The Oxygen Sensing Mechanism in Retinal Endothelial Cells as a Novel Target to Suppress Ischemic Neovascularization

项目摘要 增殖性糖尿病视网膜病变(PDR)是成人致盲的主要原因，主要原因是 缺血和缺氧性视网膜组织中的新生血管。虽然每月注射抗血管内皮生长因子可以缓解病情， 它与视网膜脱离等主要副作用有关，超过三分之一的患者是难治性的。 到抗体治疗。该项目的长期目标是研究内皮细胞氧气是如何 传感机制调节视网膜血管生成，并开发新的策略来抑制新生血管 缺血的视网膜组织。通常，低氧通过旁分泌机制推动血管生成，其中低氧- 诱导因子-α蛋白上调血管生成因子的表达，从而激活其同源因子 邻近内皮细胞(EC)上的受体。在氧气充足的组织中，低氧诱导因子-α蛋白迅速降解 在通过含有Pro羟基酶结构域的蛋白质(PHD1， PHD2和PHD3)。因此，PhDS的缺乏在分子水平上模拟了缺氧，因为它允许HIF-α蛋白 积累。事实上，我们发表的研究表明，全球或非产后PHD2缺乏症 内皮细胞促进血管生长或存活。在最近的初步研究中，我们做了相当多的 意外观察到EC特异性PHD2缺陷(Phd2EC-/-)抑制而不是促进视网膜 血管发育，尽管高水平积累HIF-1α和HIF-2α。另一方面，Flt-1是 上调，这是已知的隔离血管内皮生长因子-A。在氧诱导视网膜病变(OIR)的小鼠模型中， 作为PDR和ROP(早产儿视网膜病变)的代表，Phd2EC-/-小鼠的表现也大大减少 新生血管。这些发现导致了我们的中心假说，视网膜血管生成是 由两种相反的氧气感知机制动态调节：在视网膜实质的细胞中， 低氧通过HIF依赖的血管生成因子的表达激活血管生成；而在视网膜 缺氧通过上调Flt-1等抗血管生成分子来抑制血管生成。目标1.调查 内皮氧感应机制如何调节视网膜血管发育。检查EC是否 HIF-1α、HIF-2α或Flt-1的特异性缺失可挽救Phd2EC-/-小鼠视网膜血管的发育； 耐氧但正常的缺氧诱导因子-1α和缺氧诱导因子-2α的积累抑制了血管生成； 缺氧诱导因子-1α和缺氧诱导因子-2α是否直接与Flt-1调控元件相互作用 氧感应机制调节发育中的血管生成也在缺血时起作用 OIR模型中的新生血管。目的3.检测慢病毒载体介导的抗血管生成策略。 将构建表达PhD siRNAs或膜锚定截短Flt-1(tFlt-1)的慢病毒载体， 在空间上均局限于视网膜内皮细胞，并可能进一步局限于视网膜缺血区的内皮细胞。一种能力 在这些载体中，抑制缺血视网膜新生血管的作用将在OIR模型中进行评估。这些 研究可能会导致一种创新的治疗方法，以抑制与PDR相关的新生血管。

Project Summary Proliferative diabetic retinopathy (PDR) is a leading cause of blindness in adults, mostly due to neovascularization in ischemic and hypoxic retinal tissues. While monthly injection of anti-VEGF provides relief, it is associated with major side effect such as retinal detachment, and over a third of the patients are refractory to the antibody treatment. The long term objective of this project is to investigate how the endothelial oxygen sensing mechanism regulates retinal angiogenesis, and develop novel strategies to curb neovascularization in ischemic retinal tissues. Typically, hypoxia drives angiogenesis by paracrine mechanisms wherein hypoxia- inducible factor-α proteins upregulate the expression of angiogenic factors, which activate their cognate receptors on nearby endothelial cells (EC). In well oxygenated tissues, HIF-α proteins degrade rapidly following oxygen dependent prolyl hydroxylation by prolyl hydroxylase domain containing proteins (PHD1, PHD2, and PHD3). Thus, deficiency in PHDs mimics hypoxia at the molecular level by allowing HIF-α proteins to accumulate. Indeed, our published studies demonstrate that post-natal PHD2 deficiency globally or in non- endothelial cells promotes vascular growth or survival. In recent Preliminary Studies, we made a rather unexpected observation that EC specific PHD2 deficiency (Phd2EC-/-) inhibited instead of promoting retinal vascular development, despite high level accumulation of HIF-1α and HIF-2α. On the other hand, Flt-1 was upregulated, which is known to sequester VEGF-A. In the mouse model of oxygen-induced retinopathy (OIR), a proxy of PDR as well as ROP (retinopathy of prematurity), Phd2EC-/- mice also displayed greatly reduced neovascularization. These findings led to our Central Hypothesis stating that retinal angiogenesis is dynamically regulated by two opposing oxygen sensing mechanisms: in the cells of the retinal parenchyma, hypoxia activates angiogenesis by HIF- dependent expression of angiogenic factors; whereas in the retinal ECs, hypoxia inhibits angiogenesis by upregulating anti-angiogenic molecules such as Flt-1. Aim 1. Investigate how the endothelial oxygen sensing mechanism regulates retinal vascular development. Examine whether EC specific deletion of HIF-1α, HIF-2α, or Flt-1 rescues retinal vascular development in Phd2EC-/- mice; whether the accumulation of oxygen resistant but otherwise normal HIF-1α and HIF-2α suppresses angiogenesis; and whether HIF-1α and HIF-2α directly interact with Flt-1 regulatory elements; Aim 2. Investigate whether the oxygen sensing mechanisms regulating developmental angiogenesis also operate during ischemic neovascularization in the OIR model. Aim 3. Test lentiviral vector-mediated anti-angiogenesis strategies. Lentiviral vectors will be constructed that express PHD siRNAs or membrane - anchored truncated Flt-1 (tFlt-1), all spatially restricted to retinal ECs and likely further restricted to ECs in ischemic retinal locations. The ability of these vectors to suppress ischemic retinal neovascularization will be evaluated in the OIR model. These studies may potentially lead to an innovative therapy to suppress neovascularization associated with PDR.