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

血管生成(已有血管的生长)和淋巴管生成(淋巴内皮细胞从淋巴囊中萌发形成淋巴管)在胚胎发育和整个生命的各种生理条件下都是重要的。两者在一系列疾病状态中都很重要，例如炎症和缺血性视网膜病变以及癌症的标志。血管生成和淋巴管生成是一个多步骤的过程，需要内皮细胞内不同信号通路的精确协调。在健康的成年人中，血管生成在生理上以及对创伤、缺氧和低血糖等刺激的反应中发生。这就需要在促血管生成因子和抗血管生成因子之间保持精确的平衡，这种平衡的任何破坏都会导致异常的血管生成和病理状况。内皮细胞是血管形成的主要构件。它们形成一层单层细胞(称为内皮细胞)，排列在血管和淋巴管的管腔内。内皮细胞分为血管内皮细胞和淋巴管内皮细胞。血管生成涉及内皮细胞与周围组织中其他类型细胞的相互作用，如血管周围细胞。血管周围细胞包括血管平滑肌细胞和周细胞，它们与全身所有器官的内皮细胞密切相关，为内皮细胞提供支持和稳定。在血管生成过程中，内皮细胞从静止、静止状态转变为增殖、迁移状态。内皮细胞过度增殖导致病理性血管生成和淋巴管生成。两个重要的内皮细胞调节因子是血管内皮生长因子受体和Tie受体(Tie 1和Tie 2)，这是两个主要局限于内皮细胞和其他一些细胞类型的受体酪氨酸激酶家族。重要的是，这些受体是各种疾病的诱人治疗靶点。因此，了解控制其表达以激活或阻止血管生成和淋巴管生成的转录调控因子是至关重要的。PRH蛋白(也称为Hex、HHEX或HHEX)是一种高度保守的寡聚转录因子，包含一个被称为同源结构域的DNA结合结构域。PRH是胚胎发育过程中多个器官发育所必需的，也是成人维持不同组织类型所必需的。PRH过表达可抑制VEGFR基因(VEGFR1、VEGFR2)以及Tie-1、Tie-2、uPA、MMP1和Endoglin等血管生成相关基因的表达，从而在成人内皮细胞中发挥抗血管生成作用。因此，深入了解这些基因是如何被PRH调控的，可能会为包括许多恶性肿瘤在内的血管生成和淋巴生成相关疾病开辟新的预后和治疗视野。本项目的目的是了解PRH如何调控涉及VEGF信号的基因，以及VEGF信号如何控制PRH的活性。我们将使用RNA测序(RNA-SEQ)来识别淋巴管内皮细胞中PRH调控的基因，并结合染色质免疫沉淀测序(CHIP-SEQ)来定位这些细胞中的PRH结合部位，并在这些细胞基因组中识别可能的PRH直接靶点。然后，我们将把这些结果与以前使用人脐静脉内皮细胞(HUVECs)获得的RNA-SEQ和CHIP-SEQ数据进行比较。通过这种方式，我们将确定直接结合的PRH调节基因，涉及血管生成或淋巴管生成所需的不同信号通路。然后我们将使用蛋白质组学来确定每种细胞类型中与PRH相互作用的蛋白质的谱系。最后，我们将在体外和血管生成的动物模型中检测选择性受体酪氨酸激酶对每种细胞类型中PRH表达和活性的调节。

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.