Nicotinamide N-methyltransferase in endothelium protects against oxidant stress-induced endothelial injury

Article abstract of DOI:10.1016/j.bbamcr.2021.119082

Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1.) plays an important role in the growth of many different tumours and is also involved in various non-neoplastic disorders. However, the presence and role of NNMT in the endothelium has yet to be specifically explored. Here, we characterized the functional activity of NNMT in the endothelium and tested whether NNMT regulates endothelial cell viability. NNMT in endothelial cells (HAEC, HMEC-1 and EA.hy926) was inhibited using two approaches: pharmacological inhibition of the enzyme by NNMT inhibitors (5-amino-1-methylquinoline – 5MQ and 6-methoxynicotinamide – JBSF-88) or by shRNA-mediated silencing. Functional inhibition of NNMT was confirmed by LC/MS/MS-based analysis of impaired MNA production. The effects of NNMT inhibition on cellular viability were analyzed in both the absence and presence of menadione. Our results revealed that all studied endothelial lines express relatively high levels of functionally active NNMT compared with cancer cells (MDA-MB-231). Although the aldehyde oxidase 1 enzyme was also expressed in the endothelium, the further metabolites of N1-methylnicotinamide (N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide) generated by this enzyme were not detected, suggesting that endothelial NNMT-derived MNA was not subsequently metabolized in the endothelium by aldehyde oxidase 1. Menadione induced a concentration-dependent decrease in endothelial viability as evidenced by a decrease in cell number that was associated with the upregulation of NNMT and SIRT1 expression in the nucleus in viable cells. The suppression of the NNMT activity either by NNMT inhibitors or shRNA-based silencing significantly decreased the endothelial cell viability in response to menadione. Furthermore, NNMT inhibition resulted in nuclear SIRT1 expression downregulation and upregulation of the phosphorylated form of SIRT1 on Ser47. In conclusion, our results suggest that the endothelial nuclear NNMT/SIRT1 pathway exerts a cytoprotective role that safeguards endothelial cell viability under oxidant stress insult.

Full text of DOI:10.1016/j.bbamcr.2021.119082

BBA - Molecular Cell Research 1868 (2021) 119082
Contents lists available at ScienceDirect
BBA - Molecular Cell Research
journal homepage: www.elsevier.com/locate/bbamcr
Nicotinamide N-methyltransferase in endothelium protects against oxidant
stress-induced endothelial injury
,
,
Roberto Campagnaa b, Łukasz Mateuszuka, Kamila Wojnar-Lasona c, Patrycja Kaczaraa,
Anna Tworzydłoa, Agnieszka Kija, Robert Bujokd, Jacek Mlynarskid, Yu Wange,
,
,c,*
Davide Sartinib, Monica Emanuellib *, Stefan Chlopickia
a Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland
b Department of Clinical Sciences, Polytechnic University of Marche, Ancona, Italy
c Jagiellonian University Medical College, Faculty of Medicine, Chair of Pharmacology, Krakow, Poland
d Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw, Poland
e State Key Laboratory of Pharmaceutical Biotechnology, Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong,
LKS Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1.) plays an important role in the growth of many different
tumours and is also involved in various non-neoplastic disorders. However, the presence and role of NNMT in the
endothelium has yet to be specifically explored. Here, we characterized the functional activity of NNMT in the
endothelium and tested whether NNMT regulates endothelial cell viability. NNMT in endothelial cells (HAEC,
HMEC-1 and EA.hy926) was inhibited using two approaches: pharmacological inhibition of the enzyme by
NNMT inhibitors (5-amino-1-methylquinoline – 5MQ and 6-methoxynicotinamide – JBSF-88) or by shRNA-
mediated silencing. Functional inhibition of NNMT was confirmed by LC/MS/MS-based analysis of impaired
MNA production. The effects of NNMT inhibition on cellular viability were analyzed in both the absence and
presence of menadione.
Nicotinamide N-methyltransferase
NNMT
Endothelium
Oxidative stress
Our results revealed that all studied endothelial lines express relatively high levels of functionally active
NNMT compared with cancer cells (MDA-MB-231). Although the aldehyde oxidase 1 enzyme was also expressed
in the endothelium, the further metabolites of N1-methylnicotinamide (N1-methyl-2-pyridone-5-carboxamide
and N1-methyl-4-pyridone-3-carboxamide) generated by this enzyme were not detected, suggesting that endo-
thelial NNMT-derived MNA was not subsequently metabolized in the endothelium by aldehyde oxidase 1.
Menadione induced a concentration-dependent decrease in endothelial viability as evidenced by a decrease in
cell number that was associated with the upregulation of NNMT and SIRT1 expression in the nucleus in viable
cells. The suppression of the NNMT activity either by NNMT inhibitors or shRNA-based silencing significantly
decreased the endothelial cell viability in response to menadione. Furthermore, NNMT inhibition resulted in
nuclear SIRT1 expression downregulation and upregulation of the phosphorylated form of SIRT1 on Ser47. In
conclusion, our results suggest that the endothelial nuclear NNMT/SIRT1 pathway exerts a cytoprotective role
that safeguards endothelial cell viability under oxidant stress insult.
Abbreviations: NNMT, nicotinamide N-methyltransferase; 5MQ, 5-amino-1-methylquinoline; JBSF-88, 6-methoxynicotinamide; AOX1, aldehyde oxidase 1;
Met2py, N1-methyl-2-pyridone-5-carboxamide; Met4py, N1-methyl-4-pyridone-3-carboxamide; NAM, nicotinamide; NAD+, nicotinamide adenine dinucleotide;
SAH, S-adenosyl-L-homocysteine; MNA, 1-methylnicotinamide; OSCC, oral squamous cell carcinoma; ccRCC, renal clear cell carcinoma; COX-2, cyclooxygenase-2;
PGI2, prostacyclin; NO, nitric oxide; CQ1, confocal quantitative image cytometer; MST, mitochondrial stress test; PGC-1a, peroxisome proliferator-activated receptor-
g coactivator-1a; FoxO, forkhead transcription factor O; NFkB, nuclear factor kB; eNOS, endothelial nitric oxide synthase; NICD, notch1 intracellular domain; LKB1,
liver kinase B1; p53, tumour suppressor protein 53; CDK5, cyclin-dependent kinase 5.
* Correspondences to: Monica Emanuelli, Department of Clinical Sciences, Polytechnic University of Marche, Ancona, Italy; Stefan Chlopicki, Jagiellonian Centre
for Experimental Therapeutics (JCET), Jagiellonian University, Krakow, Poland.
