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\begin{document}

\title{Ebselen reduces cigarette smoke-induced vascular endothelial dysfunction
in mice}



\author[1]{Kurt Brassington}%
\author[1]{Stanley Chan}%
\author[1]{Huei Seow}%
\author[1]{Aleksandar Dobric}%
\author[1]{Steven Bozinovski}%
\author[1]{Stavros Selemidis}%
\author[1]{Ross Vlahos}%
\affil[1]{RMIT University}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background and Purpose: It is well established that both smokers and
patients with COPD are at a significantly heightened risk of
cardiovascular disease (CVD), although the mechanisms underpinning the
onset and progression of comorbid CVD are largely unknown. Here, we
explored whether cigarette smoke (CS) exposure impairs vascular function
in mice and given the well-known pathological role for oxidative stress
in COPD, whether the antioxidant compound ebselen prevents CS-induced
vascular dysfunction in mice. Experimental Approach: Male BALB/c mice
were exposed to either room air (sham) or CS generated from 9 cigarettes
per day, 5 days a week for 8 weeks. Mice were treated with ebselen
(10mg/kg, oral gavage once daily) or vehicle (5\% w/v CM cellulose in
water) 1 h prior to the first CS exposure of the day. Upon sacrifice,
bronchoalveolar lavage fluid (BALF) was collected to assess pulmonary
inflammation and the thoracic aorta was excised to investigate vascular
endothelial and smooth muscle dilator responses ex-vivo. Key Results: CS
exposure caused a significant increase in lung inflammation which was
reduced by ebselen. CS also caused significant endothelial dysfunction
in the thoracic aorta which was attributed to a downregulation of eNOS
expression and increased vascular oxidative stress. Ebselen abolished
the aortic endothelial dysfunction seen in CS-exposed mice by reducing
the oxidative burden and preserving eNOS expression. Conclusion and
Implications: Targeting CS-induced oxidative stress with ebselen may
provide a novel means for treating the life-threatening pulmonary and
cardiovascular manifestations associated with cigarette smoking and
COPD.%
\end{abstract}%



\sloppy


\emph{}

\emph{Introduction}

Chronic obstructive pulmonary disease (COPD) is a major incurable global
health burden and is currently the 4\textsuperscript{th} largest cause
of death in the world (WHO, 2018). Approximately 50\% of COPD patients
will die from a cardiovascular event (Sin \& Man, 2005; TLF, 2017) and
consequently the pathobiological mechanisms linking COPD to
cardiovascular disease are now an area of intensive research. Each puff
of cigarette smoke contains \textgreater{}10\textsuperscript{16} free
radicals per puff, driving oxidative stress and tissue damage (Bartalis,
Chan \& Wooten, 2007) This oxidative stress and inflammation, have been
shown to alter pulmonary blood vessel structure, through driving
vascular remodelling, and promoting arterial stiffness and
atherosclerosis (Sin, Anthonisen, Soriano \& Agusti, 2006). Vascular
tone is controlled by vasoactive substances such as NO and
prostaglandins, their secretion which can be maintained by circulating
oxygen levels (Chan \& Vanhoutte, 2013). Under hypoxic conditions like
those seen in COPD, dysregulation of this vascular homeostatic balance
occurs, due to oxidative damage to the vascular endothelial cells (VECs)
leading to impaired NO production, thereby promoting endothelial
dysfunction (Chan \& Vanhoutte, 2013). NO is a key vasodilator produced
by endothelial nitric oxide synthase (eNOS). Under normal physiological
conditions, increased sheer stress on the vascular endothelium
stimulates mechanosensitive ion channels, triggering a rapid influx of
Ca\textsuperscript{2+} into the cytoplasm of the VECs. This increases
eNOS activity via myoendothelial gap junctions, that transmit
vasodilatory NO signals to the underlying smooth muscle cells. VECs
appear to be sensitive to oxidative damage, which may be the result of
the conversion of NO to peroxynitrite (ONOO\textsuperscript{-}) in the
presence of the harmful ROS; superoxide
(O\textsubscript{2}\textsuperscript{-}) that ultimately reduces vascular
NO bioavailability (Endemann \& Schiffrin, 2004; Kolluru, Bir \& Kevil,
2012; Tabit, Chung, Hamburg \& Vita, 2010). An altered oxidative balance
in VECs has been demonstrated to promote cardiovascular manifestations
such as atherosclerosis, myocardial infarction (MI) and stroke
(Brassington, Selemidis, Bozinovski \& Vlahos, 2019; Endemann \&
Schiffrin, 2004; Kolluru, Bir \& Kevil, 2012; Tabit, Chung, Hamburg \&
Vita, 2010).

