Cellular Mechanisms of Retinopathy: Role of Arginase

描述（由申请人提供）：该项目涉及缺血性视网膜病变期间的神经血管损伤。虽然这种情况与早期神经血管功能障碍有关，但常规治疗的目标是临床上显著的黄斑水肿或新血管形成，这发生得更晚。预防/逆转缺血性视网膜损伤的治疗是一个关键的未满足的需求。该项目的目标是描绘视网膜病变过程中血管和神经元损伤的机制，并确定新的治疗策略。研究者对缺血性视网膜病变模型的研究表明，尿素循环酶抑制剂在血管和神经元损伤中起着关键作用。精氨酸酶代谢L-精氨酸形成脯氨酸、多胺和谷氨酸。过量的精氨酸酶活性降低了一氧化氮合酶（NOS）的L-精氨酸供应，导致其解偶联并产生超氧化物和更少的NO。超氧化物和NO迅速反应并形成有毒的氧化剂过氧亚硝酸盐。谷氨酸和多胺氧化的分解代谢产物可引起更多的氧化应激和DNA损伤，这两者都可导致线粒体损伤和过早衰老。初步数据显示，视网膜病变期间的神经血管损伤与视网膜色素酶表达/活性增加、NO、多胺氧化减少、超氧化物和过氧亚硝酸盐形成增加、线粒体损伤和过早衰老相关。此外，胞质异构体丝氨酸蛋白酶1（A1）与血管内皮细胞（EC）的过早衰老和功能障碍有关，而线粒体异构体丝氨酸蛋白酶2（A2）似乎与神经元功能障碍/损伤有关。因此，据推测，激活的NOS途径引起神经血管损伤解偶联NOS和诱导多胺氧化和谷氨酸形成，从而减少NO和增加氧化应激，导致线粒体功能障碍，EC衰老和血管和神经元功能障碍。目的1将使用动物和组织培养模型来测试（A）限制A1表达是否将通过阻断NOS解偶联、减少氧化应激和防止线粒体功能障碍和EC衰老来防止血管功能障碍;（B）限制A2表达是否将通过阻断多胺氧化和谷氨酸形成、减少氧化应激和防止线粒体和神经元功能障碍来防止神经元损伤。目的2将确定旨在限制酶活性，恢复NO可用性和减少氧化应激的新疗法对神经血管功能障碍和损伤的影响。创新：该应用将首次研究视网膜神经血管损伤中的视网膜色素变性酶的作用。这些研究将使用分子方法来操纵A1和A2的表达，结合实时血管成像，视网膜电图和神经元和血管损伤的形态测定分析。还将测试限制酶活性和增加NO的治疗效果。这项研究有望显著提高对视网膜神经血管损伤机制的认识，并促进缺血性视网膜病变预防和治疗新策略的发展。

DESCRIPTION (provided by applicant): This project addresses neurovascular injury during ischemic retinopathy. While this condition is associated with early neurovascular dysfunction, conventional therapies target clinically significant macula edema or neovascularization, which occur much later. Therapy to prevent/reverse ischemic retinal injury is a critical unmet need. The project goal is to delineate mechanisms of vascular and neuronal injury during retinopathy and identify novel therapeutic strategies. The investigators' studies in models of ischemic retinopathy have revealed that the urea cycle enzyme arginase is critically involved in both vascular and neuronal injury. Arginase metabolizes L-arginine to form proline, polyamines and glutamate. Excessive arginase activity reduces the L-arginine supply for nitric oxide synthase (NOS), causing it to become uncoupled and produce superoxide and less NO. Superoxide and NO react rapidly and form the toxic oxidant peroxynitrite. Glutamate and the catabolic products of polyamine oxidation can induce more oxidative stress and DNA damage, both of which can cause mitochondrial injury and premature senescence. Preliminary data show that neurovascular injury during retinopathy is associated with increased arginase expression/activity, decreased NO, polyamine oxidation, increased formation of superoxide and peroxynitrite, mitochondrial injury and premature senescence. Furthermore, the cytosolic isoform arginase 1 (A1) is implicated in premature senescence and dysfunction of vascular endothelial cells (EC), whereas the mitochondrial isoform arginase 2 (A2) appears to be involved in neuronal dysfunction/injury. Thus, it is hypothesized that activation of the arginase pathway causes neurovascular injury by uncoupling NOS and inducing polyamine oxidation and glutamate formation, thereby reducing NO and increasing oxidative stress, leading to mitochondrial dysfunction, EC senescence and vascular and neuronal dysfunction. Aim 1 will use animal and tissue culture models to test whether (A) limiting A1 expression will prevent vascular dysfunction by blocking NOS uncoupling, reducing oxidative stress and preventing mitochondrial dysfunction and senescence of ECs; (B) limiting A2 expression will prevent neuronal injury by blocking polyamine oxidation and glutamate formation, reducing oxidative stress and preventing mitochondrial and neuronal dysfunction. Aim 2 will determine the effects on neurovascular dysfunction and injury of novel therapies designed to limit arginase activity, restore NO availability and reduce oxidative stress. Innovation: This application will, for the firt time, investigate the role of arginase in retinal neurovascular injury. The studies will use molecular approaches to manipulate A1 and A2 expression in combination with real-time vascular imaging, electroretinography and morphometric analyses of neuronal and vascular injury. Therapeutic effects of limiting arginase activity and increasing NO will also be tested. Th research is expected to significantly advance the mechanistic understanding of retinal neurovascular injury and facilitate development of novel strategies for prevention and treatment of ischemic retinopathy.