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（由适用提供）：脑损伤已在中风后在脑损伤中实现了脑微血管功能障碍，但是，基本机制尚不清楚。一氧化氮合酶（NOS）具有内皮（ENOS）和神经元（NNOS）同工型，这些同工型以最初鉴定的位置命名。我们的初步研究首次使用PCR和免疫印迹技术从大鼠，小鼠和人类的新鲜分离的大鼠脑微血管和脑微血管内皮细胞（BMEC）中鉴定出NNO。我们发现内皮NNO在结构和功能上与eNOS和神经元中表达的nNO不同。因此，我们将内皮nnos命名为ennos。我们的初步研究表明，神经元中BMEC或NNOS中eNOS的抑制增加了超氧化物的水平，没有增加水平。此外，eNOS抑制会导致线粒体储备呼吸能力降低。同样，神经元中NNOS的抑制增加了超氧化物水平，没有增加水平。相比之下，ENNOS抑制导致超氧化物水平降低，NO水平升高并增强了线粒体储备呼吸系统。这与神经元起源和eNOS的nnos不同，Ennos存在于未耦合的状态下。初步研究还表明，ENNOS在BMEC中显着促进了基线以及血管紧张素II诱导的超氧化物水平，而BMEC与NADPH氧化物相当。最后，在氧葡萄糖剥夺和氧化（OGD-R）期间，对所有NOS同工型的抑制作用从细胞质和线粒体来源产生了超氧化物（OGD-R），从而提高了BMEC的存活，这表明BMEC在BMEC中的物理意义。 eNOS中，实验性中风引起的脑损伤较大，但在NNOS敲除中减少，但是，从未检查过NNOS抑制作用的确切机制，从未检查过神经保护。我们假设ENNOS在功能上与神经元起源的eNOS和NNOS不同。我们进一步假设ENNOS是OGD-R损伤对BMEC的主要介体，并且是缺血后BBB破坏的重要调节剂。 AIM 1将证明ENNOS在产生超氧化物与NO方面与ENOS和NNO在培养的BMEC和神经元中OGD-R后调节线粒体功能方面与ENOS和NNO不同。 AIM 2将确定ENNOS和ENOS在BMEC的后R-R生存力和结构完整性上的功能意义。 AIM 3将确定ENNOS和ENOS在缺血后BBB完整性和微血管功能障碍中的差异作用。拟议的研究将从根本上提高我们对NOS的机械理解，NOS是神经血管单元的最重要调节剂，并将为目标ENNOS提供突破性的发现，以治疗中风中的微血管功能障碍。