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

eNOS-caveolin-1 系统的自然老化和加速老化

• 批准号: RGPIN-2022-04815
• 负责人: Bernatchez, Pascal
• 金额: $ 2.04万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743501
• 关键词: Natural; accelerated; aging; eNOS; caveolin

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 However, unpublished observations revealed that the near complete (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.

内皮源性一氧化氮 (NO) 是平滑肌细胞 (SMC) 收缩的关键负调节因子。我的研究项目的总体目标是更好地了解内皮一氧化氮合酶 (eNOS/NOS3)（内皮细胞来源）的调节。我们主要发表了 eNOS 如何受到小凹外壳蛋白 Caveolin-1 (Cav-1) 及其负调控的研究。 20 个氨基酸支架结构域 (CSD)，甚至具有“显性失活”样特性的突变型细胞渗透性 CSD 肽 CavNOxin 如何通过阻止 Cav-1 对 eNOS 的抑制钳来显着增加血管舒张性 NO 释放。未发表的观察结果表明，CavNOxin 对血管收缩力的近乎完全 (>80%) 的 NO 依赖性抑制仅在 >10 个月大的小鼠的老化血管中观察到，而来自 2 个月大的小鼠的血管显示此外，最近使用完整血管进行的整体显微镜实验表明，eNOS 的核周高尔基体定位在老化的血管系统中发生了显着改变，呈现出更弥散和点状的外观，让人想起 Cav-1。老年组织与年轻组织的这些黑白差异表明衰老脉管系统中的 eNOS-Cav-1 系统存在深刻的变化，这是一个新概念。更重要的是，我们假设老化的血管内皮会经历其高尔基体的有计划的断裂（这是其他领域的常见话题），导致 eNOS 错误定位并改变与其一些关键辅助因子（包括抑制性 Cav-1）的相互作用。这一系列事件是否影响其他内皮血管舒张剂的释放尚不清楚，我们将解决的重要知识空白：我们将确定血管老化是否导致。计划的内皮高尔基体碎裂，以及这如何与 eNOS 定位以及与 Cav-1/caveolae 系统相互作用的变化相关甚至引起变化，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，从而影响因果关系。我们提出以下目标： 1：确认血管衰老与内皮高尔基体更大的破碎、eNOS 错误定位以及与 的更大相互作用相关。 Cav-1，导致内皮血管舒张功能降低和 CavNOxin 反应增强 2：表明血管紧张素 II 输注和其他加速血管老化的模型会加速 eNOS 错误定位、eNOS/Cav-1 功能障碍和高尔基体碎裂。强迫 eNOS NO 释放可能会激活反馈回路，从而防止血管老化和下游高尔基体破碎。我们的长期愿景包括更好地理解。内皮细胞稳态如何随年龄变化以及这如何与血管张力控制的改善联系起来 - 这将有利于加拿大老龄化人口。