Although cardiovascular comorbidities are the largest cause of mortality
in COPD, the detrimental effects of CS and its associated oxidative
stress on the systemic vasculature remain largely unknown. Given the
deleterious role of oxidative stress in COPD, antioxidant treatment may
be a viable therapeutic approach to treat the cardiovascular
manifestations associated with this disease. We have previously shown
that the antioxidant ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)one),
an organoselenium glutathione peroxidase (Gpx) mimetic, inhibits
CS-induced lung inflammation in mice (Duong, Seow, Bozinovski, Crack,
Anderson \& Vlahos, 2010). Ebselen treatment may also be an effective
therapeutic in chronic diseases such as atherosclerosis, thrombosis, and
stroke where oxidative stress and inflammation play a crucial role (Azad
\& Tomar, 2014; Sarker et al., 2003; Sarma \& Mugesh, 2008; Takasago,
Peters, Graham, Masayasu \& Macrae, 1997). Moreover, studies have shown
that Gpx-1 deficient mice have enhanced pulmonary inflammation (Duong,
Seow, Bozinovski, Crack, Anderson \& Vlahos, 2010), as well as worsened
cardiovascular outcomes including a larger infarct volume following
ischemic stroke (Crack et al., 2001; Duong, Seow, Bozinovski, Crack,
Anderson \& Vlahos, 2010), suggesting that Gpx-1 (or compounds which
mimic its actions, like ebselen) may exhibit protective effects.

Of interest, Gpx-1 activity has been shown to be elevated in smokers as
a potential mechanism to counteract the harmful oxidative stress.
However, Gpx-1 has been reported to be severely depleted in the lungs of
COPD patients, resulting in an overstated inflammatory response and
oxidative burden (Kluchova, Petrasova, Joppa, Dorkova \& Tkacova, 2007;
Santos et al., 2004; Tkacova, Kluchova, Joppa, Petrasova \& Molcanyiova,
2007; Vlahos et al., 2010). A study by Chew \emph{et.al} has showcased
the therapeutic effect of ebselen in Gpx-1 knockout mice, with the study
finding that synthetic repletion of Gpx activity in these diabetic mice
produced athero-protective effects \emph{in vivo} (Chew et al., 2010).
Exogenous repletion of Gpx-1 with compounds like ebselen may have
therapeutic potential in not only treating the pulmonary manifestations
of COPD but perhaps its cardiovascular comorbidities.

In the present study we investigated whether chronic CS exposure in a
preclinical mouse model of COPD impairs vascular function and whether
ebselen can prevent CS-induced vascular dysfunction in mice.

\emph{Methods}

\textbf{Animals}

All experiments were conducted in accordance with the Australian Code of
Practice for the Care of Experimental Animals, the ARRIVE Guidelines and
with RMIT University Animal Ethics Committee approval (Animal Ethics
Application Number 1521). 7-week-old male Balb/c mice
(\textasciitilde{}20g body weight) were obtained from the Animal
Resource Centre Pty. Ltd (Perth, Australia). Mice were housed in
micro-isolator cages (Able Scientific, Australia) at 21\selectlanguage{ngerman}°C with \emph{ad
libitum} access to water and standard mouse chow (Glen Forest Speciality
Foods, Australia).

\textbf{Cigarette smoke exposure \& ebselen treatment}

Mice were placed into an 18-litre Perspex chamber (The Plastic Man,
Huntingdale, Victoria, Australia) in a standard fume cabinet (Aircare
Extraction Systems LTD, Clayton, Victoria, Australia) and exposed to
cigarette smoke generated from 9 Winfield Red Cigarettes (total
particulate matter of 419 mg m\textsuperscript{-3}, 16 mg or less of
tar, 1.2 mg or less of nicotine and 15 mg or less of CO, Philip Morris,
Moorabbin, Australia) for 5 days a week for 8 weeks. Mice were exposed
to CS delivered 3 times per day with 3 cigarettes delivered at 9 AM, 12
noon and 3 PM, over a 1h time-period. CS was generated in 60 mL tidal
volumes over 10 s, via a timed draw-back to mimic normal smoking
inhalation and burn rates. We have previously shown that this CS
exposure protocol in Balb/C mice replicates key clinical traits of early
stage COPD in humans, including lung inflammation and pathology (mucous
hypersecretion and impaired lung function), increased lung and systemic
oxidative stress and comorbidities including skeletal muscle wasting
(Austin, Crack, Bozinovski, Miller \& Vlahos, 2016; Chan et al., 2019;
Vlahos \& Bozinovski, 2014). Thus, this model provides a robust
clinically relevant platform to test therapies for COPD and its
comorbidities. Sham-exposed mice are placed into an identical 18-liter
Perspex chamber, but do not receive cigarette smoke. Mice were weighed
every second day (prior to initial CS exposure) up to and including the
day of cull. For the ebselen treatment studies, mice were administered
10 mg kg\textsuperscript{-1} of ebselen (Sapphire Bioscience, Australia)
prepared in 5\% w/v CM-cellulose in water (Sigma-Aldrich, USA) or
vehicle treated with 5\% CM-cellulose in water alone. Treatments were
administered via oral gavage once daily, 1h prior to the initial CS
exposure.

\textbf{Bronchoalveolar lavage and lung collection}

Animals were euthanised at the end of the experimental protocol via
intraperitoneal injection of sodium pentobarbitone (240 mg
kg\textsuperscript{-1}; Virbac, NSW, Australia). Lungs were then
lavaged\emph{in situ} via a surgical tracheotomy with 0.4 mL of chilled
PBS initially followed by 0.3 mL PBS thrice, with \textasciitilde{}1mL
of BAL fluid (BALF) retrieved per mouse as previously described (Vlahos
et al., 2006) . Total viable cell numbers in the BALF were determined
using 50\selectlanguage{greek}μ\selectlanguage{english}L of BALF diluted with 50 \selectlanguage{greek}µ\selectlanguage{english}l of acridine orange/ethidium
bromide (AO/EB) (Invitrogen, USA). Cell counting was carried out using a
standard Neubauer haemocytometer, under fluorescent light on an Olympus
BX53 microscope (Olympus, Japan). Right ventricular perfusion with 6-7
mL of PBS was then performed to clear whole lungs from blood, and the
lungs then excised, rinsed in PBS, snap-frozen in liquid nitrogen and
stored at -80\selectlanguage{ngerman}°C until required.

