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)是世界范围内导致视力丧失和损害的主要原因。糖尿病黄斑水肿 血视网膜屏障(BRB)破坏导致的DME是DR导致失明的主要并发症。 尽管抗血管内皮生长因子(VEGF)治疗DME取得了成功，但人们对此知之甚少 胎盘生长因子在血管内皮细胞生长因子亚家族成员中的BRB分解中的功能作用 家族是一种多功能的细胞因子，具有病理性的血管生成特性。最近的研究强调了PlGF的作用 在二甲基醚的溴化氢分解中。我们最近表明，PlGF基因敲除(KO)小鼠可以预防糖尿病- 通过上调几种保护蛋白而导致BRB的分解。新出现的临床研究表明， 同时阻断PlGF和VEGF的药物afLibercept防止了DME患者BRB的分解。尽管 这些最新的进展有几个问题有待解决。1)选择性抑制PlGF是否足以 预防糖尿病引起的BRB分解？2)靶向对BRB功能的特定细胞类型的影响是什么 PlGF？3)PlGF是否通过与血管内皮生长因子不同的机制调节BRB？4)两者之间是否存在相互作用 PlGF和VEGF(PlGF-VEGF异源二聚体)共同导致DR中BRB的崩溃？5)什么是 血管内皮生长因子受体(VEGFR1)信号在人视网膜内皮细胞屏障调节中的作用 功能呢？这些重要问题的答案将更好地确定PlGF在糖尿病诱导中的因果作用 BRB分解将导致为DME患者设计更精确的靶向治疗。因此， 这项提议的目的就是解决这些问题。我们的总体假设是靶向PlGF 通过上调保护蛋白、破坏PlGF-VEGF来预防糖尿病引起的BRB分解 周细胞中VEGFR1的失活。为了检验这一假说，本文提出了三个具体目标。AIM-1 是通过靶向PlGF来确定关键的生存或抗氧化蛋白在BRB保护中的作用 发夹(Sh)RNA和单抗在体内或体外都能沉默或阻断PlGF。BRB的保护由 存活或抗氧化蛋白将通过HREC培养来阐明。目标2是确定以下因素的贡献 PlGF-VEGF异源二聚体对Dr.PlGF KO小鼠BRB分解的影响将被用来确定PlGF是否 血管内皮细胞生长因子对类DR功能至关重要。胰岛素将治疗糖尿病小鼠以确定低血糖是否会导致 PlGF-VEGF二聚化。显性阴性的PlGF变异体，可与血管内皮细胞生长因子异源二聚，但不结合 VEGFR1，将确定PlGF-VEGF在糖尿病诱导的BRB分解中的作用。目标3是确定 VEGFR1信号在周细胞中的作用及其在旁分泌调节BRB功能中的作用建议数 旁分泌机制(S)将被破译：高糖触发周细胞血管内皮生长因子受体信号级联 (HG)上调血管内皮细胞生长因子和/或血管内皮细胞生长因子-B的表达并诱导血管内皮细胞生长因子1 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)-ĸB)。 受损的周细胞通过破坏视网膜EC屏障之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。