Regulation of blood-retina barrier by placental growth factor

胎盘生长因子对血视网膜屏障的调节

• 批准号: 10187438
• 负责人: Hu Huang
• 金额: $ 7.53万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10187438
• 关键词: ANGPT1 gene; Address; Adhesions; Angiopoietin-2; Antibodies; Antioxidants; Apoptosis; Background Diabetic Retinopathy; Binding; Blindness; Blood Glucose; Blood-Retinal Barrier; Cattle; Cell Culture Techniques; Cells; Clinical Research; Complications of Diabetes Mellitus; Conditioned Culture Media; Diabetes Mellitus; Diabetic Retinopathy; Diabetic mouse; Dimerization; Dominant-Negative Mutation; Electrical Resistance; Endothelial Cells; Endothelial Growth Factors Receptor; Equilibrium; Event; Extravasation; Family; Functional disorder; Gene Silencing; Glucose; Glutathione S-Transferase P; Growth Factor Gene; Growth Factor Inhibition; Heterodimerization; Human; Hypoglycemia; Immunoglobulin G; In Vitro; Incubated; Insulin; KDR gene; Knockout Mice; Ligands; Mannitol; Measurement; Measures; Mediating; Monoclonal Antibodies; Mus; NF-kappa B; Nuclear; Oxidative Stress; PF4 Gene; PGF gene; Pathologic; Patients; Pericytes; Permeability; Pharmaceutical Preparations; Phosphorylation; Point Mutation; Pregnancy Proteins; Production; Property; Proteins; Receptor Signaling; Regulation; Retina; Role; SHH gene; Signal Transduction; Site-Directed Mutagenesis; Small Interfering RNA; Testing; Time; Tyrosine; Up-Regulation; Variant; Vascular Endothelial Growth Factor B; Vascular Endothelial Growth Factors; Visual impairment; activating transcription factor; adeno-associated viral vector; base; cadherin 5; cell type; cytokine; design; diabetic; dimer; electric impedance; fluorescein isothiocyanate dextran; in vivo; intravitreal injection; macular edema; member; monolayer; oxidative damage; paracrine; peroxiredoxin; prevent; protein expression; receptor function; receptor-mediated signaling; small hairpin RNA; success

Project Summary Diabetic retinopathy (DR) is a leading cause of vision loss and impairment worldwide. Diabetic macular edema (DME) as a result of blood-retinal barrier (BRB) breakdown is a major complication of DR leading to blindness. Despite the success of anti-vascular endothelial growth factor (VEGF) therapy on DME, little is known about placental growth factor (PlGF)'s functional role in BRB breakdown in DR. PlGF, a member of the VEGF sub- family, is a multifunctional cytokine with pathological angiogenic properties. Recent studies highlight PlGF's role in BRB breakdown in DME. We recently showed that PlGF knockout (KO) mice were protected from diabetes- caused BRB breakdown by upregulating several protective proteins. Emerging clinical studies showed that the drug aflibercept, which blocks both PlGF and VEGF, prevented BRB breakdown in DME patients. Despite these recent advances several questions remain to be addressed. 1) Is selective PlGF inhibition sufficient to prevent diabetes-caused BRB breakdown? 2) What is the cell type-specific effect on BRB function by targeting PlGF? 3) Does PlGF regulate BRB by mechanisms distinct from VEGF? 4) Are there interactions between PlGF and VEGF (PlGF-VEGF heterodimers) that together contribute to BRB breakdown in DR? 5) What is the role of VEGF receptor (VEGFR1) signaling in the regulation of human retinal endothelial cell (HREC) barrier function? The answers to these important questions will better define PlGF's causal role in diabetes-induced BRB breakdown that will lead to the design of better precision-targeted treatments for DME patients. Therefore, the objective of this proposal is to address these questions. Our overall hypothesis is that targeting PlGF prevents diabetes-caused BRB breakdown via upregulation of protective proteins, disruption of PlGF-VEGF dimers, and inactivation of VEGFR1 in pericyte. Three specific aims are proposed to test the hypothesis. Aim-1 is to determine the role of key survival or antioxidant proteins in BRB protection by targeting PlGF in DR. Small hairpin (sh) RNA and monoclonal antibody will silence or block PlGF in vivo or in vitro. The BRB protection by survival or antioxidant proteins will be elucidated with HREC culture. Aim-2 is to determine the contribution of PlGF-VEGF heterodimers on BRB breakdown in DR. PlGF KO mice will be used to determine if PlGF is essential for DR-like features by VEGF. Insulin will treat diabetic mice to determine if hypoglycemia induces PlGF-VEGF dimerization. A dominant-negative PlGF variant, which can heterodimerize with VEGF but not bind VEGFR1, will determine the role of PlGF-VEGF in diabetes-induced BRB breakdown. Aim-3 is to determine the role of VEGFR1 signaling in pericyte and its role in paracrine regulation of BRB function. The proposed paracrine mechanism(s) will be deciphered: pericyte VEGFR1 signaling cascades triggered by high glucose (HG) leads to upregulation of VEGF, PlGF, and/or VEGF-B expression and induction of VEGFR1 phosphorylation or activation events that mediate HG-induced pericyte apoptosis (via nuclear factor (NF)-ĸB). The damaged pericytes contribute to retinal EC barrier dysfunction by disrupting the balance between Angiopoietin (Ang)-1 and Ang-2.

