Neuronal guidance molecules control revascularization in retinopathy

描述(申请人提供)：糖尿病视网膜病变(DR)和早产儿视网膜病变(ROP)的特征是血管丢失，随后在非血管化缘出现高血管新生。这些血管不能生长到和挽救缺氧的视网膜，并被错误地定向到玻璃体，形成收缩带和视网膜脱离。如果缺氧性视网膜血管重建良好，就不会发生破坏性新生血管(NV)。误导NV被认为是由于高水平的促血管生成因子从缺氧的视网膜释放到玻璃体中。然而，在缺氧的视网膜中，这一水平甚至更高。我们假设，来自应激神经元的血管排斥因子阻止了血管重建。神经引导线索、信号素(SEMA)及其受体Neuropilin(NRP)可能与血管共享，以指导这种错误的生长。NRP-1结合相反的因子Sema3A和VEGF。Sema3A可诱导内皮细胞凋亡，抑制血管内皮生长因子依赖的趋化作用，并可介导血管与神经元之间的串扰。假设：应激神经节细胞(RGC)产生Sema3A，它通过EC凋亡促进血管丢失，并将生长的新生血管从缺氧的视网膜排斥到玻璃体。抑制Sema3A可促进血管重建，预防视网膜病变。我们初步发现，在高血糖应激性视网膜节细胞和氧源性视网膜病变(OIR)中均可诱导Sema3A的表达。玻璃体中Sema3A升高-GT；25X；增生性DR。在OIR中阻断RGCs中的Sema3A可抑制血管丢失(VO)，改善缺氧视网膜的血管重建，并减少病理性NV，提示一种治疗视网膜病变的根本新方法。我们将在糖尿病模型中、在OIR、体外(RGCs和ECs)和体外用O2和葡萄糖应激的主动脉移植来验证这一假设，模拟视网膜病变的I、II期。我们将：目标1对DR、OIR患者的视网膜Sema3A、Nrp1、VEGF进行时间定量和定位。目的2在DR，OIR中，确定在视网膜或RGC中抑制Sema3A是否抑制突变NRP-1结合VEGF而不是Sema3A的小鼠视网膜病变，以及在WT小鼠中慢病毒(Lv)驱动的针对RGCs中Sema3A的shRNA是否抑制视网膜病变。目的3体外检测Sema3A与血管内皮生长因子(VEGF)在血管导向和内皮细胞凋亡方面的竞争，探讨其分子信号转导机制。拟议的研究具有开创性，因为它们将确定神经元对血管退行性变的影响，以及血管排斥信号在视网膜病变中的重要性。我们的结果表明，Sema3A-NRP轴可能是一个有吸引力的治疗靶点，用于促进视网膜病变和其他病理疾病(如癌症和脑血管梗塞)的血管重建，在这些疾病中，血管重新生长是损伤面积的关键决定因素。

DESCRIPTION (provided by applicant): Diabetic retinopathy (DR) and retinopathy of prematurity (ROP) are characterized by vessel loss followed by hyper-vascularization at the non-vascularized border. These vessels fail to grow into and rescue hypoxic retina and are misdirected towards the vitreous with formation of contractile bands and retinal detachment. If hypoxic retina could revascularize properly, destructive neovascularization (NV) would not occur. The misdirected NV is thought to result from high levels of pro-angiogenic factors released from hypoxic retina into the vitreous. Yet levels are even higher in hypoxic retina. We hypothesize that vaso-repulsive factors from stressed neurons prevent revascularization. Neuronal guidance cues, Semaphorins (Sema) and their receptor Neuropilin (Nrp) might be shared with vessels to direct this misguided growth. Nrp-1 binds opposing factors Sema3A and VEGF. Sema3A provokes EC apoptosis, inhibits VEGF dependant chemotaxis and could mediate cross talk between vessels and neurons. Hypothesis: Stressed ganglion cells (RGC) produce Sema3A which promotes vessel loss via EC apoptosis and repels growing neovessels from hypoxic retina towards vitreous. Suppressing Sema3A promotes revascularization and prevents retinopathy. Preliminarily we find that Sema3A is induced in stressed RGCs by hyperglycemia as well as in oxygen- induced retinopathy (OIR). Sema3A is elevated >25X in vitreous with proliferative DR. Blocking Sema3A in RGCs in OIR suppresses vascular loss (VO), improves revascularization of hypoxic retina and decreases pathological NV, suggesting a fundamentally new approach to treat retinopathy. We will test this hypothesis in diabetes models, in OIR, in vitro (RGCs and ECs) and ex vivo with aortic explants with O2 and glucose stress simulating phases I, II of retinopathy. We will: AIM 1 temporally quantify and localize retinal Sema3A, Nrp1, VEGF in DR, OIR. AIM 2 in DR, OIR determine if Sema3A suppression in retina or in RGCs suppresses retinopathy in mice with mutant Nrp-1 binding VEGF but not Sema3A and in WT mice with lentivirus (Lv)-driven shRNA targeting Sema3A in RGCs. AIM 3 in vitro determine competition between Sema3A and VEGF on vessel guidance and EC apoptosis and determine molecular signaling mechanisms. The proposed studies are pioneering as they would establish neuronal influence on vaso-degeneration and the importance of vascular repulsive cues in retinopathy. Our results suggest that the Sema3A-Nrp axis is likely to be an attractive therapeutic target to promote revascularization in retinopathy and other pathologies such as cancer and cerebral- vascular infarcts where vascular re-growth is a key determinant of area of injury.