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脱细胞毛细血管的形成，导致视网膜缺氧和缺血，是DR发生的重要原因。我们已经证明，健康动物的造血干细胞和内皮前体细胞（EPC）很容易回到视网膜上，但糖尿病动物和人类的EPC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脱细胞毛细血管的发展提供了一种新的机制。我们的数据表明，在EPCs中，一氧化氮（NO）在调节肌动蛋白聚合介质，血管扩张剂刺激磷酸化蛋白（VASP）中起关键作用。在糖尿病中，由于生长因子/细胞因子受体平衡的改变、下游信号传导或NO合成酶（NOS）激活的下调，NO的生物利用度会降低。减少NO可以改变肌动蛋白聚合，影响EPC迁移及其进行内皮修复的能力。我们的假设是，在糖尿病患者中，一氧化氮生物利用度降低导致EPC迁移、附着和侵袭减少，导致内皮修复和非细胞毛细血管发育不足。最先进的成像、荧光活化细胞分选分析、新型体外培养试验和糖尿病动物模型将结合生化和分子生物学技术来验证我们的假设。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将无法回到视网膜并重新填充非细胞毛细血管，而正常人的EPCs则可以。已经形成非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内皮祖细胞，并检查血管修复的程度。具体目标2：我们的假设预测NO调节EPC迁移。直接通过促进细胞骨架的变化。我们将确定哪些因素调节EPC NOS异构体的表达和NO的产生，并描绘NO信号通路调节糖尿病EPCs的细胞骨骼功能障碍。特异性目的3：我们的假设预测糖尿病CD34+ 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的固有特性或局部基质环境，并评估人类糖尿病和非糖尿病CD34+细胞对正常和糖化基质的附着。我们的假设令人兴奋的含义是，EPC迁移缺陷的早期修复可能会阻止增殖性糖尿病视网膜病变的后续发展。

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.