P180Notch-dependent regulation of endothelial-to-mesenchimal transition is impaired in aortic endothelial cells of BAV patients

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Abstract

Purpose: Endothelial-to- mesenchimal transition (EndoMT) is an important process for cardiac outflow tract and aortic valve formation. Notch signaling is known as one of the regulators of EndoMT in development but its role in regulation of human heart development remains obscure due to difficulties of studying human development. It has been shown in our as well as other previous studies, that patents with defects of left ventricular outflow tract may have mutations in Notch1 gene as well as in other genes of Notch pathway. Thus, it has been supposed that a dysfunction in Notch pathway could lead to mistakes in development leading to malformations of LVOT, in particular, to BAV. In this study we recapitulated early cardiac development by inducing EndoMT in human endothelial aortic cells (HAEC).The purpose of this study was to compare the efficiency of Notch-dependent EndoMT in aortic endothelium of BAV patients and healthy donors.

Methods: HAEC were isolated from tissue fragments of BAV-associated thoracic aortic aneurysm patients after surgery and from healthy donors. EndoMT was induced by different stimuli: 1) TGFβ; 2) NICD (activated domain of Notch1; 3) Notch ligands – Dll1, Dll4, Jag1, Jag2. Effectiveness of EndoMT was estimated by loss of endothelial and gain of mesenchimal markers by immunocytochemistry and qPCR.

Results: We studied EndoMT induction in control and TAA-BAV group. We show that the extent of EndoMT induction in aortic endothelial cells is strongly dependent on stimuli used: the strongest inducer was TGFFβ. However HAEC co-culture with the cells bearing Notch ligands Dll1, Dll4, Jag1, Jag2 also lead to the induction of EndoMT. Finally we observed the differences in the extent of EndoMT induction between the HAEC of healthy donors and HAEC from TAA-BAV patients: NICD induced EndoMT in HAEC of patients less efficiently comparing to normal cells.

Conclusions: We developed an effective in vitro system which allows studying EndoM directly using the patients' primary cells (HAEC) thus recapitulating processes seen in early cardiogenesis. Our results provide evidence that Notch-dependent EndoMT regulation is impaired in HAEC of BAV patients. Our results for the first time directly confirm the hypothesis that Notch pathway disruption could be responsible for LVOT malformations and, in particular, may lead to BAV.

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