Mechanism and Assessment of Hypoxia-Induced Islet Death

描述（由申请人提供）：人类胰岛移植具有治疗I型糖尿病的潜力。然而，胰岛在分离过程中和移植后的死亡一直是影响胰岛高效移植的主要问题。胰岛存活的一个关键因素可能是缺氧，在胰岛移植的各个阶段，包括胰岛的储存、分离、纯化和培养以及移植后，胰岛都不同程度地遭受缺氧。缺氧可导致胰岛细胞死亡，但目前尚不清楚是什么机制介导了这一过程。我们假设，即使是短时间的缺氧也会诱导胰岛细胞凋亡，并且胰岛死亡过程可能在缺氧损伤后24-48小时发生。细胞凋亡和坏死之间的区别是临床相关的，因为针对这两个过程的治疗策略是不同的。

DESCRIPTION (provided by applicant): Transplantation of human islets has the potential to cure Type I diabetes. However, death of human islets during isolation and after transplantation has been a major problem in the efficient transplantation of islets. A critical factor in islet survival may be hypoxia, which islets are subjected to at various degrees during all stages of islet transplantation including the storage of the pancreas, isolation, purification and culture of the islets, and after transplantation. Hypoxia results in death of islet cells, however it is not known what mechanism(s) mediate the process. We hypothesize that even short periods of hypoxia induce apoptosis in the islet cell, and that the islet death process may occur 24-48 hours after the hypoxic insult. The distinction between apoptosis and necrosis is clinically relevant since therapeutic strategies targeting the two processes would be different. In Specific Aim 1, we first quantify the time course of islet cell death and apoptosis in relation to different degrees and durations of hypoxia. It will be determined whether standard markers for apoptosis including activation of caspase 3 and translocation of cytochrome c from the mitochondria to the cytosol are observed prior to necrosis. In Specific Aim 2, a systems physiology approach to mitochondrial function will be applied to the assessment of islet viability. Sophisticated non-invasive detection technology combined with our recently developed flow culture system will be utilized to continuously assess oxygen consumption, redox state of cytochromes, and insulin secretion that will quantify transition states akin to vital signs in whole organisms. Mitochondrial function and the progression of apoptosis are linked, and our data shows that continuous assessment of metabolic state may distinguish the initiation and progression of apoptosis and necrosis. We will test the hypothesis that a metabolic threshold can be empirically derived that when exceeded will irreversibly result in islet cell death, loss of insulin secretory function and thereby transplant failure. This hypothesis embodies our notion that metabolical viability will be a better predictor of insulin secretory function of islets after transplantation than in vitro tests of insulin secretion which have been shown not to correlate with transplant success. Finally, we will use the assessment approach to evaluate whether improved oxygenation of the islets by flow culture prevents hypoxia-induced cell death observed in statically cultured islets. The benefits of the proposed research are evaluation of the contribution of hypoxia to islet death in islet isolation thereby laying the foundation for cytoprotective therapies against hypoxia induced islet death. In addition, the development of an islet flow culture system that both assesses and optimally maintains islets will be an important advancement in the field of transplantation based on its ability to predict and improve transplantation efficacy.