Mechanisms of Angiogenesis of Retinopathy of Prematurity

氧波动和吸氧量增加(高氧)是严重视网膜病变的危险因素。 早产(ROP)。我们的假设是，体内与人类严重ROP相关的氧气应激增加 无血管视网膜，这是严重ROP发生的前提和必要条件。这个 无血管视网膜的增加通过两种不同的事件发生：血管生成障碍和内皮细胞 细胞凋亡。具体地说，氧波动和相对组织缺氧上调Mueller细胞血管内皮生长因子和 增加VEGF-VEGFR2信号在迁移前沿分裂内皮细胞(ECs)以定向有丝分裂 EC裂解平面并干扰正常的视网膜血管生成，从而增加外周无血管视网膜。 此外，氧波动或高氧导致NADPH氧化酶不同程度的激活 释放活性氧物质，触发内皮细胞和内皮前体细胞的凋亡，降低视网膜 血管发育和增加无血管视网膜，而在氧气设置中补充氧气 波动将进一步激活NADPH氧化酶，触发细胞骨架事件的信号转导到无序切割 平面定向，干扰正常的血管生成。 我们将在啮齿类动物身上使用氧气诱导的视网膜病变(OIR)模型，这些啮齿类动物接受视网膜血管 出生后发育。我们将使用基因可操纵的小鼠OIR模型来研究机制 高氧或相对组织缺氧，或模拟氧波动的大鼠50/10 OIR模型 早产儿所经历的那些发展为更常见形式的严重ROP(区II， 第三阶段ROP)。在特定的目标1中，测试增加的血管内皮生长因子信号将扰乱卵裂方向的预测 血管和无血管视网膜交界处内皮细胞的分裂平面及其对视网膜内的干扰 血管形成，我们将使用microRNA到VEGFA使大鼠50/10 OIR中Mueller细胞来源的VEGF164沉默 或包装成慢病毒载体的表达盒中的VEGF164。在具体目标2中，我们将测试 预测NADPH氧化酶的差异激活会触发不同的信号事件，从而导致 分裂中内皮细胞的凋亡或骨骼事件和裂面方向的改变 内皮细胞。为了做到这一点，我们将使用基因敲除小鼠p47Phox，NADPH氧化酶的一个亚单位或 NADPH氧化酶的药理抑制剂。我们将通过以下方式研究EC来源的NADPH氧化酶的作用 耗尽巨噬细胞的动物。为了改变NADPH氧化酶的激活程度，我们还将使用 在高氧中获救的50/10 OIR型号与在室内空气中获救的标准OIR型号50/10 OIR相比。 方法包括：用共聚焦显微镜观察视网膜平铺片以显示交界处的内皮细胞。 血管和无血管视网膜，以量化凋亡细胞、毛细血管密度、血管和无血管视网膜，并 计数有丝分裂的随机有丝分裂面数；冰冻切片 对于磷酸化的VEGFR1和2，共标记细胞的凋亡(TUNEL，裂解的caspase-3)；激光捕获 实时定量聚合酶链式反应和原位杂交检测血管内皮细胞生长因子的基因定位 异构体，血管内皮生长因子受体1和2，神经粘连蛋白；酶联免疫吸附试验和蛋白质印迹检测蛋白(血管内皮生长因子；裂解 Caspase-3)；免疫沉淀和探针检测磷酸化的血管内皮生长因子受体，蛋白激酶II， JAKS，STATS；构建针对VEGFA或VEGF164的microRNA，将其包装成带有CD44的慢病毒载体 促进剂；视网膜下注射；全身性NADPH氧化酶抑制剂或氯膦酸盐；反应性测定 氧物种(如二氢乙锭)；和NADPH氧化酶的激活。

Oxygen fluctuations and increased inspired oxygen (hyperoxia) are risk factors for severe retinopathy of prematurity (ROP). Our hypothesis is that in vivo oxygen stresses, relevant to human severe ROP, increase avascular retina, which precedes and is a necessary prerequisite for the development of severe ROP. The increased avascular retina occurs through two different events: disordered angiogenesis and endothelial apoptosis. Specifically, oxygen fluctuations and relative tissue hypoxia upregulate Mueller cell VEGF and increases VEGF-VEGFR2 signaling in dividing endothelial cells (ECs) at the migrating front to disorient mitotic EC cleavage planes and interfere with normal retinal angiogenesis, thus increasing peripheral avascular retina. Furthermore oxygen fluctuations or hyperoxia cause different degrees of activation of NADPH oxidase to release reactive oxygen species that trigger apoptosis of ECs and endothelial precursor cells reducing retinal vascular development and increasing avascular retina, whereas supplemental oxygen in the setting of oxygen fluctuations will further activate NADPH oxidase to trigger signaling of cytoskeletal events to disorder cleavage plane orientation and interfere with normal angiogenesis. We will use oxygen induced retinopathy (OIR) models in rodents, which undergo retinal vascular development after birth. We will use either the genetically-manipulable mouse OIR model to study mechanisms of hyperoxia or relative tissue hypoxia, or the rat 50/10 OIR model in which the oxygen fluctuations mimic those experienced by preterm human infants that develop the more common form of severe ROP (zone II, stage 3 ROP). In Specific Aim 1, to test the prediction that increased VEGF signaling will disorient cleavage planes of dividing endothelial cells at the junction of vascular and avascular retina and interfere with intraretinal vascularization, we will silence Mueller cell derived VEGF164 in the rat 50/10 OIR using a microRNA to VEGFA or VEGF164 in an expression cassette packaged into a lentiviral vector. In Specific Aim 2, we will test the prediction that differential activation of NADPH oxidase triggers different signaling events leading to either apoptosis of endothelial cells or alteration of skeletal events and cleavage plane orientation in dividing endothelial cells. To do this we will use knockout mice to p47phox, a subunit of NADPH oxidase or pharmacologic inhibitors of NADPH oxidase. We will study the effects of EC-derived NADPH oxidase by depleting animals of macrophages. To change the degree of activation of NADPH oxidase, we will also use the 50/10 OIR model rescued in hyperoxia compared to the standard 50/10 OIR model rescued in room air. Methods include: confocal microscopy of retinal flat mounts to visualize endothelial cells at the junction of vascular and avascular retina to quantify apoptotic cells, capillary density, vascular and avascular retina, and to count the number of random mitotic planes of dividing phospho-histone stained endothelial cells; cryosections for phosphorylated VEGFR1 and 2, apoptosis (TUNEL, cleaved caspase-3) of co-labeled cells; laser capture microdissection; real-time-PCR to quantitate and in situ hybridization to detect location of mRNA of VEGF isoforms, VEGF receptors 1 and 2, neuropilins; ELISA and Western blot to measure protein (VEGF; cleaved caspase-3); immunoprecipitation and probing to detect phosphorylated VEGF receptors, protein kinase ¿II, JAKs, STATs; construction of microRNAs to VEGFA or VEGF164 packaged into lentiviral vectors with a CD44 promoter; subretinal injections; systemic NADPH oxidase inhibitors or clodronate; measurement of reactive oxygen species (e.g., dihydroethidium); and NADPH oxidase activation.