Mechanisms of Angiogenesis in Retinopathy of Prematurity

描述(申请人提供)：我们感兴趣的因果关系是氧气的反复波动与早产儿视网膜病变(ROP)和既往无血管视网膜血管形成(VPAR)的玻璃体内新生血管(IVNV)有关。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血管内皮生长因子(VEGF)异构体和受体的失调，创造了一个促进不必要的IVNV和后来失明的微环境。我们认为，在视网膜血管发育的早期，氧气的波动导致血管生成抑制效应，部分原因是血管生成激动剂血管内皮生长因子和血管生成抑制因子色素上皮衍生因子(PEDF)的调节失衡。后来，反复的氧波动导致氧化损伤、细胞凋亡，并通过gp130受体亚单位/转导蛋白下游的信号通路导致进一步的血管内皮生长因子亚型的失调(尤其是病理性的血管内皮生长因子164)。所有这些活动都在宣传IVNV。 我们将采用已建立的“50/10 OIR”模型(新生大鼠在可控的24小时氧气波动条件下饲养并返回室内空气)。具体地说，我们将确定1a)用抗体特异性地阻断主要病理异构体VEGF164是否会减少IVNV并促进VPAR，1b)改变VEGF和PEDF是否会在早期时间点导致血管生成抑制效应(例如，PEDF增加和/或减少)，从而导致延迟的视网膜血管形成，以及在稍后的时间点，在IVNV期间，平衡将发生改变，从而发生血管生成刺激(例如，增加的VEGF164和/或减少的PEDF)，2a)在血管和无血管视网膜的交界处出现的活性氧物种(ROS)增加并导致内皮细胞凋亡，维持外围血管畅通和干扰VPAR，3a)激活gp130激活JAK/STAT通路，增加VEGF164、VEGFR2的表达，促进IVNV；3b)白介素6(IL-6)减少内皮细胞凋亡，促进VPAR，而白血病抑制因子(LIF)则增加内皮细胞的凋亡和IVNV。 方法将包括RT-PCR检测生长因子(VEGF异构体，PEDF)和受体(VEGFR1和VEGFR2)mRNAs；ELISA和Western印迹检测生长因子(VEGF)和磷酸化受体或蛋白(LIFR、gp130、IL-6R、STATS、JAK2、SoCs)的蛋白；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)；测量脂质过氧化和超氧化物损伤(二氢乙锭)；细胞凋亡(激活的caspase-3)和共聚焦显微镜；以及血管和视网膜的定量以及IVNV的时钟。 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.