Keap-1-mediated S-nitrosylation of NOX4 hampers oxidative damagein endothelium

Aleksandra Kopacz (PhD student)1, Damian Kloska (PhD student)1, Dominik Cysewski (PhD student)2, Bartosz Proniewski (PhD)3, Nicolas Personnic (MSc)1, Aleksandra Piechota-Polanczyk (PhD)1, Patrycja Kaczara (PhD)3, Agnieszka Zakrzewska (PhD)3, Jozef Dulak (Prof.)1, Alicja Jozkowicz (Prof.)1, Anna Grochot-Przeczek (PhD)1*

1Department of Medical Biotechnology, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland

2Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Science, 02-106 Warsaw, Poland

3Jagiellonian Centre for Experimental Therapeutics, Jagiellonian University, 30-348 Krakow, Poland

*corresponding author: Anna Grochot-Przeczek, Department of Medical Biotechnology, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland. e-mail: anna.grochot-przeczek@uj.edu.pl

Short title: Unrestrained Keap1 determines endothelial fate

Total word count: 7758

Subject codes: Endothelium/Vascular Type/Nitric Oxide, Aging, Mechanisms

1

ABSTRACT

Rationale Premature senescence is conducive to aging and cardiovascular diseases. Nrf2 transcription factor, the master orchestrator of adoptive response to cellular stress, has been implicated in regulation of premature senescence in fibroblasts, neural and mesenchymal stem cells by transactivation of antioxidant gene expression. However, as we show here, human primary endothelial cells (ECs) devoid of Nrf2 and murine Nrf2 transcriptional knockout (tKO) aortas are senescent but do not encounter oxidative stress and damage, what contradicts this mechanism. Moreover, a molecular switch between normal, senescent and apoptotic fate remains unknown.

Objective To elucidate the mechanism of Nrf2-related premature senescence of vascular system, to understand why Nrf2 deregulation does not cause oxidative stress exclusionary in ECs and to indicate a molecular switch determining ECs fate.

Methods and Results Herein we evidence that ECs deficient in Nrf2 protein, or with limited Nrf2 activity in shear stress conditions, exhibit excessive S-nitrosylation of proteins. It is also a characteristic of Nrf2 tKO murine aortas, as determined by biotin switch assay in situ. Mass spectrometry analysis reveals that NOX4 is S-nitrosylated exclusively in ECs devoid of Nrf2. A functional role of S-nitrosylation is protection of ECs from death by inhibition of NOX4-mediated oxidative damage. As a result Nrf2-deficient ECs preserve oxidative balance but are redirected to premature senescence. The same phenotype is seen in Nrf2 tKO aortas. These effects are mediated by Keap1, a direct binding partner of Nrf2 and repressor of its transcriptional activity, remaining in cytoplasm unrestrained by Nrf2. S-nitrosylation, followed by senescence, can also be triggered in smooth muscle cells (SMCs) by EC-derived paracrine induction of iNOS.

Conclusions Collectively, Keap1-dependent S-nitrosylation of NOX4 hampers oxidative detriment in ECs with disturbed Nrf2 signaling and may provide defence in the adjacent aortic cells. Overabundance of unrestrained Keap1 in the cytoplasm determines fate of ECs.

Keywords: endothelial cells/NOX4/Nrf2/oxidative stress/S-nitrosylation

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INTRODUCTION

Cellular senescence is a state of permanent cell cycle arrest with concomitant maintained metabolic activity and viability, which might be caused by DNA damage, response to oncogene activation, tumour-suppressor loss or cellular stress. It is well evidenced that premature senescence contributes to aging and age-related diseases.1 Such dependency also applies to vascular system, what manifests in an increased risk of cardiovascular disorders (CVDs).2 Endothelium is a tissue particularly exposed to adverse signals, therefore it may undergo premature senescence becoming dysfunctional, what facilitates CVD onset.3

Recent studies demonstrated that Nrf2, encoded by NFE2L2 (nuclear factor (erythroid-derived 2)-like 2) is one of the key players maintaining healthy endothelial phenotype.4–6 Nrf2 is a transcription factor mediating the adaptive response to cellular stress. A number of genes encoding detoxifying enzymes (glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase (NQO1)), stress-responsive proteins (heme oxygenase-1 (HMOX1)), reactive oxygen species (ROS) scavenging enzymes (glutathione peroxidase (GPX), superoxide dismutase (SOD)) are directly regulated by Nrf2, upon liberation from Keap1 cytoplasmic repression, through the antioxidant response element (ARE) located in their promoters.7 Not surprisingly, inhibition of Nrf2 activity was shown to make the mice susceptible to oxidative damage.8–11 Moreover, it was observed that Nrf2 level decreases with age, 12,13 what corresponds to ineffective Nrf2-dependent antioxidant defence in vascular cells in the elderly.14

Senescent-associated mechanisms for declined Nrf2 activity are, however, unclear. Moreover, the molecular mechanisms of senescence driven by Nrf2 inhibition, especially in endothelial cells (ECs), persist also unknown.

One of the key molecule modulating responses and function of ECs and vascular system is nitric oxide (NO). The classical pathway activated by NO transduces signal through soluble guanyl cyclase and cGMP, what results in the regulation of vasodilatation of vascular smooth muscle cells (VSMCs) and modulation of blood pressure.15 Noteworthy, NO can also influence the cellular response through protein S-nitrosylation,16 which is an addition of NO moiety mostly to cysteines. It enables fast, posttranslational modification of many proteins which are involved in regulation of apoptosis, migration, transport or cell division.17 A ‘switch-off’ mechanism is a denitrosylation, which is performed mainly by S-nitrosoglutathione reductase (GSNOR) and thioredoxin reductases (TrxRs). A number of proteins are constitutively nitrosylated in the cell, and denitrosylation serves as a mechanism for their activation.18

Recently we found that loss of Nrf2 transcription factor leads to induction of p53-dependent premature senescence in primary human aortic endothelial cells (HAECs).19 The molecular mechanism that triggers the senescence onset when Nrf2 signaling is absent and also its relevance in vivo remain, however, to be clarified. In this paper, we provide evidence that in ECs lack of holding Keap1 in check by Nrf2 causes excessive protein S-nitrosylation, what provides protection from NOX4-mediated oxidative damage and apoptosis, but redirects them to senescence.

