The focus of the present study was to analyse the gene and protein expression of ENTPD1/CD39 and NT5E/CD73 and the effect of BAY 41-2272, a stimulator of sGC, in lung tissue in an animal model of emphysema. To the best of our knowledge, this is the first study to analyse the effect of an sGC stimulator on the expression of genes and proteins associated with purinergic signaling in experimental COPD.
In our animal model of emphysema, we demonstrated a lower gene expression of both ENTPD1 and NT5E in the lungs of both smokers (CS group) and former smokers (Ex-CS group) compared to the control group (SHAM group). Although our findings were not statistically significant, except for NT5E in the CS + VH group, the results are in line with those of other studies indicating that purinergic signaling is involved in the development of COPD. Differences in the expression pattern of certain genes associated with the purinergic pathway have been reported in the lung tissues of COPD patients and smokers without obstruction . These changes may be responsible for the pulmonary inflammation that characterizes this disease.
Our group previously observed reductions in lung expression and activity of CD39 in patients in the initial stages of COPD . Consistent with our findings, Kratzer et al.  found a significant downregulation of CD39 in the lungs of rats with emphysema caused by CS exposure. In contrast, other studies have reported an upregulation in CD39 in peripheral blood samples from COPD patients with an acute exacerbation compared to patients with stable COPD and healthy patients . Lazar et al. also found increased cell levels of CD39 in BALF and sputum cells from COPD patients compared to those from smokers and non-smokers . These contradictory findings may be attributable to the differences in the samples used in the studies, or to the fact that patients were at different stages of the disease. The influence on CD39 expression of an acute condition such as a respiratory infection in the exacerbation of COPD or active tobacco consumption may be different from that of chronic lung damage.
The role in COPD of CD73, another key enzyme in purinergic signaling, is still not well understood. Its encoding gene, NT5E, seems to be overexpressed in the lungs of smokers without obstruction but not in COPD patients, suggesting that adenosine generated by the hydrolysis of AMP by CD73 may contribute to a decrease in the inflammatory environment in the lungs . These findings for NT5E/CD73 support the idea that the upregulation of ENTPD1/CD39 and NT5E/CD73 could be a compensatory mechanism in acute lung damage (active smoking and acute exacerbation) [15, 16].
A very interesting finding in the present study was the fact that treatment with a sGC stimulator tends to restore the expression of ENTPD1 in all groups, including the control group (SHAM + VH vs SHAM + BAY). These results suggest that treatment with BAY 41-2272 could act by enhancing ENTPD1 expression and could revert the downregulation of ENTPD1 and NT5E observed after CS exposure. On the other hand, the effect of BAY 41-2272 treatment on the expression of NT5E remains unclear since its expression remained almost unchanged in the SHAM and Ex-CS groups after treatment. In contrast, the expression of NT5E was partially restored in the smoker group (CS + VH vs CS + BAY). These differences in NT5E may be due to the length of exposure to CS (3 months in Ex-CS group vs 6 months in CS group). The results of the protein expression analysed by immunohistochemistry and western blot were in line with those of the gene expression studies, as the protein expression of NTPDase1/CD39 and Ecto-5’-NT/CD73 increased after treatment with BAY 41-2272.
Treatment with a sGC stimulator has been previously studied in COPD, where it has been shown to reduce oxidative stress and attenuate the inflammatory response in the lungs induced by CS [24–26]. The sGC stimulator treatment also has beneficial effects on lung vasculature, reducing pulmonary arterial pressure and right ventricular hypertrophy. Our findings may suggest a new beneficial effect of treatment with an sGC stimulator in COPD that upregulates ENTPD1/CD39 and NT5E/CD73 expression in lung tissues, thus reducing ATP levels and inflammation. However, further studies of this issue are required.
Data on lung morphometry and inflammatory infiltrates in lung tissue from this animal model have been reported previously . In our study, we failed to find a significant correlation between lung morphometry, inflammatory cell counts and the gene expression of ENTPD1 and NT5E based on the analysis of individual data. However, Paul et al.  reported an increase in inflammatory cells in the lung tissue of animals exposed to tobacco smoke which had developed emphysema as well as an inhibitory effect of BAY 41-2272 on inflammatory cell infiltration.
Several studies have described an interaction between nitric oxide (NO) and ATP. For example, Ruiz-Stewart et al. showed that NO and sGC are involved in ATP supply/demand as mediators, with changes in ATP levels regulating NO signaling through sGC . Moreover, ATP and ADP have been shown to induce endothelial nitric oxide synthase (eNOS) via P2 receptors (also involved in the purinergic pathway), increasing NO generation .
Although it is not well established how purinergic and NO signaling interact in modulating ATP levels, or whether the two pathways share some signaling mechanisms, our findings suggest that sGC stimulation could activate the purinergic pathway, thereby enhancing ENTDP1/CD39 and NTE5/CD73 expression in lung tissues and consequently reducing ATP levels and leading to a decreased inflammatory environment in COPD. More studies are required to shed more light on the relationship between the purinergic and NO signaling pathways in COPD.
The main limitation of this study was the large number of groups, which meant that each group produced only a limited number of samples. This made it difficult to draw conclusions about the effect of BAY 41-2272 on the genes and proteins associated with purinergic signaling. Secondly, we only analyzed the gene and protein expression of the main purinergic enzymes. In future work it would be interesting to extend the study to other molecules of the purinergic pathway, using new molecular techniques to better understand the interaction of this complex signaling pathway.