1. Computational Modelling of Epithelial to Mesenchymal Transition Tariq Abdulla1, Lucile Houyel2, Ryan Imms1, Jean-Marc Schleich3, Ron Summers1 1.Dept Electronic & Electrical Engineering, Loughborough University, UK 2. Hospital Marie Lannelongue, Paris, France 3. LTSI, University of Rennes 1, France T.Abdulla@lboro.ac.uk http://www-staff.lboro.ac.uk/~lsrs1 Compucell3D & SBW Workshop 2011
2. Outline Introduction Heart Development – what happens? Anatomy, Tissue, Cell, Protein Current Simulations In-vitro EMT, Mitosis Future Directions
5. F. Bajolle, S. Zaffran and D. Bonnet, Genetics and embryological mechanisms of congenital heart diseases Archives of Cardiovascular Diseases,Volume 102, Issue 1, January 2009, Pages 59-63
14. In vitro EMT Wildtype Notch1 BMP2 L. Luna-zurita et al. “Integration of a Notch-dependent mesenchymal gene program and Bmp2-driven cell invasiveness regulates murine cardiac valve formation,” The Journal of Clinical Investigation, vol. 120, 2010.
For more information, visit: http://www-staff.lboro.ac.uk/~elta2/index.htm
The team is formed from a collaboration between Loughborough University in the UK and Universite de Rennes 1.By bringing together engineers with modelling skills with clinical and imaging experts, we aim to unravel some of the mysteries of heart formation.(click)We illustrate some of the work done at each university, with the Loughborough team focussing on modelling. At the top is a protein level model. In the middle a cell level simulation. They are particularly interested in the linking of models at different levels of scale. The bottom figure shows their use of “ontologies” to annotate model parameters – which helps with linking between scales.(click)In Rennes we are conducting CT acquisition and 3D reconstruction of heart specimens with tetralogy of Fallot. We aim to better understand the spatial arrangement of heart structures in this disease, and to improve understanding of the embryological causes.
The motivation for looking in more detail at heart development is to understand how congenital heart defects occur.Many different mechanisms can interact and contribute towards several different types of congenital heart defect.To really understand the risk factors behind CHD, and provide early warning, it is not sufficient just to identify genes associated with CHD. We need to understand how each of these processes work.(click)Each of these processes are complex in their own right – and the work presented here focuses only on the EMT.
1) At an early stage the heart is a tube. It has no valves but is somehow pumping blood in one direction, perhaps using impedance pumping. In the third and fourth week of human development, heart looping takes place, and we end up with the four chambered double pump. Let’s look at this in more detail.2) One of the key things that happens is heart looping and wedging. There’s a lot of jargon here, so let’s break it down a little bit. The long thing, which is looping behind is known as the Outflow Tract (OFT) or conotruncus. You can divide it into two parts the conus and the truncus. Now while it is doing this looping, the Atrioventricular canal is septating (this means a wall is growing to divide it). Also, a septum is growing in the conotruncus. Because the conus is looping, the shape of this septum is spiral. When the OFT wedges into the AV canal, in normal development the conal septum lines up with the AV septum. This allows blood to flow from right ventricle to pulmonary artery (to the lungs) and from left ventricle to the aorta and around the body.3) In this bottom image we see two Scanning Electron Micrograph (SEM) images of mouse hearts during looping. The first one has looped in the correct direction – clockwise, while the second one has looped in the direction – anticlockwise. In this case, we have genetically induced situsinversus – where the organs develop on the opposite side of the body. Images: [1] http://www.helmholtz-muenchen.de/en/ieg/group-functional-genetics/deltanotch-pathway/index.html[2] Kirby et al. Cardiac Development[3] J. Schleich, C. Almange, J. Dillenseger, and J. Coatrieux, "Understanding normal cardiac development using animated models," IEEE Computer Graphics and Applications, vol. 22, 2002, pp. 14-19.
This slide shows what is happening in the heart between embryonic day 24 and embryonic day 32.Much of the inner structure of the heart is the result of the growth of endocardial cushions.These grow in the Outflow tract and the Atrioventricular canal by EMT. In both locations, they fuse by Day 32.Because the Outflow tract rotates, the fusing cushions form a helical shaped septum, which divides the aorta from the pulmonary artery.In the AV canal. Upper and lower cushions fuse to form the AV septum. They also contribute flaps to the Mitral and Tricuspid valves, as shown.