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METHODS

Animals. Nrf2-transcriptional knockout (tKO) C57BL/6 mice, initially developed by Masayuki Yamamoto as described previously,20 were kindly provided by Antonio Cuadrado (Universidad Autonoma de Madrid) together with WT mice. 8-week old animals were used in the study. All animal work was approved by the Local Ethical Committee for Animal Research at the Jagiellonian University. In tKO animals, DNA binding domain of Nrf2 was replaced by LacZ gene20 what results in the appearance of fusion protein Nrf2-β-galactosidase.21,22 Therefore, a question was raised if it is possible, as there is an activity of the transgene driven β-gal in these mice, 21 to distinguish its activity from the senescence-related one. That is why we performed additional experiments on lung fibroblast isolated from WT and tKO mice. Importantly, the activity of β-gal of LacZ origin can be detected only at pH 7,23 while senescence-associated-β-gal (SA-β-gal) activity is detected at pH 6.24,25 Our data showed, that, indeed, β-gal activity at pH 7 is detected above background only in lysates from tKO fibroblasts stimulated with doxorubicin (Fig. S1A). Importantly, it remains undetectable in in situ staining at pH 7 (Fig. S1B), what evidences that detection of LacZ-derived β-gal activity can be only achieved in lysates at pH 7. Accordingly, the blue colour of cells stained in situ at pH 6 may come only from SA-β-gal activity (Fig. S1C).

Please see the Online Supporting Document available at http://circres.ahajournals.com for all methods related to this study.

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RESULTS

Inhibition of Nrf2 transcriptional activity results in premature senescence, but not oxidative damage, in murine aorta. To verify if disruption of Nrf2 signaling drives senescent phenotype of vascular cells in vivo, aortas of 8-week old wild type (WT) and transcriptional knock out (tKO) mice were stained for SA-β-gal activity. We found that it was significantly increased in tKO aortas, compared to WT animals, and present throughout the whole length of the vessels (Fig. 1A). En face projection showed the appearance of SA-β-gal positive cells at the luminal surface of the vessels, which were also massively scattered across the whole depth of the aortic wall, including tunica intima and tunica adventitia (Fig. 1B). Moreover, a profound rise in p21 level, the senescence-mediating protein, was seen in the aortas of tKO mice (Fig. 1C). Considering the key role of Nrf2 signaling in the regulation of oxidative defence,7 and the mechanism of premature senescence induction by oxidative stress,26 an oxidative status of WT and tKO aortas was assessed. No differences in glutathione peroxidase (Gpx1), manganese superoxide dismutase (Sod2) and catalase (Cat) expression were detected between the aortic tissue of WT and tKO animals (Fig. 1D-F). Although the superoxide anion level was elevated in the aortas of tKO mice (Fig. 1G), this was not associated with the excessive lipid peroxidation (Fig. 1H), which is the hallmark of oxidative stress and damage. In addition, tKO aortas were similarly susceptible to the oxidative detriment when challenged with iron overload, as those isolated from WT mice (Fig. 1H). These data demonstrate that inhibition of Nrf2 transcriptional activity does not cause oxidative damage in the murine aorta, but results in the senescence onset.

Loss of Nrf2 protein in primary human ECs is not associated with excessive oxidative stress. Similarly to Nrf2 tKO aortas, peroxidation of lipids remained low in siNFE2L2-transfected HAECs, with the level comparable to siMock cells (Fig. 2A). Iron overload caused an even increase in lipid peroxidation in both inspected groups (Fig. 2A), what shows that control HAECs and those devoid of Nrf2 are equally susceptible to oxidative stress. No changes in the expression of GPX1 (Fig. 2B), but significant downregulation of SOD2 (Fig. 2C) and CAT (Fig. 2D) mRNA were observed upon silencing of NFE2L2 in HAECs. Even so, neither total (Fig. 2E) nor mitochondrial (Fig. 2F) ROS levels, measured by fluorescent probes, were found to be elevated in Nrf2 deficient cells and their total antioxidant capacity was preserved (Fig. 2G). Although HPLC-based analysis showed a significant increase in superoxide anion amount in siNFE2L2 HAECs (Fig. 2H), associated with decreased total glutathione level (Fig. 2I), still oxidized form of glutathione was undetectable. Such results may be indicative of maintained oxidative balance in HAECs lacking Nrf2, what reaches beyond commonplace scheme of Nrf2 deficiency being inseparable from uncontrolled oxidative stress. Finally, we made an attempt to rescue the senescent phenotype of Nrf2 deficient HAECs (Fig. 2J,K) by incubation with N-acetyl cysteine (NAC), a known glutathione precursor capable also of ROS scavenging.27 NAC triggered only a partial, up to 40%, attenuation of SA-β-gal activity in HAECs with siRNA-mediated knock down of Nrf2 (Fig. 2J,K). This suggests that the redox status is not severely affected by Nrf2 deficiency in ECs. Collectively, these data show that Nrf2 deficient HAECs do not encounter oxidative stress and damage, though their microenvironment shows slight disequilibrium towards oxidation. Nonetheless, HAEC senescence driven by knock down of Nrf2 is not linked solely to redox status of the cells.

Lack of Nrf2 transcriptional activity induces premature senescence but only in shear stress conditions. The animal model of Nrf2 deficiency used in this study is a transcriptional knockout, in which DNA binding domain of Nrf2 had been removed, but the N-terminal fragment, containing Keap1 binding domain Neh2, of Nrf2 is preserved.19,20 On the other hand, in siRNA-based in vitro experimental setting (Fig. 2) the availability of Nrf2 protein is reduced. 19 Moreover, our recent study indicates that Nrf2 activity may reach beyond transactivation of gene expression, as Nrf2 also serves as a protein sequestering Keap1 to prevent podosome disassembly in ECs.19 Therefore, to confront the findings from in vivo setting (Fig. 1) with the in vitro experiments (Fig. 2), HAECs were transduced with adenoviral vectors harbouring dominant negative NFE2L2, coding for 403-605 aa of Nrf2. The non-toxic titer of AdNFE2L2DN vectors (MOI 50) led to ~60% inhibition of Nrf2 transcriptional activity (Fig. 3A,B), however it did not result in the onset of senescence (Fig. 3C),

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unless the cells were subjected to the shear stress conditions (Fig. 3C). Noteworthy, inhibition of Nrf2 transcriptional activity at this level did not evoke any cytotoxic effects to HAECs. The use of higher concentration of AdNFE2L2DN vectors (MOI 70), however, blunted NQO1 and GCLM expression by more than 80% (Fig. 3D,E), leading to strong lipid peroxidation (Fig. 3F) and cell death (Fig. 3G). These adverse effects were fully counteracted by shear stress conditions (Fig. 3F,G). Taken together, inhibition of Nrf2 transcriptional activity leads to oxidative damage, unless the cells are subjected to shear stress. Moreover, loss of Nrf2 signaling combined with shear stress conditions induces senescence onset in vitro in HAECs and in vivo in the murine aorta, but ECs devoid of Nrf2 protein itself are protected from oxidative damage and develop senescent phenotype even in static conditions.

Nrf2-dependent senescence onset is associated with potent protein S-nitrosylation. Recent studies have demonstrated that S-nitrosylation may protect the cells from oxidative stress28 and when overabundant, it impairs multiple cellular processes, thus leading to cellular senescence. 3,29,30 We hypothesized that S-nitrosylation could be responsible for the induction of senescence and protection from oxidative damage in cells with disordered Nrf2 signaling.

