FR 4239/1-1 Die Rolle der Matrixsteifigk

Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 439891283

Endothelial cells (ECs) recognize and respond to mechanical forces through their cell-cell junctions and cell-matrix adhesions and translate physical stimuli into biological responses in a process called mechanotransduction. The extracellular matrix (ECM) surrounds ECs and composition and mechanical properties of the ECM differ along vascular trees, in various organs, during development and in disease states.I have recently shown a fundamental function of ECM stiffness in controlling lymphatic development. Here, we aim to demonstrate that ECM stiffness functions as a general regulator of ECs in vascular morphogenesis, homeostasis and disease.For the first aim, we will determine the physiological ECM stiffness experienced by ECs at different developmental stages, in different types of vessels and vascular beds to correlate in vitro ECM stiffness studies more precisely to the actual in vivo situation. To measure ECM stiffness, we have established ex vivo atomic force microscopy in combination with fluorescence microscopy. Additionally, for our in vitro ECM stiffness studies, we will generate fluorescent stiffness sensors that allow for live visualization of stiffness changes in ECs using confocal and super-resolution live imaging. In preliminary studies, we have performed differential RNA sequencing of blood (B) and lymphatic (L) ECs cultured on soft and stiff matrices to identify ECM stiffness-regulated genes and to compare relative importance of signaling pathways driven by ECM stiffness in LECs versus BECs. Expression of 3231 genes was similarly regulated in BECs and LECs in response to changes in matrix stiffness while 3475 genes were differently regulated. For the second and third aim, we will focus on the detailed analysis of two selected ECM stiffness-regulated genes, which have been previously reported to be involved in regulating cell-cell adhesion and endothelial junctions. We have identified the expression of a specific receptor tyrosine kinase (RTK) to be regulated by ECM stiffness in LECs and BECs. Preliminary in vivo studies suggested a specific role of the RTK signaling in lymphatic homeostasis and junctional regulation. We will analyse mechanisms of matrix stiffness-regulated RTK signaling with implications for lymphatic junctions and differential regulation and importance of the specific stiffness-regulated RTK signaling in LECs and BECs. Furthermore, we have identified the expression of a specific phosphodiesterase (PDE) to be regulated by ECM stiffness in LECs but not in BECs. Our preliminary studies showed impaired lymphatic development in PDE-deficent embryos. We aim to decipher matrix stiffness-regulated PDE function in ECs during vascular morphogenesis and homeostasis. Taken together, we aim to provide novel targets to modulate ECM stiffness-regulated EC signaling pathways with implications for treatment of edema and other disease states associated with ECM alterations and vascular dysfunction.