项目摘要 糖尿病性视网膜病（DR）是全球视力丧失和障碍的主要原因。糖尿病黄斑水肿 （DME）由于血红视网膜屏障（BRB）的破坏是导致失明的主要并发症。 尽管抗血管内皮生长因子（VEGF）在DME上取得了成功，但对 胎盘生长因子（PLGF）在DR中的BRB分解中的功能作用。 PLGF，VEGF子的成员 家族是具有病理性血管生成特性的多功能细胞因子。最近的研究突出了PLGF的角色 在DME中的BRB分解中。我们最近表明，PLGF敲除（KO）小鼠受到保护免受糖尿病的保护。 通过上调几种受保护的蛋白质引起BRB分解。新兴的临床研究表明 阻止PLGF和VEGF的药物Aflibercept阻止了DME患者的BRB分解。尽管 这些最近的进步仍有一些问题待解决。 1）选择性PLGF抑制足以 防止糖尿病引起的BRB崩溃？ 2）通过靶向对BRB功能的细胞类型特异性影响是什么 PLGF？ 3）PLGF是否通过与VEGF不同的机制调节BRB？ 4）是否之间存在相互作用 PLGF和VEGF（PLGF-VEGF异二聚体）共同导致DR中的BRB分解？ 5）什么是 VEGF受体（VEGFR1）信号在人类永久内皮细胞（HREC）屏障调节中的作用 功能？这些重要问题的答案将更好地定义PLGF在糖尿病引起的因果关系 BRB分解将导致设计为DME患者设计更好的精确靶向治疗方法。所以， 该提案的目的是解决这些问题。我们的总体假设是针对PLGF 通过上调受保护的蛋白质，PLGF-VEGF的破坏，可以防止糖尿病引起的BRB分解 二聚体和周围vegfr1的灭活。提出了三个特定目标来检验该假设。 AIM-1 是通过针对DR中的PLGF来确定关键生存或抗氧化剂蛋白在BRB保护中的作用。小的 发夹（SH）RNA和单克隆抗体将在体内或体外阻止PLGF。 BRB保护 将用HREC培养阐明生存或抗氧化剂蛋白。 AIM-2是确定 DR中的BRB分解的PLGF-VEGF异二聚体。 PLGF KO小鼠将用于确定PLGF是否为 VEGF的类似DR的功能必不可少的。胰岛素将治疗糖尿病小鼠，以确定低血糖影响是否影响 PLGF-VEGF二聚体。一个主导的阴性PLGF变体，可以与VEGF异二聚体，但不能结合 VEGFR1将确定PLGF-VEGF在糖尿病诱导的BRB分解中的作用。 AIM-3是确定 VEGFR1信号在周细胞中的作用及其在BRB功能的旁分泌调节中的作用。提议 将决定旁分泌机制：高葡萄糖触发的周细胞VEGFR1信号传导级联 （HG）导致VEGF，PLGF和/或VEGF-B表达的上调以及VEGFR1的诱导 介导Hg诱导的周细胞凋亡的磷酸化或激活事件（通过核因子（NF）-YB）。 受损的周细胞通过破坏在 Angiopietin（Ang）-1和Ang-2。