E-mail addresses: m.emanuelli@staff.univpm.it (M. Emanuelli), stefan.chlopicki@jcet.eu (S. Chlopicki).
https://doi.org/10.1016/j.bbamcr.2021.119082
Received 1 December 2020; Received in revised form 26 May 2021; Accepted 14 June 2021
Available online 19 June 2021
0167-4889/© 2021 Published by Elsevier B.V.

R. Campagna et al.
BBA - Molecular Cell Research 1868 (2021) 119082
1. Introduction
beneficial effects of exogenous MNA have been reported, including
PGI2-mediated anti-inflammatory [40], gastroprotective [41], hep-
atoprotective [42,43], neuroprotective [44,45] as well as anti-
metastatic effects [46,47]. Furthermore, the anti-atherosclerotic activ-
ity of MNA [48] was associated with an improvement in PGI2- and nitric
oxide (NO)-dependent endothelial function, the latter effect confirmed
also using functional 3D MRI-based assessment of endothelial function
in vivo [49].
Nicotinamide-N-methyltransferase (NNMT) (E.C. 2.1.1.1) catalyzes
the N-methylation of nicotinamide (NAM) and structurally related
compounds utilizing S-Adenosyl methionine (SAM) as the methyl donor
to produce S-adenosyl-L-homocysteine (SAH) and 1-methylnicotina-
mide (MNA). MNA is further oxidized by aldehyde oxidase 1 (AOX1)
into two related compounds, N1-methyl-2-pyridone-5-carboxamide
(Met2py) and N1-methyl-4-pyridone-3-carboxamide (Met4py) [1,2].
Nicotinamide (NAM) is a key precursor for the synthesis of nicotinamide
adenine dinucleotide (NAD+) and, therefore, NNMT activity is linked to
the metabolism of NAD+, which is a master regulator of energy meta-
bolism and influences cell life span [3,4]. NNMT is predominantly
expressed in the liver but was also detected in other tissues, including
the kidney, heart, lung, brain, placenta, striated muscle tissue and adi-
pose tissue [1,5,6]. The role of the NNMT/MNA metabolic pathway
seems to be distinct in various organs and cells and was most studied in
the liver, adipose tissue as well as in cancer cells.
Taken together, exogenous MNA positively modulates endothelial
and vascular function, but it is still not known whether the functional
NNMT is present in endothelial cells and whether endothelial NNMT
activity modulates endothelial response to injury. Therefore, the aim of
this work was to determine whether endothelial cells display an active
NNMT/AOX1 pathway and whether NNMT inhibition modulates cell
viability in response to oxidative stress.
2. Materials and methods
In the liver, NNMT expression regulates serum cholesterol and tri-
glycerides, and this effect was attributed to MNA, which enhances SIRT1
protein levels and activity [7]. The cytoprotective role of NNMT against
oxidative injury and endoplasmic reticulum stress, induced by iron
overload, was also reported [8]. On the other hand, it was found that
transgenic mice that overexpress NNMT displayed fatty livers and
fibrosis, particularly after NAM supplementation [9]. Furthermore,
increased circulating MNA levels positively correlated with obesity and
diabetes [10,11] and in a recent study, NNMT inhibitor (JBSF-88)
reduced body weight, improved insulin sensitivity and normalized
glucose tolerance in mice fed a high-fat diet [12]. Similarly, another
NNMT inhibitor, 5-amino-1-methylquinoline (5MQ), was shown to
reverse high-fat diet-induced obesity in mice [13]. As in human adipose
tissue, the NNMT expression positively correlates with adiposity and
insulin resistance, and the effects of NNMT inhibition in vivo preventing
fat accumulation and improving glucose tolerance in C57BL6 mice (fed a
high-fat diet) might be related to the inhibition of NNMT in adipose
tissue not in the liver [10,14,15].
2.1. Cell culture
The human endothelial cell line EA.hy926, purchased from the
American Type Culture Collection (ATCC, Rockville, MD, USA), was
cultured in DMEM high-glucose medium (4.5 g/l), supplemented with
10% foetal bovine serum (FBS), glutamine 2 mM and gentamicin 50 μg/
ml. The human aortic endothelial cell line (HAEC) and the human
microvascular endothelial cell line (hLMVEC) were purchased from
Lonza (Basel, Switzerland) and were cultured in a 2% FBS endothelial
growth medium (EGM)-2 Bullet Kit (Lonza), according to the manu-
facturer's instructions, with the addition of 50 μg/ml of gentamicin.
All experiments were performed using HAECs between the 2nd and
5th passages. The human microvascular endothelium cell line HMEC-1,
purchased from the ATCC, was cultured in MCDB131 medium (Gibco)
supplemented with 10% FBS, 10 mM glutamine, 1 μg/ml hydrocortisone
and 10 ng/ml epidermal growth factor (EGF). All cells were grown at
37 ◦C in a humidified 5% CO2 incubator.
Numerous reports have described the pathophysiological role of
upregulated NNMT in various types of solid tumours, including oral
squamous cell carcinoma (OSCC) [16], papillary thyroid cancer [17],
lung cancer [18], gastric cancer [19], pancreatic cancer [20], colorectal
cancer [21], renal clear cell carcinoma (ccRCC) [22,23], bladder cancer
[24], ovarian clear cell carcinoma [25], cutaneous and oral malignant
melanoma [26,27]. NNMT overexpression has been linked to increased
tumorigenicity and poor prognosis. NNMT knockdown inhibited the
proliferation and/or metastasis in ccRCC, pancreatic cancer cell Panc-1,
OSCC and other cancers [28–30]. Although several studies have
confirmed the functional pro-tumorigenic role of NNMT in these tu-
mours, the mechanisms involved were not fully elucidated. Recently,
Ulanovskaya et al. suggested that NNMT may act as a methyl donor sink
because SAM is a co-substrate required by all methyltransferases,
including NNMT and histone methyltransferases [31].
