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血管生成和血管生成这两个复杂而仍鲜为人知的过程发生的。这些过程虽然不完美，但在与多种眼部疾病(如早产儿视网膜病变、老年性黄斑变性、糖尿病视网膜病变等)相关的缺血性损伤的背景下进行了概括。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，其中新血管复合体的细胞成分部分来源于骨髓(BM)来源的循环血管前体细胞。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)是一种新的细胞外基质相关的肝素和整合素结合蛋白，能刺激骨髓来源的造血祖细胞CD34+细胞的血管生成表型。然而，当被培养的视网膜周细胞包裹并调节视网膜毛细血管的生长时，Cyr61通过失巢凋亡诱导它们死亡，这表明Cyr61在病理性血管生成中发挥了作用。目前的建议包括一种策略，即梳理出Cyr61蛋白在体外和体内的这些不同的活性。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(IGFBPs)结构域、von Willebrand因子C型(VWFC)重复序列、凝血酶反应蛋白I型(TSP1)重复序列和C末端(CT)半胱氨酸结基序。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-末端区域保留了Cyr61相对于CD34+细胞的血管生成潜能，并促进了其黏附、迁移、分化和血管生成活性。相反，含有TSP1和/或CT结构域的C-末端区域通过其抗黏附特性诱导视网膜周细胞凋亡。我们将揭示这些活性，在特定目标1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。在具体目标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新生小鼠氧诱导视网膜病变模型，以确定给予Cyr61或Cyr61模块是否通过促进内皮祖细胞向缺血损伤的视网膜部位募集来促进血管修复。同样，我们将确定，当BM来源的CD34+细胞与Cyr61的N-末端结构域一起启动时，是否整合到受损的视网膜血管中，并促进血管恢复。我们的研究将有助于建立治疗缺血性视网膜病变血管生成能力减弱/受限的替代方案，以及截短的ECM蛋白对EPC动员和募集的特殊贡献。 公共卫生相关性：视网膜新生血管是一种高度普遍且可能致盲的疾病，其特点是多种眼病，包括糖尿病视网膜病变、老年性黄斑变性、视网膜中央静脉阻塞和早产儿视网膜病变。我们的研究致力于细胞外环境的特定成分在视网膜血管中的作用及其促进或拮抗视网膜血管修复/拯救的能力。我们的研究结果将确定如何使用/修饰这种细胞外化合物的特定部分，以允许形成功能正常的视网膜血管，并改进几种视网膜疾病的药物治疗。

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.