Regulation of transcription factor activity by receptor tyrosine kinase signalling during angiogenesis

Angiogenesis (the growth of blood vessels from the pre-existing vasculature) and lymphangiogenesis (the sprouting of lymphatic endothelial cells from lymph sacs to form lymphatic vessels) are important in various physiological conditions during embryogenesis and throughout life. Both are important in a wide array of disease states such as inflammation and ischemic retinopathy and the hallmark of cancers. Angiogenesis and lymphangiogenesis are multi step processes that require precise coordination of different signalling pathways in endothelial cells. In the healthy adult, angiogenesis happens physiologically as well as in response to stimuli such as wounding, hypoxia and hypoglycemia. This requires precise balance between pro-angiogenic and anti-angiogenic factors and any disturbance in this balance results in aberrant angiogenesis and pathological conditions.
Endothelial cells (ECs) are the main building block for the formation of the vasculature. They form a single layer of cells (called endothelium) that lines the lumen of both blood and lymphatic vessels. Endothelial cells are divided into vascular endothelial cells and lymphatic endothelial cells. Angiogenesis involves the interaction between ECs and other cell types in the surrounding tissue such as perivascular cells. Perivascular cells including vascular smooth muscle cells and pericytes, are closely associated to the endothelium in all organs throughout the body and they provide support and stabilization for ECs. During angiogenesis, ECs switch from their quiescent, immotile state to a proliferating, migrating state. Excessive proliferation of endothelial cells causes pathological angiogenesis and lymphangiogenesis. Two important regulators of endothelial cells are the Vascular endothelial growth factor (VEGF) receptors and the Tie receptors (Tie 1 and Tie 2), which are two families of receptor tyrosine kinases mainly restricted to the ECs as well as a few other cell types. Importantly, these receptors are attractive therapeutic targets in various diseases. Therefore, it is crucial to understand the transcriptional regulators that control their expression to either activate or block angiogenesis and lymphangiogenesis.
The PRH protein (known also as Hex, Hhex or HHEX) is a highly conserved oligomeric transcription factor that contains a DNA-binding domain known as the homeodomain. PRH is required for the development of multiple organs during embryogenesis and also plays a role in the maintenance of different tissue types in the adult. PRH overexpression has an anti-angiogenic role in adult endothelial cells because it represses the expression of VEGFR genes (VEGFR1, VEGFR2) and other angiogenesis related genes including, Tie-1, Tie-2, uPA, MMP-1 and endoglin. Thus, a thorough understanding of how these genes are controlled by PRH may open novel prognostic and therapeutic horizons for angiogenic- and lymphogenic-related diseases including many malignant cancers.
The aim of this project is to understand how PRH regulates genes involved in VEGF signalling and how VEGF signalling controls PRH activity. We will use RNA-sequencing (RNA-seq) to identifying PRH regulated genes in lymphatic endothelial cells and combining chromatin immunoprecipitation sequencing (ChIP-seq) to map PRH binding sites in these cells and identify putative direct targets of PRH in these cells genome wide. Then we will compare these results with previously generated RNA-seq and ChIP-seq data obtained using Human umbilical vein endothelial cells (HUVECs). In this way we will identify the directly bound PRH regulated genes involved in different signalling pathways required for angiogenesis or lymphangiogenesis. We will then use proteomics to determine the repertoire of PRH-interacting proteins in each cell type. Finally, we will examine the regulation of PRH expression and PRH activity in each cell type by selected receptor tyrosine kinases in vitro and in animal models of angiogenesis.