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The integration of cell signalling and mechanical forces in vascular morphology

Periodic Reporting for period 2 - ForceMorph (The integration of cell signalling and mechanical forces in vascular morphology )

Reporting period: 2019-09-01 to 2021-02-28

Cardiovascular disease is the main cause of death worldwide. Artery structure and vascular health is controlled by the mechanical stimulus of blood flow and signaling between cells of the vessel wall. Vascular disease typically involves both abnormal mechanical stress, such as high blood pressure, and defects in artery structure. How exactly mechanical forces and cell signaling come together to determine vessel architecture is not yet well understood and the complexity of arterial cell organization, mechanical cues and biochemical factors pose a challenge for vascular studies.

Arteries comprise two cell types: endothelial cells, which line the lumen of the vessel, and vascular smooth muscle cells, which form a thicker cell layer around the endothelial cells. Vascular smooth muscle cells can exist in different forms, ranging from a contractile type typically present in normal, healthy vessels and a synthetic form present predominantly in arteries actively remodeling their structure in response to injury, inflammation or abnormal mechanical forces. A main regulator of the switch between the different vascular smooth muscle cell types is the Notch signaling pathway. It functions through cell-cell contacts and has a major role in both development and maintenance of vascular tissue. Accordingly, manipulation of this signaling pathway has potential in treatment of arterial diseases and engineering of vascular tissue grafts. To use this potential, we need to understand how mechanical forces in arteries control Notch and how Notch and mechanics together regulate vessel structure and remodeling.

The overall goals of the project are to develop new physiologically relevant tools for vascular studies, to decipher how mechanical cues affect Notch signaling between arterial cell layers and to better understand how signaling and mechanics together control vascular form and function. The novelty of the project is in its wide interdisciplinary approach where newly engineered devices, computational modelling, animal studies and cell biological experiments are combined. The project has also three detailed objectives. Firstly, the creation and use of a novel 3D Artery-on-Chip device mimicking the cell organization and mechanical environment present in a real-life vessel. The second aim is to create computational models able to predict how the combination of signaling and changing mechanical conditions controls vessel remodeling in health and disease. The third objective is to use animal models to understand how signaling perturbations and blood flow dynamics affect artery structure.
We have created and characterized two new tools to for studies on isolated vascular cells: the Artery-on-Chip and a Dish-in-a-Dish. 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 recapitulate the mechanical forces experienced by 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 proteins or collected for subsequent analysis. The advantage of the device is that the different vascular cell types are present in their physiological 3-dimensional organization under strictly controlled hemodynamic conditions and can be monitored in real-time. The Dish-in-a-Dish is a platform designed for easy implementation in any bioscience laboratory for shear stress studies on cultured cells. In the traditional setting, cells growing on a plastic dish are rotated, causing the fluid above to exert shear on the cells. However, cells at different areas of the plate experience very different levels of shear, creating a mixed population of cells and greatly affecting analysis results. The Dish-in-a-Dish contains a small inner dish mounted within a larger dish, to produce a consistent level of shear across a cell layer. Optimal plate sizes, orbital speed and fluid height were determined through computational simulations and characterized experimentally.

We have generated both a 1D and a 2D computational model of mechanosensitive Notch signaling in the artery wall. The models predict the fate of vascular smooth muscle cells in a vessel wall of increasing thickness while taking into account Notch signaling changes induced by mechanical cues. To create the 1D model, 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 revealed a switch-type behavior where at a certain wall thickness smooth muscle cells transition from an actively dividing synthetic form to a contractile form, guided by Notch signaling status. Importantly, 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 the vasculature of these individuals. The subsequent 2D model builds upon the 1D model by taking a higher number of neighboring cells into consideration. This is of importance as the Notch pathway relies upon direct cell-cell contacts for activation.

Additionally, we have combined animal work, cell biological laboratory experiments and computational modeling to identify vimentin, an important structural component of vascular cells, as a mediator of mechanosensitive Notch signaling and arterial remodeling. We used genetically modified mice deficient of vimentin to show that this protein regulates vascular smooth muscle cell state via Notch signaling and is involved in controlling vascular remodeling in response to increased mechanical stress. Using computational simulations, we were able to predict the specific Notch signaling events involved in adverse remodeling of vimentin-deficient arteries. We have also shown the central Notch signaling component Jagged to be sensitive to mechanical changes.
We will use our newly developed tools to further study mechanosensitive signaling in vascular cells. Genetically modified cells, cells isolated from mice exhibiting vascular abnormalities or Notch perturbations as well as mechanical stress conditions related to disease states will be experimented on with the Artery-on-Chip and Dish-in-a-Dish. The computational model arteries will be refined further to produce a 3D model of mechanosensitive Notch controlling vascular remodeling. We are currently using zebrafish embryos as an animal model to study the effects of Jagged and vimentin perturbations on cardiovascular development. We are also looking into how endocytosis, the uptake of extracellular molecules into cells, is controlled by mechanical stress in endothelial cells and how these cells themselves exert mechanical forces onto their surroundings, especially during the growth of new blood vessels. Overall, we expect this multifaceted approach to provide a much clearer picture of the signaling events mediating structural changes of the vasculature in response to mechanical cues.