Anti-VEGF-mediated barrier closure

该项目的长期目标是阐明抗VEGF（即VEGF（血管内皮细胞）的急性中和）如何在血管内皮细胞中发挥作用。 内皮生长因子））阻止长期暴露于过量VEGF的血管渗漏。抗 VEGF抑制血管通透性的能力改善和/或维持了患者的生活质量 患有广泛的、不可治愈的疾病，如增殖性糖尿病视网膜病变（PDR）、糖尿病黄斑病变（DAF）、糖尿病视网膜病变（DR）和糖尿病视网膜病变（DR）。 水肿（DME）和新生血管性年龄相关性黄斑变性（nAMD）。这种临床观察 证明抗VEGF的有效性，并提出抗VEGF如何平静病理血管的问题。 我们认为，慢性升高的VEGF改变了血管内皮细胞的基因表达， 一种松弛内皮屏障的方式，而抗VEGF逆转了这种变化。 目标1。检验抗VEGF在转录上起作用以关闭屏障的假设。 我们的初步数据显示，长时间暴露于VEGF会改变许多基因的表达， 包括编码已知的内皮细胞屏障调节因子的那些。这些发现支持了我们的 工作假说，即抗VEGF的转录作用，以关闭屏障。这一假设将被检验为： 如下最初，基于RNAseq的阶段将比较具有或具有以下特征的细胞中的基因表达谱： 未用抗VEGF处理，从而鉴定抗VEGF差异表达基因（DEG）。 候选DEG将基于其已知的功能进行优先排序，然后根据其对VEGF-/抗VEGF/ 将确定VEGF介导的屏障功能控制。我们将确定抗VEGF依赖的基因 关闭被VEGF破坏的内皮细胞屏障。 目标二。研究抗VEGF对患者源性视网膜内皮细胞的作用机制。 目的1将用来自健康成人供体的原代人视网膜内皮细胞（HREC）进行。目标2将 重复目标1，除了使用PRECs，从病理血管分离的视网膜内皮细胞 在PDR患者中发展的。这是抗VEGF治疗的靶细胞类型。确定 抗VEGF在这些细胞中的作用机制将提供临床相关信息。 目标3。确定抗VEGF如何平静患者的病理血管。 在目标3中，我们将通过比较新鲜的VEGF基因表达谱来了解抗VEGF在患者中的作用。 从未经治疗和抗VEGF治疗的患者的病理血管分离内皮。在 此外，我们将比较所有3个目的的结果，以确定哪种抗VEGF介导的作用， 通过对培养的PREC（目的2）或HREC（目的1）进行抗VEGF治疗，忠实地模拟了患者中发生的血管紧张素转换酶（VEGF）。 该项目将揭示抗VEGF治疗益处的分子介质。这些信息将 消除目前开发生物标志物和抗VEGF替代品的障碍， 满足患有各种致盲疾病（如DME、PDR和nAMD）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.