Natural and accelerated aging of the eNOS-caveolin-1 system

内皮源性一氧化氮（NO）是平滑肌细胞（SMC）收缩的重要负性调节因子。我的研究计划的总体目标是更好地了解内皮NO合酶（eNOS/NOS 3）的调节，激动剂诱导的NO释放的内皮源。我们已经广泛发表了关于eNOS如何受到小窝外壳蛋白小窝蛋白-1（Cav-1）及其20个氨基酸的支架结构域（CSD）的负调控，甚至如何通过阻止Cav-1对eNOS的抑制性钳夹，使一种称为CavNOxin的具有“显性负性”性质的突变细胞可渗透CSD肽大幅增加血管舒张NO释放。然而，未发表的观察结果显示，CavNO新对血管收缩性的几乎完全（>80%）的NO依赖性抑制仅在来自> 10月龄小鼠的老化血管中观察到，而来自2月龄小鼠的血管没有显示（0%）血管舒张作用。此外，最近使用完整血管的整体封片显微镜实验已经表明，eNOS的充分记录的核周高尔基体定位在老化的脉管系统中急剧改变，呈现出更弥散和点状的外观，这让人想起Cav-1染色。老年组织与年轻组织中的这些黑白白色差异表明，在老化血管系统中存在eNOS-Cav-1系统的深刻变化，这是内皮生物学中的一个新概念。更重要的是，我们假设老化的血管内皮细胞经历了高尔基体的有计划的碎片化-这是其他领域的一个共同话题-导致eNOS错误定位和与其一些关键辅因子（包括抑制性Cav-1）相互作用的变化。这一系列事件是否影响其他内皮血管扩张剂的释放尚不清楚。我们将解决的重要知识差距：我们将确定血管老化是否会导致计划的内皮细胞高尔基体片段化，以及这与eNOS定位的变化以及与Cav-1/caveolae系统的相互作用如何相关，甚至导致eNOS定位的变化。我们还将研究这一系列事件如何直接影响对CavNOxin的动态内皮反应，从而暗示因果关系。我们提出了以下结论：1：证实血管老化与更大的内皮高尔基体片段化、eNOS错误定位和与Cav-1更大的相互作用相关，导致内皮血管舒张功能降低和CavNOxin反应增强。第二章：显示血管紧张素II输注和其他加速血管老化的模型加速eNOS错误定位、eNOS/Cav-1功能障碍和高尔基体碎裂。3：确定是否强制eNOS NO释放可以激活防止血管老化和下游高尔基体断裂的反馈回路。我们的长期愿景包括更好地了解内皮细胞稳态如何随着年龄的增长而变化，以及这如何与血管张力控制的改善有关-这将使加拿大人口老龄化受益。

Endothelium-derived nitric oxide (NO) is a critical negative regulator of smooth muscle cell (SMC) constriction. The OVERALL OBJECTIVE of my research program is to better understand the regulation of the endothelial NO synthase (eNOS/NOS3), the endothelial source of agonist-induced NO release. We have extensively published on how eNOS is negatively regulated by caveolae coat protein caveolin-1 (Cav-1) and its 20 amino acid scaffolding domain (CSD), and even how a mutant cell-permeable CSD peptide called CavNOxin with `dominant-negative'-like properties can drastically increase vasodilatory NO release by preventing the inhibitory clamp of Cav-1 on eNOS. However, unpublished observations revealed that the near complete (>80%) NO-dependent inhibition of vascular contractility with CavNOxin is exclusively observed in aged vessels from >10mo old mice, whereas vessels from 2mo old mice show no (0%) vasodilatory effect. Moreover, recent whole mount microscopy experiments using intact vessels have shown that eNOS' well-documented perinuclear Golgi localization is drastically altered in aging vasculature, taking a more diffuse and punctate appearance that is reminiscent of Cav-1 staining. These black-and-white differences in old vs young tissues suggest the presence of profound changes to the eNOS-Cav-1 system in aging vasculature, a novel concept in endothelial biology. More importantly, we postulate that the aging vascular endothelium undergoes planned fragmentation of its Golgi apparatus - a common topic in other fields - resulting in eNOS mislocalization and changes to interactions with some of its critical co-factors including  inhibitory Cav-1. Whether this chain of events affects the release of other endothelial vasodilators is unknown. THE IMPORTANT KNOWLEDGE GAPS we will address: we will determine whether vascular aging causes planned endothelial Golgi fragmentation, and how this correlates with or even causes changes in eNOS localization and interaction with the Cav-1/caveolae system. We will also study how this chain of events may directly affect the dynamic endothelial response to CavNOxin and therefore imply causality. We propose the FOLLOWING OBJECTIVES: 1: Confirm that vascular aging correlates with greater endothelial Golgi fragmentation, eNOS mislocalization and greater interaction with Cav-1, resulting in lower endothelial vasodilatory function and enhanced CavNOxin response. 2: Show that angiotensin II infusion and other models of accelerated vascular aging expedite eNOS mislocalization, dysfunction of the eNOS/Cav-1 and Golgi apparatus fragmentation. 3: Determine whether forced eNOS NO release may activate a feedback loop that prevents vascular aging and downstream Golgi fragmentation. Our LONG-TERM VISION includes a better understanding of how endothelial cell homeostasis changes with age and how this might be linked to improvements in vascular tone control - which will benefit an aging Canadian population.