV. eNOS protein expression values are normalized to beta‐actin protein expression. *=significance at P<0.05; AU indicates arbitrary units. Data are presented as mean (bars)±standard error of the mean (error bars).

Figure 5. Endothelial nitric oxide synthase (eNOS) expression is altered at the concavity of the aortic wall compared with the convexity in dilated tricuspid aortic valve (TAV) aortas.

Graphs depicting the endothelial nitric oxide synthase (eNOS) mRNA expression (left) and protein expression (right) in nondilated (normal) aortas (A) and dilated aortas (B) from patients with a TAV. eNOS expression is compared between the convex and the concave wall of the aorta at the level of the sinotubular junction (STJ). C, Graphs depicting the eNOS mRNA expression (left) and protein expression (right) in dilated aortas from patients with a TAV. eNOS expression is compared between the convex and the concave wall of the aorta at the level of the mid‐ascending aorta (Asc. Ao.). *=significance at P<0.05; AU indicates arbitrary units. Data are presented as mean (bars)±standard error of the mean (error bars).

Figure 6. There are no regional differences in endothelial nitric oxide synthase (eNOS) expression in the aortic wall in dilated bicuspid aortic valve (BAV) aortas.

A‐B, Graphs depicting the eNOS mRNA expression (left) and protein expression (right) in dilated aortas from patients with a BAV. eNOS expression is compared between the convex and the concave wall of the aorta at the level of (A) the sinotubular junction (STJ) and (B) the mid‐ascending aorta. C, Graphs depicting eNOS mRNA expression between BAV aortas with right and left coronary cusp (R‐L) fusions and right noncoronary cusp (R‐N) fusions at the convexity and the concavity from the sinotubular junction (STJ) and the mid‐ascending aorta (Asc. Ao.) in BAV aortas. *=significance at P<0.05; AU indicates arbitrary units. Data are presented as mean (bars)±SEM (error bars).