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.