Role of NFAT in the vascular endothelium

The calcineurin-NFAT signaling pathway is involved in many important processes in our bodies. In particular, we know a lot about the crucial role this pathway plays in the immune system, where it switches on and off many key genes. However, less is known about what it does in other parts of the body. I am interested in understanding the control of blood vessel formation. This work focuses on endothelial cells, which form the inner lining of all vessels and are the first part of blood vessels to form. When NFAT signaling is inhibited, blood vessels do not form correctly. We recently examined mouse embryos that had lost NFAT signaling in the endothelium, and found that they have healthy arteries but defective veins. Arteries transport oxygen-rich blood to capillaries, while veins return the blood to the heart. Although vein endothelial cells are already known to be very different from their arterial counterparts, we do not understand how vein formation and growth is regulated. Based on the observations that veins were not formed correctly in NFAT mutant embryos, we looked at the binding pattern of the NFAT protein NFATc1. This revealed that NFATc1 binds directly to the enhancers (on-off switches) of many genes that are required for vein formation, suggesting that NFAT signaling may directly switch these on. In the project, we will analyse mouse and zebrafish embryos with defective NFAT signaling to establish why veins do not form properly. We will also investigate the enhancers that bind NFATc1 to both understand which genes are directly bound and activated by NFAT, and to find the other proteins which help NFAT function. Lastly, we will also find out how NFATc1 is itself able to switch on specifically in vein endothelial cells.

Calcineurin-NFAT signaling plays a crucial role in the regulation of many cellular processes. In the immune system, where it is particularly well studied, it plays a key role in the transcriptional regulation of many cytokines, chemokines and growth factors. However, the role of this pathway in the vasculature is still not fully delineated, although it has been implicated in the maturation of coronary valves and lymphatic vessels. We recently re-examined the consequences of loss of NFAT signaling in the endothelium. This revealed a previously undetected defect in venous development in these mice. Although vein endothelial cells are molecularly and functionally distinct from their arterial and lymphatic counterparts, the signalling and transcriptional pathways controlling of venous differentiation are not well characterized. Analysis of publicly available NFATc1 ChIP-seq data in vein endothelial cells demonstrated direct NFATc1 binding to enhancer regions of numerous key venous differentiation genes, suggesting that NFAT factors may directly regulation venous identity. In the project, we will use a combination of mutational analysis and enhancer characterization in zebrafish, mouse and in vitro models to determine the exact role of calcineurin-NFAT signaling in venous development, identify the direct target and partner proteins of NFATs factors, and establish the upstream regulators controlling vein-specific expression of this transcription factor family.

Our incomplete knowledge of the regulators of blood vessel growth significantly hampers our ability to understand, and to intervene in, numerous disease states. These include pathologies directly caused by aberrant vessel growth or identity (e.g. craniofacial arteriovenous malformations, pulmonary arterial hypertension), those in which inadequate or excessive vessel growth contribute to the severity of symptoms (e.g. the insufficient neovessel growth after ischemic injury in the heart), and those in which modulating vessel growth can have secondary effects on disease progression (e.g. normalizing tumour vessels can increase the efficacy of chemotherapy, while ablating these vessels entirely can starve the tumour).

This grant aims to increase our understanding the role of the calcineurin-NFAT signalling pathway on the differentiation of endothelial cells. Specifically, our pilot data strongly indicates that this pathway is crucial for correct arterial-venous differentiation and function. A greater understanding of the signalling and transcriptional cascades controlling EC heterogeneity would therefore:

- Assist in establishing the aetiology of some cardiovascular diseases: This grant will determine the expression patterns of NFAT factors in both healthy and diseased vasculature, and identify the protein partners of NFAT in venous EC. This information would benefit health professionals and researchers when investigating the underlying causes of cardiovascular diseases, especially those where patterning is affected, or communications between EC and matrix are deficient (as arterial and venous EC have different relationships to matrix). For example, this information could be used to examine whether arterial ECs associated with pulmonary arterial hypertension are losing some of their arterial identity, or acquiring some aspects of ectopic venous identity.

- Improve our ability to study pathological conditions involving incorrect vascularization: We currently lack sufficient markers of arteriovenous differentiation. Further, many of the factors known to be involved in this process are widely expressed (e.g SMAD4 and SMAD1/5 is found in both arterial and venous EC at different time-points), display different patterns of expression in adult tissue (e.g EPHB4 does not mark venous EC in adult lung) or have not been thoroughly studied in adult or pathological vasculature. This research will thoroughly describe NFAT expression patterns in normal and disordered adult vessels, identify NFAT cofactors, and find and characterize numerous vein-specific gene enhancers. Researchers will be able to use the enhancer:reporter mice and fish for studies investigating the behaviour of the venous NFAT pathway, and shared regulatory pathways, in any pathological model. Further, the identification and description of NFAT and associated protein expression patterns can be used determine the arteriovenous differentiation status in many different human pathologies also.

- Lead to the development of new therapeutic interventions to inhibit, modulate or encourage vessel growth: Due to its role in the immune system, there are already a large number of calcineurin-NFAT signalling inhibitors approved for use on humans. The work proposed here will now determine the precise role of this pathway in the vasculature. This has the potential to enable academic researchers, pharmaceutical companies and healthcare professionals to repurpose NFAT signalling inhibitors to modulate vessel growth. This may involve combination therapy with other established therapeutic approaches, such as BMP or VEGFA inhibitors, or inhibitors against the novel protein partners we will identify here. Human diseases that may benefit from such therapies include cancer (solid tumours), arteriovenous malformations, coronary vessel diseases including grafts, limb ischemia, eye disorders such as wet macular degeneration and hypertension.