2.2. Assessment of NNMT and AOX1 expression in endothelium by
western blotting
Samples containing 50
μg protein were subjected to sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using
a
running gel of 12% polyacrylamide. Proteins were transferred to poly-
vinylidene fluoride (PVDF) membranes at 0.08 mA overnight, using a
wet transfer method. PVDF membranes were blocked overnight at 4 ◦C
in 1× phosphate-buffered saline (PBS) solution containing 5% non-fat
dry milk and 0.05% tween-20. After washing three times with 1× PBS
containing 0.05% tween-20, the membranes were incubated with rabbit
polyclonal antibody against NNMT (Sigma-Aldrich, St. Louis, MO, USA)
or AOX1 (Thermo Fisher Scientific, Waltham, MA, USA) (1:1000 dilu-
tion) for 2 h, followed by incubation with horseradish peroxidase (HRP)-
conjugated goat anti-rabbit IgG (Thermo Fisher Scientific) (1:2000
dilution) for 1 h. Protein signals were visualized using an enhanced
SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher
Scientific). The chemiluminescent signal of NNMT protein detected in
blots was acquired using a ChemiDoc XRS + System (Bio-Rad
Laboratories).
Alterations in NNMT expression and activity were found to be
upregulated not only in cancer but also in several non-neoplastic dis-
orders, including Parkinson's disease [32,33], metabolic syndrome [34]
chronic obstructive pulmonary disease [35] and pulmonary hyperten-
sion [36]. However, it is still not clear whether the upregulated NNMT/
MNA pathway in these diseases contributes to the pathology or repre-
sents a compensatory mechanism [37,38].
2.3. Assessment of NNMT activity by LC/MS/MS based-MNA
measurements
Both positive and detrimental roles of endogenous NNMT enzyme
activity, depending on the experimental model and tissue studied, have
been suggested. However, exogenous MNA has been repeatedly shown
to afford vasoprotective pharmacological activities. For instance, MNA
displayed anti-thrombotic activity in rats with arterial thrombosis
through a mechanism involving cyclooxygenase-2 (COX-2) and prosta-
cyclin (PGI2) [39]. Accordingly, MNA could be regarded as the first class
of molecule to boost the endogenous COX-2/PGI2 pathway. Several
Cells were seeded in 24-well plates to have 90% confluence on the
day of the experiment. After 24 h, the medium was removed, and cells
were washed twice by PBS 1×. Cells were then incubated with 500
μ
l
l
Krebs buffer for 4 h at 37 ◦C. Krebs buffer was then replaced with 300
μ
of fresh buffer with nicotinamide 100
μM and SAM 10
μM. To measure
NNMT activity in the liver, male 12–16-week-old C57Bl/6J mice were
2

R. Campagna et al.
BBA - Molecular Cell Research 1868 (2021) 119082
used (Jackson Laboratories, Bar Harbor, ME, USA). Mice were housed in
colony cages in a temperature-controlled environment (22–25 ◦C) with a
12-hour light/dark cycle and had free access to food and water. Mice
were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg)
administered intraperitoneally, and mice livers were perfused by PBS 1×
and used for ex vivo studies. Mice liver aliquots (20 mg) were incubated
in Krebs buffer for 1 h at 37 ◦C.
K2CO3 (0.332 g, 2.40 mmol) was added to a solution of 5-cyano-2-
methoxypyridine 2 (0.648 g, 4.75 mmol) in DMSO (15 mL). The
mixture was cooled to 10 ◦C, and 30% H2O2 (4.8 ml) was added after 5
min, maintaining the temperature below 35 ◦C. When the addition was
completed, the suspension was stirred at RT for 2 h. The mixture was
poured into water (20 ml), extracted with AcOEt (5 × 20 ml), and the
combined extracts were washed with water (3 × 20 ml), dried and
evaporated. The crude product was washed with CHCl3 (3 × 5 ml) to
give 0.435 g (60%) of amide 3 as a white solid. 1H NMR (500 MHz,
CDCl3): δ 8.68 (d, J = 2.4 Hz, 1H), 8.13 (dd, J = 8.7, 2.4 Hz, 1H), 7.96
(bs, 1H), 7.37 (bs, 1H), 6.87 (d, J = 8.7 Hz, 1H), 3.91 (s, 3H). 13C NMR
(125 MHz, CDCl3): δ 166.1, 165.1, 147.3, 138.3, 123.5, 110.0, 53.5.
HRMS–ESI+ (m/z, MeOH): calcd for C7H9N2O2 [M + H]+, 153.0664;
found: 153.0660. Calcd for C7H9N2O2%C, 55.26;%H, 5.30,%N, 18.60,
found %C, 55.24;%H, 5.11,%N, 18.16.
At fixed time-points, 50 μl of extracellular fluid taken from endo-
thelial cells or liver pieces were collected, and in each sample, the MNA
concentration was analyzed by LC/MS/MS as described previously
[36,48]. Briefly, an aliquot of 50 μl of extracellular fluid was spiked with
5
μ
l of internal standard (deuterated analogues of analytes) at a con-
centration of 25 g/ml. Samples were subjected to deproteinization with
100 l of acidified acetonitrile (0.1% formic acid), vortexed, cooled at
μ
μ
4 ◦C for 15 min and centrifuged (15,000 ×g, 15 min, 4 ◦C). Supernatants
were injected into an LC column. Chromatographic analysis was per-
formed using an UltiMate 3000 LC system (Thermo Scientific Dionex,
Sunnyvale, CA) consisting of a pump (DGP 3600RS), column compart-
ment (TCC 3000RS), autosampler (WPS-3000TRS) and SRD-3600 sol-
vent rack (degasser). Chromatographic separation was carried out on an
Aquasil C18 analytical column (4.6 mm × 150 mm, 5 mm; Thermo
Scientific). The mobile phase consisted of acetonitrile (A) and 5 mM
ammonium formate (B) in isocratic elution (80:20 v/v) at a flow rate of
0.8 ml/min.
5-Amino-1-methylquinolinium iodide (5MQ, compound 5) was ob-
tained from commercially available 5-amino-quinoline 4 according to
an improved literature protocol [52].
Detection was performed using a TSQ Quantum Ultra mass spec-
trometer equipped with a heated electrospray ionization interface
(HESI-II Probe) (Thermo Scientific, Waltham, MA, US). The mass spec-
trometer was operating in the positive ionization using selected re-
actions monitoring mode (SRM), monitoring the transition of the
protonated molecular ions m/z 153–110 for Met2PY, m/z 156 → 113 for
Met2PY-d3, m/z 153–136 for Met4PY, m/z 156 → 139 for Met4PY-d3,
m/z 123–80 for NA, m/z 127–84 for NA-d4 and m/z 137–94 for MNA,
m/z 140 → 97 for MNA-d3. Data acquisition and processing were
accomplished using Xcalibur 2.1 software.
