A. Dastmaltschi(1), C. InYeop Kim(1), T. Bozoglu(1), T. Ziegler(1), V. Rajendran(1), K.-L. Laugwitz(1), T. Ziegler(1), C. Kupatt(1)

1: Klinik und Poliklinik für Innere Medizin I, Klinikum rechts der Isar der Technischen Universität München, München

Introduction:

Endothelial damage, a key factor in cardiovascular disease, is heavily influenced by blood pressure and vascular tone. While in vitro methods exist to measure forces on endothelial cells, in vivo measurements remain challenging. This project utilizes FRET between the fluorophores mTFP1 and Venus, linked by a flagelliform peptide that stretches under force, reducing FRET efficiency. By integrating this sensor into Cadherin-5 and Vinculin, we can measure forces between endothelial cells as well as between cells and the extracellular matrix. To enable in vivo application, we use a dual rAAV strategy, splitting the constructs for reconstitution via mRNA trans splicing (REVeRT) in target cells.

Methods:

To compare REVeRT with split intein-based protein trans splicing, we developed a split luciferase assay and measured bioluminescence after transient transfection in HEK293T cells. Tension sensor constructs for Cadherin-5 and Vinculin were split and modified with splice elements, producing vector pairs (5′ and 3′) for in-cell reconstitution. A tail-less control containing only the C-terminal tension sensor module was created as a zero-force reference.

Results:

Luciferase assays showed reconstitution by both REVeRT and split inteins. Due to REVeRT’s versatility, we selected it for further use. RT-PCR confirmed mRNA-level reconstitution, and imaging analysis verified full-length sensor expression and FRET signals. Ongoing work will explore endothelial force dynamics in vivo following pathological stimuli in mice.