Dysfunction of endothelial precursor subtypes dictates the outcomes of diabetic r

描述（申请人提供）：内皮祖细胞（EPC）功能障碍可能在DR的发病机制中具有关键作用。由培养的单核细胞（MNC）产生的两个EPC群体是晚期生长内皮细胞（OEC），其显示克隆表型并且属于CD 34 + CD 45-群体;以及早期EPCs（eEPCs），其表现出单核细胞表型（CD 14 +/CD 45+），但在体外和体内显示出内皮样标志物和行为。为了解决这些细胞在糖尿病视网膜病变（DR）中的作用，我们假设在DR中，eEPCs仍然是OEC的中心调节剂;然而，OEC群体在非增殖性糖尿病视网膜病变（NPDR）中短暂丢失，但它再次出现为更具侵略性和增殖性的群体，触发“血管生成”转换和增殖性（PDR）的发作。相比之下，eEPC群体从未完全消失，而是其活性水平发生变化，在PDR中更具炎症性，而在NPDR中较少。在这两个群体中，一氧化氮合酶（NOS）的失调是这些表型转变的核心，这进一步受到骨髓（BM）微环境变化的影响。具体目的1：检测eEPC和OEC的增殖潜力是否取决于视网膜病变的阶段，并确定从糖尿病个体分离的eEPC和OEC是否产生更多的活性氧，含有更多的内源性NOS抑制剂，并分泌与对照细胞不同的细胞因子谱。具体目标二：为了测试非糖尿病（健康）eEPC和OEC的组合是否将具有比在视网膜缺血-再灌注损伤模型中或在糖尿病SCID小鼠的脱细胞毛细血管中单独的群体更大的修复功能。具体目标3：测试糖尿病中的eEPC和OEC功能障碍是否是由于BM微环境中的缺陷（肥胖增加、造血减少和祖细胞功能障碍）所致。这些研究将使我们能够确定eEPCs和OECs是否代表细胞治疗的理想祖细胞群体，以改善糖尿病眼睛中血管系统的健康。 公共卫生相关性：内皮前体细胞（EPCs）是骨髓来源的细胞，已被用作目前缺乏有效治疗选择的缺血性疾病的新疗法。这些细胞的鉴定、操作和移植是优化其使用的关键;然而，糖尿病患者具有缺陷的EPC，其不能用于细胞治疗，例如用于修复糖尿病视网膜病变的血管变性阶段（脱细胞毛细血管）。本申请的重点是1）鉴定用于修复的最佳EPC群体，2）表征糖尿病EPC中的缺陷，3）修复缺陷，4）优化EPC注射的时间和剂量，并鉴定糖尿病骨髓微环境的变化。这些研究将使用来自患有不同程度视网膜病变的糖尿病患者的人EPCs，糖尿病动物模型和具有影响骨髓微环境的独特特征的基因敲除小鼠进行。

DESCRIPTION (provided by applicant): Endothelial progenitor cell (EPC) dysfunction may have a key role in the pathogenesis of DR. Two populations of EPCs that arise from cultured mononuclear cells (MNC) are the late outgrowth endothelial cells (OECs), which display a clonal phenotype and belong to a CD34+CD45- population; and early EPCs (eEPCs) which exhibit a monocyte phenotype (CD14+/CD45+) but demonstrate endothelial-like markers and behavior in vitro and in vivo. To address the role of these cells in diabetic retinopathy (DR) we hypothesized that in DR, eEPCs remain as the central modulator of the OECs; however, the OEC population is transiently lost in nonproliferative diabetic retinopathy (NPDR) but it reappears as a more aggressive and proliferative population which triggers the "angiogenic" switch and the onset of proliferative (PDR). The eEPC population, in contrast, never disappears entirely but rather shifts in its level of activity, being more inflammatory in PDR and less in NPDR. In both populations, dysregulation of nitric oxide synthase (NOS) is central to these phenotypic transitions, which are further influenced by the changing bone marrow (BM) microenvironment. We are testing this hypothesis by the following aims: Specific Aim 1: To test whether the proliferative potential of eEPCs and OECs depends on the stage of retinopathy and determine whether eEPCs and OECs isolated from diabetic individuals produce more reactive oxygen species, contain more endogenous NOS inhibitors, and secrete a distinct cytokine profile than cells from controls. Specific Aim 2: To test whether the combination of nondiabetic (healthy) eEPCs and OECs will have a greater reparative function then either population alone in models of retinal ischemia-reperfusion injury or in acellular capillaries in diabetic SCID mice. Specific Aim 3: To test whether eEPC and OEC dysfunction in diabetes is due to defects (increased adiposity, reduced hematopoiesis and progenitor dysfunction) in the BM microenvironment. These studies will allow us to determine whether eEPCs and OECs represent ideal progenitor populations for cellular therapy to improve the health of the vasculature in the diabetic eye. PUBLIC HEALTH RELEVANCE: Endothelial precursors cells (EPCs) are bone marrow derived cells that have been used as novel therapies for ischemic conditions currently lacking effective treatment options. The identification, manipulation and transplantation of these cells are key to optimizing their use; however, diabetic patients have defective EPCs that cannot be used for cellular therapy such for repair of the vasodegenerative phase (acellular capillaries) of diabetic retinopathy. The focus of this application is to 1) identify the optimal EPC populations for repair, 2) characterize the defects in diabetic EPCs, 3) repair the defects, 4) optimize the timing and dosage of the EPC injections and identify the changes in the diabetic bone marrow microenvironment. These studies will be performed using human EPCs from diabetics with varying degrees of retinopathy, animal models of diabetes and knockout mice with unique characteristics that impact the bone marrow microenvironment.