Vascular peroxidase 1 mediates hypoxia-induced pulmonary artery smooth muscle cell proliferation, apoptosis resistance and migration.

Reactive oxygen species (ROS) play essential roles in the pulmonary vascular remodelling associated with hypoxia-induced pulmonary hypertension (PH). Vascular peroxidase 1 (VPO1) is a newly identified haeme-containing peroxidase that accelerates oxidative stress development in the vasculature. This study aimed to determine the potential role of VPO1 in hypoxia-induced PH-related vascular remodelling. The vascular morphology and VPO1 expression were assessed in the pulmonary arteries of Sprague-Dawley (SD) rats. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) and VPO1 expression and HOCl production were significantly increased in hypoxic rats, which also exhibited obvious vascular remodelling. Furthermore, a hypoxia-induced PH model was generated by exposing primary rat pulmonary artery smooth muscle cells (PASMCs) to hypoxic conditions (3% O2, 48 h), which significantly increased the expression of NOX4 and VPO1 and the production of HOCl. These hypoxic changes were accompanied by enhanced proliferation, apoptosis resistance, and migration. In PASMCs, hypoxia-induced changes, including effects on the expression of cell cycle regulators (cyclin B1 and cyclin D1), apoptosis-related proteins (bax, bcl-2, and cleaved caspase-3), migration promoters (matrix metalloproteinases 2 and 9), and NF-κB expression, as well as the production of HOCl, were all inhibited by silencing VPO1 with small interfering RNAs. Moreover, treatment with HOCl under hypoxic conditions upregulated NF-κB expression and enhanced proliferation, apoptosis resistance, and migration in PASMCs, whereas BAY 11-7082 (an inhibitor of NF-κB) significantly inhibited these effects. Collectively, these results demonstrate that VPO1 promotes hypoxia-induced proliferation, apoptosis resistance, and migration in PASMCs via the NOX4/VPO1/HOCl/NF-κB signalling pathway.

You B ，Liu Y ，Chen J ，Huang X ，Peng H ，Liu Z ，Tang Y ，Zhang K ，Xu Q ，Li X ，Cheng G ，Shi R ，Zhang G ... -  《-》

Vascular peroxidase 1 promotes phenotypic transformation of pulmonary artery smooth muscle cells via ERK pathway in hypoxia-induced pulmonary hypertensive rats.

Vascular peroxidase 1 (VPO1) plays an important role in mediation of vascular remodeling with pulmonary arterial hypertension (PAH). This study aims to determine whether VPO1 can promote phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) and the underlying mechanisms. Sprague-Dawley (SD) rats were exposed to 10 % O2 for 21 days to establish the model of vascular remodeling in pulmonary arterial hypertension. PASMCs were incubated with 3 % O2 for 48 h to induce phenotypic transformation. Western blot was performed to detect the expressions of target proteins. The 5-ethynyl-2'-deoxyuridine (EdU) assay was conducted to measure the proliferation of PASMCs. In the rats exposed to hypoxia, there were increases in right ventricular systolic pressure, pulmonary vascular remodeling and phenotypic transformation of PASMCs (the down-regulated contractile proteins of α-smooth muscle actin, smooth muscle 22α while the up-regulated synthetic proteins of osteopontin, cyclinD1), accompanied by up-regulation of VPO1, increase of hypochlorous acid (HOCl) production and elevation of the phosphorylation of ERK. In the cultured PASMCs exposed to hypoxia, similar results were achieved but they were reversed by VPO1 small interfering RNA (VPO1 siRNA) or HOCl inhibitor. Replacement of hypoxia with NaOCl could induce PASMCs phenotypic transformation and activate the ERK signaling. Furthermore, ERK inhibitor (PD98059) could also attenuate hypoxia-induced PASMCs phenotypic transformation. VPO1 play a pivotal role in promotion of phenotypic transformation of PASMCs under hypoxic condition through activation of VPO1/HOCl/ERK pathway. It might serve as a potential target for prevention of pulmonary vascular remodeling.

Li T ，Liu B ，Li NS ，Luo XJ ，Peng JW ，Peng J ... -  《-》

Coordination between NADPH oxidase and vascular peroxidase 1 promotes dysfunctions of endothelial progenitor cells in hypoxia-induced pulmonary hypertensive rats.

Previous studies have demonstrated that NADPH oxidase (NOX)/vascular peroxidase (VPO1) pathway - mediated oxidative stress plays an important role in the pathogenesis of multiple cardiovascular diseases. This study aims to evaluate the correlation between NOX/VPO1 pathway and endothelial progenitor cells (EPCs) dysfunctions in hypoxia-induced pulmonary hypertension (PH). The rats were exposed to 10% hypoxia for 3 weeks to establish a PH model, which showed increases in right ventricle systolic pressure, right ventricular and pulmonary vascular remodeling, acceleration in apoptosis and impairment in functions of the peripheral blood derived - EPCs (the reduced abilities in adhesion, migration and tube formation), accompanied by up-regulation of NOX (NOX2 and NOX4) and VPO1. Next, normal EPCs were cultured under hypoxia to induce apoptosis in vitro. Consistent with the in vivo findings, hypoxia enhanced the apoptosis and dysfunctions of EPCs concomitant with an increase in NOX and VPO1 expression, hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production; these phenomena were attenuated by NOX2 or NOX4 siRNA. Knockdown of VPO1 showed similar results to that of NOX siRNA except no effect on NOX expression and H2O2 production. Based on these observations, we conclude that NOX/VPO1 pathway-derived reactive oxygen species promote the oxidative injury and dysfunctions of EPCs in PH, which may contribute to endothelial dysfunctions in PH.

