Cell Biology of Diabetic Microvascular Complications

描述(申请人提供)：我们提出糖尿病的许多视网膜、肾脏和心血管病变是由于高血糖(HG)引起的二酰甘油(DAG)和蛋白激酶C(PKC)的激活，特别是？1/2和d亚型通路的激活。在糖尿病动物和患者培养的血管细胞和血管组织(视网膜、肾脏、肾小球、动脉和心肌)中的大量证据表明，DAG/PKC通路被激活，并与细胞因子表达或活性(血管内皮生长因子、ET-1、PDGF或转化生长因子、细胞间黏附分子)、毛细血管通透性、基质蛋白产生、信号分子(MAP激酶和AKT)的变化以及通过NADPH氧化酶激活的氧化应激增加有关。临床研究表明，PKC？亚型选择性抑制剂Ruboxistaurin(RBX)在高血糖存在的情况下可以保护视力、肾功能和内皮功能。然而，对于糖尿病视网膜病变(DR)，RBX治疗并未阻止进展为增殖性视网膜病变，但加速了黄斑水肿的消退。基础和临床结果均提示，PKC亚型激活可能选择性地影响内皮功能。我们提供的初步数据表明，高血糖促进周细胞凋亡，这与PKC d亚型和p38MAPK激活平行，即使在细胞恢复到正常血糖状态后仍保持高水平。因此，这些结果表明，周细胞模型可能是一种临床研究中观察到的“代谢记忆”模型。因此，我们认为，高血糖诱导的PKC？1/2和d亚型的不同激活分别导致视网膜内皮细胞和周细胞不同和特定的细胞变化。因此，为了防止DR的进展，PKC？1/2和d亚型可能都需要正常化。为了验证这一新假说，我们将：(1)确定PKC？1/2和d-亚型在视网膜内皮细胞(激活ERK1/2 MAPK-occludin及其他蛋白)和周细胞(p38 MAPK，NADPH氧化酶)等中介导不同作用的特定信号机制；(2)确定PKC？1/2和d亚型在内皮细胞和周细胞中的各种生化、血流动力学和病理参数变化中的选择性作用；(2)通过使用C57/BL6小鼠和糖尿病PKC？/-和d-/-小鼠和内皮细胞靶向过表达PKCP2来确定PKC？1/2和d-/-小鼠和内皮细胞靶向过表达PKCP2的作用。最后，需要PKC？/d亚型的双重激活才能诱发DR的想法将通过用PKC？特异性抑制剂RBX治疗糖尿病PKC d-/-小鼠来检验，以确定是否可以预防糖尿病中的大多数视网膜病变。

DESCRIPTION (provided by applicant): We proposed that many of the retinal, renal and cardiovascular pathologies in diabetes are due to the activation of diacylglycerol (DAG) and protein kinase C (PKC), especially ¿1/2 and d isoform pathways induced by hyperglycemia (HG). A large body of evidence in cultured vascular cells and vascular tissues (retinal, renal, glomeruli, arteries and myocardium) from diabetic animals and patients have shown that DAG/ PKC pathways are activated and associated with increases in cytokine expression or activities (VEGF, ET-1, PDGF ¿ orTGF ¿, ICAM's), capillary permeability, matrix protein production, changes in signaling molecules (MAP kinases and AKT) and elevation of oxidative stress via NADPH oxidase activation. Clinical studies have shown that PKC ¿ isoform selective inhibitor ruboxistaurin (RBX) preserved visual acuity, renal function and endothelial functions in the presence of hyperglycemia. However, for diabetic retinopathy (DR), treatment with RBX did not prevent the progression to proliferative retinopathy, but accelerated the resolution of macular edema. Both basic and clinical results suggest that PKC ¿ isoform activation may selectively affect endothelial functions. We have provided preliminary data to show that hyperglycemia enhanced pericyte apoptosis, which parallels with PKC d isoform and p38 MAPK activation, remained elevated even after the cells were returned to normal glycemic condition. Thus, these results suggest that the pericyte cell model could be a model of "Metabolic Memory" as observed in clinical studies. Thus, we are proposing that the differential activation of PKC ¿ 1/2 and d isoforms induced by hyperglycemia, are causing different and specific cellular changes in retinal endothelial cells and pericytes, respectively. Thus, to prevent the progression of DR, both PKC ¿ 1/2 and d isoforms may need to be normalized. To test this new hypothesis, we will: (1) identify the specific signaling mechanisms by which PKC ¿ 1/2 and d isoforms are mediating their different effects in retinal endothelial cells (activation of ERK1/2 MAPK-occludin and others) and pericytes (p38 MAPK, NADPH oxidases) ¿ and others; (2) define the isoform selective roles of PKC ¿ 1/2 and d isoforms on the various changes in biochemical, hemodynamic and pathological parameters observed in the endothelial cells and pericytes by using control and diabetic PKC ¿-/- and d-/- mice and endothelial cell targeted overexpression of PKCP2 in C57/BL6 mice. Finally, the idea of dual activation of PKC ¿ / d isoforms are needed to induce DR will be tested by treating diabetic PKC d-/- mice with PKC ¿ specific inhibitor RBX to determine whether most of the retinal pathologies in diabetes can be prevented.