We have developed three novel tools for experimentation on isolated vascular cells: an Artery-on-Chip, a Dish-in-a-Dish, and a chip with patterns of Notch signalling molecules guiding blood vessel formation. In the Artery-on-Chip, endothelial cells and vascular smooth muscle cells are cultured on opposite sides of a porous membrane, enabling the cells to be in contact and communicate with each other but preventing the two cell layers from mixing. Fluid flow over the endothelial cell layer and stretching of the cell compartments mimic the mechanical forces experienced by cells that form blood vessels. The device is transparent to allow live imaging of the cell layers under a microscope. Cells inside the chip can also be treated with therapeutic substances, stained to detect specific molecules or structures, or collected for subsequent analysis.
The Dish-in-a-Dish is a platform designed for easy implementation in any bioscience laboratory for shear stress studies on cultured cells. Like traditional systems, it is based on rotation of cell culture plates with fluid flow exerting mechanical stress on the cells. However, it uniquely allows users to treat cells with a specific level of consistent shear and therefore greatly promotes the comparison of different stress conditions. In the Notch-patterned chip for blood vessel growth studies, alternative Notch signalling molecules are printed onto a glass plate as parallel lines. Endothelial cells are placed into a channel perpendicular to the printed lines, and finally both are covered with a gel. The cells invade and break down the gel to form blood vessel-like structures, whose growth pattern can be monitored and analysed to decipher the effects of each signalling molecule. Importantly, the platform can be used to guide regenerative medicine and tissue engineering approaches, as the formation of functional new blood vessels is a major limiting factor for tissue growth.
We have created four computational models in collaboration with other bioengineering experts to mimic human blood vessels and simulate the effects of Notch signalling and mechanical forces on artery structure. To create an initial model of a 1-dimensional blood vessel, we performed stretch experiments on vascular cells to determine changes in Notch-related gene expression and incorporated these results into an existing computational framework. The model successfully predicted vessel thickness at different arterial locations in individuals from different age groups when given information on the mechanical stress conditions in their vasculature. The subsequent 2-dimensional model builds upon the 1D model by taking a higher number of neighbouring cells into consideration. This is of importance as the Notch pathway relies upon direct cell-cell contacts for activation. The third model can simulate arteries experiencing high blood pressure while the fourth one incorporates data from further experiments with vascular smooth muscle cells and considers the turnover of other structural artery components. This model successfully mimics the changes seen in the vasculature of animal models under high blood pressure and allows simulation of other disease states as well, such as vascular deficiencies caused by Notch mutations. Importantly, the models suggest that artery structure can be maintained or manipulated via introduction of specific Notch signalling modulators.
Additionally, we have shown mechanical forces to control the level, cellular organization, and function of the Notch signalling protein Jagged1, and identified the structural protein vimentin as a mediator of mechanosensitive Notch signalling and blood vessel remodelling. We used genetically modified mice deficient of vimentin to show that this protein regulates vascular smooth muscle cell state via Jagged1, and through computational simulations we were able to predict the specific signalling events involved in the adverse remodelling seen in vimentin-deficient arteries. The vimentin-Jagged1 axis presents a novel regulator of vascular homeostasis under mechanical stress.