Iodomethane (0.160 ml, 0.365 g, 2.6 mmol) was added to a solution
of 5-aminoquinoline 4 (0.277 g, 1.9 mmol) in THF (7 ml) and the so-
lution was stirred at RT for 1 d when red solid precipitated. The product
was filtered-off, washed with THF (2 × 5 ml) and dried in a vacuum to
give 0.240 g of compound 5. The filtrate was evaporated, the residue
was dissolved in THF (4 ml), iodomethane (0.090 ml) was added, and
the mixture was stirred at RT for another day. Product was filtered-off,
washed with THF and dried to give 0.228 g (86%) of 5. 1H NMR data
were identical to that previously published [52].
The following reagents were used as measurement standards: NAM
and MNA obtained from Sigma-Aldrich and N-methyl-4- pyridone-3-
carboxamide (Met-2PY) and N-methyl-2-pyridone-5-carboxamide
(Met-4PY) purchased from TLC PharmaChem (Vaughan, ON, Canada).
Deuterated analytes were used as internal standards. NA-d4 and NAM-
d4 were purchased from Sigma-Aldrich and Dr. Ehrenstorfer GmbH
(Augsburg, Germany), respectively. Met2PY-d3 and Met-4PY-d3 were
obtained from TLC PharmaChem, and MNA-d3 was synthesized by Dr.
Adamus (Technical University, Lodz, Poland). LCMS–grade acetonitrile,
ammonium formate and formic acid were purchased from Sigma-
Aldrich. Ultrapure water was obtained from a Millipore system
(Direct-Q 3UV).
2.5. NNMT gene silencing and stable cell strain selection
Cells grown to 80–90% confluence were removed from the T25 flask
using 0.025% trypsin/EDTA (Gibco) at room temperature. 2 × 106 cells
were resuspended in Optimem (Gibco) + 10% FBS. Twenty micrograms
of shRNA plasmids against NNMT (pLKO.1-330, 1-448 and 1-711) or of
empty pLKO.1 puro (pLKO.1 Ø) vector (Sigma-Aldrich, St. Luis, MO)
were mixed with the cell suspension to a final volume of 400 μl and
incubated for 10 min at +4 ◦C. Control EA.hy926 cells (mock) were
treated with transfection reagents only.
The cell suspension was placed in a 4 mm-gap electroporation
2.4. Synthesis of NNMT inhibitors
cuvette (BTX) and electroporated at capacitances of 900
nation with 200 V.
μF in combi-
6-Methoxy-3-pyridinecarboxamide (JBSF-88, compound 3) was
synthesized from commercially available 2-chloro-5-cyanopyridine (1).
5-Cyano-2-methoxypyridine 2 was prepared by substitution of
chlorine with methoxide anion in MeOH according to a slightly modified
literature protocol [50]. 6-Methyoxy-3-pyridinecarboxamide 3 was
synthesized from compound 2 in a reaction with H2O2 in the presence of
solid K2CO3 in DMSO [51], according to the following scheme:
The electroporated cells were left in the cuvette for 10 min at room
temperature and then transferred in T75 flasks with complete growth
medium. Forty-eight hours after transfection, clones stably down-
regulating NNMT were selected by maintaining cells in complete me-
dium containing puromycin (0.4 μg/ml), with medium changes every
48 h. All subsequent experiments involving these clones were performed
by growing cells in complete medium containing puromycin.
3

R. Campagna et al.
BBA - Molecular Cell Research 1868 (2021) 119082
2.6. Determination of NNMT silencing by real time-PCR
USA). Secondary antibodies used were Alexa Fluor 647-conjugated goat
anti-rabbit and Cy3-conjugated goat-anti-mouse (Jackson Immuno,
Cambridgeshire, UK). For nuclei counterstaining, Hoechst 33258 solu-
tion was used. Images of immunostained cells were taken using a CQ1
(Yokogawa, Musashino, Tokyo, Japan) and CQ 1.04 software, then
analyzed automatically with Columbus 2.4.2 software (Perkin Elmer,
Waltham, MA, USA). Nuclear expression (SIRT1 and pSIRT) was quan-
tified based on counterstaining and specially-design algorthitms of the
Columbus 2.4.2 software. For data normalization, each immunostaining
(n = 6) was performed using cells with similar confluence (≥90%), the
same primary and secondary antibody concentration and constant in-
cubation time for each staining step. As a negative control, cells treated
only with secondary antibodies were used to estimate the background
signal.
Total RNA was isolated through the SV Total RNA Isolation System
(Promega, Madison, WI, USA), according to the manufacturer's protocol.
Total RNA (2 μg) was reverse-transcribed in a total volume of 25 μl for
60 min at 37 ◦C with M-MLV Reverse Transcriptase (Promega, Madison,
WI, USA), using random primers. cDNA samples were used to perform
subsequent real-time PCR analysis. To evaluate NNMT mRNA expression
quantitatively, a real-time PCR assay was performed using a CFX96
Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA,
USA). cDNA, generated as described above, was used as a template. The
primers used were 5′-GAA TCA GGC TTC ACC TCC AA-3′ (forward) and
5′-TCA CAC CGT CTA GGC AGA AT-3′ (reverse) for NNMT, and 5′-TCC
TTC CTG GGC ATG GAG T-3′ and 5′-AGC ACT GTG TTG GCG TAC AG-3′
for β-actin.
Both genes were run in duplicate for 40 cycles at 94 ◦C for 30 s and
58 ◦C for 30 s, using a SsoFast EvaGreen Supermix (Bio-Rad Labora-
tories, Hercules, CA, USA). All samples were tested in triplicate using the
β-actin gene for data normalization. Direct detection of PCR products
was monitored by measuring the fluorescence produced by EvaGreen
dye binding to double-stranded DNA after every cycle. These measure-
ments were then plotted against cycle numbers. The parameter
threshold cycle (Ct) was defined as the cycle number at which the first
detectable increase above the threshold in fluorescence was observed.
Following NNMT gene silencing in EA.hy926 cells, fold changes in
relative gene expression were calculated by 2ꢀ Δ(ΔCt), where ΔCt = Ct
(NNMT) - Ct (β-actin) and Δ(ΔCt) = ΔCt (cells transfected with plasmid
vectors) - ΔCt (mock).
