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.

Individuals with bicuspid aortic valve (BAV) are at significantly higher risk of developing serious aortic complications than individuals with tricuspid aortic valves (TAV). Studies have indicated an altered aortic blood flow in patients with BAV; however, the extent to which altered flow influences the pathological state of BAV aorta is unclear. In the present study, we dissected flow-mediated aortic gene expression in patients undergoing elective open heart surgery. A large collection of public microarray data sets were firstly screened for consistent co-expression with five well-characterized flow-regulated genes (query genes). Genes with co-expression probability of >0.5 were selected and further analysed in expression profiles (127 arrays) from ascending aorta of BAV and TAV patients. Forty-four genes satisfied two filtering criteria: a significant correlation with one or more of the query genes (R > 0.40) and differential expression between patients with BAV and TAV. No gene fulfilled the criteria in mammary artery (88 arrays), an artery not in direct contact with the valve. Fifty-five percent of the genes significantly altered between BAV and TAV patients showed differential expression between two identified flow regions in the rat aorta. A large proportion of the identified genes were related to angiogenesis and/or wound healing, with pro-angiogenesis genes downregulated and inhibitory genes upregulated in patients with BAV. Moreover, differential expression of ZFP36, GRP116 and PKD2 was confirmed using immunohistochemistry. Implementing a new strategy, we have demonstrated an angiostatic gene expression signature in patients with BAV, indicating impaired wound healing in these patients, potentially involved in BAV-associated aortopathy.

Disturbed flow has been suggested to contribute to aneurysm susceptibility in bicuspid aortic valve (BAV) patients. Lately, flow has emerged as an important modulator of DNA methylation. Hear we combined global methylation analysis with in vitro studies of flow-sensitive methylation to identify biological processes associated with BAV-aortopathy and the potential contribution of flow. Biopsies from non-dilated and dilated ascending aortas were collected from BAV (n = 21) and tricuspid aortic valve (TAV) patients (n = 23). DNA methylation and gene expression was measured in aortic intima-media tissue samples, and in EA.hy926 and primary aortic endothelial cells (ECs) isolated from BAV and TAV exposed to oscillatory (±12 dynes/cm2) or laminar (12 dynes/cm2) flow. We show methylation changes related to epithelial-mesenchymal-transition (EMT) in the non-dilated BAV aorta, associated with oscillatory flow related to endocytosis. The results indicate that the flow-response in BAV ECs involves hypomethylation and increased expression of WNT/β-catenin genes, as opposed to an angiogenic profile in TAV ECs. The EMT-signature was exasperated in dilated BAV aortas. Aberrant EMT in BAV aortic walls could contribute to increased aneurysm susceptibility, and may be due to disturbed flow-exposure. Perturbations during the spatiotemporally related embryonic development of ascending aorta and semilunar valves can however not be excluded.

Background Acetylsalicylic acid (ASA) therapy has been associated with a reduced prevalence and growth rate of abdominal as well as intracranial aneurysms, but the relationship between ASA and ascending aortic aneurysm formation remains largely unknown. The aim of the present study was to investigate whether ASA therapy is associated with a lower prevalence of ascending aortic aneurysm in a surgical cohort. Methods and Results One thousand seven hundred patients undergoing open-heart surgery for ascending aortic aneurysm and/or aortic valve disease were studied in this retrospective cross-sectional study. Aortic dilatation was defined as an aortic root or ascending aortic diameter ≥45 mm. Medications were self-reported by the patients in a systematic questionnaire. Cyclooxygenase gene expression was measured in the intima-media portion of the ascending aorta (n=117). In a multivariable analysis, ASA was associated with a reduced prevalence of ascending aortic aneurysm (relative risk, 0.68 [95% CI, 0.48-0.95], P=0.026) in patients with tricuspid aortic valves, but not in patients with bicuspid aortic valves (relative risk, 0.93 [95% CI, 0.64-1.34], P=0.687). Intima-media cyclooxygenase expression was positively correlated with ascending aortic dimensions (P<0.001 for cyclooxygenase-1 and P=0.05 for cyclooxygenase-2). In dilated, but not nondilated tricuspid aortic valve aortic specimens, ASA was associated with significantly lower cyclooxygenase-2 levels (P=0.034). Conclusions Our findings are consistent with the hypothesis that ASA treatment may attenuate ascending aortic aneurysmal growth, possibly via cyclooxygenase-2 inhibition in the ascending aortic wall and subsequent anti-inflammatory actions.

Individuals with a bicuspid aortic valve (BAV) are at significantly higher risk of developing aortic complications than individuals with tricuspid aortic valves (TAV) and defective signaling during the embryonic development and/or life time exposure to abnormal hemodynamic have been proposed as underlying factors. However, an explanation for the molecular mechanisms of aortopathy in BAV has not yet been provided. We combined proteomics, RNA analyses, immunohistochemistry, and electron microscopy to identify molecular differences in samples of non-dilated ascending aortas from BAV (N = 62) and TAV (N = 54) patients. Proteomic analysis was also performed for dilated aortas (N = 6 BAV and N = 5 TAV) to gain further insight into the aortopathy of BAV. Our results collectively showed the molecular signature of an endothelial/epithelial-mesenchymal (EndMT/EMT) transition-like process, associated with instability of intimal cell junctions and activation of RHOA pathway in the intima and media layers of ascending aorta in BAV patients. We propose that an improper regulation of EndMT/EMT during the spatiotemporally related embryogenesis of semilunar valves and ascending aorta in BAV individuals may result in aortic immaturity and instability prior to dilation. Exasperation of EndMT/EMT state in post embryonic life and/or exposure to non-physiological hemodynamic could lead to the aneurysm of ascending aorta in BAV individuals.