Mechanisms of Angiogenesis in Retinopathy of Prematurity

描述（由申请方提供）：我们对早产儿视网膜病变（ROP）和既往无血管视网膜血管化（VPAR）中氧浓度反复波动与玻璃体内新生血管化（IVNV）之间的因果关系感兴趣。我们的总体假设是，氧气的反复波动导致发育中的视网膜区域内的血管内皮生长因子（VEGF）亚型和受体的失调，从而产生促进不必要的IVNV和随后的失明的微环境。我们提出，在视网膜血管发育的早期，氧气的波动导致血管生成抑制作用，部分原因是血管生成激动剂VEGF和血管生成抑制因子色素上皮衍生因子（PEDF）的调节平衡失调。随后，反复的氧波动引起氧化损伤、细胞凋亡和通过gp130受体亚基/转导蛋白下游途径的信号传导，从而引起VEGF同种型（尤其是病理性VEGF 164）的进一步失调。所有这些活动都促进了IVNV。 我们将使用已建立的"50/10 OIR"模型（新生大鼠在受控的24小时氧气波动条件下饲养，并返回室内空气）。具体地，我们将确定：1a）用抗体特异性阻断主要病理性同种型VEGF 164是否会降低IVNV并促进VPAR，1b）VEGF和PEDF被改变，导致在早期时间点（例如，增加的PEDF和/或减少的VEGF），导致延迟的视网膜血管形成，并且在稍后的时间点，在IVNV期间，平衡将转移从而发生血管生成刺激（例如，增加的VEGF和/或减少的PEDF），2a）增加的活性氧物质（ROS）发生在血管和无血管视网膜的连接处并引起内皮细胞凋亡，维持外周无血管性并干扰VPAR，并且ROS将增加VEGF 164和VEGFR2的表达，促进IVNV，3a）gp130的激活将激活JAK/STAT途径以增加VEGF 164、VEGFR2的表达，白细胞介素-6（IL-6）可减少内皮细胞凋亡和IVNV，促进VPAR，而白血病抑制因子（LIF）可增加内皮细胞凋亡和IVNV。 方法将包括RT-PCR以测量生长因子（VEGF亚型，PEDF）和受体（VEGFR1和VEGFR2）mRNA; ELISA和Western印迹法测量生长因子（VEGF）蛋白和磷酸化受体或蛋白（LIFR、gp130、IL-6R、STAT、JAK2、SOCS）;眼内注射LIF、IL-6或抗体（VEGF164、VEGFR2）;全身注射抗氧化剂（n-乙酰半胱氨酸）或JAK2抑制剂（AG490）;脂质过氧化物和超氧化物损伤的测量（二氢乙锭）;细胞凋亡（活化的半胱天冬酶-3）和共聚焦显微镜;以及定量无血管和血管视网膜和IVNV的时钟小时。 了解这些导致病理性VEGF 164表达的机制将为预防IVNV、促进IVNV消退和促进VPAR提供潜在的策略。

DESCRIPTION (provided by applicant): We are interested in the causal relationships that tie repeated fluctuations in oxygen to intravitreous neovascularization (IVNV) in retinopathy of prematurity (ROP) and vascularization of previously avascular retina (VPAR). Our overall hypothesis is that repeated fluctuations in oxygen cause dysregulation of the vascular endothelial growth factor (VEGF) isoforms and receptors within regions of the developing retina creating a microenvironment that promotes unwanted IVNV and later blindness. We propose that early in retinal vascular development, fluctuations in oxygen result in an angiogenic inhibitory effect due, in part, to a disturbed balance in the regulation of the angiogenic agonist, VEGF, and angiogenic inhibitory factor, pigment epithelium-derived factor (PEDF). Later, repeated oxygen fluctuations cause oxidative injury, cell apoptosis, and signaling through pathways downstream of the gp130 receptor subunit/transducing protein causing further dysregulation of VEGF isoforms (especially the pathologic VEGF164). All of these events promote IVNV. We will use the established "50/10 OIR" model (newborn rats reared under conditions of controlled 24-hour oxygen fluctuations and return to room air). Specifically we will determine whether 1a) specific blocking of the predominant pathological isoform, VEGF164, with antibody will reduce IVNV and promote VPAR, lb) VEGF and PEDF are altered leading to an angiogenic inhibitory effect at early time points (e.g., increased PEDF and/or decreased VEGF) resulting in delayed retinal vascularization, and that, at later time points, during IVNV, the balance will shift so that angiogenic stimulation occurs (e.g., increased VEGF and/or reduced PEDF), 2a) increased reactive oxygen species (ROS) occur at the junction of vascular and avascular retina and cause endothelial cell apoptosis, maintaining peripheral avascularity and interfering with VPAR, and ROS will increase expression of VEGF164 and VEGFR2, promoting IVNV, 3a) activation of gp130 will activate JAK/STAT pathways to increase expression of VEGF164, VEGFR2, and promote IVNV, and 3b) interleukin-6 (IL-6) will reduce endothelial cell apoptosis, IVNV, and promote VPAR, whereas leukemia inhibitory factor (LIF) will increase endothelial cell apoptosis and IVNV. Methods will include RT-PCR to measure growth factor (VEGF isoforms, PEDF) and receptor (VEGFR1 and VEGFR2) mRNAs; ELISA and Western blot to measure protein of growth factors (VEGF) and phosphorylated receptors or proteins (LIFR, gp130, IL-6R, STATs, JAK2, SOCS); intraocular injections of LIF, IL-6, or antibodies (VEGF164, VEGFR2); systemic injections of antioxidants (n-acetylcysteine) or a JAK2 inhibitor (AG490); measurement of lipid peroxides and superoxide damage (dihydroethidium); apoptosis (activated caspase-3) and confocal microscopy; and quantification of avascular and vascular retina and clock hours of IVNV. Understanding these mechanisms that cause pathologic VEGF164 expression will provide potential strategies to prevent IVNV and to facilitate regression of IVNV and promote VPAR.