Research ArticleRESPIRATORY DISORDERS

Targeted inhibition of Gq signaling induces airway relaxation in mouse models of asthma

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Science Translational Medicine  13 Sep 2017:
Vol. 9, Issue 407, eaag2288
DOI: 10.1126/scitranslmed.aag2288
  • Fig. 1. FR inhibits Gq protein signaling in hBSMCs.

    (A) Immunohistochemical staining of Gq proteins (red) in hBSMCs. Scale bar, 50 μm. (B) Original traces of DMR measurements in response to bradykinin with or without FR pretreatment (quantified as change in reflected wavelength in picometers). (C) DMR measurements for a bradykinin dose-response curve with or without FR. AUC, area under the curve. (D) DMR measurements for a histamine dose-response curve with or without FR. (E) Bioimpedance measurements for a bradykinin dose-response curve with or without FR pretreatment [quantified as change in impedance of extracellular current (dZ)]. (F) Induction of cAMP production by isoprenaline with or without FR pretreatment. (G) Induction of cAMP production by forskolin with or without FR pretreatment. (H) Inhibition of cAMP production by methacholine after forskolin stimulation with or without FR pretreatment. (I) IP1 production induced by bradykinin with or without FR pretreatment. (J) Original traces of single-cell Ca2+ imaging experiments in hBSMCs without FR treatment. CPA served as control. (K) Original traces of single-cell Ca2+ imaging experiments in hBSMCs with FR treatment. Each line represents one cell. (L) Statistical analysis of single-cell Ca2+ imaging experiments in hBSMCs without (−) FR (n = 11; 252 cells in total) or with (+) FR (n = 10; 233 cells in total). (L) ***P < 0.001, unpaired two-tailed t test.

  • Fig. 2. FR reduces airway tone in mouse tracheas ex vivo.

    (A) Original trace of isometric force measurements in mouse trachea during a methacholine dose-response curve without FR. (B) Original trace of isometric force measurements in mouse trachea during a methacholine dose-response curve in the presence of FR. (C) Comparison of force production in mouse trachea in response to escalating methacholine doses with (+) or without (−) FR. (D) Original trace of isometric force measurements during an FR dose-response curve after preconstriction of mouse trachea with methacholine. (E) Dose-response curve for FR after methacholine preconstriction of mouse trachea, with increasing doses of FR applied every 2 min. (F) Statistical analysis of FR-induced relaxation of mouse trachea after methacholine-induced constriction compared to control groups. (G) Original trace of isometric force measurements in mouse trachea after a single-dose application of FR after preconstriction of mouse trachea with methacholine. (H) Statistical analysis of single-dose application of FR compared to vehicle. (I) Top: Phase-contrast microscopy images of a small intrapulmonary mouse airway. Images represent time points in the trace during perfusion with the respective compounds indicated above the traces. Scale bar, 20 μm. Bottom: Original trace of the change in lumen area of a small intrapulmonary airway before and during perfusion with FR (I) or vehicle (J). (K) Statistical analysis of airway relaxation by FR in mouse lung slices. *P < 0.05, ***P < 0.001. (F) One-way analysis of variance (ANOVA), Tukey’s multiple comparison test. (H and K) Unpaired two-tailed t test.

  • Fig. 3. FR induces relaxation of pig and human airways ex vivo.

    (A) Comparison of carbachol dose-response curves with or without FR treatment on constriction of porcine airways ex vivo. (B) Original trace of FR dose-response curve after carbachol-induced constriction of porcine airways ex vivo. (C) FR dose-response curve after carbachol-induced constriction of porcine airways ex vivo; increasing doses of FR were applied when the force plateau was reached or 10 min after the respective dose was applied. (D) Carbachol-induced airway constriction after different durations of FR treatment and washout [same porcine airways as shown in (C)]. (E) Comparison of histamine dose-response curves with or without a low concentration of FR (30 nM) in porcine airways ex vivo after 60 min of incubation. (F) Time course of the effects of FR after application of histamine. (G) FR dose-response curve after histamine-induced (1 mM) constriction of human airways ex vivo. Inset shows a representative FR dose-response experiment after histamine-induced constriction of human airways ex vivo. *P < 0.05, **P < 0.01. (A and D) One-way ANOVA, Dunnett’s multiple comparison test. (E) Unpaired two-tailed t test.

  • Fig. 4. Local application of FR provides prolonged protection against Gq-dependent airway constriction in normal and OVA-sensitized mice in vivo.

