Introduction: Vascular tone plays a central role in the regulation of endothelial biology as well as flow and tissue perfusion. FRET-based tension sensors allow for the assessment of force across specific proteins. To this end, the fluorescent proteins mTFP1 and Venus are separated by a flageliform linker, which stretches under force. The resulting separation of the fluorescent proteins reduces the energy transfer between them, resulting in reduced FRET-efficacy in proportion to the applied force. VE-Cadherin and Vinculin constructs carrying the FRET-based tension sensors have successfully been tested in cell culture conditions and were able to demonstrate flow dependent changes in the force applied between endothelial cells (with VE-Cadherin as a tension sensor) as well as between cells and the extracellular matrix (by measuring force across the Integrin adaptor molecule Vinculin). Cell culture systems, however, suffer from specific disadvantages: the rigid surface artificially elevates the tension applied on structural proteins. Furthermore, interaction between cell types as well as flow related changes in force cannot be accounted for in a meaningful way in cell culture systems. To enhance our understanding in the applied forces across the proteins VE-Cadherin as well as Vinculin in vivo we modified VE-Cadherin and Vinculin tension sensors to incorporate those into recombinant adeno-associated viral vector systems capable of specifically targeting endothelial cells.

Methods: VE-Cadherin tension sensors (CDH5-TS) and a tail-less control variant (CDH5-TL) as well as Vinculin tension sensors (Vin-TS with Vin-TL as a control construct) were split into a N-terminal and C-terminal part equipped with intein-sequences to enable intracellular recombination. The intein-split was introduced in the mTFP1 sequence of the respective protein. The N-terminal and C-terminal sequences of each sensor were packaged into rAAV vectors coated with PAMAM-nanoparticles as well as endothelial targeting peptides. To further restrict transgene expression to endothelial cells, transcription was regulated by a Endoglin promoter. To test transduction efficacy as well as intracellular recombination, HUVEC-cells were transfected with 2 rAAVs encoding for the respective halves of CDH5-TS, CDH5-TL, Vin-TS and Vin-TL.

Results: Using HUVECs as an initial test system we were not only able to demonstrate the successful transduction of both parts of the tension sensor system but we were also able to measure intracellular recombination. Here we showed the presence of functioning mTFP1-protein in cells transfected with both tension-sensor parts, which was lacking in cells transduced with either the N-terminal or C-terminal sequences alone. Furthermore, we were able to detect FRET across the mTFP1/Venus pair of fluorescent proteins. In addition, treatment of HUVEC cells with LPS resulted in a significant increase in FRET-efficacy in CDH5-TS transduced cells, indicating reduced force transduction upon LPS-mediated barrier disruption in endothelial cells. The here described system will in the future allow for the measurement of force across VE-Cadherin as well as Vinculin in vivo using MPLS-microscopy allowing for a better understanding of force transduction in endothelial cells in different disease states.