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

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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 However, unpublished observations revealed that the near complete (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.