    (A) Dose-response curves of respiratory system resistance (Rrs) in response to methacholine after inhalation of FR, salbutamol, or vehicle in healthy CD1 wild-type mice. (B) Dose-response curves for respiratory system resistance in response to methacholine treatment 24 hours after intratracheal application of FR in healthy CD1 wild-type mice. (C) Systemic systolic blood pressure (P) in healthy CD1 wild-type mice before (−) and 1 or 10 min after (+) inhalation of 2.5 μg of FR, control solution, or 5 μmol of the NO donor SNAP. (Note that 2.5 μg of FR was the amount that was used for aerosol application and that reduced respiratory system resistance.) (D) Systemic systolic blood pressure in healthy CD1 wild-type mice before (−) and after (+) intravenous injection of 2.5 or 12.5 μg of FR. (E) Tissue and plasma concentrations of FR 10 min after inhalation of 2.5 μg of FR by healthy CD1 wild-type mice. (F) Methacholine dose-response curves for respiratory system resistance after inhalation of vehicle or FR by OVA-treated Balb/c mice. *P < 0.05, **P < 0.01, ***P < 0.001. (A, B, and F) Two-way ANOVA, Bonferroni’s multiple comparison test. (C and E) One-way ANOVA, Tukey’s multiple comparison test (FR, control). (C and D) Paired two-tailed t test (SNAP). *P < 0.05, **P < 0.01, ***P < 0.001 FR versus control (A, B, and F), +P < 0.05 (FR versus salbutamol), ###P < 0.001 (salbutamol versus control).

  • Fig. 5. FR reduces airway hyperresponsiveness in the HDM mouse model of asthma but has no effect on pulmonary inflammation.

    (A to C) Hematoxylin and eosin staining of lung sections from control (A), HDM-exposed (HDM) (B), and FR-treated HDM-exposed (C) Balb/c mice. (D to F) Periodic acid–Schiff staining of lung sections from control (D), HDM-exposed (E), and FR-treated HDM-exposed (F) Balb/c mice. Scale bar, 50 μm. (G) Cell counts in BAL fluid for control, HDM-exposed, and FR-treated HDM-exposed Balb/c mice. Macro, macrophages; Eosino, eosinophils; Neutro, neutrophils; Lympho, lymphocytes. (H) Dose-response curves for respiratory system resistance in response to inhaled methacholine in control and FR-treated Balb/c mice exposed to HDM. (I to K) Sirius red staining of lung sections from control (I), HDM-exposed (J), and FR-treated HDM-exposed (K) Balb/c mice to assess collagen deposition. Scale bar, 20 μm. (L) Quantitation of collagen deposition in lung sections from control, HDM-exposed, and FR-treated HDM-exposed mice. Collagen area per micrometer length of cell basement membrane is displayed. *P < 0.05, ***P < 0.001. (H) Two-way ANOVA, Bonferroni’s multiple comparison test. (L) One-way ANOVA, Tukey’s multiple comparison test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/407/eaag2288/DC1

    Materials and Methods

    Fig. S1. FR inhibits Gq protein signaling in Ca2+ imaging experiments in mTSMCs, and in DMR and bioimpedance measurements in hBSMCs.

    Fig. S2. FR does not affect mouse heart rate when applied either locally or systemically.

    Fig. S3. FR applied before OVA protects against airway hyperreactivity in mice.

  • Supplementary Material for:

    Targeted inhibition of Gq signaling induces airway relaxation in mouse models of asthma

    Michaela Matthey, Richard Roberts, Alexander Seidinger, Annika Simon, Ralf Schröder, Markus Kuschak, Suvi Annala, Gabriele M. König, Christa E. Müller, Ian P. Hall, Evi Kostenis, Bernd K. Fleischmann,* Daniela Wenzel*

    *Corresponding author. Email: dwenzel{at}uni-bonn.de (D.W.); bernd.fleischmann{at}uni-bonn.de (B.K.F.)

    Published 13 September 2017, Sci. Transl. Med. 9, eaag2288 (2017)
    DOI: 10.1126/scitranslmed.aag2288

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. FR inhibits Gq protein signaling in Ca2+ imaging experiments in mTSMCs, and in DMR and bioimpedance measurements in hBSMCs.
    • Fig. S2. FR does not affect mouse heart rate when applied either locally or systemically.
    • Fig. S3. FR applied before OVA protects against airway hyperreactivity in mice.

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