Endothelial precursor dysfunction in development of diabetic acellular capillary

描述（由申请人提供）：糖尿病视网膜病变（DR）是西方世界工作年龄成年人失明的主要原因。早期周细胞脱落和内皮细胞死亡导致无细胞毛细血管的形成，其引起视网膜缺氧和缺血，并且是DR发展的重要贡献者。我们已经证明，在健康动物中，造血干细胞和内皮前体细胞（EPC）容易归巢到视网膜，但糖尿病动物和人类的EPC具有迁移缺陷。我们的初步研究表明，EPC功能障碍可能为糖尿病视网膜病变中无细胞毛细血管的发育提供了一种新的机制。我们的数据表明，在内皮祖细胞，一氧化氮（NO）在调节肌动蛋白聚合介质，血管舒张刺激磷蛋白（VASP）的关键作用。在糖尿病中，由于生长因子/细胞因子受体平衡、下游信号传导或NO合酶（NOS）活化的下调的改变，可发生NO的生物利用度降低。减少NO可以改变肌动蛋白聚合，影响EPC迁移和进行内皮修复的能力。我们的假设是，在糖尿病患者中，NO生物利用度降低导致EPC迁移、附着和侵袭减少，从而导致内皮修复不足和无细胞毛细血管的发育。最先进的成像技术、荧光激活细胞分选仪分析、新型体外培养试验和糖尿病动物模型将与生化和分子生物学技术结合使用，以验证我们的假设。我们提出以下具体目标：具体目标1：我们的假设预测，糖尿病EPCs将无法回家视网膜和重新填充脱细胞毛细血管，而正常人的EPCs。已形成脱细胞视网膜毛细血管的糖尿病小鼠将全身注射来自非糖尿病啮齿动物的EPC，并检查血管修复的程度。目的2：我们的假说预测NO通过促进细胞骨架的改变直接调节EPC的迁移。我们将确定哪些因素调节内皮祖细胞NOS亚型表达和NO的产生，并描绘NO信号通路，调节糖尿病内皮祖细胞的细胞骨架功能障碍。具体目标3：我们的假设预测，糖尿病CD 34 + EPC细胞有缺陷的附着和侵袭。我们将确定EPC附着是否依赖于EPC或局部基质环境的固有特性，并评估人类糖尿病和非糖尿病CD 34+细胞与正常和糖化基质的附着。我们的假设令人兴奋的含义是，早期修复的EPC迁移缺陷可能会阻止随后的发展增殖性糖尿病视网膜病变。

DESCRIPTION (provided by applicant): Diabetic retinopathy (DR) is a primary cause of blindness of working age adults in the Western world. Early pericyte dropout and endothelial cell death result in the formation of acellular capillaries, which cause retinal hypoxia and ischemia and are important contributors to DR development. We have shown have that hematopoietic stem and endothelial precursor cells (EPC) readily home to the retina in healthy animals, but that EPC of diabetic animal and humans have a migratory defect. Our preliminary studies demonstrate that EPC dysfunction may provide a novel mechanism for development of acellular capillaries in diabetic retinopathy. Our data suggest that within EPCs, nitric oxide (NO) plays a key role in regulating the actin polymerization mediator, vasodilator-stimulated phosphoprotein (VASP). In diabetes, reduced bioavailability of NO can occur as a result of alterations in growth factor/cytokine receptor balance, downstream signaling, or downregulation of NO synthase (NOS) activation. Reduced NO can alter actin polymerization affecting EPC migration and their ability to carry out endothelial repair. Our hypothesis is that in diabetic individuals reduced NO bioavailability results in decreased EPC migration, attachment and invasion contributing to inadequate endothelial repair and development of acellular capillaries. State-of-the- art imaging, fluorescent activated cell sorter analysis, novel in vitro culture assays and diabetic animal models will be used in combination with biochemical and molecular biological techniques to test our hypothesis. We propose the following specific aims: Specific Aim 1: Our hypothesis predicts that diabetic EPCs will be unable to home to the retina and repopulate acellular capillaries whereas EPCs from normals will. Diabetic mice that have developed acellular retinal capillaries will be injected systemically with EPCs from nondiabetic rodents and the degree of vascular repair examined. Specific Aim 2: Our hypothesis predicts that NO regulates EPC migration .directly by promoting cytoskeletal changes. We will determine which factors regulate EPC NOS isoform expression and NO generation and delineate the NO signaling pathway that regulates cytoskeletal dysfunction in diabetic EPCs. Specific Aim 3: Our hypothesis predicts that diabetic CD34+ EPC cells have defective attachment and invasion. We will determine whether EPC attachment is dependent upon inherent properties of the EPC or the local matrix environment and evaluate human diabetic and nondiabetic CD34+ cell attachment to normal and glycated matrix. The exciting implication of our hypothesis is that early repair of the EPC migratory defect may deter the subsequent development of proliferative diabetic retinopathy.