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的发病机制中起关键作用。来源于培养的单个核细胞(MNC)的两类EPC是晚期生长的内皮细胞(OECs)，显示克隆表型，属于CD34 CD45-群体；以及早期EPC(EEPC)，呈现单核细胞表型(CD14/CD45)，但在体外和体内显示内皮样标记和行为。为了阐明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，内皮祖细胞仍然是嗅鞘细胞的中枢调节器；然而，在非增殖性糖尿病视网膜病变(NPDR)中，OEC群体暂时消失，但它以更具侵袭性和增殖性的群体重新出现，这触发了血管生成开关和增殖性(PDR)的开始。相比之下，EEPC群体从未完全消失，而是在其活动水平上发生了变化，在PDR中炎症性更强，在NPDR中更少。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，而这些表型转变进一步受到不断变化的骨髓微环境的影响。我们通过以下目标验证这一假说：具体目标1：测试eEPC和OECs的增殖能力是否取决于视网膜病变的阶段，并确定从糖尿病患者分离的eEPC和OECs是否比来自对照组的细胞产生更多的活性氧，含有更多的内源性NOS抑制物，并分泌不同的细胞因子。具体目标2：测试非糖尿病(健康)内皮祖细胞和嗅鞘细胞的组合是否比在糖尿病SCID小鼠视网膜缺血再灌注损伤模型中单独或在无细胞毛细血管中具有更大的修复功能。具体目标3：测试糖尿病患者的eEPC和OEC功能障碍是否与骨髓微环境缺陷(肥胖增加、造血量减少和祖细胞功能障碍)有关。这些研究将使我们能够确定内皮祖细胞和嗅鞘细胞是否代表了细胞治疗的理想祖细胞群体，以改善糖尿病眼血管系统的健康。 公共卫生相关性：内皮前体细胞(EPC)是一种骨髓来源的细胞，目前缺乏有效的治疗方案，已被用作治疗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，糖尿病患者有缺陷的内皮祖细胞不能用于细胞治疗，如修复糖尿病视网膜病变的血管退行性变阶段(无细胞毛细血管)。这一应用的重点是1)确定适合修复的EPC群体，2)表征糖尿病EPC的缺陷，3)修复缺陷，4)优化EPC注射的时机和剂量，并确定糖尿病骨髓微环境的变化。这些研究将使用来自不同程度视网膜病变的糖尿病患者、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的人内皮祖细胞进行。

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.