Mitochondrial Carbonic Anhydrases and Diabetic Blood-Brain Barrier Disruption

描述(申请人提供)：糖尿病与脑微血管及其形成的血脑屏障(BBB)的恶化有关。血脑屏障可防止血浆蛋白不受限制地渗入大脑。血管血脑屏障是由特殊修饰的脑内皮细胞(BECs)物理形成的，但其他类型的细胞与其相互作用形成神经血管单位(NVU)。面对血糖升高时，周细胞在维持血脑屏障功能方面尤其重要。我们在体外已经证明，除非BEC与周细胞共培养，否则在面对高糖浓度时，胰岛素转运体会丢失；星形胶质细胞共培养不能保留BEC的胰岛素转运体功能。然而，周细胞本身容易受到葡萄糖毒性和伴随的氧化应激的影响。对视网膜周细胞和血-视网膜屏障(BRB)的研究表明，高血糖诱导视网膜周细胞凋亡；随着周细胞的丧失，BRB恶化。线粒体碳酸氢酶(CA)在高血糖时产生的活性氧簇(ROS)和随后的氧化应激反应中起重要作用。简而言之，线粒体CA产生线粒体碳酸氢盐，这是葡萄糖氧化分解代谢所必需的，葡萄糖是ROS的主要来源。高血糖会加速这一过程，增加ROS的产生。阻止线粒体CaS通过厌氧代谢穿梭丙酮酸，从而防止过量的ROS产生。我们推测，抑制线粒体碳酸氢酶(CA)将保护周细胞和BECs免受ROS诱导的细胞凋亡，并减缓STZ诱导的糖尿病血脑屏障破坏的发展。我们将在两个具体目标上检验这一假设。SA1将在从线粒体CA KO&WT小鼠分离的脑周细胞和BEC的原代培养中，以及在过度表达CA或用临床使用的CA抑制剂治疗的周细胞和BEC中，测量高糖介质诱导的氧化应激以及由此导致的线粒体泄漏和凋亡。SA2将在糖尿病小鼠和对照组小鼠中测量来自CA KO小鼠、WT小鼠和使用SA1的CA抑制剂治疗的BEC中的BBB破坏和氧化应激。

DESCRIPTION (provided by applicant): Diabetes mellitus is associated with deterioration of the brain's microvasculature and the blood-brain barrier (BBB) that it forms. The BBB prevents the unrestricted leakage of plasma proteins into the brain. The vascular BBB is physically formed by specially modified brain endothelial cells (BECs), but other cell types interact with it to form the neurovascular unit (NVU). Pericytes in particular are important in maintaining BBB function in the face of glycemic insult. We have shown in vitro that the insulin transporter is lost in the face of high glucose concentrations unless BECs are co-cultured with pericytes; astrocyte co-culture does not preserve BEC insulin transporter function. However, pericytes are themselves susceptible to glucose toxicity and the accompanying oxidative stress. Work with retinal pericytes and the blood-retinal barrier (BRB) shows that hyperglycemia induces apoptosis in retinal pericytes; with pericyte loss the BRB deteriorates. Mitochondrial carbonic anhydrases (CAs) are important in the generation of reactive oxygen species (ROS) and the subsequent oxidative stress that arises and is accelerated during hyperglycemia. In brief, mitochondrial CAs generate mitochondrial bicarbonate that is necessary for the oxidative catabolism of glucose, the major source of ROS. Hyperglycemia accelerates this process, increasing production of ROS. Blocking mitochondrial CAs shuttles pyruvate through anaerobic metabolism and so prevents excessive ROS production. We hypothesize that inhibition of mitochondrial carbonic anhydrases (CAs) will protect pericytes and BECs from ROS induced apoptosis and slow the development of disruption of the BBB in STZ-induced diabetes. Wewill test this hypothesis in two specific aims. SA1 will measure oxidative stress induced by high glucose media and the resulting mitochondrial leakage and apoptosis in primary cultures of brain pericytes and BEC isolated from mitochondrial CA KO & WT mice and in pericytes and BEC that overexpress CA or are treated with clinically used CA inhibitors. SA 2 will measure in diabetic and control mice BBB disruption and oxidative stress in BEC from CA KO mice, WT mice, and mice treated with the CA inhibitors of SA1.