In comparison to WT, tKO aortas exhibited an increased level of S-nitrosylation (Fig. 4A), together with the significant downregulation of genes coding denitrosylating enzymes, Adh5 (Fig. 4B), coding GSNOR and Txnrd1 (Fig. 4C), coding TrxR1. Analysis of NO metabolism showed lower nitric oxide functional release in aortic wall upon stimulation with calcium ionophore (Fig. 4D), decreased amount of nitrosylated haemoglobin in erythrocytes (Fig. 4E) together with unchanged nitrites (Fig. 4F), but raised nitrates, the products of nitrite oxidation (Fig. 4G), in plasma of Nrf2 tKO mice.

Likewise, silencing of NFE2L2 expression in HAECs resulted in a potent S-nitrosylation of proteins (Fig. 5A). Nitrotyrosines, however, were undetectable, what excludes the presence of uncontrolled nitrosative stress in these cells. Membrane NO concentration, in mock and NFE2L2 siRNA transfected HAECs, was equal between compared groups (Fig. 5B), whereas the level of secreted nitrites, direct products of NO decomposition, was lower in Nrf2-deficient HAECs (Fig. 5C). Although expression of NOS3 (eNOS) decreased both on the mRNA and protein level (Fig. 5D,E), eNOS activating phosphorylation of serine 1177 was strengthened (Fig. 5E). NOS2 gene expression remained unchanged (Fig. 5F), however, iNOS protein was strongly elevated in Nrf2-deficient cells (Fig. 5G). Finally, a profound downregulation of ADH5 (Fig. 5H) and TXNRD1 (Fig. 5I) expression was observed in HAECs devoid of Nrf2.

Overexpression of NFE2L2DN (MOI 50) in HAECs in static conditions only subtly increased protein S-nitrosylation (Fig. 5J,K), what was accompanied by an unchanged level of ADH5 mRNA (Fig. 5L) and a significant decrease of TXNRD1 (Fig. 5M). Subjecting of HAECs to shear stress conditions induced protein S-nitrosylation both in GFP- and NFE2L2DN-transduced cells, however, the level of this modification was much stronger in AdNFE2L2DN HAECs (Fig. 5K). Analysis of gene expression of denitrosylating enzymes revealed that shear stress conditions downregulated ADH5 expression only in AdNFE2L2DN cells (Fig. 5L), while TXNRD1 mRNA level was lower in AdNFE2L2DN cells in comparison to AdGFP regardless of flow conditions (Fig. 5M).

Collectively, these results evidence the presence of an exacerbated S-nitrosylation in Nrf2-deficient HAECs, NFE2L2DN overexpressing HAECs subjected to shear stress conditions and in Nrf2 tKO aortas, so only in those cases where the senescence onset is visible. Moreover, intensified S-nitrosylation and senescence occur only when the expression of both genes coding denitrosylating enzymes, ADH5 and TXNRD1, is downregulated concomitantly. Therefore, taking the next step, we aimed to determine the functional significance of S-nitrosylation in Nrf2-deficient ECs.

S-nitrosylation provides protection from NOX4-mediated oxidative stress and death in Nrf2-deficient cells. The role of S-nitrosylation in endothelial physiology remains elusive. It is an essential protein modification to maintain endothelial homeostasis17 and is supposed to prevent excessive oxidative stress.28 On the other hand, it contributes to the impairment of various cellular processes, thus leading to senescence.31 To address this issue, the cells were incubated with dithiothreitol (DTT), the reducing agent, which completely abolished protein S-nitrosylation (Fig. 6A). Such treatment led to oxidative damage assessed by lipid peroxidation (Fig. 6B) and sudden Nrf2

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deficient cell death (Fig. 6C), what was not the case in siMock HAECs (Fig. 6A-C). This shows that S-nitrosylation protects Nrf2-deficient ECs from oxidative injury and death.

In the pursuit of the possible mechanism of S-nitrosylation based ECs protection, mass spectrometry analysis was performed to identify modified proteins. 28 SNO-proteins, which appear preferentially only in siNFE2L2 HAECs (Table S1) included NAPDH oxidase 4 (NOX4), one of the main sources of reactive oxygen species in the cells.32 As S-nitrosylation was shown to inhibit NOX4 activity,33 we hypothesized that it provides protection from oxidative damage in HAECs devoid of Nrf2. To figure it out, concomitant silencing of NOX4 and NFE2L2 was performed (Fig. 6D,E). Inhibition of NOX4 expression fully prevented denitrosylation-induced lipid peroxidation in HAECs cells lacking Nrf2 (Fig. 6F), what successfully hampered DTT-induced cell death (Fig. 6G). Alike, stimulation of the cells with another reducing agent, sodium ascorbate, reversed S-nitrosylation of proteins (Fig. S2A) and induced oxidative damage in siNFE2L2 HAECs (Fig. S2B), unless the cells were additionally devoid of NOX4 (Fig. S2C,D). Altogether, we identify S-nitrosylation of NOX4 as a reason for the maintenance of oxidative balance and protection from oxidative damage in Nrf2-deficient ECs.

Unrestrained Keap1 triggers protein S-nitrosylation and premature senescence in ECs. A prerequisite for the occurrence of an excessive S-nitrosylation and senescence is a lack of Nrf2 protein in static conditions or a loss of Nrf2 transcriptional activity in shear stress conditions both in vitro and in vivo. Our group has recently shown that Keap1 has to be restrained by Nrf2 in ECs to prevent angiogenic dysfunction and that its level is increased in ECs devoid of Nrf2 19. On the other hand, it is known that exposure of ECs to oscillatory shear stress, used in our study, induces translocation of Nrf2 into the nuclei, although it remains transcriptionally inactive, 34 what may cause Keap1 to be left unkept by Nrf2 in the cytoplasm, unless it is degraded. Indeed, Keap1 level was increased in HAECs overexpressing dominant negative NFE2L2 cultured in shear stress conditions (Fig. 7A). Therefore, we hypothesized that unrestrained and overabundant Keap1 is responsible for S-nitrosylation and premature senescence of ECs. Concomitant silencing of NFE2L2 and KEAP1 in HAECs decreased protein S-nitrosylation (Fig. 7B), restored cell proliferation (Fig. 7C), lowered p21 level (Fig. 7D), and finally reversed senescent phenotype of Nrf2-deficient cells (Fig. 7E). Interestingly, it did not bring back expression of ADH5 (Fig. 7F), while restoration of TXNRD1 expression was statistically significant, though still low (Fig. 7G). Such a mild restoration of TXNRD1 expression raises doubts, whether it can be responsible for the reversal of S-nitrosylation in siNFE2L2/siKEAP1 cells.

