Regulation of retinal angiogenesis and vascular integrity by the oxygen sensing mechanism

 描述(申请人提供)：血管生成的研究主要是通过检测细胞外配体对内皮细胞的刺激(或抑制)来进行的，而对内皮细胞与其周围组织环境之间的联系的关注要少得多。在本项目中，我们将重点研究氧感应机制如何在视网膜血管形态发生过程中协调星形细胞和血管通讯，并探索其在保护氧诱导视网膜病变(OIR)和糖尿病视网膜病变两种模型中保护视网膜微血管完整性的治疗潜力。在初步研究中，我们获得了新生的视网膜血管驱动视网膜星形细胞前体细胞(APC)分化为成熟星形胶质细胞(MASCS)并形成星形细胞网络的具体证据。具体地说，我们假设来自视网膜血管内皮细胞的白血病抑制因子(LIF)和循环中的氧气可能作用于APC上调Pro羟基酶结构域蛋白2(PHD2)的表达和活性，PHD2反过来催化HIF-a蛋白的Pro羟基化，标记它们进行多泛素化和蛋白酶体降解。此外，我们认为HIF-2a和TLX(孤儿核受体家族的转录因子)可能形成一个正反馈环，其中TLX抑制多泛素化和羟化HIF-2a的降解，而HIF-2a促进TLX基因的转录。因此，PHD2下调HIF-2a蛋白丰度可能会触发HIF-2a和TLX水平的螺旋式下降，导致星形胶质细胞从其祖细胞成熟。另一方面，我们的研究表明，在OIR或糖尿病模型中，PHD2的丢失可能通过稳定HIF-2a来帮助保护视网膜血管的完整性。我们将从三个具体目标来调查这些问题。目的1.确定LIF是否通过氧感应机制调节星形胶质细胞和血管的发育。我们将在体内评估LIF是否上调PHD2的表达，促进APC(促血管生成)向MASCS(非血管生成)分化，从而抑制视网膜血管生成。目的2.研究HIF-2a和TLX之间的正反馈环是否促进APC状态和视网膜血管生成。目的3.探讨氧感受通路在小鼠OIR和糖尿病视网膜病变模型中的治疗潜力。我们将确定MASC特异性PHD2缺陷、全局或EC特异性LIF缺陷或LIF拮抗剂是否保护视网膜微血管免受高氧或糖尿病诱导的损伤。综上所述，这些研究旨在提供对血管和星形胶质细胞通讯的机械性见解，并揭示治疗早产儿和糖尿病视网膜病变的新的治疗机会。

 DESCRIPTION (provided by applicant): Angiogenesis has been mostly studied by examining stimulation (or inhibition) of endothelial cells (ECs) by extracellular ligands, with much less attention being paid to communications between ECs and their surrounding tissue environment. In this project we will focus on how the oxygen sensing mechanism coordinates astrocytic and vascular communication during retinal vascular morphogenesis and explore its therapeutic potentials in protecting retinal microvascular integrity in two models including oxygen induced retinopathy (OIR) and diabetic retinopathy. In preliminary studies we obtained concrete evidence that nascent retinal blood vessels drive retinal astrocytic progenitors (APCs) to differentiate into mature astrocytes (mASCs) and form an astrocytic network. Specifically, we hypothesize that leukemia inhibitory factor (LIF) from retinal vascular ECs and oxygen from the circulation may act on APCs to upregulate the expression and activity of prolyl hydroxylase domain protein 2 (PHD2), which in turn catalyzes prolyl hydroxylation of HIF-a proteins, tagging them for polyubiquitination and proteasomal degradation. Furthermore, we propose that HIF-2a and Tlx (a transcription factor in the orphan nuclear receptor family) may form a positive feedback loop wherein Tlx suppresses polyubiquitination and degradation of hydroxylated HIF-2a whereas HIF-2a promotes the transcription of the Tlx gene. Thus, downregulation of HIF-2a protein abundance by PHD2 might trigger a downward spiral of both HIF-2a and Tlx levels, leading to astrocyte maturation from their progenitors. On the other hand, our studies indicate that loss of PHD2 may be targeted to help preserve retinal vascular integrity in OIR or diabetes models, likely by stabilizing HIF-2a. We will investigate these issues in three specific aims. Aim 1. Determine if LIF regulates astrocyte and vascular development through oxygen sensing mechanisms. We will evaluate in vivo if LIF upregulates Phd2 expression, promotes the differentiation of APCs (proangiogenic) to mASCs (nonangiogenic), thus suppressing retinal angiogenesis. Aim 2. Investigate if a positive feedback loop between HIF-2a and Tlx promotes APC status and retinal angiogenesis. Aim 3. Explore therapeutic potentials of the oxygen sensing pathway in mouse OIR and diabetic retinopathy models. We will determine if mASC specific PHD2 deficiency, global or EC specific LIF deficiency, or LIF antagonist protects retinal microvessels from hyperoxia or diabetes-induced injuries. In summary, these studies are designed to provide mechanistic insights into vascular and astrocyte communication and reveal novel therapeutic opportunities to treat retinopathy of the prematurity and diabetic retinopathy.