At the tissue level, the embryonic heart is composed of two layers – outer myocardium (heart muscle) inner endocardium (an epithilium). These are seperated by an extracellular matrix termed cardiac jelly. In the
At the cell level, we need to consider changes in cell type – or state. Notch signalling creates a feedback loop whereby cells either highly express notch or a ligand for notch (eg delta). So one state is whether they are primary or secondary. In the myocardium notch has a secondary effect on expression of VEGF. We might have cell level states like the level of adhesiveness between different types of cell. These can be preferential, so you may need to specify every possible combination. Here we are looking at the same thing, but closer up. We can see the intracellular signalling that takes place within a single cell.So to properly understand these processes, we really do need to start linking between the scales. Only making a model of what the cells do, doesn’t really explain why they do it. Likewise, a protein interaction model of the pathways involved, doesn’t tell you a lot about EMT on its own, because In the Endocardium... Notch signalling has pre-sepecified cells that may undergo EMT. In combination with BMP and TGF-beta signalling from the myocardium, this increases the expression of Snail protein, which in turn reduces the expression of VE-Cadherin. This causes endocardial cells to lose their adhesiveness and undergo EMT, which leads to growth of the cushions.We will return to this later, when I talk about multiscale modelling.
Without going into too much detail, part of the explanation for this is that Notch is downstream of BMP2, as illustrated here.(click)So the endocardial cells also lose their adhesion to one another, but something else also happens to induce them to invade the collagen gel. The hypothesis tested in these initial simulations is that this could be due to a simultaneous increase in the adhesion between the endocardial cells and the collagen gel / cadiac jelly.
We apply this framework for multiscale modelling to heart development – which is illustrated here for endocardial cushion growth. We are interested in processes at the nanometer scale (protein interaction) which take place within each cell, as well as the cell behaviour and tissue interaction at the micrometer scale, and the overall development of the heart at the milimeter scale.There are different modelling approaches applicable to each level of scale, and for this initial work, we are really interested in the middle level (cell-tissue level) (click)
We base our simulations on this in vitro study – which induced EMT in mouse endocardial cells. They cells were taken from mouse ventricles, so are not disposed to undertake EMT. In their wildtype state, they remain in a monolayer floating on the collagen gel.By the introduction of Notch protein, the endocardial cells lose their adhesion as expected. However this is not sufficient to induce a full EMT – instead cells only scatter on the surface of the collagen gel, without invading it.(click)With the introduction of BMP2 – a protein which is secreted by the myocardium in the cushion forming regions – cells both scatter on the surface of the gel, and invade into it.(click)
So our simulations use a 3D lattice, at the bottom you can see a cross section. In the base case, our simulated endocardial cells float on the collagen gel in a monolayer.
By increasing the surface energy parameter between endocardial cells (e.g. Reducing their cohesion) we have a 2D scattering on the surface of the simulated collage gel.
By simulating a simultaneous reduction in endocardial – to – endocardial adhesion, and an increase in endocardial – collagen gel adhesion, we observe fully invasive behavior. This supports the hypothesis that the mesenchymal phenotype is achieved in part by an increase in adhesion to the extracellular matrix.
VEGF regulates the level of endocardial mitosis. Higher VEGF = more mitosis(click)But the level of VEGF expression is lower in the cushion forming regions.(click)If the level of VEGF is too high, there will be a reduction in EMT – with consequently malformed endocardial cushions.
The hypothesis investigated here (in an abstract way) is this:>Endocardial cells have strong adhesion to each other>With a high rate of mitosis, there is no possibility for small gaps between endocardial cells.>It this factor – no small gaps – that prevents cells from migrating into the ECM.This was simulated in an abstract way. We used the same parameters as the previous simulations. On the left we use the parameters for 2D separation, on the right the parameters for 3D separation . We use contact inhibited mitosis, which means that cells divide when the they have less than a certain percentage of their surface area adjacent to other cells. The inclusion of mitosis preserves the monolayer, and this action could plausibly inhibit EMT, and explain why a higher level of VEGF prevents EMT from happening.
