Cyr61/CCN1-Induced Angiogenesis and Vasculogenesis in the Retina

描述（由申请人提供）：视网膜血管的发育和重塑是通过复杂的血管生成和血管生成过程发生的，这一过程目前尚不清楚。这些过程在与许多眼部疾病相关的缺血性损伤（例如，早产儿视网膜病变、年龄相关性黄斑变性、糖尿病视网膜病变等）的背景下得到了概括，尽管不完美。这些视网膜新生血管疾病的发病机制是由病理性血管生成导致异常血管的形成，这些血管进入玻璃体，以及血管生成不足/有限，其中新血管复合物的细胞成分部分来自骨髓（BM）衍生的循环血管祖细胞。血管细胞外基质（ECM）的组成部分协调多种血管生成和血管生成活动的执行。PI实验室最近的工作表明，富含半胱氨酸的蛋白61 (Cyr61)，一种新的与ecm相关的肝素和整合素结合蛋白，刺激脑卒中衍生的造血祖细胞CD34+细胞的血管生成表型。然而，当暴露于培养的视网膜周细胞（保护和调节视网膜毛细血管的生长）时，Cyr61通过anoikis诱导其死亡，这表明Cyr61在病理性血管生成中起作用。目前的建议包括在体外和体内梳理Cyr61蛋白的这些不同活动的策略。我们的目标是确定Cyr61的多种活性与其由4个不同结构域组成的多模块组织有关：胰岛素样生长因子结合蛋白（igfbp）结构域，血管性血液病因子C型（vWFC）重复序列，血栓反应蛋白I型（TSP1）重复序列和C末端（CT）胱氨酸结基。我们预测含有IGFBP和/或具有整合素结合能力的vWFC结构域的n端区域保留了Cyr61对CD34+细胞的血管生成潜力，并促进了它们的粘附、迁移、分化和血管生成活性。相反，含有TSP1和/或CT结构域的c端区域凭借其抗粘附特性诱导视网膜周细胞凋亡。在Specific Aim 1中，我们将使用未分化的造血CD34+细胞和培养的视网膜周细胞暴露于重组产生的Cyr61结构域或表达n端或C端区域的慢病毒载体来揭示这些活性。在Specific Aim 2中，我们将利用以视网膜中央毛细血管床血管闭塞为特征的氧诱导视网膜病变的新生小鼠模型来确定Cyr61或Cyr61模块是否通过增强EPCs在缺血性损伤视网膜部位的募集来促进血管修复。同样，我们将确定，当转染Cyr61的N端结构域时，bm来源的CD34+细胞是否整合到受损的视网膜血管中，并改善血管恢复。我们的研究将有助于为缺血性视网膜病变的血管生成能力减弱/受限建立治疗方案，以及截断的ECM蛋白对EPC动员和募集的特殊贡献。

DESCRIPTION (provided by applicant): Development and remodeling of retinal blood vessels occurs through the complex and still poorly understood processes of vasculogenesis and angiogenesis. These processes are recapitulated, though imperfectly, in the context of ischemic injury associated with numerous ocular diseases, (e.g., retinopathy of prematurity, age-related macular degeneration, diabetic retinopathy, etc). The pathogenesis of these retinal neovascular disorders is driven by a pathological angiogenesis leading to the formation of abnormal vessels, which break into the vitreous, and an insufficient/limited vasculogenesis in which the cellular components of the new vessel complex originate in part, from bone-marrow (BM) derived circulating vascular progenitor cells. Components of the vascular extracellular matrix (ECM) coordinate the execution of multiple angiogenic and vasculogenic activities. Recent work in the PI's laboratory have revealed that the cysteine-rich protein 61 (Cyr61), a novel ECM-associated, heparin- and integrin-binding protein, stimulates the angiogenic phenotype of the BM-derived hematopoeitic progenitors, CD34+ cells. However, when exposed to cultured retinal pericytes, which sheath and regulate the growth of retinal capillaries, Cyr61 induces their death by anoikis suggesting a role of Cyr61 in pathological angiogenesis. The current proposal consists of a strategy to tease out these disparate activities of the Cyr61 protein both in vitro and in vivo. We will aim to determine that the multiple activities of Cyr61 are linked to its multi-modular organization consisting of 4 distinct domains: an insulin-like growth factor binding protein (IGFBPs) domain, the von Willebrand factor type C (vWFC) repeat, the thrombospondin type I (TSP1) repeat and a C-terminal (CT) cystine-knot motif. We predict that the N-terminal region containing IGFBP and/or vWFC domains with integrin-binding capabilities retains the angiogenic potential of Cyr61 vis-`-vis the CD34+ cells and promote their adhesion, migration, differentiation, and angiogenic activity. Conversely, the C-terminal region containing the TSP1 and/or CT domains induces apoptosis of retinal pericytes by virtue of its anti-adhesive properties. We will reveal these activities, in Specific Aim 1 using undifferentiated hematopoeitic CD34+ cells and cultured retinal pericytes exposed to recombinantly produced domains of Cyr61 or lentiviral vectors expressing the N-terminal or C- terminal regions. In Specific Aim 2, we will utilize the neonate mouse model of oxygen-induced retinopathy characterized by vaso-obliteration of the central retinal capillary bed to determine whether administration of either Cyr61 or Cyr61 modules promotes vascular repair by enhancing recruitment of EPCs to retinal sites of ischemic injury. Similarly, we will establish whether, BM-derived CD34+ cells, when primed with the N- terminal domains of Cyr61, integrate into damaged retinal vessels and improve vascular recovery. Our studies will help establish treatment alternatives for the diminished/limited vasculogenic capability in ischemic retinopathy and the specific contribution of truncated ECM proteins to EPC mobilization and recruitment. PUBLIC HEALTH RELEVANCE: Neovascularization of the retina is a highly prevalent and potentially blinding disorder characteristic of a variety of ocular diseases including diabetic retinopathy, age-related macular degeneration, central retinal vein occlusion, and retinopathy of permaturity. Our studies address the role of a specific component of the extracellular environment in retinal vessels and its ability to either facilitate or antagonize retinal vascular repair/rescue. Results of our studies will determine how specific portions of such extracellular compound can be used/modified to allow the formation of normally functioning retinal blood vessels and improve the pharmacotherapy of several retinal diseases.