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: 7012255
• 负责人: RICHARD Ira WEINER
• 金额: $ 31.51万
• 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 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7012255
• 关键词: angiogenesis; angiogenesis inhibitors; apoptosis; biological signal transduction; caveolins; cell proliferation; fluorescence microscopy; fluorescence resonance energy transfer; laboratory mouse; prolactin; retina circulation; retina circulation disorder; retinopathy of prematurity; terminal nick end labeling

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：视网膜的异常新血管形成是与糖尿病性视网膜病有关的成人失明的主要原因。人催乳素（16K HPRL）的16 kDa片段是一种有效且特异性的抗血管生成因子，可抑制视网膜诱导的视网膜病变（OIR）模型中视网膜的新血管形成。在各种内皮细胞模型中，16K HPRL抑制VEGF和BFGF诱导的内皮细胞增殖并激活凋亡。我们将确定负责16K HPRL在牛肾上腺毛细管内皮细胞（BAEC）和人视网膜内皮细胞（HREC）（HREC）和小鼠OIR模型中体内体内的16K HPRL的抗血管生成作用的信号传导途径。通过抑制RAS激活，介导了16K HPRL对VEGF诱导的内皮细胞增殖的抑制。我们假设16K HPRL通过刺激RAS与两种RAS抑制蛋白，Ras-GAP和Sprouty2（SPRY）的关联来抑制RAS激活。在体外，我们将通过测量细胞隔室中的荧光共振能量转移（FRET）强度来研究Live BAEC和HREC中RAS-GFP与RAS-GAP-RFP和SPRY2-RFP之间的蛋白质蛋白相互作用。细胞将用VEGF和VEGF + 16K HPRL处理，并实时测量的信号传导相互作用。我们假设信号分子的组织发生在Calveolae内。 Caveolin-1（CAV-1-CFP）将鉴定钙纤维素，以及与GFP和RFP供体/受体对中测量的信号分子的共定位和FRET。从细胞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诱导的视网膜新血管形成抑制是通过诱导凋亡和MAPK信号抑制的介导的。我们将确定玻璃体内注射的腺病毒（AD）（AD）是否表达16K HPRL（16k-AD）或NULI-AD中的一只眼睛会激活通过TUNEL分析和caspase-3测定法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模型中视网膜整体坐骑区域的MAPK激活。 MAPK激活将通过最近确定的测定法进行评估，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提供基础，以治疗视网膜的新自由度化。