Demographic, Pulmonary Function And Morphological Findings In Copd Patients
We have collected 27patients, who were underwent the excision of peripheral solitary pulmonary nodule or pulmonary lesion. 9 cases are normal nonsmokers, and 9 cases were non-COPD smokers. The last 9 cases were smokers with spirometry-defined COPD. There was no significant difference in the gender ration or age among the three groups (Table 1). The smoking history of non-COPD smokers and COPD smokers was analyzed, and this study did not find a difference in smoking history between the two groups (P = 0.23, Table 1).
Table 1
Anthropometric characteristics, spirometry values of the subjects.
| Nonsmoker n = 9 | Non-COPD Smoker n = 9 | COPD smoker n = 9 | P value |
Gender (M/n) | 66.7% (6/9) | 77.8% (7/9) | 77.8% (7/9) | 0.825 # |
Age (years) | 56.22 ± 10.99 | 57.44 ± 5.72 | 57.67 ± 6.50 | 0.92* |
Smoking (pack-year) | 0 | 40.33 ± 13.61 | 39.56 ± 14.87 | 0.23$ |
FEV1/Pre (%) | 85.11 ± 3.82 | 85.56 ± 4.22 | 71.78 ± 3.96 | ༜0.01* |
FEV1/FVC (%) | 81.33 ± 5.57 | 81.11 ± 5.82 | 75.30 ± 9.83 | ༜0.01* |
# P by Chi-square test, *P by one-way ANOVA test, and $ P by Student's t test. |
Compared with the other two groups, the COPD smokers showed destroyed pulmonary architecture and function (P < 0.05 by one-way ANOVA and least significant difference (LSD) test, Table 1 and Fig. 1A). Histologically, the nonsmokers presented a well-fixed normal pulmonary parenchyma with normal airways (Fig. 1A). Non-COPD smokers did not exhibit emphysematous changes, but presented a thicker alveolar septum than nonsmokers (mean alveolar septal thickness, MAST; Fig. 1B). COPD patients showed significant emphysematous disorder with increased mean linear intercept (MLI) and destructive index (DI; Fig. 1C, D).
Aggravated pulmonary apoptosis and oxidant stress in lungs of COPD patients
Compared to that in the lungs of nonsmokers and smokers without COPD, the apoptosis index (AI) was increased in the lungs of COPD patients (Fig. 2A, B). Interestingly, there was no difference in the pulmonary AI (%) between the nonsmokers and smokers without COPD (3.73 ± 1.57 vs. 3.99 ± 1.75, P < 0.01 by the Kruskal-Wallis test, Fig. 2A, B). This suggests that CS is not the only reason for apoptosis in COPD, and there might be mechanisms beyond direct CS damage. The level of reactive oxygen species (ROS) in tissues from COPD patients was higher than that in tissues from nonsmokers (Fig. 2C), indicating that there was a higher oxidant burden in COPD. In contrast, the smokers without COPD did not show a significant change in ROS level (Fig. 2C).
Dysregulation of Bcl-2 and DNMT1 expression in lungs from COPD patients
Lung tissues from nonsmokers, non-COPD smokers and COPD patients were analyzed for the expression of Bcl-2, a well-known apoptosis regulator, by western blotting. Bcl-2 protein in lung tissue from COPD patients was lower than that in lung tissue from nonsmokers (Fig. 3A, B). To discover whether DNA methylation is involved in COPD, DNMT1 protein, the DNA methyltransferase, was analyzed in lung tissues (Fig. 3C). Immunoblotting showed that DNMT1 expression in lung tissue from non-COPD smokers and COPD patients was higher than that in lung tissue from nonsmokers (0.44 ± 0.12 and 0.73 ± 0.06 vs. 0.29 ± 0.11, P < 0.01 by one-way ANOVA and LSD test, Fig. 3C, D). In addition, the COPD group was presented higher DNMT1 expression than the non-COPD smoker group (P < 0.01 by one-way ANOVA and LSD test, Fig. 3C, D).
Hypermethylation of the Bcl-2 promoter in COPD patients
Given the higher expression of DNMT1 and lower expression of Bcl-2 in COPD patients than healthy controls, we conducted bisulfite sequencing PCR (BSP) to detect the methylation status of the Bcl-2 promoter. The sequence results demonstrated that the COPD patients had an elevated level of Bcl-2 promoter methylation (Fig. 4). As the results of Bcl-2 protein detection, the BSP showed that there was no significant difference in Bcl-2 methylation levels between the nonsmoker and non-COPD smoker groups (P = 0.756, Fig. 4). Considering the simultaneously increased DNMT1 expression and methylation level, it is possible to assume that the upregulated DNMT1 expression leads to the hypermethylation of the Bcl-2 promoter in COPD patients.
