Effect of cigarette smoke extract and hypoxia on vasoactive gene expression and mediator release in human pulmonary artery smooth muscle and endothelial cells

Abstract

Abstract Background: Pulmonary hypertension is a common and serious complication of COPD. Studies suggest that hypoxia and cigarette smoke (CS) can cause vascular remodelling in COPD as a result of cell proliferation; however, the underlying cause is not fully understood. In addition, it is suggested that the use of e-cig may lead to vascular diseases, but no data are available on what effect e-cig may have on dysfunction of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs). Thus, we hypothesise that cigarette smoke extract (CSE) and hypoxia can induce imbalanced vasoactive gene expression and mediator release, and then contribute to cell proliferation and apoptosis. Furthermore, e-cig vapour extract with and without nicotine (ECVE-/+n) may have similar effects as CSE. Methods: Western blotting and real-time RT-PCR were used to assess the protein and mRNA expression of vasoactive genes, respectively. ELISA was used to assess the prostanoids and endothelin production. Nitric oxide levels were assessed by nitrate/nitrite assay. WST-1 and CCK-8 assays were used to assess cell proliferation. Results: We found that CSE induced cyclooxygenase-2 (COX-2) and thromboxane synthase (TXAS) and reduced prostaglandin (PG) I synthase at mRNA levels in PASMCs and PAECs. CSE treatment reduced PGI synthase protein expression and PGI2 production in PASMCs (but not in PAECs). Interestingly, CSE had the ability to reduce and increase microsomal prostaglandin E synthase-1 (mPGES-1) protein expression/PGE2 production in PASMCs and PAECs, respectively. Despite the undetected TXAS protein expression, CSE increased thromboxane A2 (TXA2) production in both cell types, likely as a result of the increased COX-2 protein expression. Although hypoxia induced COX-2 mRNA expression in PASMCs (but in PAECs), it had no effect on the mRNA levels of PGIS and TXAS in both cell types. We also demonstrated that hypoxia induced TXA2, PGE2, and PGI2 production as a result of the upregulated TXAS and COX-2 and unchanged PGIS protein expression in PASMCs. Interestingly, hypoxia had no effect on the protein expression of TXAS VII and COX-2 and the production of PGI2 and PGE2 in PAECs, but it induced the production of TXA2. Both CSE and hypoxia had no effect on endothelial nitric oxide synthase (eNOS) mRNA expression, but reduced eNOS protein expression/nitric oxide production in PAECs. Although hypoxia failed to affect mRNA endothelin-1 levels and endothelin production in both cell types, CSE increased mRNA endothelin- 1 levels and reduced endothelin production in PAECs (but not in PASMCs). Intriguingly, hypoxia did not enhance CSE-induced effects and ECVE+/-n had no similar effects as CSE. The results also showed CSE-induced proliferation of both cell types and hypoxia-induced proliferation of PASMCs were inhibited by the stable PGI2 analogue, selective COX-2 inhibitor and TXA2 receptor antagonist and nitric oxide donor (but not by mPGES-1 inhibitor and ET-A and ET-B receptor antagonist), whereas hypoxia-induced PAEC proliferation was only reduced by nitric oxide donor. Although hypoxia failed to cause apoptosis, CSE induced apoptosis only in PAECs and this was unlikely mediated by altered release of vasoactive mediators. Conclusion: Our results suggest that CSE (but not ECVE+/-n) and hypoxia to a lesser extent can induce imbalanced vasoactive gene expression and mediator release; however, it is unlikely that hypoxia can further amplify CSE-induced effects. The CSE- and hypoxia-induced imbalanced release of vasoactive mediators (particularly prostanoids (e.g. TXA2) and nitric oxide) may be critically involved in the contribution to vascular remodelling via the induction of cell proliferation. These data provide new insights into our understanding of CS- and hypoxia-induced vascular