2.9. Cell proliferation assay using electric cell-substrate impedance
sensing
Cells were grown on the surface of planar golden electrodes of an
electric cell-substrate impedance sensing set-up (ECIS), and the resis-
tance of the cell-covered electrode was measured continuously at a
frequency of 4000 Hz. Because of the insulating properties of cell
membranes, the resistance increases with increasing coverage of the
electrode. HAECs and HMEC-1 cells were seeded in 96-well, gold-film
electrode-coated 96W1E + PET arrays (Applied Biophysics). The resis-
tance parameter was constantly monitored up to 125 h after seeding.
2.10. Mitochondrial stress test
2.7. Cell viability assay in menadione-induced endothelial cell injury
For the mitochondrial stress test (MST), HAEC cells were seeded into
Seahorse XFe 96-well plates at a density of 1.5 × 103 per well to produce
confluency on the next day. The cells were treated with JBSF or 5MQ or
not treated for 72 h, and the medium was exchanged after 24 and 48 h
before MST. An assay medium for MST was prepared on the day of the
experiment by supplementation of Seahorse DMEM XF Base medium
(Agilent) with glucose (1 g/l; Merck), glutamine (2 mM; Sigma-Aldrich)
and sodium pyruvate (1 mM; Sigma-Aldrich) and adjusting the pH to 7.4
using 0.1 M NaOH (Sigma-Aldrich). Immediately prior to MST, the cells
were washed twice with the assay medium, then fresh assay medium
was added and cells were incubated for 1 h at 37 ◦C without CO2.
Changes in oxygen consumption rate (OCR) and extracellular acidifi-
cation rate (ECAR) were recorded over time. After four basal measure-
To assess cell viability, cells were cultivated in full medium and
treated with menadione (Sigma Aldrich, Saint Louis, MO, USA) for 24,
48 or 72 h and 5MQ or JBSF-88 when indicated. To confirm that effects
of NNMT inhibitors are not related to their toxicity 5MQ and JBSF-88
(3–15 uM) incubated with HAEC and HMEC-1 for 2, 24 and 72 h were
tested by MTT assay and there was no statistically significant effects on
the cell viability for all the concentrations and time-points used in this
study. Additionally, cells were treated with specific SIRT1 inhibitor EX-
527 (Sigma Aldrich, Saint Louis, MO, USA) to examine the role of
sirtuin-dependent pathways in menadione-induced endothelial injury.
Cells were washed with PBS before fixing (with a solution of 4% para-
formaldehyde at room temperature) or before live cell imaging and
incubated with a solution of YO-PRO™-1 Iodide (Thermo Fisher Scien-
tific, USA) diluted 1:1000 in the dark, at 37 ◦C for 30 min. For nuclei
counterstaining, a Hoechst 33258 solution (Sigma Aldrich, Saint Louis,
MO, USA) was used. Imaging was performed with a confocal quantita-
tive image cytometer (CQ1) (Yokogawa, Musashino, Tokyo, Japan).
Images were analyzed via Columbus 2.4.2 software (Perkin Elmer,
Waltham, MA, USA) and the number of cells and/or number of cells
positive to the YO-PRO™-1 iodide stain was calculated.
ments, oligomycin (1
followed by three measurements, then FCCP (1.5
was added from port B, followed by three measurements, and finally,
μ
g/ml; Calbiochem) was added from port A,
μM; Sigma-Aldrich)
rotenone (0.5 μM; Sigma-Aldrich) with antimycine A (0.5 μM; Sigma-
Aldrich) were added from port C, followed by three measurements.
Concentrations of oligomycin, FCCP, rotenone and antimycine A had
been optimized in preliminary experiments. After the assay, cells were
washed once with DPBS (Gibco), 10 μl of 0.1% Triton X-100 (Sigma-
Aldrich) in DPBS was added per well, and cells were frozen at ꢀ 80 ◦C.
On the next day, the protein content was measured using a Bicinchoninic
Acid Protein Determination Kit (Sigma-Aldrich). The data were
normalized for protein content and analyzed using the Seahorse XF Cell
Mito Stress Test Report Generator (Agilent). The following parameters of
mitochondrial function were analyzed: basal respiration, proton
leakage, ATP production, maximal respiration, spare respiratory ca-
pacity and non-mitochondrial oxygen consumption.
2.8. Expression of SIRT1, pSIRT1, p53, acetyl-p53 and NNMT by
immunocytochemistry
Cells were plated in 96-well format on black Corning multiplates
with a clear bottom. After the treatment, cells were fixed with a 4%
formalin solution for 10 min, washed with PBS and then incubated with
a blocking solution containing 5% normal goat serum (Sanquin,
Amsterdam, Netherlands) to minimalize non-specific binding of anti-
bodies. For indirect immunohistochemical detection, cells were incu-
bated overnight with the following primary antibodies: NNMT
polyclonal antibody, p53 monoclonal antibody, Acetyl(Lys382)p53
monoclonal antibody (Thermo Fisher Scientific, Waltham, MA, USA),
SIRT1 monoclonal antibody (Merck Millipore, Burlington, MA, USA),
Phospho-SIRT1 (Ser47) polyclonal antibody (Cell Signaling Technology,
2.11. Statistical analysis
Data were analyzed using GraphPad Prism software version 6.00 for
Windows (GraphPad Prism Software, San Diego, CA, USA). Differences
between groups were determined using the Mann-Whitney nonpara-
metric test, or Kruskal-Wallis one-way ANOVA, followed by a post-hoc
Dunn test. A p-value <0.05 was considered statistically significant.
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Fig. 1. NNMT and AOX1 expression levels in endothelial cells. Protein lysates obtained from HAEC, HMEC-1, HUVEC, hLMVEC, EA.hy926, as compared with MDA-
MB-231 and MCF-7 cells were analyzed by Western blot to evaluate NNMT (panel A) and AOX1 (panel B) expression levels.
Fig. 2. Inhibition of NNMT activity and MNA production by 5MQ and JBSF-88 in endothelial cells. HMEC-1 cells (A), HAEC cells (C) and aliquots of mice liver (20
mg, D) were incubated in Krebs buffer with nicotinamide 100
μ
M, SAM 10
μ
M and 5MQ or JBSF-88 for 2 h. HMEC-1 cells (B) were incubated for 24 h in full medium
with 5MQ or JBSF-88 and nicotinamide 100 μM, SAM 10 μM.