SNO-proteins formation can be achieved by transnitrosylation due to activity of proximal SNO-modified proteins – transnitrosylases, which include hemoglobin, thioredoxin, caspase 3, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and protein disulphide isomerase (PDI).35 We have looked for such proteins in Keap1 pulled down pellet. GAPDH and PDI-A3 subunit were identified with high score (Table S2). Unlike PDI (Fig. S3), GAPDH strongly colocalized with Keap1 both in siMock and siNFE2L2 cells (Fig. 7H), and the level of GAPDH decreased in cells devoid of Nrf2 and Keap1. The question remained, why denitrosylation of proteins in Nrf2 deficient cells redirects them from senescence to apoptosis (Fig. 6), while silencing of Keap1, the protein responsible for induction of S-nitrosylation and senescence in these cells, restores their normal phenotype (Fig. 7). Such reliance implicates that NOX4 can be active only when it is denitrosylated and Keap1 is present. As depicted in Fig. 7I, NOX4 protein is abolished in siNFE2L2/KEAP1 cells, what solves this puzzle.

Collectively, these data show that overabundance of unrestrained Keap1 causes protein S-nitrosylation and ECs senescence. In addition, it determines normal, senescent or apoptotic fate of ECs.

Paracrine induction of iNOS in smooth muscle cells drives the senescence onset. Detailed macroscopic analysis revealed the increased SA-β-gal activity at the luminal surface of Nrf2 tKO aortas, but also its appearance across the full thickness vessel wall, reaching up to adventitia (Fig. 1B). The question raises then, whether the senescent phenotype is developed in the particular aortic layers of Nrf2 tKO animals independently or which cells devoid of Nrf2 signaling initiate development of senescence in the adjacent layers.

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Senescent cells were shown to secrete a wide range of soluble factors associated with inflammation and growth, termed the senescence associated secretory phenotype (SASP), which may paracrinely induce senescence in the surrounding cells.36 An inflammasome was identified as a critical regulator of the SASP and paracrine senescence mediator.37 To verify the influence of Nrf2 inhibition on smooth muscle cell (SMCs) phenotype, primary human aortic smooth muscle cells (HASMCs) were transfected with siRNA against NFE2L2. Silencing of NFE2L2 in HASMCs (Fig. S4A) did not result in significant increase in the onset of senescence (Fig. S4B), however, it rendered the cells more susceptible to iron induced oxidative damage (Fig. S4C). It shows that, in contrast to ECs, SMCs deficient in Nrf2 are not protected from oxidative damage. In order to investigate whether Nrf2 deficient ECs are able to induce senescent phenotype in a paracrine manner, HAECs and HASMCs were treated with conditioned media from HAECs transfected either with siMock or siNFE2L2. In case of both cell types conditioned medium from HAECs lacking Nrf2 promoted SA-β-gal activity, indicating for induction of premature paracrine senescence (Fig. S4D-G). Silencing of Nrf2 in HAECs significantly increased production of IL-6 (Fig. S4H) and sICAM-1 (Fig. S4I), known SASP mediators,38 while IL-1β remained undetectable. Moreover, incubation of HASMCs with paracrine media from Nrf2-deficient HAECs resulted in elevated NOS2 expression (Fig. S4J) and abundant protein S-nitrosylation (Fig. S4K). Counteracting the NOS2 induction in response to siNFE2L2 conditioned media in SMCs by genetic depletion of NOS2 in SMCs (Fig. S4L) prevented the appearance of SNO-proteins (Fig. S4M). Taken together, these results indicate that ECs trigger the senescent phenotype in SMCs through the paracrine activation of iNOS resulting in protein S-nitrosylation.

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DISCUSSION

Studies of the past decade suggested that Nrf2 plays a protective role in aging.39 Associations of this transcription factor with senescence and aging exhibit consistency across species, including human, rat, mouse, Drosophila and C. elegans (CncC and SKN-1 are Nrf2 homologs in flies and worms, respectively).40 But the mechanisms of senescence related to Nrf2 pathway remain still open to question, especially in respect of the cardiovascular system.40 We have found that Keap1, overabundant and unrestrained by Nrf2 in the cytoplasm, determines normal, senescent or apoptotic fate of ECs, by regulation of NOX4 level and its S-nitrosylation (Fig. 8).

The overall mechanisms of vascular aging are outlined, indicating oxidative stress as the major reason for age-related vascular impairment.3 Taking into account that Nrf2 is the main factor transactivating the expression of antioxidant genes, one could suppose that its exclusion would result in the oxidative stress and damage. In case of ECs, however, not only is there no difference in total and mitochondrial ROS level between control and Nrf2-deficient cells, but the latter are not more susceptible to oxidative damage control ones (Fig. 2). The mechanism providing this protection is mediated by S-nitrosylation of NOX4 (Fig. 6). The maintenance of oxidative balance, despite of Nrf2 deregulation, seems to be a specific feature of ECs, as for example HSCs from Nrf2 tKO mice do not differ from their littermate counterparts in the level of ROS, however, they are more prone to suffer from oxidative agent stimulation.11 These results are in line with our observations in HASMCs (Fig. 7C).

Although Nrf2 deficient ECs are fully protected from oxidative damage (Fig. 2A), total antioxidant capacity remains unchanged (Fig. 2G) and the oxidized glutathione undetectable, it should be emphasized that the level of superoxide anion is increased to some extend in these cells (Fig. 2H). Such mild ROS accumulation, not exceeding buffering capacity of the cells, may activate protective mechanisms, including anti-oxidative systems or autophagy, which counteract aging.41 In case of Nrf2 deficient ECs, elevated level of superoxide anion may be responsible for the observed slight shift in the redox status of the cell towards more oxidative, but still not harmful, microenvironment. Such conditions may facilitate the covalent attachment of NO· to cysteines, which are oxidized at first in the reaction of S-nitrosylation.42 Therefore, partial rescue of siNFE2L2 HAEC phenotype by NAC might be attributed to inhibition of cysteine oxidation. As our data show, S-nitrosylation provides protection from oxidative damage and death (Fig. 6, S2). On the dark side, however, accumulation of S-nitrosylated proteins redirects the cell to senescence, so the protection against oxidative damage provided by S-nitrosylation has its price for the cell. S-nitrosylation is considered as a fateful posttranslational modification due to the impairment of various cellular processes such as autophagy,31 actin polymerisation43 and protein folding.44 It is known that this modification may induce senescent phenotype.31,43 In accordance, we observed the coexistence of senescence and S-nitrosylation in all investigated experimental models (Fig. 4,5). On the other hand, S-nitrosylation of caspases inhibit their enzymatic activity.45 So it can have a defensive role, similarly to S-glutathionylation, as it indisposes further protein modifications by oxidative stress.28,46 By using DTT, a denitrosylating agent,47 we showed that S-nitrosylation of NOX4, which represents an inhibitory modification for NOXes,33 protects Nrf2-deficient ECs from oxidative damage and death. Moreover, sudden apoptosis of DTT-treated siNFE2L2 cells may be additionally facilitated by denitrosylation and subsequent activation of caspases.

