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）：我们对因果关系感兴趣，这些因果关系将氧气中的重复波动与早产性（ROP）和先前血管性视网膜的血管化（VPAR）的血管化（VPAR）的血管化（ROP）和血管化相关。我们的总体假设是，氧气中反复的波动会导致血管内皮生长因子（VEGF）同工型和受体的失调，在发育中的视网膜区域内产生了微环境，从而促进了不必要的IVNV及更高的盲目性。我们提出，在视网膜血管发育的早期，氧气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的调节的平衡造成的，色素上皮源性因子（PEDF）。后来，反复的氧气波动会导致氧化损伤，细胞凋亡以及通过GP130受体亚基/转导蛋白下游的途径传导，从而导致VEGF同工型的进一步失调（尤其是病理VEGF164）。所有这些事件都会促进IVNV。 我们将使用已建立的“ 50/10 OIR”型号（在受控的24小时氧气波动的条件下饲养的新生大鼠并恢复空气）。 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会发生变化，使血管生成刺激发生（例如，VEGF和/或PEDF减少），2A），2A）增加活性氧（ROS）发生在血管和血管视网膜的连接处，并导致内皮细胞凋亡，保持周围性血管性，并促进VPAR和ROS的表达，并促进VEGFFF2的表达，3. 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. 方法将包括用于测量生长因子（VEGF同工型，PEDF）和受体（VEGFR1和VEGFR2）mRNA的RT-PCR； ELISA和Western印迹以测量生长因子（VEGF）和磷酸化受体或蛋白质（LIFR，GP130，IL-6R，STATS，JAK2，SOCS）的蛋白质； LIF，IL-6或抗体的眼内注射（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（二乙基）；凋亡（激活的caspase-3）和共聚焦显微镜；以及量化血管和血管视网膜以及IVNV的时钟小时。 了解这些引起病理VEGF164表达的机制将提供预防IVNV并促进IVNV回归并促进VPAR的潜在策略。