Introduction: Vascular tone plays a crucial role in the regulation of endothelial biology as well as vascular flow and tissue perfusion. In vivo force measurements have thus far been difficult to perform although these in vivo measurements would be far more meaningful than cell culture studies. 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. Here we utilize a VE-Cadherin and Vinculin tension sensors. To overcome the size-restrictions of the intended rAAV-vector, we used a dual vector system and an intein-split mechanism.

Methods: VE-Cadherin (CDH5-TS) and Vinculin (Vin-TS) tension sensors were split into an N-terminal and C-terminal part flanked by intein peptide sequences. 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 cell. 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 or injected into mice via the tail-vein.

Results: In cultured cells we were able to demonstrate the successful recombination of both parts of the tension sensor module, their proper subcellular localization and function. In in vivo studies, we showed similarly the intein-split recombination as well as their function as tension sensors. We could also measure a decrease in endothelial force transduction after stimulation with Ang-2.