The in vitro observations fully translate into in vivo setting. Increased lipid peroxidation was not detected in the aortas of Nrf2 tKO mice, neither were they more prone to oxidative damage induced by iron overload (Fig. 1H). Similarly to in vitro model, the overabundant S-nitrosylation was found in these aortas (Fig. 4). Also the presence of senescent phenotype and increased production of superoxide anion (Fig. 1) was alike. Of note, 40% of Nrf2 transcriptional activity seems to be enough to provide normal cell function (Fig. 3A-C). Inhibition of Nrf2 activity by 80%, however, induces cellular death accompanied by massive lipid peroxidation, unless the cells are cultured in shear stress conditions (Fig. 3D-G). Accordingly, Nrf2 tKO animals, which possess Keap1 binding domain Neh2,19 exhibit increased protein S-nitrosylation and oxidative protection within aortas, accompanied by senescent phenotype (Fig. 1,4). Thus, we conclude that Nrf2 deficiency or disruption of Nrf2 signaling in shear stress conditions are oxidatively detrimental neither to ECs nor to aorta in vivo.

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S-nitrosylation and premature senescence driven by Nrf2 dysfunction appeared in two cases in our study. Either when Nrf2 protein was lost or when Nrf2 signaling was absent in the oscillatory shear stress conditions. Inhibition of Nrf2 activity by 60% in static conditions did not induce the senescence, what indicates that lack of Nrf2 protein itself, but not its transcription factor function is responsible for the senescence onset, unless the cells are subjected to shear stress conditions in vitro and in vivo. In the light of our recent paper, showing the additional transcription-independent function of Nrf2 in angiogenesis, which unveil that Nrf2 serves as a Keap1 restraining protein,19 and also the papers showing nuclear translocation of Nrf2 in ECs in response to shear stress in vitro,34 and in aorta in vivo,48 it becomes very likely that the protein mediating cell senescence and S-nitrosylation of Nrf2-deficient ECs is Keap1 unbound to Nrf2. Keap1, indeed, is overabundant is ECs devoid of Nrf2 19 and in ECs transduced with AdNFE2L2DN cultured in shear stress conditions (Fig. 7A). Each of these conditions facilitates liberation of Keap1 from Nrf2 repression. In support of the hypothesis, concomitant silencing of Keap1 in Nrf2-deficient cells fully rescued the phenotype of HAECs (Fig. 7). Intriguingly, deletion of Keap1 in Nrf2-deficient cells, caused inhibition of S-nitrosylation (Fig. 7B) and brought back the normal phenotype of ECs, while DTT-induced denitrosylation subjected these cells to apoptosis (Fig. 6). A simple explanation of these extremely contrasting phenotypes, both achieved by protein denitrosylation, is the regulation of NOX4 level by Keap1 (Fig. 7). Hence, HAECs devoid of Nrf2 and Keap1 had normal phenotype, in contrast to Nrf2-deficient HAECs treated with DTT. Having look at the murine model, double Nfe2l2/Keap1 mutant mice also exhibit healthy phenotype.49

Although the senescence of Nrf2 tKO aortas is clearly visible (Fig. 1), and associated with potent S-nitrosylation of proteins (Fig. 4), the origin of this phenotype, in regard of the cells inducing such a phenotype, remains unclear. Basing on the in vitro studies, we suppose that ECs exert paracrine effect on the surrounding cells, through secretion of proinflammatory cytokines which may induce iNOS in the neighbouring smooth muscle cells and lead to S-nitrosylation. In accordance, overactivity of iNOS in SMC was reported to inhibit cell proliferation through p21 upregulation.50 In order to unambiguously resolve this issue, further EC-specific Nrf2 tKO research is needed.

Activation of Nrf2 or its homologues can prevent eNOS uncoupling and nitrosative stress,51–54

which is manifested by excessive level of posttranslational modifications, such as protein S-nitrosylation and nitration. Our data show that silencing of Nrf2 in ECs leads to profound protein S-nitrosylation, indeed, but we do not report presence of nitrated tyrosines. As they are detected only in the presence of strong nitrosative and oxidative stress,55,56 this supports the conclusion that redox buffering capacity of Nrf2 devoid cells is sufficient to preserve balance. Still, in these cells, protein S-nitrosylation can be facilitated by slight oxidative shift in redox status and may result from overproduction of nitric oxide, presence of peroxynitrate, inhibition of denitrosylating enzymes or regulation of transnitrosylating proteins by Keap1.18,57 The measurement of level of membrane nitric oxide revealed no changes between both groups, however lower nitric oxide secretion in Nrf2-deficient cells (Fig. 5), and also in Nrf2 tKO aortas (Fig. 4), could imply the retention of the molecule inside of the cells and usage for S-nitrosylation. We also observe eNOS activation by phosphorylation of serine 1177,58 accompanied by increased level of iNOS and downregulation of denitrosylating enzymes mRNA in Nrf2-deficient cells. The latter seems to be however not the primary reason for observed abundant S-nitrosylation in ECs with disturbed Nrf2, as Keap1 inhibition in Nrf2 deficient cells does not reverse expression of ADH5, while the TXNRD1 mRNA transcript still remains low (Fig. 7), while the phenotype is rescued. Though, only inhibition of both enzymes, both for in vitro and in vivo data, led to the occurrence of S-nitrosylation. Thus, development of Nrf2-dependent S-nitrosylation can be facilitated by concomitant decrease in TrxR1 and GSNOR, which no longer keep nitrosylation/de-nitrosylation balance in check.

In conclusion, this study reveals that in the vascular system lack of Nrf2 protein or inhibition of its activity in shear stress conditions causes Keap-1-dependent S-nitrosylation of proteins, which is an oxidative damage escaping mechanism relying on NOX4, redirecting cells from death to senescence, propagating most likely from ECs. These data are the second example 19 evidencing that Nrf2 serves not only as a transcription factor, but also as a protein scavenging Keap1. We unveil a new non-cannonical role of Keap1, a protein facilitating transnitrosylation of proteins, in addition to an oxidation sensor system, Nrf2 repressor and substrate adaptor protein for Cul3.

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SOURCES OF FUNDING

This work was supported by the National Science Centre grants OPUS No. 2012/07/B/NZ3/02912 (AGP) and SONATA BIS No. 2016/22/E/NZ3/00405 (AGP), as well as the Ministry of Science and Higher Education grants IUVENTUS PLUS No. 0138/IP1/2015/73 (AGP). Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education. The equipment used was sponsored in part by the Centre for Preclinical Research and Technology (CePT), a project co-sponsored by European Regional Development Fund and Innovative Economy, The National Cohesion Strategy of Poland.

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DISCLOSURES

None.

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REFERENCES

1. Campisi J, d’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol. 2007;8:729–740.

2. Nilsson PM. Early vascular aging (EVA): consequences and prevention. Vasc Health Risk Manag. 2008;4:547–552.

3. Erusalimsky JD. Vascular endothelial senescence: from mechanisms to pathophysiology. J Appl Physiol Bethesda Md 1985. 2009;106:326–332.