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3. Results
MNA production by >90% and >70%, respectively (Fig. 2A). Similarly, in
HAECs, both inhibitors at a concentration of 30 μM lowered MNA by >90%
3.1. NNMT expression in endothelial cells
(Fig. 2B). Longer incubation with inhibitors (24 h incubation) in HMEC-1
also resulted in a profound inhibition of NNMT activity (by 75.90% and
As shown in Fig. 1, endothelial cells expressed NNMT, but the level of
expression was not uniform for all endothelial cells tested. Surprisingly,
NNMT expression in HAECs was comparable to the expression of NNMT
in human MDA-MB-231 breast cancer cells. With the exception of EA.
hy926, in other types of endothelial cells (HMECs-1, HUVECs and
hLMVECs), the expression of NNMT was higher as compared with
HAECs (by approximately 4.0, 6.4 and 1.8 folds, respectively). NNMT
protein was not detected in MCF-7 cells, used here as a negative control.
In contrast to the different expression of NNMT in various endothe-
lial cells lines, the expression level of AOX1 was comparable for tested
endothelial cells (HAEC, HMEC-1, HUVEC, hLMVEC EA.hy926) was
approximately two folds lower than in MDA-MB-231 breast cancer cells
(Fig. 1B).
59.79% for 30
(Fig. 2C).
μM concentration of 5MQ and JBSF-88, respectively)
A similar degree of NNMT inhibition by 5MQ and JBSF-88 was
achieved in the liver because a 30 μM concentration of the 5MQ or JBSF-
88 inhibited NNMT by >90% (Fig. 2D).
3.3. Effect of NNMT inhibition on endothelial cell viability in response to
menadione-induced oxidative stress
To examine the effects of NNMT inhibition on endothelial response to
oxidative stress, HAEC and HMEC-1 cells were incubated with increasing
concentrations of menadione in the absence or presence of NNMT in-
hibitors (5MQ or JBSF-88). Both inhibitors were given at a concentration of
30 μM, inhibiting NNMT activity >90% (Fig. 2). As shown in Fig. 3, in the
3.2. Pharmacological inhibition of NNMT in endothelial cells
presence of NNMT inhibitors, menadione significantly decreased the cell
viability as compared to the cells treated with menadione alone. A similar
pattern of potentiated menadione-induced injury by NNMT inhibition was
observed in HAEC (Fig. 3A, B) and HMEC-1 (Fig. 3C, D) cells.
Both NNMT inhibitors, 5-Amino-1-methylquinoline (5MQ) and 6-
methoxynicotinamide (JBSF-88) incubated for 1 h inhibited NNMT activ-
ity in a concentration-dependent manner in HMEC-1 (Fig. 2A) and HAECs
(Fig. 2B), as evidenced by a progressive, concentration-dependent decrease
in MNA production. In HMEC-1, the efficiency of inhibition was slightly
higher for 5MQ as compared with JBSF-88, but both inhibitors at a con-
To confirm that the observed reduction in cell viability was a
consequence of the NNMT inhibition and not a non-specific effect, the
same experiment was performed on EA.hy926 cell lines in which the
NNMT was efficiently silenced by shRNAs (Fig. 4A, B). NNMT-silenced
endothelial cell lines displayed a marked reduction in cell viability in
centration of 30 μM profoundly inhibited NNMT activity and decreased
Fig. 3. Effect of NNMT inhibition on endothelial cells viability after menadione-induced injury. HAECs (A, B) and HMEC-1 (C, D) were incubated for 24 h with
different concentrations of menadione together with 5MQ or JBSF-88, and cell viability was assessed by counting surviving cells through the automatic CQ-1 station
and subsequent Columbus software analysis. Values are reported as mean ± standard deviation (n = 6, *p < 0.05, **p < 0.01).
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Fig. 4. Effect of NNMT knockdown on cell viability after menadione-induced injury in endothelial cells. EA.hy926 cells were transfected with 3 shRNA plasmids
against NNMT (1–330, 1–448 and 1–711) or with empty plasmid vector (pLKO.1Ø) or with transfection reagent only (CTRL). NNMT expression levels were evaluated
by real-time PCR (A) and western blot (B). NNMT-silenced lines were incubated for 24 h with different concentrations of menadione, and cell viability was assessed
by counting surviving cells through the automatic CQ-1 station and subsequent Columbus software analysis (n = 3, *p < 0.05).
response to menadione as compared to the empty plasmid-transfected
control, thus confirming that NNMT exerts a protective role in the
endothelium against oxidative stress (Fig. 4C).
3.5. NNMT inhibition alters nuclear pSIRT1/SIRT1 expression ratio in
menadione-induced oxidant stress
In the presence of the NNMT inhibitor JBSF-88, low concentrations
of menadione triggered a moderate but significant increase in the per-
centage of Yo-Pro1 iodide-positive cells (Fig. 5A). Interestingly, when
HAECs were incubated with NNMT inhibitors alone for a longer time
As shown in Fig. 7, inhibition of NNMT by 5MQ or JBSF-88 resulted
in a significant downregulation of nuclear SIRT1 expression, whereas
phosphorylation of nuclear SIRT1 at S47 was significantly elevated
(Fig. 7). Noticeably, the ratio pSIRT1/SIRT1 increased in NNMT-
inhibited endothelial cells as compared to control cells (Fig. 7). Alto-
gether, these results revealed that NNMT inhibition triggered a decrease
in nuclear SIRT1 expression associated with an increase of nuclear
SIRT1 phosphorylation at S47 in endothelial cells. Menadione alone
induced a parallel increase in the nuclear expression of SIRT1 enzyme,
together with its phosphorylated form at Ser47 (pSIRT1) (Fig. 8). Inhi-
bition of SIRT1 by EX-527 potentiated the effect of menadione on
cellular viability (Fig. 9A) and increased menadione-induced expression
of p53 in HAEC cells (Fig. 9B), while the combination of menadione and
EX-527 had no effect on the expression of acetylated p53 (Fig. 9C).
Interestingly menadione-induced endothelial cell injury was associated
with NNMT enzyme expression upregulation as evidenced by immuno-
cytochemistry (Fig. 8), and an inverse correlation was found between
cell viability and NNMT expression (Fig. 8A, B). Indeed, cells treated
with increasing concentrations of menadione (Fig. 8A) displayed a
parallel increase in NNMT expression. Altogether these data suggests
that increased activity of NNMT in menadione-treated cells represents a
compensatory response to menadione-induced oxidant stress response.
period of 72 h (5MQ or JBSF-88 3-50
μM), there was a clear
concentration-dependent increase in Yo-Pro1 iodide-positive cells from
approximately 4.05% to 8–10% (Fig. 5B, C), suggesting that NNMT in-
hibition alone affects plasma membrane integrity.
