Neuronal guidance molecules control revascularization in retinopathy

描述（由申请人提供）：糖尿病视网膜病变（DR）和早产儿视网膜病变（ROP）的特征是血管损失，随后是非血管化边缘的过度血管化。这些血管不能生长到缺氧的视网膜中并拯救缺氧的视网膜，并且错误地指向玻璃体，形成收缩带和视网膜脱离。缺氧性视网膜如果能适当地血管化，就不会发生破坏性的新生血管（NV）。被误导向的NV被认为是由缺氧视网膜释放到玻璃体中的高水平促血管生成因子引起的。然而，在缺氧视网膜中的水平甚至更高。我们推测，血管排斥因子从应激神经元阻止血管重建。神经元引导线索，脑信号蛋白（Semaphorins，Sema）及其受体神经纤毛蛋白（Neuropilin，Nrp）可能与血管共享，以指导这种误导性的生长。Nrp-1结合相反的因子Sema 3A和VEGF。Sema 3A可引起EC凋亡，抑制VEGF依赖的趋化性，并可介导血管和神经元之间的串扰。假设：应激的神经节细胞（RGC）产生Sema 3A，其通过EC凋亡促进血管损失，并将生长的新血管从缺氧的视网膜排斥到玻璃体。抑制Sema 3A可促进血管再生并预防视网膜病变。因此，我们发现Sema 3A在高血糖应激的RGC中以及在氧诱导的视网膜病变（OIR）中被诱导。在具有增殖性DR的玻璃体中，Sema 3A升高> 25倍。在OIR中阻断RGCs中的Sema 3A抑制血管损失（VO），改善缺氧视网膜的再血管化并减少病理性NV，这表明了治疗视网膜病变的全新方法。我们将在糖尿病模型、OIR、体外（RGC和EC）和离体使用O2和葡萄糖应激模拟视网膜病变I、II期的主动脉外植体来检验这一假设。我们将：AIM 1在时间上定量和定位DR、OIR中的视网膜Sema 3A、Nrp 1、VEGF 13 DR、OIR中的AIM 2确定视网膜或RGCs中的Sema 3A抑制是否抑制具有结合VEGF但不结合Sema 3A的突变Nrp-1的小鼠和具有靶向RGCs中的Sema 3A的慢病毒（Lv）驱动的shRNA的WT小鼠中的视网膜病变。目的3体外研究Sema 3A和VEGF在血管导向和内皮细胞凋亡中的竞争作用，探讨其分子信号转导机制。拟议的研究是开创性的，因为它们将建立神经元对血管变性的影响以及血管排斥线索在视网膜病变中的重要性。我们的结果表明，Sema 3A-Nrp轴可能是一个有吸引力的治疗靶点，以促进视网膜病变和其他病理学（如癌症和脑血管梗死）的血管再生，其中血管再生是损伤面积的关键决定因素。 公共卫生相关性：糖尿病视网膜病变和早产儿视网膜病变是导致失明的主要原因，并严重降低生活质量。目前的激光治疗破坏缺氧的神经视网膜，以防止血管生长因子的释放。我们需要从根本上解决视网膜病变的病理学问题，以促进正常的血管生长。这项拟议的工作将解决操纵神经元的指导线索，以促进视网膜血管重建。

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. PUBLIC HEALTH RELEVANCE: Diabetic retinopathy and retinopathy of prematurity are leading causes of blindness and severely reduce the quality of life. Current laser treatment destroys hypoxic neural retina to prevent release of vascular growth factors. We need to more fundamentally address the pathology of retinopathy to promote normal vascular growth. This proposed work will address manipulation of neuronal guidance cues to promote revascularization of the retina.