NO Dysregulation of the Peripheral Clock in Diabetic Complications

描述(申请人提供)：使用2型糖尿病(T2D)的大鼠模型，我们发现在T2D的2个月时，糖尿病骨髓(BM)的骨髓祖细胞(BMPC)释放减少是由BM神经病变引起的，并且这些变化先于糖尿病视网膜病变(DR)的发展。BM神经病变与BMPC自身的时钟基因表达显著减少有关，这导致这些细胞的修复能力减弱，4个月的T2D导致了糖尿病视网膜病变(DR)的显著特征，即无细胞视网膜毛细血管。糖尿病患者的BMPC功能障碍通过增加生物可利用的一氧化氮(NO)水平达到正常的非糖尿病患者水平而得到纠正。由于NO调节Clock基因的表达，生物可利用NO水平的降低会导致Clock基因表达的改变。纤溶酶原激活物抑制物(PAI-1)是造血干细胞成熟和从骨髓中释放的重要调节因子，是时钟基因调控的直接下游代谢调节因子。PAI-1在健康人群中表现出强大的昼夜节律模式，但这种模式在糖尿病患者中会发生变化，糖尿病患者的内皮细胞(EC)会产生升高的水平。我们认为这种非调控的时钟基因功能障碍导致PAI-1的昼夜节律丧失，是DR和动脉粥样硬化的潜在机制和治疗靶点，这两种疾病分别是糖尿病患者失明和死亡的主要原因。在这一应用中，我们提出了以下假设：在糖尿病中，内皮细胞和骨髓基质细胞中生物可用NO的减少导致时钟蛋白BMAL1和PER-2振荡模式的幅度和频率降低，导致PAI-1的昼夜调节丧失，从而进一步降低EC和BMPC中的NO水平。NO生物利用度的降低和PAI-1在血管神经中的持续增加导致骨髓神经病变的发生，从而导致EPC动员缺陷，进一步加重终末器官并发症。为了验证我们的假设，我们提出了以下目标：1)确定NO的生物利用度是否能够影响血管内皮细胞和骨髓基质细胞的分子时钟和昼夜节律，并确定NO水平是否通过BMAL1和PER-2等必需时钟蛋白的S亚硝化而直接或间接影响时钟基因的表达；2)确定特定的时钟基因敲除内皮和骨髓基质细胞是否概括了糖尿病血管加速损伤和修复减少的表型；以及3)确定血管神经内NO和PAI-1的调节失调是否导致骨髓神经病变和骨髓基质细胞昼夜节律释放到循环中的丧失。 公共卫生相关性：这一建议代表了我们对糖尿病微血管和大血管并发症发病机制的理解的范式转变。我们正在测试昼夜节律振荡器是否在内皮细胞和骨髓前体细胞的动态平衡中发挥关键作用。我们认为，当它们在糖尿病中被破坏时，必须在实现血管健康之前恢复。我们明确指出纤溶酶原激活物抑制物可作为糖尿病治疗的新靶点，以恢复血管功能和血管和骨髓细胞中一氧化氮的昼夜振荡。

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.