TRPV4 inhibition attenuates stretch-induced inflammatory cellular responses and lung barrier dysfunction during mechanical ventilation

Abstract

Mechanical ventilation is an important tool for supporting critically ill patients but may also exert pathological forces on lung cells leading to Ventilator-Induced Lung Injury (VILI). We hypothesised that inhibition of the force-sensitive transient receptor potential vanilloid (TRPV4) ion channel may attenuate the negative effects of mechanical ventilation. Mechanical stretch increased intracellular Ca2+ influx and induced release of pro-inflammatory cytokines in lung epithelial cells that was partially blocked by about 30% with the selective TRPV4 inhibitor GSK2193874, but nearly completely blocked with the pan-calcium channel blocker ruthenium red, suggesting the involvement of more than one calcium channel in the response to mechanical stress. Mechanical stretch also induced the release of pro-inflammatory cytokines from M1 macrophages, but in contrast this was entirely dependent upon TRPV4. In a murine ventilation model, TRPV4 inhibition attenuated both pulmonary barrier permeability increase and pro-inflammatory cytokines release due to high tidal volume ventilation. Taken together, these data suggest TRPV4 inhibitors may have utility as a prophylactic pharmacological treatment to improve the negative pathological stretch-response of lung cells during ventilation and potentially support patients receiving mechanical ventilation.

Fig 1. Concentration-dependent inhibition of TRPV4 effect on Ca²⁺ response.

Representative Ca²⁺-Influx measurement in NCI-H292 cells stimulated with the TRPV4 agonist GSK1016790A (2 nM) and challenged against different concentration of the TRPV4 antagonist GSK2193874 (0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM and 300 nM) preincubated for 15 min before agonist addition in the FLIPR^TETRA. Concentration-dependent inhibition of the agonist effect through TRPV4 antagonism with an IC50 of 48,67 nM. Data are mean ± SEM; n = 3.

Fig 2. TRPV4 mediated calcium influx after stretch.

Ca2+ response in NCI-H292 cells 10 s after a single uni-axial cell-stretch to 80% length increase and back to relaxation within 800 ms. Cells were loaded with the Ca²⁺ dye fluo-4 (2μM) and 0.2% Pluronic F127 (Molecular Probes; Karlsruhe, Germany) and the average fluorescence values of each cell before and 10 s after the strain were determined. The strain-induced change after stimulation was expressed as the % change intensity compared to baseline signal before stretch. (A) Ca²⁺ response 10 s after stretch for 60 seconds. (B) Summary of the mean % Ca²⁺ response from the 60 sec after stretch, a 2.5 fold increase in the [Ca2+]_i was observed that was significantly decreased by 26% with the TRPV4 antagonist (1 μM) GSK2193874 (184.5 ± 5.07 vs 248.6 ± 10.17). For (A) and (B) data are mean ± SEM; (control n = 121; GSK2193874 n = 94, summary of 13 experiments; ****p ˂ 0.0001 vs control; Unpaired two-tailed t test).

Fig 3. TRPV4 mediated cytokine release.

Representative experiment of NCI-H292 cells incubated for 24 h in the presence or absence of the TRPV4 agonist GSK1016790A [3nM] (ag) with or without pre-treatment with the TRPV4-Antagonist GSK2193874 [1μM] (ant). (A,B) release of IL-6 and IL-8 through TRPV4 activation compared to medium (ctrl) and DMSO control (veh) that could be blocked by the TRPV4 antagonist. (C,D) TRPV4 mediated release of IL-1α and MDC. Data are mean ± SEM; (n = 6; ****p ˂ 0.0001 vs agonist control; one-way ANOVA Tukey's multiple comparisons test).

Fig 4. TRPV4 mediated stretch-induced cytokine release.

Repesentative experiment of NCI-H292 cells seeded on silicoelastic membranes and exposed to cyclic equibiaxial stretch (cyclic 30% strain with 1.25 hz) for 24 h in the presence or absence of the TRPV4-Antagonist GSK2193874 [1μM]. (A) Stretch induced release of IL-8 compared to unstretched control (ctrl) reduced by 34% with the TRPV4 antagonist (ant) and reduced by 86% with Ruthenium Red (RR). (B) IL-6 release via stretch that was reduced through TRPV4 antagonism by 33% (ant) and reduced by 80% with Ruthenium Red addition (RR). Data are mean ± SEM; (n = 3; *p ˂ 0.05; ****p ˂ 0.0001 vs stretch control; one-way ANOVA Tukey's multiple comparisons test).

Fig 5. TRPV4 mediated stretch-induced cytokine release in macrophages M1 and M2.

Representative experiment of Macrophages seeded on silicoelastic membranes and exposed to cyclic equibiaxial stretch (cyclic 30% strain with 1.25 Hz) for up to 48 h in the presence or absence of the TRPV4-Antagonist GSK2193874 [1μM]. (A-E) Stretch induced cytokine release in M1 macrophages compared to unstretched control (ctrl) that could be blocked with the TRPV4 antagonist (ant). Stretch induced release of MCP-1 and TNF-α in M2 macrophages that could be blocked by TRPV4 inhibition (F,G). Data are mean ± SEM; (n = 3; *p ˂ 0.05; **p ˂ 0.01; ***p ˂ 0.001; ****p ˂ 0.0001 vs stretch control; one-way ANOVA Tukey's multiple comparisons test).

Fig 6. TRPV4 antagonist effect on ventilation induced cytokine release and protein concentration in BALF.

Balb/c mice were anesthetized and mechanically ventilated (VT) in presence or absence of the TRPV4 antagonist GSK2193874 [90mg/kg] with different ventilation protocols; with tidal volumes of 20 ml/kg and 30ml/kg with a frequency of 75/min and a control group (ctrl) ventilated with a normal tidal volume of 6.5ml/kg and a frequency of 150/min for 3h and a non-ventilated control group (ctrl). (A) Increased protein concentration in BALF was observed at 30 ml/kg ventilation that could be blocked by TRPV4 inhibition (ant). (B,C) Increase release of the cytokine KC/GRO and IL-6 after a 30 ml/kg ventilation that could be blocked by TRPV4 antagonism (ant). Data are mean ± SEM; (n = 8); *p ˂ 0.05; ***p ˂ 0.001 vs 30 ml/kg VT control; one-way ANOVA Tukey's multiple comparisons test).