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 糖尿病视网膜病变（DR）是全球范围内视力丧失和损害的主要原因。糖尿病性黄斑水肿 (DME)由于血-视网膜屏障（BRB）的破坏是导致失明的DR的主要并发症。 尽管抗血管内皮生长因子（VEGF）治疗DME取得了成功，但人们对它知之甚少。 胎盘生长因子（PlGF）在DR中BRB分解中的功能作用。 是一种具有病理性血管生成特性的多功能细胞因子。最近的研究强调了PlGF的作用 在DME中BRB故障。我们最近表明，PlGF敲除（KO）小鼠免受糖尿病- 通过上调几种保护性蛋白质导致BRB分解。新兴的临床研究表明， 阻断PlGF和VEGF的药物aflibercept可防止DME患者的BRB破裂。尽管 这些最新进展中仍有若干问题有待解决。1)选择性PlGF抑制是否足以 预防糖尿病引起的BRB崩溃？2)靶向BRB功能的细胞类型特异性影响是什么 PlGF？3)PlGF是否通过不同于VEGF的机制调节BRB？4)是否有相互作用， PlGF和VEGF（PlGF-VEGF异源二聚体）共同导致DR中BRB分解？5)是什么 血管内皮生长因子受体1信号在人视网膜内皮细胞屏障调节中的作用 功能？这些重要问题的答案将更好地定义PlGF在糖尿病诱导的糖尿病中的因果作用。 BRB故障，这将导致更好的精确针对性治疗DME患者的设计。因此，我们认为， 本提案的目的是解决这些问题。我们的总体假设是靶向PlGF 通过上调保护性蛋白、破坏PlGF-VEGF， 二聚体和周细胞中VEGFR 1的失活。提出了三个具体目标来检验这一假设。Aim-1 是通过靶向DR中的PlGF来确定关键存活或抗氧化蛋白在BRB保护中的作用。 发夹（sh）RNA和单克隆抗体将在体内或体外沉默或阻断PlGF。BRB保护， 存活或抗氧化蛋白将用HREC培养物阐明。目标-2是确定 PlGF-VEGF异二聚体对DR中BRB分解的影响。 VEGF对DR样特征至关重要。胰岛素将治疗糖尿病小鼠以确定低血糖是否诱导 PlGF-VEGF二聚化。一种显性阴性PlGF变体，可与VEGF异源二聚化但不结合 VEGFR 1，将决定PlGF-VEGF在糖尿病诱导的BRB破坏中的作用。目标-3是确定 VEGFR 1信号在周细胞中的作用及其在BRB功能旁分泌调节中的作用。拟议 旁分泌机制将被破译：高葡萄糖触发的周细胞VEGFR 1信号级联反应 (HG)导致VEGF、PlGF和/或VEGF-B表达的上调和VEGFR 1的诱导 磷酸化或活化事件介导HG诱导的周细胞凋亡（通过核因子（NF）-κ B）。 受损的周细胞通过破坏细胞间的平衡而导致视网膜EC屏障功能障碍。 血管生成素（Ang）-1和Ang-2。