Endothelial precursor dysfunction in development of diabetic acellular capillary

描述(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，从而导致视网膜缺氧和缺血，是DR发生的重要因素。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)很容易定位于视网膜，但糖尿病动物和人类的EPC存在迁移缺陷。我们的初步研究表明，内皮祖细胞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展提供了一种新的机制。我们的数据表明，在内皮祖细胞中，一氧化氮(NO)在调节肌动蛋白聚合介质血管扩张剂刺激的磷酸蛋白(VASP)中起着关键作用。在糖尿病中，由于生长因子/细胞因子受体平衡的改变、下游信号转导或一氧化氮合酶(NOS)活性的下调，可能导致NO的生物利用度降低。还原的NO可以改变肌动蛋白聚合，影响内皮祖细胞的迁移和执行内皮修复的能力。我们的假设是，在糖尿病患者中，NO的生物利用度降低会导致EPC迁移、附着和侵袭减少，从而导致内皮修复和无细胞毛细血管的发育不足。最先进的成像技术、荧光激活细胞分选仪分析、新型体外培养分析和糖尿病动物模型将与生化和分子生物学技术相结合来验证我们的假设。我们提出了以下具体目标：具体目标1：我们的假设预测，糖尿病内皮祖细胞将不能定位于视网膜并重新填充无细胞的毛细血管，而正常人的内皮祖细胞将会。已经形成无细胞视网膜毛细血管的糖尿病小鼠将被系统地注射来自非糖尿病啮齿动物的EPC，并检查血管修复的程度。具体目标2：我们的假设预测，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一氧化氮合酶亚型的表达和NO的产生，并描绘调控糖尿病内皮祖细胞骨架功能障碍的NO信号通路。具体目标3：我们的假设预测糖尿病CD34 EPC细胞具有缺陷的附着和侵袭。我们将确定EPC附着是否取决于EPC的固有属性或局部基质环境，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是，EPC移行缺陷的早期修复可能会阻止随后的增殖性糖尿病视网膜病变的发展。

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.