Endothelial Expression of Neuronal Nitric Oxide Synthase

 描述（由申请人提供）：脑微血管功能障碍与卒中后的脑损伤有关，然而，其潜在机制尚不清楚。一氧化氮合酶（NOS）具有内皮型（eNOS）和神经型（nNOS）亚型，它们以它们首次被鉴定的位置命名。我们的初步研究首次利用PCR和免疫印迹技术在来自大鼠、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）中鉴定了nNOS。我们发现内皮型nNOS在结构和功能上不同于eNOS和神经元中表达的nNOS。因此，我们将内皮nNOS命名为eNOS。我们的初步研究表明，抑制BMEC中的eNOS或神经元中的nNOS，增加超氧化物的水平，降低NO的水平。此外，eNOS抑制导致线粒体储备呼吸能力降低。类似地，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抑制导致减少超氧化物水平，增加NO水平，并增强线粒体储备呼吸能力。因此，与神经元来源的nNOS和eNOS不同，eNOS以解偶联状态存在。初步研究还表明，eNOS显著促进BMEC中基线以及血管紧张素II诱导的超氧化物水平，其与NADPH氧化酶相当但独立于NADPH氧化酶。最后，在氧-葡萄糖剥夺和再氧合（OGD-R）期间抑制所有NOS同工型减少了来自胞质溶胶和线粒体来源的超氧化物的产生，导致BMEC的存活增加，这表明enNOS在BMEC中的生理意义。实验性中风引起的脑损伤在eNOS中更大，但在nNOS敲除中减少，然而，从未检查过nNOS抑制提供神经保护的确切机制。我们假设，eNOS是功能不同的eNOS和神经元来源的nNOS。我们进一步假设，eNOS是OGD-R损伤BMEC的主要介质，是缺血后BBB破坏的重要调节剂。目的1将证明eNOS和nNOS在产生超氧化物和NO以及在培养的BMEC和神经元中OGD-R后调节线粒体功能方面在功能上不同。目的2将确定eNOS和eNOS对OGD-R后BMEC活力和结构完整性的功能意义。目的3探讨内皮型一氧化氮合酶（enNOS）和一氧化氮合酶（eNOS）在缺血后血脑屏障完整性和微血管功能障碍中的作用。拟议的研究将从根本上推进我们对NOS（神经血管单位的单一最重要调节因子）的机制理解，并将为靶向enNOS治疗卒中微血管功能障碍提供突破性发现。

 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.