\textbf{Differential cell counting}

To differentiate the various cell populations in the BALF,
cytocentrifuged cell preparations (Shandon Cytospin 3, 400 RPM, 10 min)
were used, with \textasciitilde{}5x10\textsuperscript{4} cells per
slide. Dried cytospots were subjected to a Shandon
Kwik-Diff\textsuperscript{®} Fixative (Thermo Fischer Scientific, USA),
followed by Hemacolour\textsuperscript{®} eosin and thiazine
differential stains (Merck, USA) as outlined in the manufacturer's
instructions. Cell types (i.e. macrophages, neutrophils and lymphocytes)
were identified according to standard morphological criteria, using the
above-mentioned microscope, with at least 500 cells counted per slide.

\textbf{Quantitative real-time PCR (RT-qPCR)}

Total RNA was extracted from approximately 10 mg of whole lung tissue
using a RNeasy\textsuperscript{®} Mini Kit (Qiagen, Germany). Isolated
mRNA was then reverse transcribed with a High Capacity RNA-to-cDNA kit
(Thermo Fisher Scientific, USA). Real time PCR reactions were performed
using Thermo Fisher Scientific pre-developed Taqman assay reagents in
triplicate using GAPDH as the internal housekeeping control. Through
utilisation of the threshold cycle (C\textsubscript{t}) value which is
the PCR cycle number out of 40 at which the fluorescence signal measured
exceeds the calculated background threshold, indicative of amplification
of the target sequence value, which is proportional to the number of
target copies within the sample and converting this result to the
threshold cycle time (\selectlanguage{english}[?]\selectlanguage{english}[?]CT). mRNA expression levels can then be
quantified and referenced against GAPDH, allowing comparisons between
treatment groups to be made.

\textbf{Vascular reactivity}

To assess the effect of both CS-exposure and ebselen treatment on vessel
function, thoracic aorta was excised from the mice and all perivascular
fat removed. Vessels were placed into carbogen-bubbled (95\%
O\textsubscript{2}, 5\% CO\textsubscript{2}) cold Krebs buffer
(composition in mmol/L: NaCl 119, KCl 4.7, MgSO\textsubscript{4} 1.17,
NaHCO\textsubscript{3} 25, KH\textsubscript{2}PO\textsubscript{4} 1.18,
CaCl\textsubscript{2} 2.5, glucose 5.5). The aortae were cut into four 2
mm rings and mounted onto the pins of the myograph system (Danish Myo
Technology A/S, Model 610M) with the resting tension increased to 5mN
which was determined to give an effective arterial wall pressure of
\textasciitilde{}100mmHg (13.3kPa) thus mimicking \emph{in
vivo}conditions. Following a 30-minute equilibration, aortic rings were
exposed to 0.5 x 10\textsuperscript{-3}M of the thromboxane A2 agonist
U46619 (Cayman Chemical, USA) to induce the maximal vascular
contraction, defined as 100\%. In each ring, both endothelial integrity
and smooth muscle function were assessed using cumulative doses of
acetylcholine (ACh, 1x10\textsuperscript{-8}M to
1x10\textsuperscript{-5}M) (Thermo Fisher Scientific, USA) and sodium
nitroprusside (SNP, 1x10\textsuperscript{-8}M to
1x10\textsuperscript{-5}M) (Thermo Fisher Scientific, USA) respectively
in sub-maximally contracted aorta (50-60\% of maximal U46619
contraction) with all experiments ran in duplicate and compared to sham
or sham + vehicle treated mice.

\textbf{Immunohistochemical staining for endothelial nitric oxide
synthase and oxidative stress}

Vascular expression of endothelial nitric oxide synthase was quantified
using eNOS/NOS3 antibody (1:100 dilution, Thermo Fisher Scientific,
USA), which is the key nitric oxide generating enzyme within the
vascular endothelium. Vascular oxidative stress was measured using
3-Nitrotyrosine (3-NT) (1:100 dilution, Thermo Fisher Scientific, USA)
as this is a specific marker for peroxynitrite production, the direct
product of the reaction between superoxide to nitric oxide. Thoracic
aorta was collected upon cull and fixed in 4\% paraformaldehyde prior to
sucrose saturation. The aortae were then paraffin embedded and 4 \selectlanguage{greek}µ\selectlanguage{english}M
sections were cut. Aortic sections were subject to standard
deparaffinisation and rehydration, followed by antigen retrieval (in
10mM citric acid, 0.05\% Tween 20, pH 6.0) and blocking (blocking
buffer; 10\% horse serum, 10\% FBS, 2\% triton-X, 1xPBS to 50mL for
1hr). Sections were then incubated overnight with either eNOS of 3-NT
primary antibodies at 4~\selectlanguage{english}C. Excessive primary antibodies were removed by
washing, the sections were then incubated at room temperature with the
fluorescently labelled secondary antibody, Alexa 488 (1:200 dilution,
Thermo Fisher Scientific, USA), then cover slipped using
Fluoromount-G\textsuperscript{TM}, with DAPI (Thermo Fisher Scientific,
USA) prior to imaging on an Olympus slide scanner VS120-SS (Olympus,
Japan). The expression of eNOS and 3-NT was quantified in the
endothelial layer of the aortae using Olympus cellSens
Dimension\textsuperscript{TM} desktop software, calculating Object Area
Fraction ROI (\%) (version 1:18, Olympus Corporation). All analysis of
immunofluorescence was completed in a blinded manner.

