Fig 1.
Immunofluorescence is employed to evaluate ROS production in cells (green) and mitochondria (red).
(A) PAECs were pretreated with CDC (inhibitor of 15-LO) or si15-LO and then exposed to 15-HETE and CM-H2DCFDA or MitoSOX to measure the effect of 15-HETE on the production of ROS. To locate the target of 15-HETE, PAECs (B) and PASMCs (C) were incubated with APO (apocynin, an NADPH oxidase inhibitor) or RE (rotenone, a mitochondria inhibitor) prior to treatment with 15-HETE. ROS were assessed by CLSM after treatment with CM-H2DCFDA and MitoSOX. Green, cellular ROS; red, mitochondrial ROS. Scale bars equal 100 μm, Nor, normoxia; Hyp, hypoxia; 15, 15-HETE.
Fig 2.
H&E and Masson staining and Nox4 expression in human and rat pulmonary vessels.
Histological analysis of rat (n = 6) and human lung tissues (n = 3): (A) H&E staining; (B) collagen deposition; scale bars equal 100 μm. (C) Nox4 expression in PAs from rat (n = 6) and human lungs (n = 3) was evaluated by immunohistochemistry. Data reflect quantitative analyses of positive staining per vascular area. Framed areas in a through d are shown at high magnification in a1 through d1. Scale bars in Ca1 through Cd1 equal 20 μm, and all others are 50 μm. Groups of rat lung tissues: a: Nor; b: Hyp; c: NADC + Hyp; d: MCT. Groups of human lung tissues; a: Nor; b: PH. After incubation with CDC, NDGA (an inhibitor of 15-LO) or si15-LO (E), Nox4 protein (D) and mRNA (G) expression levels in PAECs were evaluated by western blot and real-time PCR. Nox4 protein levels in mitochondria (F) were also examined (n = 4). All of the values reflect the means ± SEM; *p<0.05, **p<0.01 versus normal; #p<0.05, ##p<0.01 versus Hyp+15. Nor, normal; Hyp, hypoxia; MCT, monocrotaline; PH, pulmonary hypertension; X-treme, siRNA Transfection Reagent.
Fig 3.
Nox4 expression is up-regulated by 15-HETE, possibly through the p38 MAPK pathway.
After pretreatment with SB203580 (10 μmol/L), PAECs were exposed to 15-HETE to measure its effects on (A) ROS production (using CM-H2DCFDA or MitoSOX assays; scale bars equal 100 μm) and Nox4 (B) protein and (C) mRNA expression (western blot and real time-PCR (n = 5)). Nor, normoxia; 15, 15-HETE. Data are presented as the means ± SEM. *p<0.05, **p<0.01 versus normal; #p<0.05, ##p<0.01 versus Hyp+15.
Fig 4.
15-HETE-induced ROS promote PAEC migration and tube formation and increase PASMC proliferation.
After scavenging ROS with NAC (ROS scavenger), the effects of hypoxia and exogenous 15-HETE on PAEC migration (A) were examined by the scratch-wound assay (n = 4). Scale bars indicate 100 μm. Tube formation in PAECs (B) was evaluated by the tube formation assay (n = 5). PCNA expression in PASMCs (n = 4) (C) was examined by western blot. 5-BrdU incorporation assays were performed to detect DNA synthesis (n = 5). All of the values are expressed as the mean ± SEM. *p<0.05, **p<0.01 versus normal; # p<0.05, ##p<0.01 versus Hyp+15.
Fig 5.
15-HETE-induced ROS promote PASMC cell cycle progression and α-tubulin polymerization in the nucleus.
After employing NAC to scavenge ROS in PASMCs, the effects of hypoxia and exogenous 15-HETE on (A) the percentages of cells in the S and G2/M phases were analyzed by flow cytometry (n = 3). (B) α-tubulin polymerization in the nucleus was detected by immunocytochemistry. Scale bars indicate 100 μm. Data are presented as the means ± SEM; *p<0.05, **p<0.01 versus normal; # p<0.05, ##p<0.01 versus Hyp+15 (n = 3). Nor, normoxia; Hyp, hypoxia; 15, 15-HETE.
Fig 6.
The p38 MAPK pathway contributes to PAEC migration and PASMC proliferation downstream of the ROS induced by 15-HETE.
After pretreating cells with H2O2 (50 μmol/L) plus SB203580 (inhibitor of p38 MAPK signaling) or with SB203580 alone, exogenous 15-HETE was applied in hypoxic conditions, and (A) PAEC migration was examined by the scratch-wound assay. Scale bars indicate 100 μm. (B) PAEC tube formation was evaluated by the tube formation assay. Scale bars represent 100 μm. (C) PCNA expression was examined by western blot (n = 4). (D) 5-BrdU incorporation assays were performed to detect DNA synthesis (n = 5). All of the values are expressed as the mean ± SEM; *p<0.05, **p<0.01 versus normal; # p<0.05, ##p<0.01 versus Hyp+15. Nor, normoxia; SB, SB203580; 15, 15-HETE; Hyp, hypoxia.
Fig 7.
Pharmacological blockade of the p38 MAPK pathway inhibits the PASMC cycle progression and α-tubulin nuclear polymerization induced by 15-HETE via ROS.
After adding H2O2 (50 μmol/L) to activate ROS and then blocking the p38 MAPK pathway with SB203580, or adding SB203580 alone, the effects of endogenous and exogenous 15-HETE on (A) PASMC cell numbers in the S and G2/M phases were analyzed by flow cytometry (n = 4). (B) α-tubulin polymerization in the nucleus was detected by immunocytochemistry. Scale bars indicate 100 μm. Data are presented as the means ± SEM; *p<0.05, **p<0.01 versus normal; # p<0.05, ##p<0.01 versus Hyp+15. Nor, normoxia; Hyp, hypoxia; 15, 15-HETE.
Fig 8.
A proposed schematic links between 15-LO/15-HETE, ROS, and proliferation in PVR under hypoxia.
The ROS induced by 15-HETE are generated in the mitochondria and by Nox4. These ROS stimulate migration, cell cycle progression, and proliferation via the p38 MAPK pathway. This process triggers pulmonary vascular remodeling to cope with hypoxic pulmonary hypertension.