Ibuprofen-Caffeine Modulation of Retinal Endothelial Tip Cell Migration

早产儿视网膜病变(ROP)在所有极低胎龄儿中高达50%(28周/1250g)。布洛芬和咖啡因已经被证明可以降低伊尔甘人和动物模型中严重ROP的风险。利用我们实验室开发的独特技术，我们将检测对人视网膜微血管内皮细胞(ECs)的特定影响，这是异常血管生成背后的驱动力。我们将研究它们与星形胶质细胞的动态相互作用，以及它们在氧化应激过程中的选择、激活和迁移。更重要的是，我们将检查布洛芬和/或咖啡因在防止它们的激活和降低它们感知血管生成信号的能力方面的有效性。这项建议的首要目标是研究氧化应激(高氧/低氧循环)下EC TIP细胞的行为及其与星形胶质细胞的关系；并确定布洛芬与咖啡因联合应用是否能维持FIP细胞的静止。利用最先进的生物分析、蛋白质组学、药物基因组学、生物信息学和成像技术，我们将使用三个相互关联的具体目标：1)研究常氧和氧化应激(短暂的高氧/低氧循环)下人视网膜微血管内皮细胞和人脑星形胶质细胞之间的关系。2)利用小干扰RNA研究VEGFR-2、VEGFR-3、NP-1、Notch 1、D1I4和Jagge1在TIP细胞选择、激活和迁移中的作用 (SiRNA)敲除人视网膜微血管内皮细胞中的这些特定基因。我们将研究星形胶质细胞的影响；以及3)确定咖啡因增强的布洛芬是否会在氧化应激下保护和保存正常的人视网膜微血管内皮细胞和星形胶质细胞的生长和功能。我们将使用最先进的技术来提供关于生物分子机制、药代动力学、药物相互作用、药物运输和新陈代谢的见解。

Retinopathy of prematurity (ROP) is afflicts as much as 50% of all extremely low gestational age neonates (ELGANs, <28 weeks/<1250g). Ibuprofen and caffeine have been shown to decrease the risk of severe ROP in ELGANs and in animal models. Using unique techniques developed in our laboratory, we will examine the specific effects on human retinal microvascular endothelial tip cells (ECs), the driving force behind aberrant angiogenesis. We will study their dynamic interaction with astrocytes, their selection, activation, and migration during oxidative stress. More importantly, we will examine the efficacy of ibuprofen and/or caffeine in preventing their activation and reducing their capacity to sense angiogenic cues. The overarching goal of this proposal is to study the behavior of EC tip cells and their relationship with astrocytes in the setting of oxidative stress (hyperoxia/hypoxia cycling); and to determine whether ibuprofen coadministered with caffeine will preserve fip cell quiescence. Using state-of-the-art bioanalytics, proteomics, pharmacogenomics, bioinformatics, and imaging techniques, we will use three interrelated specific aims: 1) to examine the relationship between human retinal microvascular ECs and human brain astrocytes in normoxia and in oxidative stress (brief hyperoxia/hypoxia cycling). We will focus on VEGF and ECM proteolysis, VEGF release and increased gradient; tip cell activation, release and recapture of VEGF, and migration; and VEGF and Notch signaling mechanisms; 2) to establish the roles of VEGFR-2, VEGFR-3, NP-1, Notch 1, D1I4, and Jagged 1 on tip cell selection, activation and migration using small interference RNA (SiRNA) knockdown of these specific genes in human retinal microvascular ECs. We will study the influence of astrocytes; and 3) to determine whether ibuprofen potentiated with caffeine will protect and preserve normal human retinal microvascular EC and astrocyte growth and function in oxidative stress. We will use state-of-the-art technologies to provide insights on the biomolecular mechanisms, pharmacokinetics, drug interactions; drug transport and metabolism.