Endothelin-1 activation of the endothelin B receptor modulates pulmonary endothelial CX3CL1 and contributes to pulmonary angiogenesis in experimental hepatopulmonary syndrome

Abstract

Hepatic production and release of endothelin-1 (ET-1) binding to endothelin B (ETB) receptors, overexpressed in the lung microvasculature, is associated with accumulation of pro-angiogenic monocytes and vascular remodeling in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). We have recently found that lung vascular monocyte adhesion and angiogenesis in HPS involve interaction of endothelial C-X3-C motif ligand 1 (CX3CL1) with monocyte CX3C chemokine receptor 1 (CX3CR1), although whether ET-1/ETB receptor activation influences these events is unknown. Our aim was to define if ET-1/ETB receptor activation modulates CX3CL1/CX3CR1 signaling and lung angiogenesis in experimental HPS. A selective ETB receptor antagonist, BQ788, was given for 2 weeks to 1-week CBDL rats. ET-1 (±BQ788) was given to cultured rat pulmonary microvascular endothelial cells overexpressing ETB receptors. BQ788 treatment significantly decreased lung angiogenesis, monocyte accumulation, and CX3CL1 levels after CBDL. ET-1 treatment significantly induced CX3CL1 production in lung microvascular endothelial cells, which was blocked by inhibitors of Ca(2+) and mitogen-activated protein kinase (MEK)/ERK pathways. ET-1-induced ERK activation was Ca(2+) independent. ET-1 administration also increased endothelial tube formation in vitro, which was inhibited by BQ788 or by blocking Ca(2+) and MEK/ERK activation. CX3CR1 neutralizing antibody partially inhibited ET-1 effects on tube formation. These findings identify a novel mechanistic interaction between the ET-1/ETB receptor axis and CX3CL1/CX3CR1 in mediating pulmonary angiogenesis and vascular monocyte accumulation in experimental HPS.

Figure 1

Effects of ET_B receptor inhibition with BQ788 on lung CX3CL1/CX3CR1 levels and monocyte accumulation after CBDL. A: mRNA levels for CX3CL1 and CX3CR1 in lung. B: Representative immunofluorescence staining for ED1 (monocyte marker, red) in lung and representative immunoblots and summary of lung ED1 protein levels. Values are expressed as means ± SEM (n = 8 animals for each group). ^∗P < 0.05 versus control; ^†P < 0.05 versus CBDL. Scale bar = 50 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

Effects of ET_B receptor inhibition with BQ788 on pulmonary angiogenesis and Akt and ERK phosphorylation after CBDL. A: Representative immunofluorescence images of lung microvessel staining (FVIII, green) and quantitation of microvessel density after CBDL. B: Representative immunoblots of p-Akt (Ser473) and p-ERK (Thr202/Tyr204) after CBDL and summary of protein levels for p-Akt/Akt and p-ERK/ERK. Values are expressed as means ± SEM (n = 8 animals for each group). ^∗P < 0.05 versus control; ^†P < 0.05 versus CBDL. Scale bar = 50 μm. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3

Effects of ET-1 on CX3CL1 production in ET_B receptor–overexpressing RPMVECs. A: CX3CL1 mRNA and supernatant protein levels in RPMVECs treated with 1, 10, and 50 nmol/L ET-1 for 4 and 8 hours, respectively. B: CX3CL1 mRNA and supernatant protein levels in 10 nmol/L ET-1–treated RPMVECs in the presence or absence of 10 μmol/L BQ788, respectively, for 4, 8, and 16 hours. Values are expressed as means ± SEM (n = 3 independent experiments in duplicate). ^∗P < 0.05 versus control; ^†P < 0.05 versus ET-1 treatment.

Figure 4

Effects of ET-1 on ERK activation in ET_B receptor–overexpressing RPMVECs. Representative immunoblots and graphs summarize ERK1/2 phosphorylation (Thr202/Tyr204) in RPMVECs treated with 1, 10, and 50 nmol/L ET-1 in the presence or absence of 10 μmol/L BQ788 for 5 minutes (A); and 10 nmol/L ET-1 in the presence or absence of 10 μmol/L MEK/ERK inhibitor U0126 and inhibitors of PLC/InsP₃/Ca²⁺/calmodulin pathway (5 μmol/L U73122, 20 μmol/L 2-APB, 5 μmol/L BAPTA-AM, and 10 μmol/L W7, respectively) (B). Values are expressed as means ± SEM (n = 3 independent experiments in duplicate). ^∗P < 0.05 versus control; ^†P < 0.05 versus ET-1 treatment.

Figure 5

Effects of Ca²⁺ and MEK/ERK pathway inhibition on ET-1–stimulated CX3CL1 production in ET_B receptor–overexpressing RPMVECs. CX3CL1 mRNA and supernatant protein levels in RPMVECs treated with 10 nmol/L ET-1 in the presence or absence of inhibitors of MEK/ERK (10 μmol/L U0126), PLC/InsP₃/Ca²⁺/calmodulin pathway (5 μmol/L U73122, 20 μmol/L 2-APB, 5 μmol/L BAPTA-AM, and 10 μmol/L W7), and Akt (0.5 μmol/L wortmannin) for 4 (A) and 8 (B) hours. Values are expressed as means ± SEM (n = 3 independent experiments in duplicate). ^∗P < 0.05 versus control; ^†P < 0.05 versus ET-1 treatment.

Figure 6

Effects of ET-1 on endothelial tube formation in ET_B receptor–overexpressing RPMVECs. Representative images of tubular structure formation and summary of relative tube length in RPMVECs treated with 10 nmol/L ET-1 in the presence or absence of inhibitors of 10 μmol/L BQ788, 10 ng/mL anti–CX3CR1-neutralizing antibody, 5 μmol/L MEK/ERK inhibitor (U0126), and Ca²⁺ pathway inhibitors (2.5 μmol/L U73122, 20 μmol/L 2-APB, and 5 μmol/L W7) for 6 hours. Values are expressed as means ± SEM (n = 3 independent experiments in duplicate). ^∗P < 0.05 versus control; ^†P < 0.05 versus ET-1 treatment. Scale bar = 200 μm.

Figure 7

Working model of the ET-1/ET_B receptor axis and the pulmonary vascular endothelial CX3CL1/CX3CR1 pathway in the development of experimental HPS.