\textbf{Data and Statistical analysis}

All data and statistical analysis comply with the recommendations on
experimental design and analysis in pharmacology (Curtis et al, 2018).
All data presented as mean \selectlanguage{ngerman}± standard error of the mean (SEM) unless
otherwise stated. Statistical differences between treatments were
determined by analysis of variance (ANOVA) followed by Tukey's multiple
comparison post-hoc tests where appropriate. One-way ANOVA were used for
three of more unmatched groups. Two-way ANOVA were used to analyse data
when response was influenced by two categorized factors of interest. For
comparisons between two groups of results, regular unpaired t-tests were
used to generate the P value. All statistical analyses were performed
using GraphPad Prism\textsuperscript{TM} for Microsoft Windows®
(Versions 8, Graphpad software\textsuperscript{®}, USA) where \emph{p}
\textless{}0.05 was accepted as significant for all cases.

\textbf{Materials}

The Suppliers of the following compounds are as follows: Winfield red
cigarettes (Phillip Morris, Australia); ebselen (Sapphire Bioscience,
Australia); Carboxymethyl Cellulose (Sigma-Aldrich, USA); sodium
pentabarbitone (Virbac, Australia); acridine orange/ethidium bromide
(Invitrogen, USA); Kwik-Diff\textsuperscript{®} reagent 1, fixative
(Thermo Fisher Scientific, USA); Hemacolour\textsuperscript{®} rapid
staining of blood smear (eosin solution) (Merck, USA);
Hemacolour\textsuperscript{®} rapid staining of blood smear (thiazine
solution) (Merck, USA); RNeasy Mini Kit (Qiagen, Germany); High Capacity
RNA-to-cDNA kit (Thermo Fisher Scientific, USA); pre-developed TaqMan
primers (Thermo Fisher Scientific, USA), CRP ELISA Kit (RayBiotech,
USA); U46619 (Cayman Chemical, USA); acetylcholine (Thermo Fisher, USA);
sodium nitroprusside (Thermo Fisher, USA); NOS3/eNOS RRID: AB\_2533121
(Thermo Fisher Scientific, USA), 3-NT RRID: AB110282 (Abcam, USA); Goat
anti-mouse IgG (H+L) secondary antibody, Alexa Flour Plus 488 RRID:
AB\_2633275 (Thermo Fisher Scientific, USA);
Fluoromount-G\textsuperscript{TM}, with DAPI (Thermo Fisher Scientific,
USA) .

\emph{Results}

Chronic CS exposure causes endothelial dysfunction in mouse thoracic
aorta

The effect of chronic CS exposure on vascular function was examined to
establish if the enhanced oxidative stress and inflammation arising from
smoking has detrimental effects on blood vessel function \emph{in vivo}
. ACh (10\textsuperscript{-8} to 10\textsuperscript{-5} M) caused an
\textasciitilde{}90\% maximal relaxation (R\textsubscript{max}) of
U46619-precontracted thoracic aorta obtained from sham-exposed mice
(Figure 1A). However, in thoracic aorta taken from CS-exposed mice,
endothelial-dependent vasodilatory responses were significantly impaired
(\textasciitilde{}40\% R\textsubscript{max}, \emph{p\textless{}0.05} )
demonstrating that chronic CS exposure causes endothelial dysfunction.
Sodium nitroprusside (10\textsuperscript{-8} to 10\textsuperscript{-5}M)
caused an \textasciitilde{}90-95\% maximal relaxation of aortae taken
from both sham and CS-exposed mice with no statistically significant
difference observed between groups (Figure 1B), suggesting CS exposure
has no effect on vascular smooth muscle function.

CS exposure drives pulmonary immune cell infiltration, pro-inflammatory
and oxidative stress gene expression

CS exposure caused a significant increase in the total number of immune
cells infiltrating the lungs when compared to sham-exposed control mice
(Figure 2A,\emph{p\textless{}0.05} ). This increase in total cell number
was attributed to a significant increase in macrophages, neutrophils and
lymphocytes) (Figures 2B, C \& D, \emph{p\textless{}0.05} ).

To better understand the mechanism(s) underlying the increased BALF
inflammation, the pulmonary expression of both pro-inflammatory and
oxidative stress genes was examined. CS exposure caused a significant
increase in mRNA expression of the pro-inflammatory cytokines TNF-\selectlanguage{greek}α
\selectlanguage{english}(2.2-fold) and IL-6 (4.7-fold), as well as the key oxidative stress
enzyme NOX-2 (1.5-fold; Figures 2, F \& G, \emph{p\textless{}0.05} ).