4. Zakkar M, Van der Heiden K, Luong LA, Chaudhury H, Cuhlmann S, Hamdulay SS, Krams R, Edirisinghe I, Rahman I, Carlsen H, Haskard DO, Mason JC, Evans PC. Activation of Nrf2 in endothelial cells protects arteries from exhibiting a proinflammatory state. Arterioscler Thromb Vasc Biol. 2009;29:1851–1857.

5. Ungvari Z, Bailey-Downs L, Gautam T, Jimenez R, Losonczy G, Zhang C, Ballabh P, Recchia FA, Wilkerson DC, Sonntag WE, Pearson K, de Cabo R, Csiszar A. Adaptive induction of NF-E2-related factor-2-driven antioxidant genes in endothelial cells in response to hyperglycemia. Am J Physiol Heart Circ Physiol. 2011;300:H1133-1140.

6. Jyrkkänen H-K, Kansanen E, Inkala M, Kivelä AM, Hurttila H, Heinonen SE, Goldsteins G, Jauhiainen S, Tiainen S, Makkonen H, Oskolkova O, Afonyushkin T, Koistinaho J, Yamamoto M, Bochkov VN, Ylä-Herttuala S, Levonen A-L. Nrf2 regulates antioxidant gene expression evoked by oxidized phospholipids in endothelial cells and murine arteries in vivo. Circ Res. 2008;103:e1-9.

7. Li W, Kong A-N. Molecular mechanisms of Nrf2-mediated antioxidant response. Mol Carcinog. 2009;48:91–104.

8. Umemura T, Kuroiwa Y, Kitamura Y, Ishii Y, Kanki K, Kodama Y, Itoh K, Yamamoto M, Nishikawa A, Hirose M. A Crucial Role of Nrf2 in In Vivo Defense against Oxidative Damage by an Environmental Pollutant, Pentachlorophenol. Toxicol Sci. 2006;90:111–119.

9. Aoki Y, Sato H, Nishimura N, Takahashi S, Itoh K, Yamamoto M. Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust. Toxicol Appl Pharmacol. 2001;173:154–160.

10. Cho H-Y, Jedlicka AE, Reddy SPM, Kensler TW, Yamamoto M, Zhang L-Y, Kleeberger SR. Role of NRF2 in protection against hyperoxic lung injury in mice. Am J Respir Cell Mol Biol. 2002;26:175–182.

11. Merchant AA, Singh A, Matsui W, Biswal S. The redox-sensitive transcription factor Nrf2 regulates murine hematopoietic stem cell survival independently of ROS levels. Blood. 2011;118:6572–6579.

12. Tomobe K, Shinozuka T, Kuroiwa M, Nomura Y. Age-related changes of Nrf2 and phosphorylated GSK-3β in a mouse model of accelerated aging (SAMP8). Arch Gerontol Geriatr. 2012;54:e1-7.

13. Kubben N, Zhang W, Wang L, Voss TC, Yang J, Qu J, Liu G-H, Misteli T. Repression of the Antioxidant NRF2 Pathway in Premature Aging. Cell. 2016;165:1361–1374.

14. Ungvari Z, Bailey-Downs L, Sosnowska D, Gautam T, Koncz P, Losonczy G, Ballabh P, de Cabo R, Sonntag WE, Csiszar A. Vascular oxidative stress in aging: a homeostatic failure due to

13

dysregulation of NRF2-mediated antioxidant response. Am J Physiol Heart Circ Physiol. 2011;301:H363-372.

15. Arnold WP, Mittal CK, Katsuki S, Murad F. Nitric oxide activates guanylate cyclase and increases guanosine 3’:5’-cyclic monophosphate levels in various tissue preparations. Proc Natl Acad Sci U S A. 1977;74:3203–3207.

16. Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, Singel DJ, Loscalzo J. S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci U S A. 1992;89:444–448.

17. Iwakiri Y. S-Nitrosylation of Proteins: A New Insight into Endothelial Cell Function Regulated by eNOS-derived NO. Nitric Oxide Biol Chem Off J Nitric Oxide Soc. 2011;25:95–101.

18. Benhar M, Forrester MT, Stamler JS. Protein denitrosylation: enzymatic mechanisms and cellular functions. Nat Rev Mol Cell Biol. 2009;10:721–732.

19. Kloska D, Kopacz A, Cysewski D, Aepfelbacher M, Dulak J, Jozkowicz A, Grochot-Przeczek A. Nrf2 sequesters Keap1 preventing podosome disassembly: a quintessential duet moonlights in endothelium. Antioxid Redox Signal. 2018. doi:10.1089/ars.2018.7505.

20. Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, Yamamoto M, Nabeshima Y. An Nrf2/Small Maf Heterodimer Mediates the Induction of Phase II Detoxifying Enzyme Genes through Antioxidant Response Elements. Biochem Biophys Res Commun. 1997;236:313–322.

21. Itoh K, Wakabayashi N, Katoh Y, Ishii T, O’Connor T, Yamamoto M. Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response to electrophiles. Genes Cells. 2003;8:379–391.

22. Itoh K, Mimura J, Yamamoto M. Discovery of the negative regulator of Nrf2, Keap1: a historical overview. Antioxid Redox Signal. 2010;13:1665–1678.

23. Bell P, Limberis M, Gao G, Wu D, Bove MS, Sanmiguel JC, Wilson JM. An optimized protocol for detection of E. coli beta-galactosidase in lung tissue following gene transfer. Histochem Cell Biol. 2005;124:77–85.

24. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A. 1995;92:9363–9367.

25. Mishima K, Handa JT, Aotaki-Keen A, Lutty GA, Morse LS, Hjelmeland LM. Senescence-associated beta-galactosidase histochemistry for the primate eye. Invest Ophthalmol Vis Sci. 1999;40:1590–1593.

26. Csiszar A, Pacher P, Kaley G, Ungvari Z. Role of oxidative and nitrosative stress, longevity genes and poly(ADP-ribose) polymerase in cardiovascular dysfunction associated with aging. Curr Vasc Pharmacol. 2005;3:285–291.

27. Zafarullah M, Li WQ, Sylvester J, Ahmad M. Molecular mechanisms of N-acetylcysteine actions. Cell Mol Life Sci CMLS. 2003;60:6–20.

28. Sun J, Steenbergen C, Murphy E. S-nitrosylation: NO-related redox signaling to protect against oxidative stress. Antioxid Redox Signal. 2006;8:1693–1705.

14

29. Brodsky SV, Gealekman O, Chen J, Zhang F, Togashi N, Crabtree M, Gross SS, Nasjletti A, Goligorsky MS. Prevention and reversal of premature endothelial cell senescence and vasculopathy in obesity-induced diabetes by ebselen. Circ Res. 2004;94:377–384.

30. Lamoke F, Shaw S, Yuan J, Ananth S, Duncan M, Martin P, Bartoli M. Increased Oxidative and Nitrative Stress Accelerates Aging of the Retinal Vasculature in the Diabetic Retina. PLoS ONE. 2015;10. doi:10.1371/journal.pone.0139664.

