Regulation of endothelial cell quiescence by Zeb1 as a regulator of angiogenesis

从原有的血管系统中生长出新的血管(血管生成)是低氧驱动的一个重要的生理过程。所有的血管内皮细胞(EC)在正常情况下形成一层光滑的单层，它不增殖，不运动，连接受到严格控制，表面不粘连，没有炎症或白细胞结合。在这种情况下，血管内皮细胞处于“静止”状态，允许有效的氧气和其他溶质交换进入间质和呼吸组织。EC通过表观遗传、表面受体和连接受体活性的组合、代谢以及关键基因的转录控制来维持这种静止的表型。当组织改变他们的新陈代谢需求时，内皮细胞就会做出反应。响应的EC可以被认为是激活的，根据激活的特异性，可以导致表型重塑，导致白细胞黏附增加，炎症标志物表达增加，增殖，运动性和信号通路的显著变化1。内皮细胞功能障碍发生在多种疾病中。糖尿病时，微环境改变导致内皮细胞功能障碍，导致缺氧和炎症，导致血管过度渗漏和生长。糖尿病视网膜病变是糖尿病的一种常见并发症，视网膜血管的生长会导致视力丧失。在心血管疾病(包括冠状动脉和外周血管疾病、中风和糖尿病)和血管相关疾病(神经退行性癌症、关节炎和败血症)中，EC被激活，疾病进展导致血管重塑，诱导新血管的生长(血管生成)或炎症条件。了解静息-激活开关是如何发生的，将使这一过程得以修改，从而达到治疗的目的：如果我们能够理解如何恢复(或产生维持)EC静默的方法，我们就能够抑制不必要的EC激活。我们最近发现ZEB1(一种已知在肿瘤进展和上皮向间充质转化中起作用的转录因子)在静止的内皮细胞中表达。这最初是在体外发现的，随后在体内使用新生血管的小鼠视网膜作为实验模型。使用可诱导的和内皮特异的ZEB1基因敲除小鼠，我们已经证明了初步证据表明，小鼠在成年视网膜自发地经历异常的内皮生长，并在脉络膜新生血管形成过程中增加溶质渗漏。这些初步数据表明，ZEB1信号的改变有助于成年小鼠视网膜血管生成和血管渗漏，这在DR的进展中可能是重要的。因此，本项目旨在研究一系列发育和病理血管生成模型中的血管生成重构。目的1：ZEB1是否改变发育中的血管生成表型？ZEB1将使用EC特异的、有条件的转基因来敲除。新生小鼠和成年小鼠的视网膜血管生成将被量化。病理性血管生成将使用后肢缺血和脉络膜新生血管模型来诱导。EC表型的特征及其与血管生成调节白细胞种群的相互作用目的2：EC ZEB1在糖尿病视网膜病变进展中的作用。将在可诱导的EC特异性KO小鼠体内诱导糖尿病，并将在体内测量视网膜通透性。EC表型的鉴定将通过免疫组织化学和分子鉴定目标3：ZEB1调控的血管生成依赖于血管内皮生长因子吗？我们将在体外探索连接VEGFR2、缺氧和糖酵解的信号通路。这也将在体内通过使用血管内皮生长因子抗体来防止EC ZEB1 KO糖尿病模型中血管内皮生长因子的刺激来进行探索。

The growth of new blood vessels from the pre-existing vasculature (angiogenesis) is an important physiological process driven by hypoxia. All blood vessels are lined by endothelial cells (EC) which under normal conditions form a smooth monolayer, which is non-proliferative, non-motile, tightly controlled junctions, non-adherent surface with no inflammation or leukocyte binding. Under these conditions the endothelium is in a state of 'quiescence' allowing efficient oxygen and other solute exchange into the interstitium and respiring tissues. The EC maintains this quiescent phenotype through the activity of a combination of epigenetic, surface receptor and junctional receptor activity, metabolic and of course transcriptional control of key genes. As tissues change their metabolic needs, the endothelium responds. A responding EC can be considered activated and depending on the specifics of the activation, can result in a phenotypic remodelling causing increased leukocyte adherence, increased inflammatory marker expression, proliferation, motility and significant changes in signalling pathways 1. Endothelial cell dysfunction occurs across a wide variety of diseases. During diabetes the altered microenvironment leads to EC dysfunction resulting in hypoxia and inflammation which results in excessive vascular leakage and growth. Diabetic retinopathy is a frequent complication of diabetes in which vessel growth in the retina results in loss of vision. In cardiovascular diseases (including coronary and peripheral vascular disease, stroke and diabetes) and vascular related diseases (neurodegeneration cancer, arthritis and sepsis) the EC is activated, and disease progression results in vascular remodelling, inducing the growth of new vessels (angiogenesis) or inflammatory conditions. Understanding how the quiescence-activation switch occurs will enable modification of this process for therapeutic benefit: if we can understand how to restore (or generate methods to maintain) EC quiescence we will be able to inhibit undesired EC activation. We have recently identified Zeb1 (a transcription factor with known roles in tumour progression and epithelial to mesenchymal transition) as being expressed in quiescent ECs. This was originally identified in vitro, and subsequently in vivo using the angiogenic mouse retina as an experimental model. Using inducible and endothelial specific Zeb1 knockout mice we have demonstrated preliminary evidence to show mice spontaneously undergo aberrant endothelial growth in adult retinae and increase solute leakage during choroidal neovascularisation. These preliminary data suggest altered Zeb1 signalling contributes to retinal angiogenesis and vessel leakage in adult mice, which could be important in the progression of DR. Therefore, this project will aim to investigate angiogenic remodelling in a series of developmental and pathological angiogenesis models. Aim 1: Does Zeb1 alter angiogenic phenotype in development? Zeb1 will be knocked out using a EC specific, conditional transgene. Retinal angiogenesis will be quantified in neonatal mice, and adult mice. Pathological angiogenesis will be induced using hindlimb ischaemia and choroidal neovascularisation models. Characterisation of EC phenotypes and interactions with angiogenesis modulating leukocyte populationsAim 2: Contribution of EC Zeb1 to the progression of diabetic retinopathy. Diabetes will be induced within the inducible EC specific KO mice and retinal permeability will be measured in vivo. Characterization of EC phenotype will be made by immunohistochemistry and molecular characterisation Aim 3: Is Zeb1 regulated angiogenesis VEGF dependent? Signalling pathways linking VEGFR2, hypoxia and glycolysis will be explored in vitro. This will also be explored in vivo with the use of VEGF antibodies to prevent VEGF stimulation within the EC Zeb1 KO diabetic model.