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

视网膜内皮细胞的氧传感机制作为抑制缺血性新生血管的新靶点

• 批准号: 10436853
• 负责人: Guo-Hua Fong
• 金额: $ 58.85万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10436853
• 关键词: Adult; Affect; American; Angiogenic Factor; Antibodies; Antibody Therapy; Apoptosis; Astrocytes; Binding; Biochemical; Biological Availability; Blindness; Blood; Blood Vessels; Cell Hypoxia; Cell Proliferation; Cells; Data; Development; Endothelial Cells; Endothelium; Extravasation; Genetic Transcription; Growth; Hepatocyte; Hydroxylation; Hypoxia; Hypoxia Inducible Factor; Injections; Innovative Therapy; Lead; Lentivirus Vector; Literature; Location; Mediating; Membrane; Mitogens; Modeling; Molecular; Mus; Mutation; Oxygen; Paracrine Communication; Pathologic; Pathologic Neovascularization; Patients; Phenotype; Point Mutation; Polyubiquitination; Procollagen-Proline Dioxygenase; Proteins; Proxy; Publishing; Refractory; Regulatory Element; Resistance; Response Elements; Retina; Retinal Detachment; Retinal Diseases; Retinal Neovascularization; Retinopathy of Prematurity; Site; Small Interfering RNA; Testing; Tissues; Traction; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors; Vision; angiogenesis; antiangiogenesis therapy; bevacizumab; design; experimental study; improved; insight; keratinocyte; mouse model; neovascular; neovascularization; novel; novel strategies; novel therapeutic intervention; overexpression; paracrine; proliferative diabetic retinopathy; receptor; response; retinal angiogenesis; retinal neuron; scaffold; side effect; vector

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.

项目摘要 糖尿病视网膜病变（PDR）是成人失明的主要原因，主要是由于 缺血和缺氧视网膜组织中的新生血管形成。虽然每月注射抗VEGF提供了缓解， 它与主要的副作用如视网膜脱落有关，超过三分之一的患者是难治性的 抗体治疗。本项目的长期目标是研究内皮氧是如何 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的形成。 缺血视网膜组织通常，缺氧通过旁分泌机制驱动血管生成，其中缺氧- 诱导因子-α蛋白上调血管生成因子的表达，从而激活其同源物， 受体在附近的内皮细胞（EC）。在氧合良好的组织中，HIF-α蛋白迅速降解 在通过含脯氨酰羟化酶结构域的蛋白质（PHD 1， PHD 2和PHD 3）。因此，PHDs的缺陷通过允许HIF-α蛋白在分子水平上模拟缺氧， 积累。事实上，我们发表的研究表明，出生后PHD 2缺乏症在全球或非 内皮细胞促进血管生长或存活。在最近的初步研究中，我们做了一个相当 EC特异性PHD 2缺陷（Phd 2 EC-/-）抑制而不是促进视网膜色素变性 血管发育，尽管HIF-1α和HIF-2α的高水平积累。另一方面，Flt-1是 上调，这是已知的螯合VEGF-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，Phd 2 EC-/-小鼠也表现出极大的降低 新生血管形成这些发现导致了我们的中心假设，即视网膜血管生成是 由两种相反的氧传感机制动态调节：在视网膜实质的细胞中， 缺氧通过HIF依赖性血管生成因子的表达激活血管生成;而在视网膜中， 在内皮细胞中，缺氧通过上调抗血管生成分子如Flt-1抑制血管生成。目标1.探讨 内皮氧传感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特异性缺失可挽救Phd 2 EC-/-小鼠视网膜血管发育; 耐氧但其他方面正常的HIF-1α和HIF-2α的积累抑制血管生成; HIF-1α和HIF-2α是否直接与Flt-1调控元件相互作用;目的2.调查是否 调节发育性血管生成的氧传感机制也在缺血性心脏病期间起作用。 OIR模型中的新血管形成。目标3.测试慢病毒载体介导的抗血管生成策略。 将构建表达PHD siRNA或膜锚定截短Flt-1（tFlt-1）的慢病毒载体， 所有这些在空间上都局限于视网膜EC，并且可能进一步局限于缺血性视网膜位置中的EC。的能力 将在OIR模型中评估这些载体抑制缺血性视网膜新生血管的作用。这些 研究可能潜在地导致抑制与PDR相关的新血管形成的创新疗法。