1Klinikum rechts der Isar der Technischen Universität München Klinik und Poliklinik für Innere Medizin I München, Deutschland
Introduction: Vascular tone plays a crucial role in the regulation of endothelial biology as well as vascular flow and tissue perfusion. Measuring forces applied by endothelial cells in vitro has been facilitated by numerous techniques. In vivo force measurements, however, have thus far been difficult to perform although these in vivo measurements would be far more meaningful. FRET-based tension sensors allow for the assessment of force across specific proteins. In our system, the fluorescent proteins mTFP1 and Venus are separated by a flageliform linker, which stretches under force, reducing FRET-efficacy. This tension sensor has been incorporated into the proteins VE-Cadherin and Vinculin. These fusion proteins have demonstrated the capacity to measure forces between endothelial cells (with VE-Cadherin as a tension sensor) as well as between endothelial cells and the extracellular matrix (by measuring force across the Integrin adaptor molecule Vinculin). To enhance our understanding of the applied forces between endothelial cells and endothelial cells and the extracellular matrix, we intend to measure tension across the proteins VE-Cadherin and Vinculin in vivo. To incorporate the tension sensors in our rAAV vector system we split the sensors with the use of an inten-extein system to be deliverd via two rAAVs.
Methods: The ORF of the VE-Cadherin (CDH5-TS) and Vinculin (Vin-TS) tension sensors were split into an N-terminal and C-terminal part with the introduction of the Intein-sequences within the corresponding mTFP1-Sequences of the full-length ORF. This lead to the generation of pairs of tension sensor modules (CDH5-TSN and CDH5-TSC / Vin-TSN and Vin-TSC) able to realign inside the targeted cell. A tail-less variant (tail-less control) consisting only of the C-terminus of the tension sensor module (c-terminal part of mTFP1, the flageliform linker and Venus) was generated as a zero-force control. rAAV-vectors of all constructs were generated, coated with dendrimer nanoparticles (PAMAM) and endothelial cell specific targeting peptides. These viruses were used in either cell culture in HUVEC cells and HEK293 cells or injected into mice via the tail-vein.
Results: In initial experiments in HEK293-cells we were able to demonstrate the successful recombination of both parts of the tension sensor module. In HUVEC-cells furthermore, we could demonstrate the proper recombination of both tension sensor constructs into a full-length protein for CDH5-TS as well as Vin-TS via imaging and Western Blot analysis. Lastly in mice, we could demonstrate the expression of the full-length proteins in endothelial cells as expected. We were furthermore able to demonstrate the functionality of the tension sensors by measuring FRET-efficacy at rest as well as after treatment with Angiopoietin-2, which is known du disrupt endothelial attachment to the underlying extracellular matrix as well as other endothelial cells. In further experiments we aim to analyze the changes in endothelial force transduction in a time dependent manner after pathological stimulation.