31. Montagna C, Rizza S, Maiani E, Piredda L, Filomeni G, Cecconi F. To eat, or NOt to eat: S-nitrosylation signaling in autophagy. FEBS J. 2016;283:3857–3869.

32. Ago T, Kitazono T, Ooboshi H, Iyama T, Han YH, Takada J, Wakisaka M, Ibayashi S, Utsumi H, Iida M. Nox4 as the Major Catalytic Component of an Endothelial NAD(P)H Oxidase. Circulation. 2004;109:227–233.

33. Selemidis S, Dusting GJ, Peshavariya H, Kemp-Harper BK, Drummond GR. Nitric oxide suppresses NADPH oxidase-dependent superoxide production by S-nitrosylation in human endothelial cells. Cardiovasc Res. 2007;75:349–358.

34. Hosoya T, Maruyama A, Kang M-I, Kawatani Y, Shibata T, Uchida K, Warabi E, Noguchi N, Itoh K, Yamamoto M. Differential responses of the Nrf2-Keap1 system to laminar and oscillatory shear stresses in endothelial cells. J Biol Chem. 2005;280:27244–27250.

35. Evangelista AM, Kohr MJ, Murphy E. S-Nitrosylation: Specificity, Occupancy, and Interaction with Other Post-Translational Modifications. Antioxid Redox Signal. 2013;19:1209–1219.

36. Kuilman T, Peeper DS. Senescence-messaging secretome: SMS-ing cellular stress. Nat Rev Cancer. 2009;9:81–94.

37. Acosta JC, Banito A, Wuestefeld T, Georgilis A, Janich P, Morton JP, Athineos D, Kang T-W, Lasitschka F, Andrulis M, Pascual G, Morris KJ, Khan S, Jin H, Dharmalingam G, Snijders AP, Carroll T, Capper D, Pritchard C, Inman GJ, Longerich T, Sansom OJ, Benitah SA, Zender L, Gil J. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat Cell Biol. 2013;15:978–990.

38. Coppé J-P, Desprez P-Y, Krtolica A, Campisi J. The Senescence-Associated Secretory Phenotype: The Dark Side of Tumor Suppression. Annu Rev Pathol. 2010;5:99–118.

39. Swamy SM, Rajasekaran NS, Thannickal VJ. Nuclear Factor-Erythroid-2-Related Factor 2 in Aging and Lung Fibrosis. Am J Pathol. 2016;186:1712–1723.

40. Kloska D, Kopacz A, Piechota-Polanczyk A, Nowak W, Dulak J, Jozkowicz A, Grochot-Przeczek A. Nrf2 in aging - Focus on the cardiovascular system. Vascul Pharmacol. 2018. doi:10.1016/j.vph.2018.08.009.

41. Zhang H, Puleston DJ, Simon AK. Autophagy and Immune Senescence. Trends Mol Med. 2016;22:671–686.

42. Di Giacomo G, Rizza S, Montagna C, Filomeni G. Established Principles and Emerging Concepts on the Interplay between Mitochondrial Physiology and S-(De)nitrosylation: Implications in Cancer and Neurodegeneration. Int J Cell Biol. 2012;2012:361872.

43. García-Ortiz A, Martín-Cofreces NB, Ibiza S, Ortega Á, Izquierdo-Álvarez A, Trullo A, Victor VM, Calvo E, Sot B, Martínez-Ruiz A, Vázquez J, Sánchez-Madrid F, Serrador JM. eNOS S-

15

nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1. PLoS Biol. 2017;15:e2000653.

44. Nakamura T, Lipton SA. Emerging roles of S-nitrosylation in protein misfolding and neurodegenerative diseases. Antioxid Redox Signal. 2008;10:87–101.

45. Melino G, Bernassola F, Knight RA, Corasaniti MT, Nistico G, Finazzi-Agro A. S-nitrosylation regulates apoptosis. Nature. 1997;388:432–433.

46. Chen Y-Y, Chu H-M, Pan K-T, Teng C-H, Wang D-L, Wang AH-J, Khoo K-H, Meng T-C. Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation. J Biol Chem. 2008;283:35265–35272.

47. Arnelle DR, Stamler JS. NO+, NO, and NO- donation by S-nitrosothiols: implications for regulation of physiological functions by S-nitrosylation and acceleration of disulfide formation. Arch Biochem Biophys. 1995;318:279–285.

48. Kim M, Kim S, Lim JH, Lee C, Choi HC, Woo C-H. Laminar flow activation of ERK5 protein in vascular endothelium leads to atheroprotective effect via NF-E2-related factor 2 (Nrf2) activation. J Biol Chem. 2012;287:40722–40731.

49. Wakabayashi N, Itoh K, Wakabayashi J, Motohashi H, Noda S, Takahashi S, Imakado S, Kotsuji T, Otsuka F, Roop DR, Harada T, Engel JD, Yamamoto M. Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation. Nat Genet. 2003;35:238–245.

50. Kibbe MR, Li J, Nie S, Watkins SC, Lizonova A, Kovesdi I, Simmons RL, Billiar TR, Tzeng E. Inducible nitric oxide synthase (iNOS) expression upregulates p21 and inhibits vascular smooth muscle cell proliferation through p42/44 mitogen-activated protein kinase activation and independent of p53 and cyclic guanosine monophosphate. J Vasc Surg. 2000;31:1214–1228.

51. Heiss EH, Schachner D, Werner ER, Dirsch VM. Active NF-E2-related factor (Nrf2) contributes to keep endothelial NO synthase (eNOS) in the coupled state: role of reactive oxygen species (ROS), eNOS, and heme oxygenase (HO-1) levels. J Biol Chem. 2009;284:31579–31586.

52. Oliveira CJR, Curcio MF, Moraes MS, Tsujita M, Travassos LR, Stern A, Monteiro HP. The low molecular weight S-nitrosothiol, S-nitroso-N-acetylpenicillamine, promotes cell cycle progression in rabbit aortic endothelial cells. Nitric Oxide. 2008;18:241–255.

53. Yagishita Y, Fukutomi T, Sugawara A, Kawamura H, Takahashi T, Pi J, Uruno A, Yamamoto M. Nrf2 protects pancreatic β-cells from oxidative and nitrosative stress in diabetic model mice. Diabetes. 2014;63:605–618.

54. Yoh K, Hirayama A, Ishizaki K, Yamada A, Takeuchi M, Yamagishi S, Morito N, Nakano T, Ojima M, Shimohata H, Itoh K, Takahashi S, Yamamoto M. Hyperglycemia induces oxidative and nitrosative stress and increases renal functional impairment in Nrf2-deficient mice. Genes Cells Devoted Mol Cell Mech. 2008;13:1159–1170.

55. Sawa T, Akaike T, Maeda H. Tyrosine nitration by peroxynitrite formed from nitric oxide and superoxide generated by xanthine oxidase. J Biol Chem. 2000;275:32467–32474.

