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

血管生成（angiogenesis）是缺氧驱动下的一个重要生理过程。所有血管都由内皮细胞（EC）排列，在正常情况下形成光滑的单层，无增殖，无运动，紧密控制连接，非粘附表面，无炎症或白细胞结合。在这种情况下，内皮处于“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、代谢，当然还有关键基因的转录控制的组合活动来维持这种静止表型。当组织改变它们的代谢需求时，内皮细胞就会做出反应。响应EC可被认为是激活的，根据激活的具体情况，可导致表型重塑，导致白细胞粘附性增加，炎症标志物表达、增殖、运动性增加和信号通路的显着变化1。内皮细胞功能障碍发生在多种疾病中。在糖尿病期间，微环境的改变导致EC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常见并发症，视网膜血管生长导致视力丧失。在心血管疾病（包括冠状动脉和周围血管疾病、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。了解休眠-激活开关是如何发生的，将有助于修改这一过程，以获得治疗益处：如果我们能够了解如何恢复（或产生维持）EC休眠，我们将能够抑制不想要的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，随后在体内使用血管生成小鼠视网膜作为实验模型。通过诱导和内皮特异性Zeb1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中自发地经历了异常的内皮生长，并在脉络膜新生血管过程中增加了溶质渗漏。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，本项目将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塑。目的1:Zeb1在发育过程中是否改变血管生成表型？将使用EC特异性的条件转基因敲除Zeb1。将对新生小鼠和成年小鼠的视网膜血管生成进行量化。采用后肢缺血和脉络膜新生血管模型诱导病理性血管生成。EC表型的特征和与血管生成调节白细胞群的相互作用目的2:EC Zeb1对糖尿病视网膜病变进展的贡献。在可诱导的EC特异性KO小鼠体内诱导糖尿病，并在体内测量视网膜通透性。EC表型的表征将通过免疫组织化学和分子表征进行。将在体外探索连接VEGFR2、缺氧和糖酵解的信号通路。这也将在EC Zeb1 KO糖尿病模型中通过使用VEGF抗体来防止VEGF刺激进行体内探索。

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.