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引起成人失明的主要原因。人催乳素的16 kDa片段（16 K hPRL）是一种有效且特异性的抗血管生成因子，其在氧诱导的视网膜病变（OIR）模型中抑制视网膜的新血管形成。在多种内皮细胞模型中，16 K hPRL抑制VEGF和bFGF诱导的内皮细胞增殖并激活凋亡。我们将确定的信号通路负责的抗血管生成作用的16 K hPRL在视网膜中的牛肾上腺毛细血管内皮细胞（BAEC）和人视网膜内皮细胞（HREC），在体外和在体内的小鼠OIR模型。16 K hPRL对VEGF诱导的内皮细胞增殖的抑制作用是通过抑制Ras活化介导的。我们假设16 K hPRL通过刺激Ras与两种Ras抑制蛋白Ras-GAP和Sprouty 2（Spry）的结合来抑制Ras活化。在体外，我们将研究Ras-GFP和Ras-GAP-RFP和Spry 2-RFP之间的蛋白质-蛋白质相互作用在活的BAEC和HREC通过测量荧光共振能量转移（FRET）强度在细胞区室。将用VEGF和VEGF +16 K hPRL处理细胞，并实时测量信号传导相互作用。我们推测，组织的信号分子发生在calveolae。将通过小窝蛋白-1（Cav-1-CFP）和在小窝中测量的信号分子与GFP和RFP供体/受体对的共定位和FRET来鉴定小窝。将使用从细胞区室纯化的蛋白质的免疫印迹来确认结果。我们推测16 K hPRL诱导的OIR模型中视网膜新生血管的抑制是通过诱导细胞凋亡和抑制MAPK信号传导介导的。我们将确定玻璃体内注射表达16 K hPRL的腺病毒（Ad）（16 K-Ad）或NulI-Ad到一只眼睛中是否激活通过TUNEL测定和半胱天冬酶-3测定测量的新血管形成区域中的细胞凋亡。我们将利用定量荧光显微镜来测量来自OIR模型的视网膜全标本中新生血管形成区域中的MAPK活化。将用最近建立的测定Ras-GFP和Raf-I-RFP的共定位和FRET强度的测定来评估MAPK活化。这些研究将为开发有效的抗血管生成因子治疗视网膜异常新生血管提供基础。

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.