Bicuspid aortic valve aortopathy is characterized by embryonic epithelial to mesenchymal transition and endothelial instability

Abstract

Bicuspid aortic valve (BAV) is the most common congenital heart malformation frequently associated with ascending aortic aneurysm (AscAA). Epithelial to mesenchymal transition (EMT) may play a role in BAV-associated AscAA. The aim of the study was to investigate the type of EMT associated with BAV aortopathy using patients with a tricuspid aortic valve (TAV) as a reference. The state of the endothelium was further evaluated. Aortic biopsies were taken from patients undergoing open-heart surgery. Aortic intima/media miRNA and gene expression was analyzed using Affymetrix human transcriptomic array. Histological staining assessed structure, localization, and protein expression. Migration/proliferation was assessed using ORIS migration assay. We show different EMT types associated with BAV and TAV AscAA. Specifically, in BAV-associated aortopathy, EMT genes related to endocardial cushion formation were enriched. Further, BAV vascular smooth muscle cells were less proliferative and migratory. In contrast, TAV aneurysmal aortas displayed a fibrotic EMT phenotype with medial degenerative insults. Further, non-dilated BAV aortas showed a lower miRNA-200c-associated endothelial basement membrane LAMC1 expression and lower CD31 expression, accompanied by increased endothelial permeability indicated by increased albumin infiltration. Embryonic EMT is a characteristic of BAV aortopathy, associated with endothelial instability and vascular permeability of the non-dilated aortic wall. KEY MESSAGES: Embryonic EMT is a feature of BAV-associated aortopathy. Endothelial integrity is compromised in BAV aortas prior to dilatation. Non-dilated BAV ascending aortas are more permeable than aortas of tricuspid aortic valve patients.

Keywords: Ascending aneurysm; Bicuspid aortic valve; EMT; Endothelial instability.

Fig. 1

Movat pentachrome staining of dilated ascending aortas (magnification × 20) from patients with BAV (n = 11) and TAV (n = 8), respectively. BAV, bicuspid aortic valve; TAV, tricuspid aortic valve

Fig. 2

ORIS migration assay using ascending aortic vascular smooth muscle cells isolated from patients with BAV and TAV, respectively, visualized by IncuCyte live cell imaging. a Area covered by cells at 0–24 h of growth and b fluorescent dye intensity at 0–10 h of growth. Red dotted lines represent the regression line for BAV (n = 5) and TAV cells (n = 3), respectively. c proliferation at 24 h, P = 0.036, Mann Whitney U-test. BAV, bicuspid aortic valve; TAV, tricuspid aortic valve

Fig. 3

a Network of BAV-specific (red), TAV-specific (green), and common (yellow) Hallmark EMT genes and their predicted miRNAs (identified using TargetScan). Gray squares and arrows mark predicted miRNAs and their targets. b Zoomed network of BAV-specific Hallmark EMT genes and their predicted miRNAs. Red arrows show negative correlation between gene and miRNA expression with corresponding P and correlation values presented in c. BAV, bicuspid aortic valve; TAV, tricuspid aortic valve

Fig. 4

a Laminin γ1 (red) expression (magnification × 10) and b quantification, non-dilated ascending aortas of TAV (n = 11) and BAV (n = 8) patients. c LAMC1/TAGLN intima-media protein expression (global protein expression measured previously using HiRIEF LC–MS/MS, described previously in Maleki et al. [13]) in relation ascending aortic diameter in patients with BAV (n = 11), P = 0.004, Pearson correlation =  − 0.818. d CD31 (green) and vWF (red) expression (magnification × 10) and e quantification, non-dilated ascending aortas of BAV (n = 7) and TAV (n = 6) patients. BAV, bicuspid aortic valve; TAV, tricuspid aortic valve; D, dilated; ND, non-dilated

Fig. 5

a Endothelial and medial albumin expression in non-dilated and dilated ascending aortas of BAV (n = 9 non-dilated, n = 5 dilated) and TAV (n = 9 non-dilated, n = 7 dilated) patients (magnification × 10). b vWF expression, non-dilated and dilated ascending aortas, BAV (n = 9 non-dilated, n = 5 dilated), and TAV (n = 9 non-dilated, n = 7 dilated) patients (magnification × 10). c, d Quantification of albumin staining in intima and media layers, respectively. BAV, bicuspid aortic valve; TAV, tricuspid aortic valve; vWF, von Willebrand factor