Anti-VEGF-mediated barrier closure

该项目的长期目标是阐明抗血管内皮细胞生长因子(即急性中和血管内皮生长因子) 内皮生长因子)可以阻止长期暴露在过量血管内皮生长因子中的血管渗漏。反- 血管内皮生长因子抑制血管通透性的能力改善和/或维持了患者的生活质量 患有无处不在的不治之症，如增殖性糖尿病视网膜病变(PDR)、糖尿病黄斑 水肿(DME)和新生血管性老年性黄斑变性(NAMD)。这样的临床观察 证明了抗血管内皮生长因子的有效性，并提出了抗血管内皮生长因子如何平息病变血管的问题。 我们推测慢性升高的血管内皮生长因子改变了血管内皮细胞的基因表达。 一种放松内皮细胞屏障的方式，而抗血管内皮生长因子逆转了这种变化。 目的1.验证抗血管内皮生长因子转录作用关闭屏障的假说。 我们的初步数据显示，长期接触血管内皮生长因子会改变许多基因的表达， 包括那些编码已知的内皮细胞屏障调节因子的基因。这些发现支持了我们的 工作假说，抗血管内皮生长因子转录作用，以关闭屏障。这一假设将被检验为 下面是。最初，基于RNAseq的阶段将比较具有或具有 未用抗血管内皮生长因子处理，从而鉴定抗血管内皮生长因子差异表达基因(DEG)。 候选人DEG将根据其已知的功能被优先排序，然后他们对血管内皮生长因子-/抗- 血管内皮生长因子对屏障功能的调控将被确定。我们将确定抗血管内皮生长因子依赖的基因 关闭已经被血管内皮生长因子破坏的内皮细胞屏障。 目的2.探讨抗血管内皮生长因子对患者视网膜内皮细胞的作用机制。 目的1用健康成人供体的原代人视网膜内皮细胞(HRECs)进行培养。目标2将 重复目标1，除了使用前内皮细胞外，视网膜内皮细胞从病理性血管中分离出来 发生在患有PDR的患者身上。这是抗血管内皮生长因子治疗的靶细胞类型。确定 抗血管内皮生长因子在这些细胞中的作用机制将为临床提供相关信息。 目的3.确定抗血管内皮生长因子对患者病变血管的抑制作用。 在目标3中，我们将通过比较新鲜人的基因表达谱来了解抗血管内皮生长因子在患者中的作用。 从单纯治疗和抗血管内皮生长因子治疗患者的病理血管中分离内皮细胞。在……里面 此外，我们将比较所有3个目标的结果，以确定哪种抗血管内皮生长因子介导的作用 以抗血管内皮生长因子治疗培养的前内皮细胞(Aim 2)或高密度脂蛋白内皮细胞(Aim 1)为模型。 本项目将揭示抗血管内皮生长因子治疗作用的分子介体。这样的信息将 消除目前开发生物标记物和抗血管内皮生长因子替代品的障碍，这是 满足患有DME、PDR和NAMD等各种致盲疾病的患者的需求。

This project’s long-term goal is to elucidate how anti-VEGF (i.e. acute neutralization of VEGF (vascular endothelial growth factor)) stops leakage of blood vessels that are chronically exposed to excess VEGF. Anti- VEGF’s ability to curb blood vessel permeability has improved and/or sustained the quality of life of patients afflicted with pervasive, incurable diseases such as proliferative diabetic retinopathy (PDR), diabetic macular edema (DME) and neovascular age-related macular degeneration (nAMD). Such clinical observations demonstrate anti-VEGF’s efficacy, and beg the question of how anti-VEGF calms pathological blood vessels. We posit that chronically elevated VEGF alters gene expression in the endothelium of blood vessels in a way that relaxes the endothelial barrier, and that anti-VEGF reverses such changes. Aim 1. Test the hypothesis that anti-VEGF acts transcriptionally to close the barrier. Our preliminary data reveal that prolonged exposure to VEGF changes the expression of many genes, including those that encode known regulators of the endothelial cell barrier. These discoveries support our work hypothesis that anti-VEGF acts transcriptionally to close the barrier. This hypothesis will be tested as follows. The initial, RNAseq-based phase will compare the gene expression profile in cells that have or have not been treated with anti-VEGF and thereby identify anti-VEGF differentially expressed genes (DEGs). Candidates DEGs will be prioritized based on their known function, and then their contribution to VEGF-/anti- VEGF-mediated control of barrier function will be determined. We will identify genes that anti-VEGF depends on to close the endothelial cell barrier that has been breached with VEGF. Aim 2. Investigate the mechanism of action of anti-VEGF on patient-derived retinal endothelial cells. Aim 1 will be done with primary human retinal endothelial cells (HRECs) from a healthy adult donor. Aim 2 will be a repeat of aim 1, except using PRECs, retinal endothelial cells isolated from pathological blood vessels that develop in patients with PDR. This is the target cell type of anti-VEGF therapy. Determining the mechanism of action of anti-VEGF in these cells will provide clinically relevant information. Aim 3. Determine how anti-VEGF calms pathological blood vessels in patients. In aim 3 we will learn how anti-VEGF acts in patients by comparing the gene expression profile in freshly isolated endothelium from pathological blood vessels of treatment naïve and anti-VEGF-treated patients. In addition, we will compare the results from all 3 aims to determine which of the anti-VEGF-mediated effects that occur in patients are faithfully modeled by anti-VEGF treatment of cultured PRECs (aim 2) or HRECs (aim 1). This project will unveil the molecular mediators of anti-VEGF’s therapeutic benefit. Such information will remove current roadblocks to developing biomarkers and alternatives to anti-VEGF, which are needed to address the needs of patients afflicted with a variety of blinding conditions such as DME, PDR and nAMD.