Antiangiogenic action 16k hPRL in retinal microvessels

描述：视网膜异常新生血管形成是糖尿病视网膜病变导致成人失明的主要原因。人催乳素16kda片段（16K hPRL）是一种有效的特异性抗血管生成因子，可抑制氧诱导视网膜病变（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）以及小鼠OIR模型中确定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的激活。在体外，我们将通过测量细胞间荧光共振能量转移（FRET）强度来研究Ras-GFP、Ras-GAP-RFP和Spry2-RFP在活体BAEC和HREC中的蛋白-蛋白相互作用。细胞将接受VEGF和VEGF + 16K hPRL治疗，并实时测量信号相互作用。我们假设信号分子的组织发生在小裂孔内。小窝蛋白-1 （Cav-1-CFP）和信号分子的共定位和FRET在小窝中测量与GFP和RFP供体/受体对。从细胞区室纯化的蛋白质免疫印迹将用于确认结果。我们假设，在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信号传导介导的。通过TUNEL试验和caspase-3试验，我们将确定在一只眼睛内玻璃体内注射表达16K hPRL （16K-Ad）或NulI-Ad的腺病毒（Ad）是否会激活新生血管区域的细胞凋亡。我们将利用定量荧光显微镜测量视网膜新生血管区域的MAPK激活。MAPK激活将通过最近建立的测定Ras-GFP和Raf-I-RFP的共定位和FRET强度的方法来评估。这些研究将为开发有效的抗血管生成因子治疗视网膜新生血管异常提供基础。

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.