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-α and IL-6 in response to CS,
which is consistent with our previously published work (Hansen et al.,
2013; Vlahos et al., 2006). TNF-α 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 ~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 ~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 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 et.alhave 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 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 TNFα 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-α protein
expression is altered following ebselen administration. Similarly, it
would be worth investigating whether ebselen can directly impede the
activity of the regulatory p47phox 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 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 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-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.