Systems-Mechanobiology of Endothelial Gap Dynamics

The vasculature is a complex system, critical to the functioning of higher-level organisms. It is composed of large vessels that branch into smaller and smaller vessels. On the smallest scale, the microvasculature consists of arterioles, venules and capillaries. Here, oxygen and nutrients are exchanged between the vessels and the tissue. Also, immune or cancer cells can transmigrate through gaps within the blood vessels into the surrounding tissues. For the immune system, this is a critical function, as immune cells need to reach sites of infection. However, high levels of transmigration may also contribute to chronic inflammation, and cancer cells transmigrate the blood vessels during metastasis. Therefore, a tight regulation of the blood vessel gaps is critical during homeostasis, and de-regulation of gaps may contribute to diseases.

Our preliminary mathematical modelling/in vitro experimental work revealed that a balance of intracellular forces in the endothelial cells, the cells that line the blood vessels, regulates the formation of gaps in between the cells. We found that these gaps occur most frequently at the vertex points between three endothelial cells, and may appear autonomously, in absence of transmigrating cells. This finding complemented earlier studies that uncovered a critical role of inflammatory signals, released by transmigrating cells, in the regulation of endothelial cells. We further showed that transmigrating cells may exploit these autonomously forming gaps by migrating towards the gaps, where they cross the endothelium. Therefore, studying the dynamic nature of the endothelium, and the resulting formation of gaps, is critical to understand the physiologically important processes of immune and cancer transmigration.

In vivo, the dynamics of the microvasculature is influenced by several further biophysical properties not present in most in vitro assays. Notably, blood flow in the vessels, interactions of endothelial cells with the surrounding extracellular matrix, and the complex geometry and topology of the microvasculature, have all been found to influence endothelial dynamics individually. In vivo these properties exist simultaneously. Systems biology models are typically employed to study cellular decision making in response to multiple stimuli. However, current systems biology models are focused on the study of multiple molecular stimuli, e.g. inflammatory cytokines, but cannot capture biophysical stimuli. Therefore, there is an urgent need to incorporate the effect of multiple biophysical stimuli into systems biology models.

In this project, we are developing an integrative modelling/experimental approach that incorporates multiple physiological biophysical properties into both mathematical models and in vitro assays. Our approach will advance models and experiments iteratively together to gain unprecedented insights into the dynamic nature of the endothelial microvasculature. The outcome will be a versatile mathematical modelling platform to study the dynamics of the microvasculature in homeostasis, and will underpin future work on the contribution of endothelial dynamics to diseases. Moreover, we will advance our recently developed engineered in vitro assays that can generate stable, perfused 3D microvasculature in complex extracellular matrices, and that is therefore ideally suited to validate our mathematical modelling predictions. The combined modelling/experimental system will be used to test several specific biological hypotheses on the complex role of major contributors to endothelial dynamics and gap formation.

Building on methodology that we recently developed to generate the preliminary data, we will iteratively build up the complexity in both of our mathematical and experimental model systems. We will first extend our recently published model of a 2D endothelial monolayer on glass to incorporate effects of realistic soft extracellular matrices. This model is a mechano-chemical model where cells and matrix are represented by a network of springs that are connected through molecular adhesion complexes (e.g. focal adhesions or adherens junctions). Crucially, binding and unbinding of adhesions depend on the forces that act on them. Next, we will extend our model into 3D to test how forces and resulting adhesions and gaps are altered in 3D. We will also develop a systems-biology model based on ordinary differential equations describing the evolution of molecular concentrations. Multiple biophysical properties (e.g. flow, extracellular matrix stiffness and forces from neighbouring cells) are inputs that affect mechanosensors in the model (e.g. Talin, FAK) and are then integrated through the RhoA and YAP/TAZ signalling pathways. To investigate how shear stresses stimulate pathway components, we will perform simulations of fluid flow in microvascular networks.

Iteratively and alongside the mathematical model, we will build up complexity in our advanced engineered microvascular in vitro assays. Initially we will focus on a 2D endothelial monolayer formed on soft extracellular matrices, where traction force microscopy will reveal how endothelial gap formation depends on forces and matrix properties. Next, we will employ our recently developed microfluidic assay of a 3D microvasculature to study effects of topology and flow on gap dynamics. For all objectives, inhibitor or overexpression studies will reveal how signalling pathways integrate multiple biophysical stimuli to regulate endothelial dynamics and gap formation.

PUBLIC
We will engage with the general public through school visits, public lectures and pub/cafe visits. Specifically, we will engage with high school students interested in mathematical and physical sciences to demonstrate that these sciences can successfully address important biological problems. Likewise, we will engage with students interested in biology to showcase how close interactions with mathematics, physics and engineering can advance biology. The aim is to inspire the next generation of students and the general public to take a broader view of science; appreciating that interdisciplinary collaborations can synergistically advance biological, mathematical and physical sciences. Moreover, we will inform the general public that our interdisciplinary biological methods (e.g. mathematical modelling and engineered in vitro assays) will, in the long term, crucially address important health problems through medical research that will adapt our methodology.

INTERDISCIPLINARY EDUCATION
Through this project, we provide direct training to two postdoctoral scientists; one from experimental biosciences and one from the mathematical modelling sciences. Likewise, a PhD student based at UoB will join this project, working at the interface of modelling and experiments. Crucially, these researchers will not only be taught cutting edge mathematical methods or experimental assays to achieve their specific objectives. Each of them will be fully immersed into our interdisciplinary research environment, where modellers and experimentalists learn from each other and iteratively advance their respective methodologies. Such interdisciplinary skills are widely sought after not only in various academic disciplines, but also by industry including pharmaceutical, biotech or chemical industries.

PHARMACEUTICAL INDUSTRY
A vast majority of drugs rely on the vasculature to deliver bioactive ingredients to the target tissues. Quantitative systems-pharmacology models employed by the industry take the vasculature into account very broadly through compartmentalization; yet specific knowledge of microvascular dynamics and organ-specific permeability in dependence on mechanics is lacking. We will involve a wide range of industries through workshops, study groups or joint PhD projects, therefore assisting the industry in advancing their pharmacological models.

HEALTH APPLICATIONS
Endothelial dynamics play a crucial role during development, progression and treatment of many diseases (e.g. cardiovascular or neurodegenerative diseases and cancer). The models arising from this project will therefore significantly contribute towards a much better understanding of the bioscience underpinning health. Specifically, the diseases affected by altered endothelial dynamics are typically associated with people of high age; this research therefore will contribute to the BBSRC priority area "Healthy aging across the life course". Moreover, physical parameters such as blood flow speed or vascular stiffness can change with age. Yet, there is a lack of computational and experimental tools to understand how these physical alterations are driving changes in the gap formation of the endothelium, and consequently, immune or cancer cell extravasation or transport of nutrients. Our project provides new insights into the systems-mechanobiology of the endothelium, and therefore underpins future research aimed at overcoming the adverse effects of mechanics on age-related diseases, including crucially, chronic inflammation, cardiovascular diseases and cancer.

While our research will primarily deliver fundamental biological insights into vascular dynamics, we will engage with clinicians and drug manufacturers that, for example, work on cardiovascular diseases, cancer immunotherapies or diabetes. We will work with these communities to help them to utilise our mathematical and in vitro models to target important diseases associated with high age.