NO Dysregulation of the Peripheral Clock in Diabetic Complications

描述（由申请人提供）：使用2型糖尿病（T2D）大鼠模型，我们发现在T2D 2个月时，糖尿病骨髓（BM）中骨髓祖细胞（BMPC）释放的减少是由BM神经病变引起的，这些变化先于糖尿病视网膜病变（DR）的发展。BM神经病变与BMPCs自身时钟基因表达的显著减少有关，这导致这些细胞的修复能力减弱，到4个月的T2D导致糖尿病视网膜病变（DR）的标志性特征，无细胞视网膜毛细血管。糖尿病BMPC功能障碍通过将生物可利用性一氧化氮（NO）水平提高到正常的非糖尿病水平而得到纠正。由于NO调节生物钟基因的表达，生物可利用NO水平的降低导致生物钟基因表达的改变。平面蛋白原激活因子抑制剂（PAI-1）是一种由时钟基因控制的直接下游代谢调节剂，是造血干细胞成熟和从骨髓中释放的重要调节剂。PAI-1在健康状态下表现出强大的昼夜节律模式，但这种模式在糖尿病患者中发生改变，糖尿病患者的内皮细胞（EC）产生PAI-1水平升高。我们认为这种no调节的时钟基因功能障碍导致PAI-1昼夜节律调节的丧失，代表了DR和动脉粥样硬化的潜在机制和治疗靶点，而DR和动脉粥样硬化分别是糖尿病患者失明和死亡的主要原因。在这个应用中，我们提出了以下假设：在糖尿病中，EC和BMPCs中生物可利用NO的减少导致时钟蛋白BMAL1和PER-2振荡模式的振幅和频率降低，导致PAI-1的昼夜节律调节丧失，随后EC和BMPCs中NO水平进一步降低。一氧化氮生物利用度的降低和血管神经中PAI-1的持续升高导致BM神经病的发展，导致EPC动员缺陷，进一步加剧了终末器官并发症。为了验证我们的假设，我们提出了以下目标：1)确定NO的生物利用度是否会影响血管内皮细胞和血管内皮细胞的分子时钟和昼夜节律，并确定NO水平是否会通过s -亚硝基化基本时钟蛋白（如BMAL1和PER-2）对时钟基因表达产生直接和间接影响；2)确定内皮和BMPC的特异性时钟基因敲除是否再现了糖尿病血管损伤加速和修复减少的表型；3)确定神经血管内NO和PAI-1的失调是否会导致BM神经病变和BMPCs进入循环的昼夜节律释放丧失。

DESCRIPTION (provided by applicant): Using a rat model of type 2 diabetes (T2D), we showed that at 2 months of T2D the decrease in bone marrow progenitor cell (BMPC) release from diabetic bone marrow (BM) is caused by BM neuropathy and that these changes precede the development of diabetic retinopathy (DR). BM neuropathy was associated with a marked reduction in clock gene expression in the BMPCs themselves which led to diminished repair by these cells and by 4 months of T2D resulted in the hallmark feature of diabetic retinopathy (DR), acellular retinal capillaries. Diabetic BMPC dysfunction was corrected by increasing levels of bioavailable nitric oxide (NO) towards normal non-diabetic levels. Since NO modulates clock gene expression, the reduced levels of bioavailable NO results in altered clock gene expression. Plaminogen activator inhibitor (PAI-1), an important modulator of hematopoetic stem cell maturation and release from the BM is an immediate downstream metabolic regulator controlled by clock genes. PAI-1 demonstrates a robust circadian pattern in health, but this pattern is altered in diabetes and elevated levels are produced by endothelial cells (EC) from diabetics. We believe this NO-modulated clock gene dysfunction leads to the loss of circadian regulation of PAI-1 and represents an underlying mechanism and therapeutic target for DR and atherosclerosis, which are the major causes of blindness and mortality in diabetics, respectively. In this application, we propose the following hypothesis: In diabetes, a reduction in bioavailable NO in ECs and BMPCs causes a diminished amplitude and frequency of the oscillatory pattern of the clock proteins, BMAL1 and PER-2, leading to a loss of circadian regulation of PAI-1 and subsequent further diminution of NO levels in EC and BMPCs. Decreasing NO bioavailability and persistent increase in PAI-1 in the vasa nervorum leads to development of BM neuropathy which results in defective EPC mobilization further exacerbating end organ complications. To test our hypothesis, we propose the following aims: 1) to determine whether NO bioavailability can affect the molecular clock and circadian rhythms in vascular ECs and BMPCs and to determine whether levels of NO will have direct and indirect effects on clock gene expression via S-nitrosylation of essential clock proteins such as BMAL1 and PER-2; 2) to determine whether specific clock gene knock-out of the endothelium and BMPC recapitulates the diabetic vascular phenotype of accelerated injury and reduced repair; and 3) to determine whether dysregulation of NO and PAI-1 within the vasa nervorum leads to BM neuropathy and loss of circadian release of BMPCs into the circulation. PUBLIC HEALTH RELEVANCE: This proposal represents a paradigm shift in our understanding of the pathogenesis of diabetic micro- and macro-vascular complications. We are testing whether circadian oscillators play a pivotal role in endothelial cell and bone marrow progenitor cells homeostasis. We believe that when disrupted in diabetes, they must be restored before vascular health is achieved. We specifically identify plasminogen activator inhibitor as a novel therapeutic target in diabetes to restore vascular function and circadian oscillation of nitric oxide in vascular and bone marrow cells.