Computational model of brain endothelial cell signaling pathways predicts therapeutic targets for cerebral pathologies
Graphical abstract
Introduction
Many pathologies of the brain are characterized by both devastating daily effects for patients and daunting challenges and limitations to treatment for physicians [1]. These conditions, which include brain tumors, neurodegenerative diseases, and stroke, vary widely in their incidence, molecular mechanisms, and progression, and yet all remain inherently difficult to treat. At least some of the challenge of treatment rests in the nature of the brain structure itself, and more specifically, the blood-brain barrier (BBB). For many years, delivery of therapeutics to the brain required risky and invasive surgery to facilitate direct injections [[2], [3], [4], [5]], or brain-wide disruption of the BBB to allow passage of drugs [6,7]. More recently, focused ultrasound (FUS) in conjunction with intravenously-injected gas-filled microbubbles has been used to facilitate drug delivery to the brain in a spatially-targeted manner. This approach can be used to deliver genes or other therapeutics to a specific structure or region of the brain non-invasively [[8], [9], [10], [11]].
The cerebral microvasculature consists of a continuous, non-fenestrated layer of brain capillary endothelial cells. Brain endothelial cells are held together by tight junctions, which serve as a barrier to paracellular passive diffusion of materials from the systemic circulation into the surrounding brain tissue [12]. The brain endothelium also expresses drug efflux pumps, such as p-glycoprotein, to continuously remove potentially harmful materials from the central nervous system [13]. Thus, in essence, brain endothelial cells are the gatekeepers to the brain. As the gatekeepers, cerebral endothelial cells play a tremendous role in the state of the brain in both physiological and pathological conditions, by dictating the blood flow, oxygenation, nutrient transport, and drug concentrations in the brain parenchyma. Brain endothelial cells experience competing cues from growth factors, neurotrophic factors, and other cytokines and paracrine signaling molecules, and integrate these diverse signals to regulate behaviors such as angiogenesis, immune cell recruitment, and apoptosis. Therefore, appropriate therapeutic strategies to modulate cerebral endothelial cell signaling must function within this complex environment of diverse signaling cues present in the healthy or diseased brain. Designing such therapies relies on understanding how cells integrate these signals, yet to date no computational models of brain endothelial cell signaling exist.
In this study we develop the first computational model of the brain endothelial cell signaling network in order to identify disease context-dependent drivers of therapeutic patterns of molecule expression or phenotypes. The model incorporates the VEGF-A signaling pathway, the BDNF and NGF neurotrophin signaling pathways, and the Wnt signaling pathway. BDNF and NGF can act through either their unprocessed, pro-neurotrophin forms, which preferentially bind sortilin and the p75NTR receptor to activate a number of pro-apoptotic pathways, or can be processed by protein convertases such as furin or plasmin into their mature neurotrophin forms, which preferentially bind the Trk receptors and induce more pro-survival signaling [14]. VEGF-A signaling drives many pro-angiogenic processes [15], and Wnt signaling in the brain endothelium has been implicated in promoting survival and BBB integrity [16]. Additionally, our model features a FUS input node, based upon prior in vivo transcriptomic studies which demonstrated that FUS-mediated BBB opening results in upregulation of cathepsin D in cerebral endothelial cells [17]. The model's outputs were selected for their relevance to brain pathologies of interest. These include the proteins which constitute tight junctions, claudin-5 and occludin, and adherens junctions, VE cadherin, between endothelial cells. Outputs also include expression of the glucose transporter GLUT1 and the efflux transporter p-glycoprotein, as well as nitric oxide production (which helps regulate vasodilation and blood flow), and apoptosis or cell death.
After validating the model at baseline (“normal” physiological conditions) against the existing literature, we simulate the altered signaling network under three disease states (glioma, Alzheimer's disease, and ischemic stroke) and predict the most influential nodes in regulating potential therapeutic responses to those specific pathologies. This analysis reveals novel potential targets (or combinations of targets) for pharmaceutical intervention using FUS as a delivery mechanism. We submit that this model represents a valuable tool to develop strategic therapeutic approaches for FUS-mediated drug and gene delivery in a range of different disease contexts in the brain.
Section snippets
Validation of computational model of brain endothelial cell signaling
A predictive computational model of brain endothelial cell signaling (Fig. 1) was manually reconstructed from previous experimental studies from the literature. The detailed procedure for literature review and network reconstruction is provided in the Materials and Methods section. The list of molecular nodes implemented in the model is provided in Table S1, and full documentation of each reaction and the experimental evidence supporting that reaction is provided in Table S2. We first predicted
Discussion
Here we manually reconstructed a literature-based network of brain endothelial cell signaling that identifies the nodes regulating the expression of BBB tight junctional and adherens junctional proteins, GLUT1, and the P-glycoprotein efflux receptor, as well as nitric oxide release and apoptosis. This network was used to develop a logic-based predictive model of endothelial cell signaling (with or without FUS treatment), which validated at a rate of 73% in comparison to independent, published
Conclusions
We present here, to our knowledge, the first computational model of brain endothelial cell signaling. We have demonstrated its utility in modeling signaling pathways in brain endothelial cells in either normal physiology or a variety of different disease states. In each of these different states, combinations of therapeutically beneficial goals can be used to design treatment scores. Maximization of these treatment scores with either overexpression or inhibition of different nodes in the
Model construction
A brain endothelial cell signaling network was manually reconstructed from previous in vitro and in vivo experimental studies from the published literature. The network integrates four key inputs of interest, chosen due to their role in angiogenesis, cell survival, or neural stem cell recruitment. These include NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), VEGF-A (vascular endothelial growth factor-A), and Wnt. Of these, NGF and BDNF can act in either or both of two forms
Declaration of Competing Interest
None.
Acknowledgements
RJP was supported by National Institutes of Health grants R01EB030409, R01EB030744, R01NS111102, and R21NS118278. JJS was supported by National Institutes of Health grants R01 HL137755 and R01 HL137100.
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