Wang EL ，Jia MM ，Luo FM ，Li T ，Peng JJ ，Luo XJ ，Song FL ，Yang JF ，Peng J ，Liu B ... -  《-》

The role of vascular peroxidase 1 in ox-LDL-induced vascular smooth muscle cell calcification.

Reactive oxygen species (ROS)-induced osteogenic differentiation of vascular smooth muscle cells (VSMCs) is associated with the pathogenesis of vascular calcification. Vascular peroxidase 1 (VPO1), a peroxidase in the cardiovascular system, utilizes the hydrogen peroxide (H2O2) produced by co-expressed NADPH oxidases to produce hypochlorous acid (HOCl) and catalyze peroxidative reactions. The aim of this study was to determine whether VPO1 plays a role in the osteogenic differentiation of VSMCs in the setting of the vascular calcification induced by oxidized low-density lipoprotein (ox-LDL). In cultured primary rat VSMCs, we observed that the expression of VPO1 was significantly increased in combination with increases in calcification, as demonstrated via increased mineralization, as well as increased alkaline phosphatase (ALP) activity and up-regulated runt-related transcription factor 2 (Runx2) expression in ox-LDL-treated cells. Ox-LDL-induced VSMC calcification and Runx2 expression were both inhibited by knockdown of VPO1 using a small interfering RNA or by an NADPH oxidase inhibitor. Moreover, the knockdown of VPO1 in VSMCs suppressed the production of HOCl and the phosphorylation of AKT, ERK and P38 MAPK. Furthermore, HOCl treatment facilitated the phosphorylation of AKT, ERK1/2 and P38 MAPK and the expression of Runx2, whereas LY294002 (a specific inhibitor of PI3K), U0126 (a specific inhibitor of ERK1/2) and SB203580 (a specific inhibitor of P38 MAPK) significantly attenuated the HOCl-induced up-regulation of Runx2. Collectively, these results demonstrated that VPO1 promotes ox-LDL-induced VSMC calcification via the VPO1/HOCl/PI3K/AKT, ERK1/2, and P38 MAPK/Runx2 signaling pathways.

Tang Y ，Xu Q ，Peng H ，Liu Z ，Yang T ，Yu Z ，Cheng G ，Li X ，Zhang G ，Shi R ... -  《-》

Serotonin and chronic hypoxic pulmonary hypertension activate a NADPH oxidase 4 and TRPM2 dependent pathway for pulmonary arterial smooth muscle cell proliferation and migration.

5-Hydroxytryptamine (5-HT)-dependent signaling mediated through its transporters and receptors plays important roles in chronic hypoxic pulmonary hypertension (CHPH), which is associated with aberrant reactive oxygen species (ROS) production. NADPH oxidase 4 (NOX4) is one of the major sources of ROS in pulmonary vasculature, and has been implicated in the development of PH. NOX4 generates H2O2, which can activate the transient receptor potential melastatin 2 (TRPM2) channels, providing Ca2+ signals for cell proliferation and migration. However, the connection between 5-HT, NOX4, ROS and TRPM2 in the context of PH has not been established. Here we examined the level of 5-HT and expression of NOX4 and TRPM2, and their roles in pulmonary arterial smooth muscle cells (PASMCs) proliferation and migration. NOX4 and TRPM2 were upregulated in pulmonary arteries of CHPH rats, which were associated with elevated levels of 5-HT and ROS, and enhanced proliferation and migration in PASMCs. The increase in ROS, and the enhanced proliferation and migration of PASMCs from CHPH rats were mimicked by treating normoxic PASMCs with 5-HT. 5-HT; and CH-induced ROS production were reversed by catalase, the NOX1/NOX4 inhibitor GKT137831, and Nox4 siRNA. 5-HT and H2O2 elicited Ca2+ responses were significantly augmented in CHPH PASMCs; and the augmented Ca2+ responses were obliterated by the 2-Aminoethoxydiphenyl borate (2-APB) and Trpm2-specific siRNA. Moreover, 5-HT and CH-induced proliferation and migration were suppressed by Nox4 or Trpm2 siRNA; and simultaneous transfection of both siRNA did not cause further inhibition. These results suggest that the 5-HT and CH-induced PASMC proliferation and migration were mediated, at least in part, by TRPM2 via activation of NOX4-dependent ROS production; and revealed a novel NOX4-ROS-TRPM2 signaling pathway for the pathogenesis of CHPH.

Song JL ，Zheng SY ，He RL ，Gui LX ，Lin MJ ，Sham JSK ... -  《-》