Ceramide-mediated mitochondrial damage in diabetic retinopathy investigated by novel microfluidic O2 sensing and bio-mimetic electrochemistry

尽管最近在治疗方法上取得了成功，但糖尿病视网膜病变(DR)仍然是 进行性视力丧失和失明的主要原因。理念和技术上的突破 寻找新的靶点和策略来治疗这种并发症是至关重要的。我们相信 糖尿病患者线粒体功能障碍的最新证据提供了这一突破 视网膜病变与来自大型临床试验的数据相结合表明 血脂异常与糖尿病视网膜病变进展的关系以及神经酰胺的发现 影响线粒体功能。 线粒体在细胞代谢中起着基石作用，甚至是轻微的修饰 线粒体功能可导致病理改变。的确，线粒体损伤先于 DR的组织病理学异常。 最近的研究表明神经酰胺和神经酰胺之间存在着复杂的联系。 线粒体功能。线粒体已被证明含有许多鞘磷脂，包括 鞘磷脂和神经酰胺，以及鞘磷脂途径的酶。神经酰胺诱导 呼吸链功能在复合体III水平上的限制，以及琥珀酸 蓄积已被证明是缺血/再灌流和中风的一个致病因素。 导致组织损伤。 除了对呼吸酶的影响外，神经酰胺还被证明对 线粒体外膜通透性通过S1P和十六烯的产生 和Bax/BAK的激活，或直接通过形成蛋白质通透性神经酰胺 线粒体外膜中的通道。这些渠道被证明在 通过将细胞色素c释放到细胞质中来诱导细胞凋亡。我们有 先前证明酸性鞘磷脂酶的激活是一种重要的早期事件。 糖尿病视网膜病变的发病机制。在这个提案中，我们将检验总体假设 ASM激活时线粒体神经酰胺水平的增加导致a) 细胞色素c的释放与细胞凋亡及b)对线粒体呼吸链的限制 糖尿病患者REC和RPE细胞的功能。作为传统的极谱或荧光 淬火(海马)方法不利于研究有限数量的可用 视网膜组织和细胞，我们正在开发一种新的微流控方法 线粒体研究。我们将利用这一新的方法来评估ASM的作用 糖尿病视网膜线粒体损伤中的激活和神经酰胺的产生。

Despite recent success in treatment approaches, diabetic retinopathy (DR) remains a leading cause of progressive vision loss and blindness. Conceptual and technical breakthroughs to identify novel targets and strategies to cure this complication are paramount. We believe that such a breakthrough is offered by recent evidence of mitochondrial dysfunction in diabetic retinopathy in combination with the data from large clinical trials demonstrating a strong association between lipid abnormalities and DR progression, and the discovery that ceramide affects mitochondrial function. Mitochondria play cornerstone role in cellular metabolism and even slight modification of mitochondrial function can lead to pathology. Indeed, mitochondrial damage precedes histopathological abnormalities in DR. Recent studies demonstrate that there is an intricate connection between ceramide and mitochondrial function. Mitochondria have been shown to contain many sphingolipids including sphingomyelin and ceramide, as well as enzymes of sphingolipid pathway. Ceramide-induced restriction of respiratory chain function at the level of complex III, as well as succinate accumulation has been shown to be a causative factor in ischemia/reperfusion and stroke- induced tissue damage. In addition to effects on respiratory enzymes, ceramides were shown to contribute to mitochondrial outer membrane permeability either through S1P and hexadecenal production and activation of BAX/BAK, or directly through the formation of protein-permeable ceramide channels in mitochondrial outer membranes. These channels are shown to play a key role in the induction of apoptosis through the release of cytochrome c into the cytoplasm. We have previously demonstrated that activation of acid sphingomyelinase is an important early event in the pathogenesis of diabetic retinopathy. In this proposal we will test the overall hypothesis that increased levels of mitochondrial ceramide upon ASM activation leads to a) cytochrome c release and apoptosis and b) restriction on mitochondrial respiratory chain function in REC and RPE cells in diabetes. As traditional polarographic or fluorescence quenching (Seahorse) methods are not conducive for studies on limited amounts of available retinal tissue and cells, we are developing a novel microfluidic method for functional mitochondrial studies. We will utilize this novel methodology to assess the role of ASM activation and ceramide production in mitochondrial damage in diabetic retina.