Abstract 264: Serotonin Signaling Through Nox1 in Human Pulmonary Artery Smooth Muscle Cells - Implications in Vascular Remodeling in Pulmonary Arterial Hypertension
Serotonin (5HT) and NADPH oxidases (Nox) have been linked to the pathology of pulmonary arterial hypertension (PAH). However, it is unclear whether 5HT plays a role in PAH through Nox-dependent mechanisms. We hypothesised that 5HT through Nox1-induced reactive oxygen species (ROS) induces PASMC damage. Human PASMC primary cultures were stimulated with 5HT (1μM) in the absence/presence of tempol (antioxidant, 10μmol/L), ML171 (Nox1 inhibitor; 1μM), and EHT1864 (Rac1 inhibitor; 100μM). ROS generation was assessed by chemiluminescence. Markers of fibrosis (fibronectin), growth (PCNA), inflammation (TRAIL, DcR2), Nrf2 regulation (Nrf2, BACH1, HO1) and MAPKs activation (ERK1/2 and p38MAPK) were analyzed by immunoblotting. Proliferation was assessed by [3H]-thymidine incorporation in the presence/absence of Nox1 inhibitor. 5HT induced increased in ROS production at 4h of stimulation (224 ± 49% vs vehicle; p<0.05) in PASMC, an effect blocked by tempol, EHT1864 and ML171. Fibronectin (192 ± 26%), PCNA (124 ± 2%), TRAIL (122 ± 4%) and DcR2 (133 ± 2%) protein levels were increased by 5HT in PASMCs (p<0.05, vs vehicle). In addition, 5HT increased phosphorylation of ERK1/2 (169 ± 10%) and p38MAPK (157 ± 15%) (p<0.05, vs vehicle). Levels of Nrf2 and its downstream target, HO-1, were decreased by 5HT stimulation (90 ± 1%; 84 ± 6% respectively vs vehicle 100%, p<0.05). BACH1 expression, a protein that negatively modulates antioxidant regulator Nrf2, was increased by 5HT in PASMCs (153 ± 8% vs vehicle; p<0.05). Nox1, but not Nox2, expression was increased in PASMC after stimulation with 5HT (139 ± 11% vs vehicle, p<0.05). 5HT induced a 3.95±0.39 fold increase in proliferation PASMCs (P<0.05), which was blocked by ML171. In conclusion, Nox1 may play a role in 5HT-induced pro-proliferative, fibrotic and inflammatory responses. These data highlight some molecular mechanisms whereby 5HT may exert deleterious vascular effects in PAH.
- © 2013 by American Heart Association, Inc.