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

Journal of Molecular Medicine https://doi.org/10.1007/s00109-023-02316-5 ORIGINAL ARTICLE Bicuspid aortic valve aortopathy is characterized by embryonic epithelial to mesenchymal transition and endothelial instability David Freiholtz1 · Otto Bergman2 · Karin Lång2 · Flore‑Anne Poujade2 · Valentina Paloschi2 · Carl Granath1 · Jan H. N. Lindeman3 · Christian Olsson1 · Anders Franco‑Cereceda1 · Per Eriksson2 · Hanna M. Björck2 Received: 15 December 2022 / Revised: 24 February 2023 / Accepted: 11 April 2023 © The Author(s) 2023 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 • Endothelial integrity is compromised in BAV aortas prior • Embryonic EMT is a feature of BAV-associated aortopathy. • Non-dilated BAV ascending aortas are more permeable to dilatation. than aortas of tricuspid aortic valve patients. Keywords Bicuspid aortic valve · Ascending aneurysm · EMT · Endothelial instability David Freiholtz and Otto Bergman contributed equally. * Hanna M. Björck 1 Section of Cardiothoracic Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden 2 Division of Cardiovascular Medicine, Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm Solna, Sweden 3 Department of Vascular Surgery, Department of Surgery, Medical Center Leiden, Leiden University, Leiden, the Netherlands List of Abbreviations AscAA Ascending aortic aneurysm AUC Area under curve BM Basement membrane BAV Bicuspid aortic valve D Dilated EMT Epithelial to mesenchymal transition GO Gene ontology ND Non-dilated TAV Tricuspid aortic valve VSMC Vascular smooth muscle cell Introduction Ascending aortic aneurysm (AscAA) is a silent, potentially life-threatening disease manifesting as a localized dilatation of the ascending aorta. Bicuspid aortic valve (BAV), the most 13 Vol.:(0123456789) Journal of Molecular Medicine common congenital heart defect with an estimated prevalence of ~ 1% [1, 2], has a strong association with AscAA with up to 50–70% of all BAV patients requiring ascending aortic surgery at some point during their lifetime [2, 3]. The mechanism behind BAV formation is unknown, but a defective epithelial-tomesenchymal transition (EMT), and a faulty signaling between different cardiac progenitor cells thereof, during semilunar valve formation has been highlighted [4–6]. This link between EMT and BAV occurrence was recently also strengthened by a large genome-wide association study, in which a deleterious missense variant in the EMT-related gene MUC4 was associated with BAV formation [7]. Mechanistical studies in zebrafish further showed that loss of MUC-4 led to a delay in cardiac valvular development, putting MUC4 in the context of aortic valve malformation. Importantly for the context of this study, in pioneering experiments performed by Epstein et al., the faulty embryonic EMT signaling was further extended to link defective valve formation with postnatal aortic abnormalities, such as AscAA formation [6, 8–10]. In line with this, we and others have previously implicated EMT in association with aortic dilatation in patients with non-familial BAV [11–13], which represents the vast majority of BAV cases, although it does not show traditional signs of post-natal EMT, such as elastin fragmentation, extracellular matrix deposition, and smooth muscle cell loss [14, 15]. Instead, the dilated BAV aortic wall seems structurally well preserved, devoid of tissue resident fibroblasts, and clearly different from that of aneurysmal tricuspid aortic valve (TAV) patients [16, 17]. Additionally, key signs of inflammation and fibrosis are lacking in BAV-associated aneurysms [18], which may allude to the fact that another type of EMT is active in BAV adult aortas, possibly originating from embryonic development and the defective valve formation (i.e., embryonic EMT in contrast to fibrotic postnatal EMT). The aim of this study was to investigate EMT in ascending aortas of patients with non-familial BAV from a subtypespecific perspective (embryonic vs. fibrotic EMT), as well as delineating the state of the endothelium, to further understand the pathology behind general BAV-associated aortic disease. We hypothesize that, in contrast to TAV AscAA, an embryonic-like EMT is active in BAV ascending aorta and contributes to aneurysm formation possibly by compromising the intimal/endothelial stability. An increased knowledge of the specific mechanism behind BAV aneurysm susceptibility is of importance to improve risk assessment, surveillance, and possible novel treatment targets for these patients. Material and methods Patients Patients from the Advanced Study of Aortic Pathology (ASAP) and Disease of the Aortic Valve, Ascending Aorta and Coronary 13 Arteries (DAVAACA) cohorts were studied. The cohorts have been described in detail elsewhere [19, 20]. In brief, all patients underwent elective open-heart surgery for aortic valve disease and/or ascending aortic aneurysm (AscAA) with or without coronary artery disease at the Karolinska University Hospital, Stockholm, Sweden. Tissue biopsies were collected from the anterior part of the ascending aorta, at the site of aortotomy a few centimeters above the aortic valve. Patients were classified according to aortic valve morphology (BAV or TAV) and aortic dilatation (non-dilated (ND) or dilated (D), where aortic diameters of > 45 mm were considered dilated and aortas < 40 mm were classified as non-dilated). Pati (...truncated)