Endocannabinoid anandamide mediates hypoxic pulmonary vasoconstriction

Abstract

Endocannabinoids are important regulators of organ homeostasis. Although their role in systemic vasculature has been extensively studied, their impact on pulmonary vessels remains less clear. Herein, we show that the endocannabinoid anandamide (AEA) is a key mediator of hypoxic pulmonary vasoconstriction (HPV) via fatty acid amide hydrolase (FAAH)-dependent metabolites. This is underscored by the prominent vasoconstrictive effect of AEA on pulmonary arteries and strongly reduced HPV in FAAH(-/-) mice and wild-type mice upon pharmacological treatment with FAAH inhibitor URB597. In addition, mass spectrometry measurements revealed a clear increase of AEA and the FAAH-dependent metabolite arachidonic acid in hypoxic lungs of wild-type mice. We have identified pulmonary vascular smooth muscle cells as the source responsible for hypoxia-induced AEA generation. Moreover, either FAAH(-/-) mice or wild-type mice treated with FAAH inhibitor URB597 are protected against hypoxia-induced pulmonary hypertension and the concomitant vascular remodeling in the lung. Thus, the AEA/FAAH pathway is an important mediator of HPV and is involved in the generation of pulmonary hypertension.

Keywords: cannabinoid; pulmonary vascular tone.

Fig. 1.

The endocannabinoid AEA increases pulmonary arterial tone in the IPL model of mice. (A) Original recording illustrates an elevation of pulmonary arterial pressure (PAP) in the IPL upon application of the potent pulmonary vasoconstrictor serotonin (5-HT, 10 μM) or AEA (10 μM). (B) The AEA-induced PAP increase is dose-dependent; the contractile response to AEA is normalized to PAP values in presence of 5-HT (10 μM). (C) Original recording of PAP in the IPL reveals almost no effect upon application of the solvent ethanol (EtOH). Control: 5-HT (10 μM). (D) Statistical analysis of PAPs normalized to the 5-HT response indicates that AEA-induced vasoconstriction is preserved in Cnr1^−/− and Cnr2^−/− mice, whereas the solvent EtOH has no effect. *P < 0.05, one-way ANOVA with Dunnett’s test.

Fig. 2.

AEA elevates PAP via the FAAH signaling pathway. (A) Original recording of PAP in the IPL illustrates only a minimal vasoconstrictive response by AEA (10 μM) in FAAH^−/− mice. Control: 5-HT (10 μM). (B) Statistical analysis of PAPs normalized to the 5-HT response shows that vasoconstriction by AEA (10 µM) is mediated by FAAH-dependent AEA metabolites because it is strongly reduced in FAAH^−/− mice and upon pharmacological inhibition of FAAH by URB597 (URB, 1 µM) in wild-type and Cnr1/2^−/− mice. In addition, the nonhydrolyzable AEA analog Meth-anandamide (Meth-AEA, 10 µM) has no effect. **P < 0.01, one-way ANOVA with Dunnett’s test. (C) PCR analysis reveals expression of cyclooxygenase1 (COX1), cyclooxygenase2 (COX2), and 5-lipoxygenase (5-LOX) in murine lung; as positive control, mouse brain was used. (D) Statistical analysis of PAPs normalized to the 5-HT response indicates that vasoconstriction by AEA (10 μM) is mediated by COX- and 5-LOX–dependent metabolites because it is diminished by the respective inhibitors Indo (10 µM) and NDGA (10 µM) or blockade of leukotriene receptors with montelukast (1 μM). The CYP450 inhibitor ODYA (1 µM) has no effect. **P < 0.01, one-way ANOVA with Dunnett’s test.

Fig. 3.

The AEA/FAAH axis is an important mediator of hypoxic pulmonary vasoconstriction (HPV). (A and B) Original recordings of PAP in the IPL demonstrate strongly reduced HPV (0% O₂) in FAAH^−/− (B) compared with WT mice (A); as control, 5-HT (10 μM) was used. (C) Statistical analysis of PAPs normalized to 5-HT reveals that genetic abrogation (FAAH^−/− mice) and pharmacological inhibition (URB, 10 μM) of FAAH and leukotriene receptor blockade by montelukast (1 μM) strongly diminish HPV. **P < 0.01, one-way ANOVA with Dunnett’s test.

Fig. 4.

Hypoxia increases AEA and AA levels in the lung. (A) LC-MRM measurements of AEA and AA levels in murine lungs yielded an increase after 2 and 6 h of hypoxic ventilation (10% O₂). The values are normalized to levels at normoxic ventilation (21% O₂). *P < 0.05, one-way ANOVA with Dunnett’s test. (B) PCR analysis reveals prominent expression of N-acyl-phosphatidyl-ethanolamine phospholipase D (NAPE-PLD) and weak expression of MAGL in murine lung tissue; as positive control, brain tissue was used. (C) Western blot analysis demonstrates strong protein expression of NAPE-PLD and FAAH in murine lung tissue but very low levels in the heart. Positive control: brain. (D–I) Immunohistochemistry of murine lung sections shows colocalization of NAPE-PLD (red) (D and F), FAAH (red) (G and I), and vascular smooth muscle cells (α-smooth muscle actin; green) (E, F, H, and I); nuclei are stained with hoechst (blue) (F and I). (Scale bar, 10 μm.)

Fig. 5.

Vascular smooth muscle cells display elevated AEA production under hypoxia. (A and B) Immunohistochemistry shows NAPE-PLD (green) and FAAH (red) expression in hPASMCs; nuclei are stained with hoechst (blue). (Scale bar, 20 μm.) (C) LC-MRM measurements reveal strongly elevated AA levels in hPASMCs after 1 h of AEA incubation (10 µM) compared with EtOH. *P < 0.05, Student’s t test. (D) AEA and AA levels in hPASMCs are strongly increased after 5 h of hypoxia (HX) compared with normoxia (NX). *P < 0.05, Student’s t test. (E) Western blot analysis shows enhanced NAPE-PLD protein expression after 5 h of HX compared with NX.

Fig. 6.

The AEA/FAAH axis is involved in the generation of hypoxia-induced pulmonary hypertension. (A) H&E stainings of intrapulmonary arteries of WT mice demonstrate increased vascular wall thickness after 3 wk of HX (10% O₂) (Right) compared with NX (21% O₂) (Left). (Scale bar, 20 μm.) (B) H&E stainings of intrapulmonary arteries of FAAH^−/− mice display no change in vascular wall thickness after 3 wk of HX (10% O₂) (Right) compared with NX (21% O₂) (Left). (Scale bar, 20 μm.) (C) H&E stainings of intrapulmonary arteries of WT mice after 3 wk of HX (10% O₂) with daily injections of URB (5 mg/kg) (Left) demonstrate reduced vascular wall thickness compared with solvent (Right). (Scale bar, 20 μm.) (D–F) Statistical analysis of vascular wall thickness (D), Fulton index (E), and RVSP (F) demonstrates increased values after 3 wk of HX compared with NX in WT mice; these changes were absent in FAAH^−/− mice and could be abrogated by URB injection in WT mice. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test.