Endothelial nitric oxide synthase expression is downregulated due to
increased vascular ROS in COPD

Given that chronic CS exposure causes endothelium-dependent vascular
dysfunction, we then went on to investigate the potential underlying
mechanism driving this impaired vascular function. Endothelial
expression of the key vascular tone regulator eNOS and a marker of
oxidative stress, 3-nitrotyrosine (3-NT), were quantified. CS exposure
significantly reduced expression of eNOS by \textasciitilde{}70\%
(Figure 3A, \emph{p} \textless{}0.05). In addition, the level of 3-NT
expression was significantly upregulated by \textasciitilde{}3.2 fold
following CS exposure (Figure 3B, \emph{p\textless{}0.05} ), indicative
of enhanced vascular oxidative stress which may be responsible for the
reduced expression of eNOS.

Ebselen prevents CS-induced endothelial dysfunction

As chronic CS exposure caused significant lung inflammation as well as
heightened lung and vascular oxidative stress, we sought to investigate
the effect of antioxidant treatment on vascular endothelial function.
Sham + Veh-treated mice showed an \textasciitilde{}90\%
R\textsubscript{max}, while CS + Veh-treated mice showed an
\textasciitilde{}40\% R\textsubscript{max} to ACh (Figure 4A),
confirming CS-induced endothelial dysfunction as also demonstrated in
Figure 1. CS-exposed mice treated with ebselen were completely protected
from the CS-induced endothelial dysfunction evidenced by the maximal
relaxation of \textasciitilde{}90\% to ACh. In addition, ebselen did not
affect vascular endothelial function in sham-exposed mice (Figure 4A),
suggesting its protective effects are specific to CS exposure. Like the
data in Figure 1, smooth muscle relaxant responses to SNP were unaltered
irrespective of CS exposure or ebselen treatment (Figure 4B).

Ebselen reduces pulmonary immune cell infiltration but has no effect on
pro-inflammatory and oxidative stress gene expression

Given that ebselen was able to protect against CS-induced vascular
dysfunction, we next sought to determine the effect of ebselen on
CS-induced lung inflammation. Consistent with Figure 2, CS caused a
significant increase in BALF total cells, macrophages, neutrophils and
lymphocytes (Figure 5A-D). Interestingly, ebselen significantly reduced
CS-induced increases in BALF total cell and neutrophil counts but not
macrophage and lymphocyte numbers (Figure 5A-D). Moreover, while CS
exposure increased TNF-\selectlanguage{greek}α \selectlanguage{english}and NOX-2 mRNA expression in the lungs, no
detectable attenuations were found with ebselen administration (Figure
5E \& F). Ebselen administration also appears to be ineffective in
preserving lung Gpx-1 mRNA expression against CS exposure (Figure 5G)
but rather mimics its antioxidant activity.

Ebselen diminishes CS-induced vascular oxidative stress and
downregulation of eNOS

We next sought to define the underlying mechanism by which ebselen
prevented CS-induced endothelial dysfunction. Mice chronically exposed
to CS had increased vascular endothelial oxidative stress as measured by
3-NT expression (Figure 6, \emph{p} \textless{}0.05). However, ebselen
administration significantly reduced CS-induced increases in 3-NT
expression to baseline sham levels (Figure 6, \emph{p} \textless{}0.05).
Moreover, ebselen treatment completely prevented the downregulation of
eNOS as a result of CS-exposure (Figure 7, \emph{p} \textless{}0.05).
Collectively, these findings suggest that ebselen reduced CS-induced
vascular oxidative stress and subsequently prevented the downregulation
of eNOS within the thoracic aorta.

\emph{Discussion}

In the present study, the mechanisms underlying systemic endothelial
dysfunction, the key driver of comorbid CVD-associated mortality are
revealed and the impact of CS-exposure on blood vessel function in our
preclinical murine model of COPD were examined. We found that 8-weeks of
CS exposure increased immune cell infiltration into the lung resulting
in significant pulmonary inflammation and oxidative stress. Pulmonary
immune cell infiltration is believed to be the underlying factor driving
increased NOX-2 expression and ROS formation. Studies from our group
have shown that mice treated with influenza A virus had increased levels
of ROS production in the lungs as a consequence of increased pulmonary
inflammation (macrophages and neutrophils) and NOX-2 expression (To et
al., 2017; Vlahos \& Selemidis, 2014; Vlahos, Stambas, Bozinovski,
Broughton, Drummond \& Selemidis, 2011). In this study we also showed
increased NOX-2 expression in the lung which is presumably a result of
increased pulmonary macrophage and neutrophil numbers in response to
cigarette smoke. We also found a significant increase in the expression
of the pro-inflammatory mediators TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-6 in response to CS,
which is consistent with our previously published work (Hansen et al.,
2013; Vlahos et al., 2006). TNF-\selectlanguage{greek}α \selectlanguage{english}is largely secreted by stimulated
macrophages (e.g. in response to CS), driving the inflammatory response
and intracellular ROS production, whilst downregulating antioxidant
activity (Mukhopadhyay, Hoidal \& Mukherjee, 2006). IL-6 has also been
implicated in the pathophysiology of pulmonary diseases, thus, these
pro-inflammatory mediators may contribute to pulmonary inflammation and
reduced lung function observed in COPD patients (Rincon \& Irvin,
2012),.

