In Vitro Angiogenic Sprout Forces: Microscopically Mapped 4D Matrix Displacements Coupled to Actomyosin-based Force Generation of Endothelial Sprouts

Angiogenesis - the formation of new blood vessels from the pre-existing vasculature - occurs in both physiological and pathological conditions and is a hallmark of a wide range of diseases. During sprouting angiogenesis, invading endothelial cells exert cellular traction forces at cell-cell and cell-matrix interaction sites. While leader cells pulling on follower cells, follower cells pushing on leader cells, or both, have previously been postulated as mechanical forces that underlie the sprout outgrowth, forces during in vitro sprouting have never been measured directly. Deciphering the forces during this angiogenic sprouting - a process that is highly actomyosin-dependent - is at the heart of understanding sprouting angiogenesis. Within the scope of this study, first an in vitro model of angiogenesis is selected. With this basic model, the respective roles of actin and myosin for sprouting angiogenesis are investigated by targeting the actomyosin force generation of endothelial sprouts with small molecule inhibitors. Next, the in vitro model is extended to relate actomyosin-dependent cellular tractions to deformations of the surrounding matrix by means of 4D Displacement Microscopy. 4D Displacement Microscopy combines fluorescence microscopy with image registration algorithms to express cell-matrix mechanical interactions in terms of matrix deformation-based metrics. The model extensions include, amongst others: (1) Microscopically mapping matrix displacements around in vitro sprouts in 4D (in x, y, z and time), (2) inducing a stress-free reference state of the matrix by chemical relaxation of the cellular traction forces, and (3) calculating absolute displacements from the microscopy data with image registration algorithms. Microscopically mapped matrix displacements are here irrefutably coupled to the cellular traction forces of endothelial sprouts growing within the matrix. Tackling the core questions of the dissertation, 4D Displacement Microscopy is then applied to spatio-temporally analyse displacement field patterns around in vitro sprouts. Patterns are examined in relation to sprout morphology and dynamics; which are possible predictors of matrix displacement magnitudes. Recurrent patterns unravel how in vitro endothelial sprouts are mechanically interacting with the matrix, and shed light on the pulling versus pushing nature of forces underlying sprout protrusion dynamics of endothelial sprouts. Further analysis of local displacement fields around retracting and extending sprout protrusions confirms the expected predominant role of sprout pulling instead of sprout pushing forces. Ultimately, mathematically calculating the magnitudes of these sprout forces - for which matrix mechanical properties are needed - will lead to an even deeper understanding of sprouting angiogenesis by allowing comparative studies of hydrogels with different mechanical properties. Therefore, the 4D Displacement Microscopy analysis is taken a step further and as a proof of concept, cellular traction forces around in vitro angiogenic sprouts are estimated for the first time. Finally, modifications of the sample design allow conducting 4D Displacement Microscopy with Selective Plane Illumination microscopy (SPIM) instead of with Confocal Laser Scanning Microscopy (CLSM). SPIM-based displacement microscopy is promising for investigating fast processes of in vitro angiogenesis - such as protrusion dynamics - as it allows imaging multiple sprouts simultaneously at high temporal resolution, while maintaining the subcellular resolution required for the registration algorithms. In summary, this research highlights the key role of actomyosin-based traction forces for in vitro sprouting angiogenesis, it couples microscopically mapped matrix displacements to the cellular traction forces of endothelial sprouts, and it contributes to deciphering how in vitro endothelial sprouts mechanically and reciprocally interact with their micro-environment. 4D Displacement Microscopy (both with CLSM and SPIM) is shown to provide a quantitative and mechanically sound approach to advance the knowledge in the field, and forces around in vitro sprouts are reported for the first time.

Jaar van publicatie:2020

Toegankelijkheid:Open