Endothelial cells (ECs) line the innermost surface of blood vessels, forming the monolayer barrier between blood and tissues. The endothelium is a spatially distributed dynamic and reactive organ, which rules nitric oxide (NO)-dependent vasodilatation and maintains the anti-thrombotic and anti-inflammatory phenotype of blood vessels. Its undisturbed function is essential for cardiovascular health. One of the most dominant detrimental factors heavily exposing cardiovascular homeostasis at risk is ageing. Along with increased life expectancy, the prevalence of cardiovascular disorders (CVDs) is a prime concern. 
The classical view of vascular impairment in many CVDs is generalised to endothelial dysfunction attributed to oxidative stress and lower nitric oxide (NO) availability due to decreased NO production. However, the mechanisms subsequent to NO formation may also compromise its bioactivity. Their contribution to age-related endothelial dysfunction and their principal molecular effectors remains undefined. We propose that the limited NO-dependent bioavailability in ageing blood vessels results from increased protein S-nitrosation, which not only traps NO inside ECs but also changes the properties of proteins and induces protein aggregation.
S-nitrosation (SNO), a covalent attachment of a nitroso group (-NO) to cysteine, is a post-translational modification catalysed by an enzymatic complex, recently discovered by our group, comprising Keap1, a known repressor of Nrf2 transcription factor, GAPDH and NO synthase. The central hypothesis of this proposal predicts that ageing-related endothelial dysfunction and vascular adverse effects may result from Keap1-dependent massive S-nitrosation of proteins, which affects their activity and triggers aggregation, thus deteriorating endothelial proteostasis. This hypothesis is supported by our preliminary data showing a profound Keap1-dependent protein aggregation in prematurely and physiologically ageing ECs and aortas. We also found similar deposits of abnormal proteins formed in ECs devoid of GSNOR, a major denitrosating enzyme, and those exposed to exogenous delivery of S-nitrosothiols. It indicates that excessive protein S-nitrosation is associated with proteostasis loss in ECs. However, the functional relevance of this vascular proteopathy is unknown. According to our observations, the level of Nrf2, a Keap1 binding partner and major protein regulating the cellular stress response, dramatically declines with age in human ECs, which causes an increase in the free Keap1 pool. Subsequently, an active SNO complex formation is probably assembled since protein aggregates can be removed from aged ECs by Keap1 depletion or treatment with sodium ascorbate, the S-nitrosothiol reductant. If the proposed mechanism of endothelial dysfunction proves to be true, Keap1 targeting may open up new perspectives in CVD prevention and therapy.
Our primary goal is to examine the functional significance and experimental therapeutic relevance of Keap1-dependent vascular proteopathy. We will accomplish this aim by meeting the following objectives: (A) Determine why Nrf2 protein level declines with age in human ECs, (B) Assess the effect of SNO-dependent protein aggregation on functional capability of ECs and blood vessels, (C) Verify if vascular complications can be counteracted by inhibition of SNO and protein aggregation by modulation of endothelial Keap1 level, treatment with a Keap1 inhibitor or a chemical chaperone. The proposal relies on in vitro and in vivo experiments. We will use human primary endothelial cells derived from young and aged donors. The in vivo experiments will be carried out on aged mice, Nrf2 knockout mice, and GSNOR knockout mice. 
The predicted insights of this study include functional translation of the newly discovered S-nitrosating enzymatic complex in pathological conditions, understanding the out-of-the-box molecular basis of vascular dysfunction, and expanding the significance of proteopathies beyond neurodegenerative diseases. The long-term expected outcome of the proposal is to address the potential therapeutic relevance of Keap1 targeting in vascular complications.