Having established that mice exposed chronically to CS had pulmonary
inflammation, enhanced oxidative stress and systemic inflammation, we
then proceeded to investigate whether this may impact on blood vessel
function. It was clear from the present study that CS exposure
significantly impaired vasodilation of mouse thoracic aorta to ACh and
that this was specifically attributed to endothelial dysfunction without
affecting smooth muscle function. However, it was unclear if this was a
secondary result of pulmonary and systemic inflammation or a direct
result of CS-induced ROS on the vasculature, and therefore further
experimentation is required to ascertain a definitive explanation.
Nevertheless, this CS-induced endothelial dysfunction may be a critical
link between the heightened risk of CVD and related mortality that
claims the lives of \textasciitilde{}50\% of COPD patients, as well as
current and ex-smokers.

It has been well characterised in models of diabetes mellitus (DM) that
increased vascular oxidative stress is the key driver of endothelial
dysfunction seen in diabetic complications (de Haan \& Cooper, 2011;
Kolluru, Bir \& Kevil, 2012; Rask-Madsen \& King, 2007; Shenouda et al.,
2011; Tabit, Chung, Hamburg \& Vita, 2010). The excess blood-glucose
from DM drives the formation of ROS; such as superoxide, which reduces
the bioavailability of NO in the vascular wall (Tabit, Chung, Hamburg \&
Vita, 2010). The increased oxidative burden in patients with DM
resembles that seen in patients with COPD, as both may lead to
post-translational modification of eNOS and increase vascular oxidative
stress, thereby resulting in endothelial dysfunction (de Haan \& Cooper,
2011). Having shown that CS causes endothelium-dependent vascular
dysfunction, we next investigated whether this was attributed to changes
in eNOS expression. We found that CS caused an \textasciitilde{}60\%
reduction in the expression of eNOS, which would likely lead to a
drastic reduction in the production of the key vasodilator NO and
reduced NO bioavailability, a hallmark feature of CVD. With studies like
that of de Hann \emph{et.al} showing that eNOS can undergo oxidative
modification under highly oxidative environments (de Haan \& Cooper,
2011), vascular expression levels of peroxynitrite were therefore
analysed using 3-NT, which specifically detects the conversion of
superoxide radical and NO to ONOO-. In agreement with this, the present
study also found enhanced vascular oxidative burden following exposure
to CS, which in turn may promote endothelial dysfunction via
post-translational modifications of eNOS and ablating the
bioavailability of NO.

Given the significant role of oxidative stress in COPD and this study,
we reasoned whether the administration of ebselen, an antioxidant drug
which has shown promising results in the context of DM-induced vascular
complications through its free radical scavenging activity, could
prevent CS-induced vascular dysfunction. Moreover, de Haan
\emph{et.al}have proposed that deficiencies in the antioxidant GPX and
an enhanced oxidative burden promotes endothelial dysfunction leading to
DM-related micro- and macrovascular complications (de Haan \& Cooper,
2011). As such, targeted antioxidant replenishment therapy using
GPX-mimetics (i.e. ebselen) may be effective in reducing the
cardiovascular manifestations in disease states such as DM. de Haan
\emph{et al} . also noted that, an increase in ROS within the vascular
endothelium, is one of the most significant factors in NO reduction (de
Haan \& Cooper, 2011).

In the present study we showed that ebselen completely prevented
endothelial dysfunction induced by CS-exposure. We found that ebselen
significantly reduced CS-induced endothelial 3-NT expression and that
ebselen was able to prevent the loss of aortic eNOS by CS-exposure. It
is well established that under normal physiological conditions,
stimulation of the vascular endothelium drives the production of NO,
diffusing to the surrounding cells, in particular the underlying
vascular smooth muscle cells inducing vasodilation, as well as
preventing the adhesion and migration of leukocytes and platelets
into/onto the arterial wall, thereby maintaining normal vascular
function (Versari, Daghini, Virdis, Ghiadoni \& Taddei, 2009). However,
vascular oxidative stress may evoke endothelial damage which may
significantly impair NO bioavailability and eNOS activity within the
vascular endothelium (de Haan \& Cooper, 2011; Versari, Daghini, Virdis,
Ghiadoni \& Taddei, 2009). This would post significant risk for the
development of CVDs, and mortality in DM patients. This data suggests
that modulation of oxidative stress may be beneficial in the clinical
treatment of CVD in the context of COPD.

Consistent with our previous studies, ebselen significantly reduced
CS-induced BALF inflammation which was largely attributed to a reduction
in neutrophilic infiltration (Duong, Seow, Bozinovski, Crack, Anderson
\& Vlahos, 2010; Oostwoud et al., 2016). Excess neutrophils play a
detrimental role in COPD particularly during periods of acute
exacerbation, as they can directly induce protease mediated tissue
damage, that has been directly correlated to worsening of emphysema in
these patients (Oostwoud et al., 2016; Pesci et al., 1998). MMP
activation drives a loss of lung integrity and an increase in
permeability which may facilitate the spill over of proinflammatory
mediators into the systemic circulation.

