Antiangiogenic action 16k hPRL in retinal microvessels

16k hPRL 在视网膜微血管中的抗血管生成作用

• 批准号: 7171798
• 负责人: RICHARD Ira WEINER
• 金额: $ 31.31万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7171798
• 关键词: Adenovirus Vector; Adenoviruses; Adrenal Glands; Adult; Affect; Aftercare; Age; Age related macular degeneration; Angiogenesis Inhibitors; Animals; Apoptosis; Apoptotic; Area; Biological Assay; Blindness; Blood Vessels; Blood capillaries; Blood-Retinal Barrier; Bos taurus; CD36 gene; Capillary Endothelial Cell; Cattle; Cell Proliferation; Cell membrane; Cell model; Cells; Child; Cicatrix; Co-Immunoprecipitations; Computer software; Cyclophosphamide/Fluorouracil/Prednisone; Degenerative Disorder; Development; Diabetes Mellitus; Diabetic Retinopathy; Disease; Edema; Elderly; Endothelial Cells; Etiology; Eye; Fibroblast Growth Factor 2; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; Hemorrhage; Histologic; Human; Image; Immunoblotting; In Vitro; Induction of Apoptosis; Injection of therapeutic agent; Label; Life; Light Coagulation; MAP Kinase Gene; Measurement; Measures; Mediating; Medicine; Membrane Microdomains; Modeling; Mus; Neonatal; Organ Culture Techniques; Oxygen; Pathology; Pathway interactions; Patients; Pharmacotherapy; Phosphorylation; Population; Principal Investigator; Prolactin; Proteins; Radioimmunoassay; Ras Inhibitor; Ras/Raf; Retina; Retinal; Retinal Diseases; Retinal Neovascularization; Retinopathy of Prematurity; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Staging; TdT-Mediated dUTP Nick End Labeling Assay; Testing; Therapeutic Agents; Time; Tissues; Treatment Factor; United States; Vascular Endothelial Growth Factors; Vascular Endothelium; Vascularization; Western Blotting; Western World; base; blood glucose regulation; capillary; caspase-3; caveolin 1; cell growth; in vivo; intermolecular interaction; middle age; model development; mutant; neovascularization; programs; proliferative diabetic retinopathy; protein protein interaction; ras GTPase-Activating Proteins; receptor; research study; retinal damage; scavenger receptor; two-photon

DESCRIPTION: Aberrant neovascularization of the retina is a major cause of adult blindness associated with diabetic retinopathy. The 16 kDa fragment of human prolactin (16K hPRL) is a potent and specific antiangiogenic factor that inhibits neovascularization of the retina in the oxygen-induced retinopathy (OIR) model. In a variety of endothelial cell models, 16K hPRL inhibits VEGF- and bFGF-induced endothelial cell proliferation and activates apoptosis. We will determine the signaling pathways responsible for the antiangiogenic action of the 16K hPRL in the retina in vitro in bovine adrenal capillary endothelial cells (BAEC) and human retinal endothelial cells (HREC), and in vivo in the mouse OIR model. The inhibition of VEGF-induced endothelial cell proliferation by 16K hPRL is mediated via inhibition of Ras activation. We hypothesize that 16K hPRL inhibits Ras activation by stimulating the association of Ras with two Ras inhibitory proteins, Ras-GAP and Sprouty2 (Spry). In vitro we will study the protein-protein interactions between Ras-GFP and Ras-GAP-RFP and Spry2-RFP in living BAEC and HREC by measuring fluorescence resonance energy transfer (FRET) intensity in cellular compartments. Cells will be treated with VEGF and VEGF + 16K hPRL and signaling interactions measured in real-time. We hypothesize that the organization of signaling molecules occurs within calveolae. The calveolae will be identified by caveolin-1 (Cav-1-CFP) and the co-localization and FRET of signaling molecules measured in calveolae with the GFP and RFP donor/acceptor pair. Immunoblotting of proteins purified from cell compartments will be used to confirm results. We hypothesize that the 16K hPRL-induced inhibition of retinal neovascularization in the OIR model is mediated by the induction of apoptosis and inhibition of MAPK signaling. We will determine if intravitreal injection of an adenovirus (Ad) expressing 16K hPRL (16K-Ad) or a NulI-Ad into one eye activates apoptosis in areas of neovascularization measured by the TUNEL assay and a caspase-3 assay. We will utilize quantitative fluorescence microscopy to measure MAPK activation in areas of neovascularization in retinal whole mounts from the OIR model. MAPK activation will be assessed with a recently established assay that measures co-localization and FRET intensity of Ras-GFP and Raf-I-RFP. These studies will provide the basis for developing potent antiangiogenic factors for the treatment of abnormal neovascualization of the retina.

描述：视网膜异常新生血管形成是与糖尿病视网膜病变相关的成人失明的主要原因。人催乳素的 16 kDa 片段 (16K hPRL) 是一种有效且特异性的抗血管生成因子，可抑制氧诱导的视网膜病变 (OIR) 模型中视网膜的新生血管形成。在多种内皮细胞模型中，16K hPRL 抑制 VEGF 和 bFGF 诱导的内皮细胞增殖并激活细胞凋亡。我们将在体外牛肾上腺毛细血管内皮细胞 (BAEC) 和人视网膜内皮细胞 (HREC) 以及体内小鼠 OIR 模型中确定负责 16K hPRL 在视网膜中抗血管生成作用的信号通路。 16K hPRL 对 VEGF 诱导的内皮细胞增殖的抑制是通过抑制 Ras 激活来介导的。我们假设 16K hPRL 通过刺激 Ras 与两种 Ras 抑制蛋白 Ras-GAP 和 Sprouty2 (Spry) 的结合来抑制 Ras 激活。在体外，我们将通过测量细胞区室中的荧光共振能量转移 (FRET) 强度来研究活体 BAEC 和 HREC 中 Ras-GFP、Ras-GAP-RFP 和 Spry2-RFP 之间的蛋白质-蛋白质相互作用。将用 VEGF 和 VEGF + 16K hPRL 处理细胞，并实时测量信号传导相互作用。我们假设信号分子的组织发生在小凹内。小凹将通过caveolin-1 (Cav-1-CFP) 进行识别，并通过 GFP 和 RFP 供体/受体对在小凹中测量信号分子的共定位和 FRET。从细胞区室纯化的蛋白质的免疫印迹将用于确认结果。我们假设 OIR 模型中 16K hPRL 诱导的视网膜新生血管抑制是通过诱导细胞凋亡和抑制 MAPK 信号传导来介导的。我们将确定向一只眼睛玻璃体内注射表达 16K hPRL (16K-Ad) 的腺病毒 (Ad) 或 NulI-Ad 是否会激活通过 TUNEL 测定和 caspase-3 测定测量的新血管形成区域的细胞凋亡。我们将利用定量荧光显微镜来测量 OIR 模型中视网膜整体中新血管形成区域的 MAPK 激活。 MAPK 激活将通过最近建立的测定法进行评估，该测定法测量 Ras-GFP 和 Raf-I-RFP 的共定位和 FRET 强度。这些研究将为开发有效的抗血管生成因子来治疗视网膜异常新生血管形成奠定基础。