After some 40 years of research and numerous failed clinical trials, only two interventions have been shown to significantly reduce the high mortality rate of the acute respiratory distress syndrome (ARDS), a reduction of tidal volume during mechanical ventilation (22%), and infusion of activated protein C (19%). These clinical studies indicate that coagulation activation renders the lung exquisitelv sensitive to stretch because the injurious lung distention producing a 22% increase in ARDS fatalities is half that in resting tidal breathing. We recentty reported that experimental Ventilator Induced Lung Injury was prevented in mice by deletion of the calcium-permeable stretch-activated transient receptor potential vanilloid-4 (TRPV4) ion channel. Recent studies indicate that the proteinase activated receptor-2 (PAR2) associates with the TRPV4 channel in neurons and greatly amplifies the TRPV4 induced calcium increase. We show (Preliminary Data) that lung epithelial cell monolayers co-cultured with alveolar macrophages activated with trypsin (PAR2), thrombin (PAR1, 3, 4) or the TRPV4 agonist, 4alpha-phoroot 12,13--didecanoate (4a-PDD) given separately actually decreased monolayer permeability, but that pre-treatment with either trypsin or thrombin followed by 4a-PDD produced a sustained permeability increase. We Hvpothesize: that coagulation induced proteases and TRPV4 induced alveolar macrophage release of proteases that activate PAR2 and PAR1, 3, and 4 which then amplify the mechanogated TRPV4 response in epithelial and endothelial cells sufficiently to cause increased lung vascular permeability. We propose these Specific Aims: 1) Test the hypotheses that: PAR1 and PAR2 activation peptides followed by 4a-PDD amplify the TRPV4 mediated calcium influx in alveolar macrophages and alveolar epithelial cells measured using whole cell patch clamp and fluorescence microscopy;and that the PAR amplification is reduced by inhibitors of PLC~, PKA, and PKC. 2) Test the hypothesis that PAR1 and PAR2 activation peptides followed by 4a-PDD increases alveolar epithelial monolayer permeability using electric cell-substrate impedance sensing, and that lung PAR1 and PAR2 activation peptidesincrease lung vascular permeability in TRPV4+/+ mice ventilated with high airway pressures but not in TRPV4-/- mice, or either genotype ventilated with low airway pressures
Ventilator induced lung injury can contribute to the mortality of patients with acute lung injury. These studies will determine the mechanisms by which alveolar macrophages amplify the mechanical stretch induced permeability response in the alveolar capillaries initiated by the transient receptor potential vanilloid-4 channel.
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