It was interesting to note that CS-induced whole lung gene expression of
the pro-inflammatory mediator TNF\selectlanguage{greek}α \selectlanguage{english}and the oxidative stress enzyme NOX-2
were not reduced by ebselen pre-treatment. Although not investigated in
the present study, it would be worth exploring whether TNF-\selectlanguage{greek}α \selectlanguage{english}protein
expression is altered following ebselen administration. Similarly, it
would be worth investigating whether ebselen can directly impede the
activity of the regulatory p47\emph{phox} and other subunits of the
NOX-2 enzyme ultimately reducing superoxide production, as this has been
previously shown (Smith et al., 2012). The ROS scavenging properties of
ebselen within the lung have also been established in the context of
asthma, with Zhang \emph{et.al} showing that following ovalbumin
challenge, guinea pigs showed significantly enhanced pulmonary
superoxide and hydrogen peroxide concentration, which was ablated in
ebselen treated animals (Zhang et al., 2002), reinforcing the powerful
antioxidant properties of ebselen. A study by Yatmaz \emph{et.al} showed
that genetic depletion of Gpx-1 causes a significant increase in BALF
cellularity in response to influenza infection (HKx31) when compared to
wild type control mice (Yatmaz et al., 2013). Interestingly,
administration of ebselen (10 mg/kg\textsuperscript{-1}) abolished this
viral induced immune cell recruitment to the lung, however, it did not
reduce either the protein or mRNA expression of pro-inflammatory
cytokines and chemokines (Yatmaz et al., 2013). Nevertheless, the
protective effects of ebselen in CS-induced lung inflammation and
oxidative stress are promising however, they yet to be determined
thoroughly.

It has been shown that there is upregulation of Gpx-1 gene expression in
the lungs of smokers (Barnes \& Celli, 2009), which may be a
compensatory antioxidant mechanism in response to noxious effects of CS.
Conversely, smokers and patients with established COPD have reduced Gpx
activity (James \& Wenzel, 2007; Versari, Daghini, Virdis, Ghiadoni \&
Taddei, 2009; Vlahos et al., 2006), contributing to an overexuberant
oxidative burden in the lungs of these patients (Geraghty et al., 2013).
In the present study we found that whole lung Gpx-1 mRNA expression was
significantly downregulated in mice exposed to CS irrespective of
ebselen treatment, further reinforcing that loss of Gpx would increase
lung oxidative stress and inflammation. Blunted Gpx expression has also
been implicated as a contributing factor in driving endothelial
dysfunction, inducing apoptosis and promoting atherosclerosis
systemically (Geraghty et al., 2013).

Ebselen treatment has shown promising effects on the vasculature in this
study by completely preventing endothelial dysfunction in CS-exposed
mice as well as reducing BALF cellularity attributed to neutrophilic
infiltration. It has been established that eNOS can undergo oxidative
modification as a direct result of the heightened oxidative burden in
smokers (Arunachalam, Yao, Sundar, Caito \& Rahman, 2010; Edirisinghe \&
Rahman, 2010; Li \& Forstermann, 2014; Zhang, Venardos, Chin-Dusting \&
Kaye, 2006). Findings from this study showed that eNOS expression as
quantified through immunofluorescent staining was significantly
downregulated as a result of CS exposure. However, pre-treatment with
ebselen prevented CS-induced downregulation of eNOS and was the likely
mechanism by which ebselen restored vascular function in CS-exposed
mice. It was also interesting to note that ebselen significantly reduced
CS-induced 3-NT staining in the thoracic aorta indicating that ebselen
completely prevented enhanced oxidative stress within the vascular
endothelium, leading to sustained eNOS levels and normal vascular
function in CS-exposed ebselen treated mice.

In conclusion, we found that chronic CS exposure in mice causes
endothelial dysfunction, as a direct result of enhanced vascular
oxidative stress leading to a downregulation of eNOS. In addition,
ebselen administration significantly reduced CS-induced lung
inflammation and vascular oxidative stress leading to restored vascular
endothelial function in CS-exposed mice. Collectively, the data from the
present study suggest that ebselen may be a novel therapeutic in the
treatment of both the pulmonary manifestations and cardiovascular
comorbidities associated with cigarette smoke-induced COPD.

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\emph{Figure Legends}

\textbf{Figure 1: CS exposure causes endothelial dysfunction in the
thoracic aorta.} Cumulative concentration response curves to (\textbf{A}
) acetylcholine and (\textbf{B} ) sodium nitroprusside
(1x10\textsuperscript{-8}M to 1x10\textsuperscript{-5}M) to assess both
endothelial-dependent and smooth muscle-dependent vasodilatory responses
in mouse thoracic aorta (n=8/group) following either chronic CS or sham
exposure, respectively. Results are expressed as mean percentage
relaxation relative to precontraction ± SEM. * indicates statistical
significance (\emph{p\textless{}0.05} ) between data sets by two-way
ANOVA with Tukey's multiple comparisons.

\textbf{Figure 2: Chronic CS exposure increases BAL fluid cellularity
and enhances both lung pro-inflammatory and oxidative stress mediator
gene expression.} The lungs of mice exposed to CS were lavaged for the
assessment of total cells (A), macrophages (B), neutrophils (C) and
lymphocytes (D) (n=10). Whole lungs excised from mice were then used to
measure mRNA expression by RT-qPCR of TNF\selectlanguage{greek}α \selectlanguage{english}(E) (n=10), IL-6 (F) (n=10)
and NOX-2 (G) (n=9). Gene expression data are expressed as fold change
relative to the sham group. All data are expressed as mean + SEM and
were analysed by students unpaired t-test with significance being
denoted by * (\emph{p\textless{}0.05)} between treatment groups.