3.4. NNMT inhibition does not affect the proliferation rate nor the
bioenergetics of endothelial cells
To determine whether the effects of NNMT inhibition on cell viability
are related to the effects on endothelial cell proliferation or to endo-
thelial bioenergetics, we analyzed how 5MQ and JBSF-88 affected cell
proliferation (ECIS assay) and mitochondrial respiration (Seahorse XF96
Analyzer). As shown in Fig. 6, the long-term NNMT inhibition by JBSF-
88 did not affect the proliferation of HAEC or HMEC-1 cells (Fig. 6E, F)
and did not significantly affect mitochondrial function (Fig. 6A-D) as
evidenced by unchanged basal and maximal mitochondrial respiration
as well as other measured parameters (not shown).
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Fig. 5. Effects of NNMT-inhibition on Yo-Pro1 positive staining in endothelial cells subjected or not to menadione-induced injury. HAEC cells were incubated for 24
h with various concentrations of menadione together with JBSF-88 3 M or 30 M (A) or for 72 h with NNMT inhibitors only (B, C). Yo-Pro1 iodide-positive cells
were detected by live imaging through the automatic CQ-1 station and subsequent Columbus software analysis (n = 6, *p < 0.05, **p < 0.01, ***p < 0.001).
μ
μ
4. Discussion
latter enzyme most likely metabolizes other substrates in the endothe-
lium [54]. Remarkably, in all our assays, the MNA concentration was
detected in the extracellular fluid, suggesting that extracellular
endothelial-derived MNA could have a biological role [39,55,56] and
could contribute to altered MNA plasma concentration in various dis-
eases [10] and conditions [57].
Despite the abundant literature regarding the role of nicotinamide N-
methyltransferase (NNMT) activity in various physiological and patho-
physiological conditions, up to now, the presence and functional role of
NNMT in the endothelial cells had not been characterized. In this work,
we demonstrated to our knowledge, for the first time, that endothelial
cells display a relatively high expression and activity of NNMT that
safeguards endothelial cell viability against oxidative stress-induced
insult.
In the present work, the functional role of NNMT in endothelial cells
was analyzed using two NNMT inhibitors; 5-amino-1-methylquinoline
(5MQ) and 6-methoxynicotinamide (JBSF-88) [12,52] and shRNA-
mediated silencing. In our hands, both inhibitors profoundly inhibited
We demonstrated that endothelial cells exhibited an NNMT activity
that was comparable to the activity of this enzyme in the liver, the organ
with high NNMT expression, and comparable or even higher expression
than in a breast cancer cell line, the phenotype of which is known to be
dependent on NNMT activity [53]. On the other hand, although endo-
thelial cells expressed aldehyde oxidase 1 (AOX1) at the protein level,
the further metabolites of MNA (Met2Py and Met4Py) were not detected
either in HAEC or in HMEC-1, suggesting that endothelial NNMT-
derived MNA is not further metabolized by AOX1 in the endothelium
or that this metabolism is negligible in our experimental setting. The
NNMT activity at 10–30 μM concentration in HMEC-1 and in HAECs
with a similar level of inhibition as in the liver tissue, supporting the
validity of a pharmacological approach to inhibit NNMT activity in
endothelial cells. Similarly, shRNA-mediated silencing based on three
different RNA sequences resulted in profound down-regulation of mRNA
for NNMT, supporting the effective silencing of NNMT. Using pharma-
cological and molecular approaches, we demonstrated that the inhibi-
tion of NNMT sensitized both HAEC and HMEC-1 cells to menadione-
induced oxidative stress, decreasing cell viability. Similar results were
obtained in the endothelial lines EA.hy926 where NNMT was silenced,
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Fig. 6. Effect of NNMT inhibition on mitochondrial function and cell proliferation in endothelial cells. Key parameters of mitochondrial function were analyzed on
HAECs using a Seahorse XF96 Cell Analyzer as described in the methods. (A) Basal respiration, (B) proton leak, (C) maximal respiration, (D) ATP production.
The rate of cell proliferation of HAECs (E) and HMEC-1 (F) incubated with NNMT inhibitors was measured in real-time on the ECIS instrument by monitoring the
resistance parameter at 4 kHz up to 150 h.
thus confirming that the reduction in cell viability in the oxidative stress
model, upon NNMT inhibition, was indeed NNMT related.
protected endothelial cells against oxidative stress, and NNMT inhibi-
tion had detrimental cell viability effects in the well-known model of
oxidative stress injury induced by menadione [62,63].
Previous studies in other cell types suggested that NNMT enhances
cellular resistance to radiation in the context of cancer [58]. The
ionizing radiation used in radiotherapy induces free radical production;
the generated ROS reacts with DNA causing single- or double stranded-
breaks, and NNMT was reported to be downregulated in a radiosensitive
bladder carcinoma cell line [59]. Moreover, nicotinamide is known to be
a radiation sensitizer and is used clinically, also acting as a PARP in-
hibitor; thus, the inhibition of NNMT resulting in accumulation of
nicotinamide could result in the attenuation of efficient single-stranded
DNA break repair [60,61]. Our results in endothelial cells agree with
some other reports in other cell types, suggesting a cytoprotective role of
NNMT. In particular, our findings show that NNMT in the endothelium
In previous works, the mechanisms of the beneficial role of NNMT were
linked to bioenergetics, the SIRT-1-dependent pathway or other mecha-
nisms [7,31,64]. Here, we verify a possible involvement of two of these
mechanisms in endothelial cells. NNMT as a major enzymatic source of
nicotinamide methylation may indeed affect the NAD+ pool, a process that
is dependent on constant re-synthesis from endogenous nicotinamide.
Downregulation or pharmacological inhibition of NNMT in mouse models
of diet-induced obesity increased NAD+ content in adipocytes [15,65],
whereas in the OVCAR-3 cancer cell line, the increased expression of
NNMT resulted in decreased parameters of mitochondrial oxygen con-
sumption (basal respiration, maximal respiration, ATP production),
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Fig. 7. Results of SIRT1 and phospho(Ser47)SIRT1 immunostaining in nucleus after 72 h incubation with NNMT inhibitor JBSF-88. Diagrams showing SIRT1- and
phospho(Ser47)SIRT1-specific fluorescence in the nuclear area after 72 h incubation with 5MQ (A) and JBSF-88 (B); representative images showing SIRT1 and
phospho(Ser47)SIRT1 expression in the nucleus after treatment with 5MQ (C) or JBSF-88 (D); representative images of immunostained phospho(Ser47)SIRT1 in the
nuclear area (C); n = 6, **p ≤ 0.01, ***p ≤ 0.001.
measured by the Seahorse assay [66]. On the other hand, the expression of
NNMT in SH-SY5Y human neuroblastoma cells, which have no endogenous
expression of NNMT, significantly increased ATP synthesis [64]. Our re-
sults did not confirm any significant change in the bioenergetic parameters
induced by prolonged pharmacological NNMT inhibition, suggesting that
in endothelial cells, this enzyme does not affect mitochondrial function.