CS-induced oxidative stress participates in emphysema, pulmonary apoptosis and lung function damage
To confirm that oxidant stress contributes to COPD pathogenesis, we treated mice with CS and vitamin E (Vit E), one of the most common antioxidant reagents. Consistent with previous studies[13–15], we found that CS exposure caused increased ROS levels in lung tissue (Fig. 5A). Antioxidant feeding with Vit E reversed the increased ROS levels in CS exposed mice (Fig. 5A). CS treated mice exhibited emphysematous changes with aggravated MLI, DI and AI (Fig. 5B, C, D, and E). Coincidentally, the mice exposed to CS showed impaired pulmonary function, including deteriorated tidal volume (TV, mL), dynamic compliance (Cdyn, mL/cm H2O) and airway resistance (RI, cm H2O/mL/min; Fig. 5F, G). Interestingly, antioxidant treatment also alleviated the pulmonary morphological and functional damage caused by CS (Fig. 5B, C, D, E, F, and G). This finding suggests that CS-induced oxidant stress plays a role in emphysema, pulmonary apoptosis and lung function damage through oxidant stress.
CS-induced oxidative stress is involved in the dysregulation of Bcl-2, cleaved caspase-3 and DNMT1 protein expression and the DNA hypermethylation of Bcl-2 promoter
Because of the increased apoptosis in COPD patients’ lungs, we detected the apoptosis regulator Bcl-2 and the apoptosis inducer caspase-3 in mouse models. Compared to the control mice, the CS exposure mice had lower Bcl-2 protein expression in the pulmonary tissue (0.62 ± 0.03 vs. 0.37 ± 0.05, P < 0.01 by one-way ANOVA and LSD test, Fig. 6A, B). Correspondingly, we also found that the expression of cleaved caspase-3 in the CS group was higher than control group (0.75 ± 0.09 vs. 0.15 ± 0.05, P < 0.01 by the Kruskal-Wallis test, Fig. 6A, C). Given the hypermethylation of the Bcl-2 promoter in the lungs from COPD patients, we detected DNMT1 protein expression and the DNA methylation level of Bcl-2 in pulmonary tissue from mouse models. Immunoblotting showed that DNMT1 expression was upregulated in the lungs from the CS group (Fig. 6A, D). BSP was subsequently conducted, and showed that CS led to the hypermethylation of the Bcl-2 promoter (Fig. 6E). In contrast, the antioxidant reagent rescued the CS-induced dysregulation of the apoptosis-associated proteins Bcl-2 and cleaved caspase-3. Moreover, it reversed the alternation in DNMT1 expression and the CS-induced epigenetic modification of Bcl-2 (Fig. 6A, B, C, D, and E). This finding indicates that CS-induced oxidative stress causes the dysregulation of apoptosis-associated proteins and gene methylation status, and ultimately initiates apoptosis.
DNMT1 gene silencing or pharmacologic antagonism ameliorates CS-induced emphysema, pulmonary apoptosis, and hypermethylation of the Bcl-2 promoter without altering ROS levels
Because of the elevated DNMT1 expression and the hypermethylation of Bcl-2 in the lung tissue of emphysema subjects, we tested whether modulation of the DNMT1 protein level or activity ameliorates emphysema, pulmonary apoptosis and Bcl-2 promoter hypermethylation in mouse models. The mice were depleted of DNMT1 intratracheally using a lentiviral delivery system [108 plaque-forming units (pfu) per mouse] and subsequently exposed to CS. Compared to the mice from the control group, the mice from the CS group had a lower MLI and DI (Fig. 7A, C, D). The lung function test showed better TV and Cdyn in the CS + DNMT1 shRNA treated mice than in the single CS-exposed mice (Fig. 7E, F, G). TUNEL staining also showed less pulmonary apoptosis in the CS + DNMT1 shRNA group than in the single CS group (Fig. 7H, I). Immunoblotting revealed that there was higher Bcl-2 and lower cleaved caspase-3 expression in the CS + DNMT1 shRNA group than in the CS group subjects (Fig. 8A, B, C). Furthermore, DNMT1 knockdown mice presented decreased methylation level of the Bcl-2 promoter in the lungs (Fig. 8E). This result indicates that DNMT1 gene silencing reversed emphysema, pulmonary apoptosis, downregulated Bcl-2 expression, increased cleaved caspase-3 levels and Bcl-2 promoter hypermethylation in CS exposed mice.
5-Aza-2-deoxycytidine (AZA) is a well-known DNMT1 antagonist that can inhibit the activity and expression of DNMT1 in vivo and in vitro. We observed whether this antagonism protected CS exposed mice from pathologic process. As in our previous study [24], we found fewer emphysematous changes and less pulmonary apoptosis, including AI and cleaved caspase-3 expression, in the AZA plus CS treated mice than in the single CS-exposed models (Fig. 7, Fig. 8). Furthermore, the AZA plus CS treatment mice showed lower DNMT1 expression and Bcl-2 promoter methylation level, but higher Bcl-2 expression than CS only exposed mice (Fig. 8E). The depletion of DNMT1 by shRNA or inhibition of DNMT1 with AZA markedly reduced the lung function damage, emphysema, pulmonary apoptosis, and Bcl-2 expression, suggesting that CS exposure causes emphysema, pulmonary apoptosis and hypermethylation through DNMT1.
To determine whether DNMT1 controls oxidative stress via feedback, we observed the ROS levels after DNMT1 gene silencing or pharmacologic inhibition. Interestingly, mice with DNMT1 knockdown still had higher ROS levels than control mice (Fig. 7B). This finding implies that inhibiting DNMT1 does not reverse CS-induced oxidant stress and supports the hypothesis that DNMT1 could be a downstream factor in oxidant stress.