Leukotriene B4 Activates Pulmonary Artery Adventitial Fibroblasts in Pulmonary Hypertension

Abstract

A recent study demonstrated a significant role for leukotriene B4 (LTB4) causing pulmonary vascular remodeling in pulmonary arterial hypertension. LTB4 was found to directly injure luminal endothelial cells and promote growth of the smooth muscle cell layer of pulmonary arterioles. The purpose of this study was to determine the effects of LTB4 on the pulmonary adventitial layer, largely composed of fibroblasts. Here, we demonstrate that LTB4 enhanced human pulmonary artery adventitial fibroblast proliferation, migration, and differentiation in a dose-dependent manner through its cognate G-protein-coupled receptor, BLT1. LTB4 activated human pulmonary artery adventitial fibroblast by upregulating p38 mitogen-activated protein kinase as well as Nox4-signaling pathways. In an autoimmune model of pulmonary hypertension, inhibition of these pathways blocked perivascular inflammation, decreased Nox4 expression, reduced reactive oxygen species production, reversed arteriolar adventitial fibroblast activation, and attenuated pulmonary hypertension development. This study uncovers a novel mechanism by which LTB4 further promotes pulmonary arterial hypertension pathogenesis, beyond its established effects on endothelial and smooth muscle cells, by activating adventitial fibroblasts.

Keywords: NADPH oxidase; fibroblasts; inflammation; leukotriene B4; p38 mitogen-activated protein kinases; pulmonary artery; vascular remodeling.

© 2015 American Heart Association, Inc.

Figures

Figure 1

5-LO/LTB4 signaling is increased around pulmonary vascular adventitial fibroblasts in PAH. A, Plasma LTB4 concentration in 10 healthy controls and 10 SSc-PAH patients. B, Demography table. C, Representative immunofluorescence images of human lung sections stained with 5-LO (green) and S100A4 (fibroblasts, red) from healthy individuals and SSc-PAH patients. D, Morphometric analysis of images in C. Number of 5-LO positive cells within 5 μm of the pulmonary adventitia. E, Representative immunofluorescence images of lung sections stained with 5-LO (green) and Vimentin (fibroblasts, red) from DMSO (negative control), SU (PH) or SU+bestatin (bestatin-treated) animals. F, Morphometric analysis of E. In A, data are presented in the scatter plots showing minimal to maximal values and all data points. In C, E, DAPI (blue) stains nuclei; differential interference contrast (DIC) highlights alveolar and vascular structures; n=5. Yellow dashed lines indicate the adventitia area. In A, D, F, data are presented as mean ± SEM. (*: p<0.05)

Figure 2

LTB4 promotes proliferation, migration and differentiation in HPAAF. Proliferation of HPAAF with increasing doses of LTB4 treatment for 72hrs was measured by A, the MTT assay, B, cell counting, C, BrdU assay. Data are presented as mean ± SEM. (*: p<0.05) D, PCNA expression was measured by western blot after treatment with LTB4 for 24hr. E, F, Migration of HPAAF after LTB4 exposure were determined and quantified by Boyden Chamber assay. Data are presented as mean ± SEM. (*: p<0.05) G, H α-SMA and p-p38 MAPK expression after LTB4 treatment for 24hr as determined by western blot. β-actin was used as a loading control. The experiments were repeated three times.

Figure 3

LTB4 induced HPAAF proliferation, migration and differentiation were inhibited by pretreatment with BLT1 blockade (U75302) or p38 MAPK inhibition (SB203580). A, HPAAF proliferation, B, cell counting, C, BrdU assay, D, PCNA expression E, F, migration and G, differentiation were determined after pretreatment of LTB4 receptor antagonist U75302 (1μM) or p38 MAPK inhibitor SB203580 (10μM) in the presence of LTB4. (#: According to the manufacturer, increased p38 phosphorylation in the SB203580-treated group in Figure 3D is likely attributable to the fact that this agent inhibits p38 catalytic activity by binding to the ATP binding pocket without affecting phosphorylation of p38 by upstream kinases). Data are presented as mean ± SEM (*: p<0.05). The experiments were repeated three times.

Figure 4

Blocking p38 MAPK signaling attenuates experimental PH. Rats were treated with the p38 MAPK inhibition (SB203580) starting at the time of SU administration. A, B, Animals were monitored by echocardiography weekly. C, D, Hemodynamic measurements were done at week 3. RVSP measurements in DMSO, SU, and p38 MAPK inhibition treatment groups were assessed at week 3 post-SU. RV hypertrophy (RVH) measurements as assessed by the RV/LV+S weight ratios. E, Survival of rats after treatment was compared with DMSO and SU rats. (n=6 per group). Data are expressed as means ± SEM. (*: p<0.05) The experiments were repeated three times.

Figure 5

Blocking LTB4 synthesis and inhibiting p38 MAPK signaling decreases fibroblast activation and Nox4 in experimental PAH. A, LTB4 concentrations in the bronchoalveolar lavage fluid (BALF) of DMSO, SU or SU+SB203580 animals. B, Immunofluorescence images of rat lung tissues stained with 5-LO (magenta), Vimentin (green) and Nox4 (red). Yellow dash lines approximate the adventitial zone. C, Representative immunofluorescence images of lung sections stained with Nox4 (green). Amplex Red (red) indicates tissue H2O2 level. DAPI (blue) stains nuclei; DIC highlights alveolar and vascular structures; n= 5. Data are expressed as means ± SEM. (*: p<0.05)

Figure 6

LTB4 induced Nox4 expression, hydrogen peroxide (H2O2) production and HPAAF activation. A, HPAAFs were treated with LTB4 at concentrations of 200 and 400nM; H2O2 production was measured by Amplex assay and B, Nox4 expression was determined by western blot. C, After pretreatment with U75302 (1μM), SB203580 (10μM) or the Nox4 inhibitor apocynin (300μM), the effects of exogenous LTB4 on HPAAFs were assayed. H2O2 production was measured by the Amplex assay. D, Western blots were used to determine the expression of Nox4, p-p38 MAPK and total p38 MAPK; β-actin was used as a loading control. E, MTT assay, F, cell counting, G, BrdU assay, H, PCNA expression, I, J, migration and K, differentiation were determined with Nox inhibition (apocynin) in the presence of LTB4. (#: Note again in Figure 6D, increased p38 phosphorylation in the SB203580-treated group likely due to this agent inhibiting p38 catalytic activity without affecting phosphorylation of p38 as described in Figure 3D). (*: p<0.05) The experiments were repeated three times.

Figure 7

Schematic of LTB4 induce p38 MAPK and Nox4 in HPAAF.