Mechanisms of Angiogenesis in Retinopathy of Prematurity

早产儿视网膜病变的血管生成机制

• 批准号: 6866803
• 负责人: Mary Elizabeth Ruth Hartnett
• 金额: $ 28.23万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-02 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6866803
• 关键词: JAK kinase; acetylcysteine; angiogenesis; apoptosis; biological signal transduction; blocking antibody; confocal scanning microscopy; cytokine receptors; enzyme linked immunosorbent assay; fluorescence; free radical oxygen; growth factor receptors; hyperoxia; interleukin 6; laboratory rat; leukemia inhibitory factor; lipid peroxides; neurotrophic factors; oxygen tension; pathologic process; polymerase chain reaction; protein isoforms; retinopathy of prematurity; transcription factor; vascular endothelial growth factors; western blottings

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.

描述（由申请人提供）：我们感兴趣的是氧的反复波动与早产儿视网膜病变（ROP）中的玻璃体内新生血管形成（IVNV）和先前无血管视网膜（VPAR）的血管形成之间的因果关系。我们的总体假设是，氧气的反复波动会导致视网膜发育区域内的血管内皮生长因子 (VEGF) 同工型和受体失调，从而形成一个微环境，促进不必要的 IVNV 和后来的失明。我们认为，在视网膜血管发育的早期，氧的波动会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) 的调节平衡被破坏。 。随后，反复的氧波动会导致氧化损伤、细胞凋亡，并通过 gp130 受体亚基/转导蛋白下游途径发出信号，从而导致 VEGF 亚型（尤其是病理性 VEGF164）进一步失调。所有这些事件都促进了 IVNV。 我们将使用已建立的“50/10 OIR”模型（在受控24小时氧气波动和返回室内空气的条件下饲养的新生大鼠）。具体来说，我们将确定 1a) 用抗体特异性阻断主要病理亚型 VEGF164 是否会减少 IVNV 并促进 VPAR，lb) VEGF 和 PEDF 发生改变，导致早期时间点的血管生成抑制作用（例如，增加 PEDF 和/或）或减少 VEGF）导致视网膜血管化延迟，并且在 IVNV 期间的稍后时间点，平衡将发生变化，从而发生血管生成刺激（例如，增加 VEGF 和/或减少 PEDF），2a) 血管和无血管视网膜交界处发生活性氧 (ROS) 增加，导致内皮细胞凋亡，维持外周无血管并干扰 VPAR，ROS 会增加VEGF164和VEGFR2，促进IVNV，3a)激活gp130会激活JAK/STAT通路，增加VEGF164、VEGFR2的表达，促进IVNV 和 3b) 白细胞介素 6 (IL-6) 将减少内皮细胞凋亡、IVNV 并促进 VPAR，而白血病抑制因子 (LIF) 将增加内皮细胞凋亡和 IVNV。 方法包括 RT-PCR 来测量生长因子（VEGF 同工型、PEDF）和受体（VEGFR1 和 VEGFR2）mRNA； ELISA 和蛋白质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、STATs、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（激活的 caspase-3）和共聚焦显微镜；无血管和血管视网膜的量化以及 IVNV 的时钟时间。 了解这些导致病理性 VEGF164 表达的机制将为预防 IVNV、促进 IVNV 消退和促进 VPAR 提供潜在策略。