Notch is highly conserved signaling pathway that regulates the development and differentiation of many types of tissues and influences major cellular processes such as cell proliferation, differentiation and apoptosis. Mutations in several Notch signaling components have been associated with a number of congenital heart defects, demonstrating an essential role for Notch both in cardiovascular system development and its maintenance during postnatal life. In particular, mutations in NOTCH1 have been linked to congenital abnormalities of aortic valve and aorta, such as bicuspid aortic valve, increasing risk of aortic dilatation and valve calcification. Therapeutic agents that may influence these disorders are absent to date and the only therapeutic decision is elective surgical intervention. Thus, understanding the mechanisms underlying left ventricular outflow tract malformations become especially important. This study aimed to investigate the role of Notch-dependent cellular and molecular mechanisms in development of aortic and aortic valve pathologies. Using primary cells, first, we sought to compare the cellular functions of endothelial and smooth muscle cells in patients with thoracic aortic aneurysm and healthy donors. Second, since Notch signaling pathway is key regulator of endothelial-to-mesenchymal transition, the process underlying valve formation, we explored whether Notch-dependent endothelial-to-mesenchymal transition is affected in aortic endothelial cells from patients with thoracic aortic aneurysm, associated with bicuspid aortic valve – common congenital heart malformation. Third, although vascular smooth muscle cells have been considered as the main target of degeneration in the aortic wall, endothelial dysfunction might also be responsible for thoracic aortic aneurysm formation. Therefore, we addressed the role of Notch and Notch-related signaling pathways in shear stress response in endothelial cells from aortic aneurysm and healthy controls. Forth, the involvement of dysregulated Notch pathway in calcification is evident. In this work we sought to reveal early Notch-dependent mechanisms of valve calcification in patients with bicuspid- or tricuspid aortic valve associated calcified stenosis. Our data demonstrate downregulation of smooth muscle as well as endothelial cell specific markers in the patient cells. Cellular proliferation, migration, and synthesis of extracellular matrix proteins are attenuated in the cells of the patients with thoracic aortic aneurysm compared to healthy controls. We show that endothelial cells from persons with aortic aneurysm and bicuspid aortic valve have downregulated Notch signaling and fail to activate Notch-dependent endothelial-to-mesenchymal transition in response to its stimulation by different Notch components. Activity of Wnt and BMP pathways was significantly elevated in endothelial cells from aneurysms. Furthermore, activation of DLL4, SNAIL1, DKK1, TCF4 and BMP2 was attenuated in cells of patients in response to shear stress, implying dysregulated Notch/BMP/WNT cross-talk. We report that the expression pattern of Notch genes is altered in the aortic valve interstitial cells of patients with calcific aortic stenosis compared to those of healthy persons. Interstitial cells from bicuspid calcified valves demonstrated significantly higher sensitivity to stimuli at early stages of induced proosteogenic differentiation and were significantly more sensitive to the activation of proosteogenic OPN, ALP and POSTIN expression by Notch activation. Notchdependent endothelial-to-mesenchymal transition was also more prominent in bicuspid valve derived endothelial cells compared to the cells from calcified tricuspid and healthy valves. This study provides the first direct functional evidence that primary aortic and valvular cells from patients with left ventricle outflow tract pathologies have impaired Notch signaling pathway comparing to healthy donors. In conclusion: 1 - both endothelial and smooth muscle cells of aneurysmal aortic wall have downregulated specific cellular markers and altered functional properties, such as growth rate, apoptosis induction, and extracellular matrix synthesis; 2 – Notchdependent endothelial-to-mesenhymal transition is attenuated in endothelial cells of patients with thoracic aortic aneurysm and bicuspid aortic valve; 3 - shearstress response is impaired in endothelial cells of the patients with thoracic aortic aneurysm due to altered Notch/BMP/WNT/β-catenin network; 4 - early events of aortic valve calcification are Notch-dependent and differ in bicuspid and tricuspid aortic valves.
|Titolo:||Role of Notch signaling pathway in pathologies of aorta and aortic valve|
Kostina, Aleksandra [Writing – Original Draft Preparation]
|Data di pubblicazione:||2018|
|Appare nelle tipologie:||07.13 Doctoral Thesis|