\textbf{Figure 3 Immunofluorescent staining of endothelial nitric oxide
synthase and 3-nitrotyrosine in the thoracic aorta of mice exposed to 8
weeks of CS or room air.} Immunofluorescent quantification of eNOS and
3-NT expression in either sham or 8-week CS-exposed mice. Green staining
indicates the presence of eNOS (A) or 3-NT (B) and blue staining denotes
the nuclear counterstain, DAPI (4', 6-diamidino-2- phenylindole,
dilactate). Representative photographs of immunofluorescent staining in
(A) sham-exposed or (B) CS-exposed mice. Expression of eNOS (n=5-6 mice
per group) and 3-NT (n=5 mice per group) both normalised to the relative
negative control and expressed as fold percentage change relative to the
sham-treated group. Scale bar represents 50\selectlanguage{greek}µ\selectlanguage{english}M. Data expressed as mean +
SEM and analysed by students unpaired t-test with significance being
denoted by; * indicating \emph{p\textless{}0.05} .

\textbf{Figure 4 Ebselen prevents CS-induced endothelial
dysfunction.}Cumulative concentration response curves to acetylcholine
(A) and sodium nitroprusside (B) (1x10\textsuperscript{-8}M to
1x10\textsuperscript{-5}M) to assess endothelial and smooth
muscle-dependent vasodilation in mouse thoracic aorta (n=6) following
either chronic CS or sham exposure in either ebselen or vehicle-treated
mice, respectively. Results are expressed as mean percentage relaxation
relative to pre-constriction \selectlanguage{ngerman}± SEM. * indicates statistical significance
(\emph{p\textless{}0.05} ) between data sets by two-way ANOVA with
Tukey's multiple comparisons.

\textbf{Figure 5: Effect of chronic CS and ebselen treatment on BAL
fluid cellularity and pulmonary pro-inflammatory and oxidative stress
gene expression.} The lungs of mice exposed to CS were lavaged for the
assessment of total cells (A), macrophages (B), neutrophils (C) and
lymphocytes (D) (n=10). Whole lungs excised from mice were then used to
measure mRNA expression by RT-qPCR of TNF\selectlanguage{greek}α \selectlanguage{english}(E), NOX-2 (F) and GPX-1(G)
(n=10). Responses are expressed as fold change relative to the sham +
vehicle-treated group, post normalisation to GAPDH (housekeeping gene).
All data are expressed as mean + SEM and were analysed by 2-way ANOVA
and Tukey's post hoc analysis with significance being denoted by *
(\emph{p} \textless{}0.05) between treatment groups.

\textbf{Figure 6: Immunofluorescent staining of 3-Nitrotyrosine in the
thoracic aorta of mice exposed to chronic CS and treated with
ebselen.}Immunofluorescent quantification of 3-NT expression in either
sham or 8-week CS-exposed mice that were treated with either vehicle or
ebselen. Green staining indicates the presence of 3-NT specific and blue
staining denotes the nuclear counterstain, DAPI (4', 6-diamidino-2-
phenylindole, dilactate). Representative photographs of
immunofluorescent staining in (A) sham-exposed vehicle treated (n=5),
(B) sham-exposed ebselen treated (n=5), (C) CS-exposed vehicle treated
(n=4) and (D) CS-exposed ebselen treated mice (n=6). Expression of 3-NT
normalised to the negative control and expressed as fold percentage
change relative to the sham vehicle-treated group. Scale bar represents
50\selectlanguage{greek}µ\selectlanguage{english}M. Data expressed as mean + SEM and analysed by two-way ANOVA with
Tukey's multiple comparisons; significance is denoted by; * indicating
\emph{p\textless{}0.05} .

\textbf{Figure 7: Immunofluorescent staining of endothelial nitric oxide
synthase in the thoracic aorta of mice exposed chronically to CS and
treated with ebselen} Immunofluorescent staining of eNOS in either sham
or 8-week CS-exposed mice with or without ebselen administration. Green
staining detects the presence of eNOS and Blue staining denotes the
nuclear counterstain, DAPI (4', 6-diamidino-2- phenylindole, dilactate).
Representative photographs of immunofluorescent staining in (A)
sham-exposed vehicle treated (n=4), (B) sham-exposed ebselen treated
(n=4), (C) CS-exposed vehicle treated (n=7) and (D) CS-exposed ebselen
treated mice (n=7). Expression of eNOS normalised to the negative
control and expressed as fold percentage change relative to the sham
vehicle-treated group. Scale bar represents 50\selectlanguage{greek}µ\selectlanguage{english}M. Data expressed as mean
\selectlanguage{ngerman}± SEM and analysed by two-way ANOVA with Tukey's multiple comparisons;
significance is denoted by * (\emph{p\textless{}0.05} ).

\textbf{Hosted file}

\verb`Brassington K BJP Final Figures Submitted 3 September 2020.pptx`  available at \url{https://authorea.com/users/356125/articles/479107-ebselen-reduces-cigarette-smoke-induced-vascular-endothelial-dysfunction-in-mice}

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