56. White PJ, Charbonneau A, Cooney GJ, Marette A. Nitrosative modifications of protein and lipid signaling molecules by reactive nitrogen species. Am J Physiol Endocrinol Metab. 2010;299:E868-878.

57. Sun J, Murphy E. Protein S-nitrosylation and cardioprotection. Circ Res. 2010;106:285–296.

16

58. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999;399:597–601.

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FIGURE LEGENDS

Fig. 1. Senescence, but no oxidative damage is present in Nrf2 tKO aortas. (A) SA-β-gal activity. n=8. Scale bar 4 mm. (B) En face projection of aortas stained for SA-β-gal activity. n=5. Scale bar 0.35 mm. (C) p21 level. n=6. Scale bar 0.2 mm. Insets – negative control. (D-F) Expression of Gpx1 (D), Sod2 (E) and Cat (F). n=7-8. (G) Superoxide anion level. ***p<0.001 vs WT, n=7-8. (H) Assessment of lipid peroxidation. ***p<0.001 vs PBS, #p<0.05 vs WT. n=5-10.

Fig. 2. Loss of Nrf2 in HAECs does not result in oxidative damage. (A) Assessment of lipid peroxidation. n=3, ***p<0.001 vs PBS. (B-D) Expression of GPX1 (B), SOD2 (C) and CAT (D). *p<0.05 vs siMock. n=17. (E-F) Total (E) and mitochondrial (F) ROS level. n=7-9. (G) Assessment of total antioxidant capacity. n=3. (H) Superoxide anion level. *p<0.05 vs siMock, n=3. (I) Total glutathione level. *p<0.05 vs siMock. n=4. (J-K) Assessment of SA-β-gal activity. Quantitative data (J) and representative pictures (K). Scale bars 75 µm. ***p<0.001 vs siMock, ###p<0.001 vs siNFE2L2. n=4.

Fig. 3. Inhibition of Nrf2 transcriptional activity together with subjection to shear stress results in a senescence onset in HAECs. (A-B) Expression of NQO1 (A) and GCLM (B) at MOI 50. ***p<0.001 vs AdGFP. n=4-5. (C) Assessment of SA-β-gal activity. n=3, ***p<0.001 vs static, ###p<0.001 vs AdGFP. (D-E) Expression of NQO1 (D) and GCLM (E) at MOI 70. ***p<0.001 vs AdGFP. n=3. (F) Measurement of lipid peroxidation. ***p<0.001 vs AdGFP, ###p<0.001 vs static, n=3. (G) Detection of cell death with propidium iodide (PI) staining. Representative pictures of 5 experiments. Scale bar 50 µm.

Fig. 4. Nrf2 tKO aortas exhibit increased level of S-nitrosylation. (A) Protein S-nitrosylation. Representative picture of 5 animals. Scale bar 175 µm. (B-C) Expression of Adh5 (B) and Txnrd1 (C). *p<0.05 vs WT. n=8. (D) Level of nitric oxide. ***p<0.001 vs DMSO. ##p<0.01 vs WT, n=5-7. (E) Level of nitrosylated haemoglobin (HbNO) in blood. *p<0.05 vs WT, n=7-8. (F-G) Level of nitrite (F) and nitrate (G) in plasma. *p<0.05 vs WT, n=8-10.

Fig. 5. Nrf2-dependent senescence onset is associated with potent protein S-nitrosylation in HAECs. (A) Protein S-nitrosylation. Representative picture of 6 experiments. (B) Membrane trapped nitric oxide level. n=3. (C) Concentration of nitrites secreted into media. **p<0.01 vs siMock, n=9. (D) Expression of NOS3. ***p<0.001 vs siMock. n=11-12. (E) Level of total and phosphor-Ser1177 eNOS. Representative example of 3 experiments. (F) Expression of NOS2. n=9. (G) iNOS immunofluorescent staining. Representative pictures of three experiments. Scale bar 10 µm. (H-I) Expression of (H) ADH5 and (I) TXNRD1. *p<0.05 vs siMock, ***p<0.001 vs siMock, n=6. (J) Protein S-nitrosylation at MOI 50. Representative picture of 3 experiments. (K) Protein S-nitrosylation at MOI 50. Representative picture of 3 experiments. Scale bar 10 µm. (L-M) Expression of ADH5 (L) and TXNRD1 (M) at MOI 50. *p<0.001 vs static, #p<0.05 vs AdGFP, ###p<0.001 vs AdGFP.

Fig. 6. S-nitrosylation provides protection from NOX4-mediated oxidative stress and death in Nrf2-deficient HAECs. (A) Protein S-nitrosylation. Representative picture of 3 experiments. (B) Measurement of lipid peroxidation. n=3, **p<0.01 vs PBS, ##p<0.01 vs siNFE2L2. (C) Detection of cell death with propidium iodide (PI) staining. Representative pictures of 5 experiments. Scale bar 75 µm. (D-E) Efficiency of (D) NOX4 and (E) NFE2L2 knock down. ***p<0.001 vs siMock/siMock, ###p<0.001 vs siMock/siNFE2L2, n=3. (F) Measurement of level of lipid peroxidation. ***p<0.001 vs control, ###p<0.001 vs siMock/siMock, $$$p<0.001 vs siMock/siNFE2L2, n=3. (G) Detection of cell death with propidium iodide (PI) staining. Representative pictures of 5 experiments. Scale bar 75 µm.

Fig. 7. Keap1 mediates protein S-nitrosylation and senescent phenotype of HAECs. (A) Keap1 level. Representative picture of 3 experiments. Scale bar 10 µm. (B) Protein S-nitrosylation. Representative picture of 3 experiments. Scale bar 10 µm. (C) PCNA staining. Qualitative analysis. n=3, *** p<0.001 vs siMock/siMock, ### p<0.001 vs siNFE2L2/siMock. (D) p21 level. Representative picture of 3 experiments. (E) SA-β-gal activity. n=3, ** p<0.01 vs siMock/siMock, ## p<0.01 vs siNFE2L2/siMock. (F-G) Expression of (F) ADH5 and (G) TXNRD1. n=3, *** p<0.001 vs

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siMock/siMock, ## p<0.01 vs siNFE2L2/siMock. (H) Colocalization between GAPDH (green) and Keap1 (red) assessed by Pearson’s correlation coefficient (R). Representative pictures and quantitative data. n=4, * p<0.05 vs siMock/siMock, ### p<0.001 siNFE2L2/siMock. (I) NOX4 level. Representative pictures of 3 experiments. Scale bar 10 µm.

Fig. 8. Proposed model of Keap1-depedent regulation of ECs fate. ECs with overabundance of unrestrained Keap1 (Nrf2 deficient cells or the cells with transcriptionally inactive Nrf2 subjected to shear stress conditions) exhibit excessive protein S-nitrosylation what triggers the senescence onset. S-nitrosylation of NOX4 provides protection from oxidative damage. Denitrosylation of NOX4, but with preserved Keap1 level, causes cell apoptosis. Removal of Keap1 rescues normal phenotype of Nrf2-deficient ECs.

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