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Mechanoregulation of Notch signaling activity in endothelial cells and its influence on angiogenic sprouting
Mechanoregulation of Notch signaling activity in endothelial cells and its influence on angiogenic sprouting
The Notch signaling pathway plays a critical role in many developmental and disease related processes. Furthermore, Notch regulates the differentiation of tip and stalk cells during angiogenesis. It is widely accepted that Notch has a mechanotransduction module that regulates cleavage of the receptor. However, the role of biomechanical properties of the cellular environment on this module and on Notch signaling in general is still poorly understood. In the first part of this thesis, the influence of substrate stiffness on the Notch signaling pathway was investigated in endothelial cells. Using stiffness-tuned PDMS substrates it could be shown that Notch signaling pathway activity inversely correlates with the physiologically relevant substrate stiffness, with increased Notch activity on softer substrates. In this context, trans-endocytosis of the Notch extracellular domain, but not the overall endocytosis, is regulated by substrate stiffness. Furthermore, Notch related adhesion pathways were studied in connection with different substrate stiffnesses. It was observed that integrin cell-matrix connections are both stiffness-dependent and influenced by Notch. Cadherin-mediated cell-cell adhesion and Notch influence each other in that basal Notch signaling is cell-cell contact-dependent. Inhibition of the Notch signaling pathway however also results in a reduction of VE-cadherin levels. In the second part of the project, 2D and 3D approaches were used to investigate the role of Notch and substrate stiffness in angiogenesis. It was shown that both overexpression of Dll4 and inhibition of the Notch signaling pathway drive sprouting, although Notch blocking led to excessive and ineffective sprout formation. By analyzing the cell positions of Dll4 overexpressing cells, it was further identified that Dll4 expression is not a selection factor for tip cell differentiation, but a consequence. In connection to the identified stiffness-dependent activation of the Notch signaling pathway, it was also shown that sprouting and sprout elongation is increased in matrices with low stiffness. In sum, the present work demonstrates a mechanosensitivity of the Notch signaling pathway likely associated with the process of trans-endocytosis, suggesting a second mechanical aspect of the Notch signaling pathway besides the pulling force generated by the ligand presenting cell. Furthermore, a new insight into the influence of stiffness on the sprouting behavior of endothelial cells as well as the role of Dll4 overexpression in tip cell selection is provided.
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Kretschmer, Maibritt Vanessa
2022
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Kretschmer, Maibritt Vanessa (2022): Mechanoregulation of Notch signaling activity in endothelial cells and its influence on angiogenic sprouting. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The Notch signaling pathway plays a critical role in many developmental and disease related processes. Furthermore, Notch regulates the differentiation of tip and stalk cells during angiogenesis. It is widely accepted that Notch has a mechanotransduction module that regulates cleavage of the receptor. However, the role of biomechanical properties of the cellular environment on this module and on Notch signaling in general is still poorly understood. In the first part of this thesis, the influence of substrate stiffness on the Notch signaling pathway was investigated in endothelial cells. Using stiffness-tuned PDMS substrates it could be shown that Notch signaling pathway activity inversely correlates with the physiologically relevant substrate stiffness, with increased Notch activity on softer substrates. In this context, trans-endocytosis of the Notch extracellular domain, but not the overall endocytosis, is regulated by substrate stiffness. Furthermore, Notch related adhesion pathways were studied in connection with different substrate stiffnesses. It was observed that integrin cell-matrix connections are both stiffness-dependent and influenced by Notch. Cadherin-mediated cell-cell adhesion and Notch influence each other in that basal Notch signaling is cell-cell contact-dependent. Inhibition of the Notch signaling pathway however also results in a reduction of VE-cadherin levels. In the second part of the project, 2D and 3D approaches were used to investigate the role of Notch and substrate stiffness in angiogenesis. It was shown that both overexpression of Dll4 and inhibition of the Notch signaling pathway drive sprouting, although Notch blocking led to excessive and ineffective sprout formation. By analyzing the cell positions of Dll4 overexpressing cells, it was further identified that Dll4 expression is not a selection factor for tip cell differentiation, but a consequence. In connection to the identified stiffness-dependent activation of the Notch signaling pathway, it was also shown that sprouting and sprout elongation is increased in matrices with low stiffness. In sum, the present work demonstrates a mechanosensitivity of the Notch signaling pathway likely associated with the process of trans-endocytosis, suggesting a second mechanical aspect of the Notch signaling pathway besides the pulling force generated by the ligand presenting cell. Furthermore, a new insight into the influence of stiffness on the sprouting behavior of endothelial cells as well as the role of Dll4 overexpression in tip cell selection is provided.