Hendrik Cuylaerts

Endothelial cells lining blood vessels constantly interact with their extracellular environment. While the chemical interactions have been extensively studied, there is a whole range of physical signals underexplored (e.g. stiffness of the ECM and shear stress from the blood flow) which elicit biological reactions. Unsurprisingly, these mechanical signals are altered in vascular diseases such as cerebral cavernous malformations (CCM), which this project will focus on. In CCM disease stacks of overgrown, dilated and hemorrhagic venous capillaries are formed by a unique layer of poorly joined ECs in the brain. This genetic disease affects 1 in 200 persons worldwide of which half are symptomatic (seizures, hemorrhage and neurological deficits). Studies into CCM have provided evidence that the endothelial cell behavior is affected by external physical forces. To mimic the extracellular CCM microenvironment, we will combine microfluidics with different hydrogels and fluorescence probes to systematic evaluate the role of mechanical and biochemical cues in the balance between cell-cell and cell-matrix interactions. The fluorescence probes are force sensitive and are called FRET-based molecular tension sensors. The critical element in molecular force sensors is the stress sensitive module, placed between the FRET donor and acceptor molecules. These sensing modules will deform and extend in length upon application of a physiological force and can be placed within relevant structural proteins, targeting the sensor to specific intracellular structures. To calculate the distance between donor and acceptor, and therefore the applied force, we perform fluorescence lifetime FRET (FRET-FLIM) where the fluorescence lifetime of the donor is recorded. The resulting data will be analyzed using phasor-FLIM, fit-free analysis is facilitated leading to more accurate and unbiased results to extract FRET efficiency. Using this, FRET-based molecular tension sensors linked to structural proteins in cell-cell and cell-ECM interactions, exposed to a range of extrinsic mechanical factors in the form of hydrogels and microfluidic chambers, further paves the way to provide a molecular force connectivity map of the vascular mechanotransduction pathways. This in turn will help us to not only better understand all factors involved in vascular diseases, but also lead to more comprehensive treatments.