Endothelial Expression of Neuronal Nitric Oxide Synthase

神经元一氧化氮合酶的内皮表达

• 批准号: 9305167
• 负责人: Prasad V Katakam
• 金额: $ 32.92万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9305167
• 关键词: Angiotensin II; Astrocytes; Blood - brain barrier anatomy; Blood capillaries; Brain; Brain Injuries; Cerebrum; Coagulation Process; Cytosol; Data; Employee Strikes; Endothelial Cells; Generations; Glucose; Human; Injury; Ischemic Brain Injury; Knock-out; Location; Mediator of activation protein; Microvascular Dysfunction; Mitochondria; Mus; NADPH Oxidase; NOS2A gene; NOS3 gene; Names; Neurons; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxygen; Pathologic; Pharmacology; Physiological; Process; Protein Isoforms; RNA Splicing; Rattus; Reactive Oxygen Species; Resolution; Role; Source; Stroke; Superoxides; Techniques; Time; Variant; brain endothelial cell; capillary; cerebral hypoperfusion; deprivation; knock-down; neuroprotection; neurovascular unit; novel; public health relevance; respiratory

 DESCRIPTION (provided by applicant): Cerebral microvascular dysfunction has been implicated in the brain injury following stroke, however, the underlying mechanisms are unclear. Nitric oxide synthase (NOS) has endothelial (eNOS) and neuronal (nNOS) isoforms that were named after the locations where they were first identified. Our preliminary studies, for the first time, identified nNOS in freshly isolated rat brain microvessels and brain microvascular endothelial cells (BMECs) from rat, mouse, and humans utilizing PCR and immunoblot techniques. We found that endothelial nNOS is structurally and functionally distinct from eNOS and the nNOS expressed in the neurons. Therefore, we named the endothelial nNOS as enNOS. Our preliminary studies revealed that inhibition of eNOS in BMECs or nNOS in neurons increased the levels of superoxide and decreased NO levels. Furthermore, eNOS inhibition results in diminished mitochondrial reserve respiratory capacity. Similarly, inhibition of nNOS in neurons increased superoxide levels and decreased NO levels. In contrast, enNOS inhibition led to diminished superoxide levels, increased NO levels, and enhanced mitochondrial reserve respiratory capacity. Thus, unlike nNOS of neuronal origin and eNOS, enNOS exists in the uncoupled state. Preliminary studies also showed that enNOS significantly contributes to baseline as well as angiotensin II- induced superoxide levels in BMECs that is comparable to but independent of NADPH oxidase. Finally, inhibition of all NOS isoforms during oxygen-glucose deprivation and reoxygenation (OGD-R) decreased superoxide generation from cytosol and mitochondrial sources resulting in increased survival of BMECs which indicates the physiological significance of enNOS in BMECs. Experimental stroke-induced brain damage is greater in eNOS but diminished in nNOS knockouts, however, the exact mechanisms underlying the nNOS inhibition afforded neuroprotection have never been examined. We hypothesize that enNOS is functionally distinct from eNOS and nNOS of neuronal origin. We further hypothesize that enNOS is the primary mediator of OGD-R injury to BMECs and is an important modulator of post-ischemic BBB disruption. Aim 1 will demonstrate that enNOS is functionally distinct from eNOS and nNOS in generating superoxide versus NO and in modulating mitochondrial function after OGD-R in cultured BMECs and neurons. Aim 2 will determine the functional significance of enNOS and eNOS on post-OGD-R viability and structural integrity of BMECs. Aim 3 will determine the differential role of enNOS and eNOS on the post-ischemic BBB integrity and microvascular dysfunction. The proposed studies will fundamentally advance our mechanistic understanding of NOS, the single most important regulator of neurovascular unit, and will provide breakthrough findings to target enNOS for treating microvascular dysfunction in stroke.

 描述（由申请人提供）：脑微血管功能障碍与中风后的脑损伤有关，然而，一氧化氮合酶（NOS）具有以一氧化氮合酶命名的内皮（eNOS）和神经元（nNOS）亚型。我们的初步研究首次在新鲜分离的大鼠脑微血管和脑微血管内皮细胞中发现了 nNOS。利用 PCR 和免疫印迹技术，我们发现内皮型 nNOS 在结构和功能上与神经元中表达的 nNOS 不同，因此，我们将内皮型 nNOS 命名为 enNOS。抑制 BMEC 中的 eNOS 或神经元中的 nNOS 会增加超氧化物水平并降低 NO 水平，此外，抑制 eNOS 会导致线粒体储备呼吸能力下降。神经元中 nNOS 的抑制增加了超氧化物水平并降低了 NO 水平，相反，enNOS 抑制导致超氧化物水平降低，NO 水平增加，并增强了线粒体储备呼吸能力。因此，与神经元起源的 nNOS 和 eNOS 不同，enNOS 存在于非偶联状态。初步研究还表明，enNOS 对 BMEC 中的基线以及血管紧张素 II 诱导的超氧化物水平有显着贡献，其与 NADPH 相当但独立。最后，在缺氧和复氧 (OGD-R) 过程中抑制所有 NOS 亚型会减少细胞质和线粒体来源的超氧化物的产生，从而导致 BMEC 的存活率增加，这表明 enNOS 在实验性中风诱导的大脑中的生理意义。 eNOS 中的损伤更大，但 nNOS 敲除中的损伤减弱，然而，我们从未研究过 nNOS 抑制提供神经保护的确切机制。 enNOS 在功能上不同于神经源的 eNOS 和 nNOS。我们进一步发现，enNOS 是 OGD-R 对 BMEC 损伤的主要调节剂，并且是缺血后 BBB 破坏的重要调节剂。在培养的 BMEC 和神经元中，eNOS 和 nNOS 相对于 NO 产生超氧化物以及 OGD-R 后调节线粒体功能的作用将决定其功能意义。 enNOS 和 eNOS 对 OGD-R 后活力和 BMEC 结构完整性的影响 目标 3 将确定 enNOS 和 eNOS 对缺血后 BBB 完整性和微血管功能障碍的不同作用。是神经血管单位最重要的调节因子，将为靶向 enNOS 治疗中风微血管功能障碍提供突破性发现。