Daniel Seeler (1,2,3), Nastasja Grdseloff (2), Claudia Jasmin Rödel (2), Charlotte Kloft (4, 3), Salim Abdelilah-Seyfried (2), Wilhelm Huisinga (1, 3)
(1) Institute of Mathematics, University of Potsdam, Germany, (2) Institute of Biology, University of Potsdam, Germany, (3) Graduate Research Training Program PharMetrX: Pharmacometrics & Computational Disease Modelling, (4) Clinical Pharmacy & Biochemistry, Freie Universität Berlin
Objectives: Cerebral cavernous malformations (CCMs) are dilated, blood-filled lesions primarily found in capillaries and small veins of the brain vasculature caused by a biallelic loss of either protein CCM1/2/3 [1]. These lesions are prone to bleed and can therefore result in hemorrhagic stroke. Currently, no drug therapy is approved for the treatment of CCMs [6]. A loss of either CCM1/2/3 alters the way endothelial cells sense fluid shear stress created by the blood flow. This leads to aberrant downstream signaling and, as a consequence, changes in cellular morphology. Cellular morphology also affects the manner in which endothelial cells experience blood flow. Due to their transparency, zebrafish embryos are a common model organism for CCMs allowing for easy visualization of vessel structures. Furthermore, since they are able to survive for several days without cardiac contractility, it is possible to examine the direct effect of blood flow.
Our objectives were to (1) identify molecular components critical to develop a mathematical model including genes essential for blood flow sensing and flow-sensitive genes interacting with CCM proteins and (2) develop a model describing the morphology of endothelial cells in wild-type zebrafish during blood vessel development. Further, we wanted to integrate the cellular morphologies into a multicellular vessel segment.
Methods: We performed extensive literature research to identify molecular components involved in both flow sensing and CCM-loss phenotypes in HUVEC (human umbilical vein endothelial cells), mice and zebrafish models for CCM disease.
We used transgenic PECAM1-EGFP as a junctional marker to visualize endothelial cells of the dorsal aorta in wild-type zebrafish embryos at 48hpf (hours post fertilization) and 72hpf using 3D confocal microscopy. The endothelial cell junctions were used to reconstruct the vessel surface by fitting cylinders with varying radii to segments of the dorsal aorta to minimize the distance of the data points. At the same time we penalized a change of vessel diameter. We then calculated endothelial cell surface area, perimeter and circularity from the resultant curved vessel surface.
Results: Endothelial cells sense flow via a complex of PECAM-1, VE-cadherin and VEGFR2/3. Possible upstream mediators of this complex are GPCRs, stretch-activated ion channels and the glycocalyx [4,5]. According to the fluid shear stress set point model, endothelial cells evaluate their experienced level of fluid shear stress by comparison with an internal set point encoded by the level of VEGFR3. Fluid shear stress below or above the set point results in vessel diameter changes [3].
KLF2 is the central flow-sensitive gene, activated by MEKK3 and physiologically inhibiting VEGFR-mediated angiogenesis and TGF-β signaling, activating anti-inflammatory pathways [2]. In CCM-loss phenotypes however, MEKK3 is no longer bound by the CCM complex and the resulting increased level of KLF2 acts pro-angiogenic via EGFL7 resulting in aberrant vessel growth and dilation [7, 9]. Highly pulsatile blood flow restores vasoprotective pathways parallel to KLF2 and thereby prevents the formation of CCM lesions in arteries [8]. CCM pathology is further mediated by increased RhoA signaling which causes leakage between endothelial cells [9].
Reconstructing the vessel surface of our in vivo data, we found a decrease in cell circularity, an increase in cell elongation and a decrease in vessel diameter between 48hpf and 72hpf. We observed no evidence of cell division in the vessel segment.
Taken together, we envision a multi-scale model of multiple endothelial cells that locally determine the vessel diameter. Each cell is characterized by its shape and the state of its inner biochemical reaction network. The input into the signaling network is the activity level of the mechanosensory complex, a function of local deviation of fluid shear stress from the cell’s set point. Changes in actin and myosin conformation are outputs of the network and then translated into a change in cell shape. Vessel surface and local diameters are updated when cell shapes change.
Conclusions: The determined molecular components allow us to develop a cellular signaling network model for the endothelial response to blood flow. Our aim is to apply the proposed multi-scale model structure to explain the changes in cell morphology we observed over the time period between 48hpf and 72hpf.
References: [1] Akers, A. L.; Johnson, E.; Steinberg, G. K.; Zabramski, J. M. & Marchuk, D. A., Biallelic somatic and germline mutations in cerebral cavernous malformations (CCMs): evidence for a two-hit mechanism of CCM pathogenesis., Human Molecular Genetics, 2009, 18, 919-930
[2] Atkins, G. B. & Jain, M. K., Role of Krüppel-like transcription factors in endothelial biology. , Circulation Research, 2007, 100, 1686-1695
[3] Baeyens, N.; Nicoli, S.; Coon, B. G.; Ross, T. D.; Van den Dries, K.; Han, J.; Lauridsen, H. M.; Mejean, C. O.; Eichmann, A.; Thomas, J.-L.; Humphrey, J. D. & Schwartz, M. A., Vascular remodeling is governed by a VEGFR3-dependent fluid shear stress set point., eLife, 2015, 4
[4] Baeyens, N. & Schwartz, M. A., Biomechanics of vascular mechanosensation and remodeling., Molecular Biology of the Cell, 2016, 27, 7-11
[5] Davies, P. F., Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology., Nature Clinical Practice. Cardiovascular medicine, 2009, 6, 16-26 [6] Li, D. Y. & Whitehead, K. J., Evaluating strategies for the treatment of cerebral cavernous malformations., Stroke, 2010, 41, S92-S94
[7] Renz, M.; Otten, C.; Faurobert, E.; Rudolph, F.; Zhu, Y.; Boulday, G.; Duchene, J.; Mickoleit, M.; Dietrich, A.-C.; Ramspacher, C.; Steed, E.; Manet-Dupé, S.; Benz, A.; Hassel, D.; Vermot, J.; Huisken, J.; Tournier-Lasserve, E.; Felbor, U.; Sure, U.; Albiges-Rizo, C. & Abdelilah-Seyfried, S., Regulation of β1 integrin-Klf2-mediated angiogenesis by CCM proteins., Developmental Cell, 2015, 32, 181-190
[8] Rödel, C. J.; Otten, C.; Donat, S.; Lourenço, M.; Fischer, D.; Kuropka, B.; Paolini, A.; Freund, C. & Abdelilah-Seyfried, S., Blood Flow Suppresses Vascular Anomalies in a Zebrafish Model of Cerebral Cavernous Malformations., Circulation research, 2019, 125, e43-e54
[9] Zhou, Z.; Tang, A. T.; Wong, W.-Y.; Bamezai, S.; Goddard, L. M.; Shenkar, R.; Zhou, S.; Yang, J.; Wright, A. C.; Foley, M.; Arthur, J. S. C.; Whitehead, K. J.; Awad, I. A.; Li, D. Y.; Zheng, X. & Kahn, M. L., Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling., Nature, 2016, 532, 122-126
Reference: PAGE () Abstr 9542 [www.page-meeting.org/?abstract=9542]
Poster: Drug/Disease Modelling - Other Topics