Accordingly, we excluded a possibility that the decrease in cell viability in
response to oxidative stress in the presence of NNMT inhibition was linked
to a collapse of the bioenergetic balance of the cell. The cytoprotective
effects of NNMT in the endothelium were also not associated with an
altered endothelial proliferation profile as also excluded in the present
work. These results underscored a distinct role of endothelial NNMT as
compared to cancer cells [67]. In turn, NNMT inhibition in the endothelium
led to the downregulation of nuclear SIRT1, associated with upregulation
of nuclear pSIRT1 resulting in an increased pSIRT1/SIRT1 ratio as
compared to the control, suggesting the involvement of nuclear SIRT-
dependent pathways in the cytoprotective effects of NNMT in the endo-
thelium in an oxidant stress-induced model. The important role of SIRT-1 in
the regulation of endothelial cell viability in our experimental setting was
confirmed by the demonstration of the enhanced cellular damage after
SIRT1 inhibition by EX-527, which was reflected by increased p53
expression. The upregulation of p53 after inactivation of SIRT1 has been
previously reported [68] and this mechanism has an indubitable effect on
cell survival under oxidative stress conditions.
SIRT1 expression and activity is associated with the progression of
endothelial dysfunction and senescence [72,73].
Many of the beneficial effects of SIRT1 in stress responses, cell survival,
senescence and metabolism [75] are mediated via histones' or other
signaling proteins' deacetylation, including peroxisome proliferator-
activated receptor-g coactivator-1a (PGC-1a), forkhead transcription fac-
tor O (FoxO), nuclear factor kB (NFkB), endothelial nitric oxide synthase
(eNOS), notch1 intracellular domain (NICD), liver kinase B1 (LKB1) and
tumoursuppressor protein53(p53)[73,76–81], the latteraffectedby SIRT1
inhibition by EX-527 in our experiments. On the other hand, the SIRT1
phosphorylation on Ser47 results in increased nuclear localization of SIRT1,
and NNMT inhibition upregulated nuclear SIRT1 phosphorylation on Ser47
and the latter response could be dependent on cyclin-dependent kinase 5
(CDK5)-mediated hyper-phosphorylation of SIRT1 at Ser47 contributing to
senescence and atherosclerosis [82]. In turn, the mammalian target of
rapamycin (mTOR)-dependent phosphorylation of Ser47 resulted in the
inhibition of SIRT1 deacetylase activity [83]. We did not specifically
investigate here whether the effects of NNMT inhibition on phosphorylation
of SIRT1 at Ser47 were mediated by CDK5, mTOR or p53. We also did not
analyze whether the NNMT inhibition affected the nuclear SIRT1 pathway
bynicotinamideaccumulationordownregulationofMNA. Previously,MNA
was shown to stabilize the activity of SIRT1 in the liver [7]. On the other
hand, increasing intracellular levels of nicotinamide may lead to the inhi-
bition of SIRT1 [84–86]. Further studies are necessary to elucidate the
mechanisms explaining the NNMT/SIRT1 interacting pathways that in vivo
settings could also involve intercellular cross-talk.
SIRT1 displays vasoprotective activity, inhibits endothelial senes-
cence and inflammation [69–71], and a decreased SIRT1 expression and
activity is associated with the progression of endothelial dysfunction
and senescence [72–74]. SIRT1 exerts vasoprotective activity, inhibits
endothelial senescence and inflammation [69–71], and a decreased
In fact, vasoprotection afforded by endothelial SIRT1 in vivo was
shown to be mediated by the intercellular cross-talk of SIRT1 in endo-
thelium with soluble guanylyl cyclase in smooth muscle [87,88]. Clearly,
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Fig. 8. NNMT, SIRT1 and pSIRT1 expression levels in menadione-treated HAECs. HAECs were incubated for 48 h with increasing concentrations of menadione (A),
and the expression of NNMT (B, E), SIRT1 (C, E) and pSIRT1 (D, E) was assessed by immunocytochemistry using the automatic CQ-1 station and subsequent Co-
lumbus software analysis (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).
Fig. 9. Cell viability, p53 and acetyl-p53 levels in SIRT1-inhibited endothelial cells treated with menadione. HAECs were incubated for 48 h with increasing
concentrations of menadione with or without the SIRT1 inhibitor EX-527, and the cell viability (A), p53 (B) and acetyl-p53 (C) were assessed by immunocyto-
chemistry using the automatic CQ-1 station and subsequent Columbus (n = 3, *p < 0.05, **p < 0.01).
11

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further studies are necessary to elucidate the mechanisms underlying the
vasoprotective NNMT pathway and its link with SIRT1-mediated path-
ways in an in vivo setting.
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Roberto Campagna, Łukasz Mateuszuk, Monica Emanuelli and Ste-
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Roberto Campagna, Łukasz Mateuszuk, Kamila Wojnar-Lason,
Patrycja Kaczara, Anna Tworzydło and Agnieszka Kij collected,
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Robert Bujok, Jacek Mlynarski, Yu Wang, Davide Sartini and Monica
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Roberto Campagna, Łukasz Mateuszuk and Stefan Chlopicki drafted
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Roberto Campagna and Stefan Chłopicki prepared the final version
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All authors approved the final version of the manuscript.
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Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Acknowledgements
This study was supported by the Polish National Centre for Science
(OPUS project No. 2015/19/B/NZ3/02302). J.M. and R.B. thank the
Foundation for Polish Science for financial support under award TEAM/
2017-4/38. R.C. greatly acknowledges the European Molecular Biology
Organization (EMBO) for providing a short-term fellowship (EMBO
Short-Term Fellowship 7274) as well as the JMRC Foundation for a
fellowship. This study was also supported by Programme Canaletto
Bilateral Exchange of Scientists Italy – Poland (PO19MO11). The open-
access publication of this article was funded by the BioS Priority
Research Area under the program “Excellence Initiative Research
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