Periodic Reporting for period 4 - ForceMorph (The integration of cell signalling and mechanical forces in vascular morphology )
Reporting period: 2022-09-01 to 2023-08-31
Cardiovascular disease is a main cause of death worldwide. Vascular health is controlled by the mechanical stimulus of blood flow and signalling 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 signalling 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 are multi-layered structures with 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 healthy vessels and a synthetic form present predominantly in arteries actively remodelling their structure in response to injury, inflammation, or abnormal mechanical forces. A main regulator of the switch between these cell forms is the Notch signalling pathway consisting of cell surface proteins which upon activation move to the cell nucleus to regulate target genes. Notch has a major role in both development and maintenance of vascular tissue. Accordingly, manipulation of this signalling 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. This has been the main objective of the project together with development new non-animal tools for vascular studies, including an Artery-on-Chip device and computational models of human blood vessels.
Arteries are multi-layered structures with 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 healthy vessels and a synthetic form present predominantly in arteries actively remodelling their structure in response to injury, inflammation, or abnormal mechanical forces. A main regulator of the switch between these cell forms is the Notch signalling pathway consisting of cell surface proteins which upon activation move to the cell nucleus to regulate target genes. Notch has a major role in both development and maintenance of vascular tissue. Accordingly, manipulation of this signalling 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. This has been the main objective of the project together with development new non-animal tools for vascular studies, including an Artery-on-Chip device and computational models of human blood vessels.
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.
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.
We have successfully created an Artery-on-Chip device as well as two other platforms allowing vascular studies on human cells, developed four computational models mimicking human arteries, identified vimentin as a mediator of the dynamic structural changes seen in blood vessels, and characterized the effects of mechanical stress on Jagged1-related Notch signalling. Importantly, the new devices and computational models presented can be used to replicate human physiology and therefore help decrease the need for animal experimentation in biomedical research. We have presented our work in dozens of events and publications for both scientific audiences and the public and compiled numerous review articles on different aspects of vascular research. Overall, we have successfully combined engineering, computational approaches, cell biology techniques and animal models to create novel tools for research and drug development purposes and to reveal new molecular details of how vascular tissues adapt to abnormal mechanical stress related to vascular disease.