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）的特征是血管丢失，随后在非血管化边界出现血管过度化。这些血管不能生长并挽救缺氧的视网膜，并被误导到玻璃体，形成收缩带和视网膜脱离。如果缺氧视网膜能正常重建血管，就不会发生破坏性的新生血管形成。错误的NV被认为是由于缺氧的视网膜释放出高水平的促血管生成因子进入玻璃体所致。然而，在缺氧的视网膜中，含量甚至更高。我们假设来自应激神经元的血管排斥因子阻止了血运重建。神经元的引导信号，信号素（Sema）和它们的受体神经匹林（Nrp）可能与血管共享，以指导这种错误的生长。Nrp-1结合对立因子Sema3A和VEGF。Sema3A可诱导EC细胞凋亡，抑制VEGF依赖性趋化，介导血管与神经元间的串扰。假设：应激神经节细胞（RGC）产生Sema3A，通过EC凋亡促进血管损失，并排斥从缺氧视网膜向玻璃体生长的新血管。抑制Sema3A促进血运重建和预防视网膜病变。我们初步发现，Sema3A在高血糖应激RGCs和氧诱导视网膜病变（OIR）中被诱导。Sema3A在玻璃体增生dr中升高bbbb25倍，阻断OIR RGCs中的Sema3A可抑制血管丧失（VO），改善缺氧视网膜的血运重建，降低病理性NV，为治疗视网膜病变提供了一种全新的方法。我们将在糖尿病模型、OIR、体外（RGCs和ECs）和体外（O2和葡萄糖应激的主动脉移植体模拟视网膜病变的I、II期）中验证这一假设。我们将：AIM 1在DR， OIR中暂时定量和定位视网膜Sema3A， Nrp1， VEGF。在DR中，AIM - 2、OIR检测视网膜或RGCs中Sema3A的抑制是否抑制了突变的Nrp-1结合VEGF但不结合Sema3A的小鼠和慢病毒（Lv）驱动shRNA靶向RGCs中Sema3A的WT小鼠的视网膜病变。AIM 3在体外检测Sema3A和VEGF对血管引导和EC凋亡的竞争，并确定分子信号机制。提出的研究具有开创性，因为它们将确定神经元对血管变性的影响以及血管排斥信号在视网膜病变中的重要性。我们的研究结果表明，Sema3A-Nrp轴可能是一个有吸引力的治疗靶点，可以促进视网膜病变和其他病变（如癌症和脑血管梗死）的血管再生，其中血管再生是损伤区域的关键决定因素。

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.