Figure 1.
IMD improved endothelial barrier function in endothelial cells.
HPMVECs grown to confluence on gold microelectrodes to measure transcellular electrical resistance (TER) were stimulated with 0.01 or 0.1 µM IMD or with solvent. IMD dose dependently improved endothelial barrier function as displayed by TER increase. (n = 6).
Figure 2.
Regulation of IMD and its receptor complexes in VILI.
Mice were ventilated with a tidal volume of 12 ml/kg for 6 h (6 h vent.). Non ventilated individuals (NV) served as controls. A) Regulation of IMD, CGRP and RAMP1-3 was quantified by qRT-PCR in the lungs of ventilated (6 h vent.) and non ventilated mice (NV). (** p<0.01, n = 5). B) Immunofluorescence analysis of subpleural peripheral lung. IMD-immunolabelling was slightly enhanced in ventilated (6 h vent.) compared to non-ventilated mice (NV) while CRLR-immunolabelling did not differ between groups. Tissue sections depicting NV and 6 h vent groups were processed simultaneously and images were taken at the same exposure time (30 ms for IMD and 150 ms for CRLR).
Figure 3.
Pulmonary distribution of IMD and CRLR was not altered in VILI.
In non ventilated mice (A) and individuals ventilated with a tidal volume of 12 ml/kg for 6 h (6 h vent.) (B) IMD- and CRLR-immunolabelling colocalized with CD31-immunoreactivity (arrows), a marker for endothelial cells. In addition, alveolar macrophages were IMD- and CRLR-positive (arrowheads). (n = 5, representative images shown).
Figure 4.
IMD reduced pulmonary vascular hyperpermeability in VILI.
Mice were ventilated with a tidal volume of 12 ml/kg for 6 h and treated with IMD 0.025 mg/kg*h (6 h vent.+IMD) or solvent (6 h vent). NV = non ventilated mice. Human serum albumin (HSA) was infused 90 min prior to termination of the experiment. HSA concentration (cHSA) in plasma and BAL were determined. Increased HSA BAL/plasma ratio indicated microvascular leakage. (*p<0.05, ## p<0.01 vs. NV, NV n = 5, 6 h vent. n = 7, 6 h vent. + IMD n = 8)
Figure 5.
IMD had no impact on oxygenation in VILI.
Mice were ventilated with a tidal volume of 12 ml/kg for 6 h and treated with IMD 0.025 mg/kg*h (6 h vent.+IMD) or solvent (6 h vent). A) Peripheral SpO2 was monitored. B) At the end of the 6 h ventilation period, SpO2 was not relevantly different between groups. C) After 6 h of MV, the P/F ratio was not different between groups. (***p<0.001, n = 15)
Figure 6.
IMD mediated pulmonary vasodilation and reversed hypoxic pulmonary vasoconstriction.
A) In precision cut lung slices (PCLS) hypoxic pulmonary vasoconstriction (HPV) was induced, and the luminal areas of single intra-acinar pulmonary arteries were continuously analyzed by planimetry. HPV was markedly reduced by 500 nM IMD (***p<0.001; n = 10). B) In isolated ventilated and perfused mouse lungs the thromboxane agonist U46619 induced a marked elevation of pulmonary artery pressure (PAP) during constant perfusion flow. PAP was lowered dose dependently by IMD bolus injection (* p<0.05, ** p<0.01, n = 5)
Figure 7.
VILI induced pulmonary leukocyte recruitment and blood leukocytosis were unaffected by IMD treatment.
Mice were ventilated with a tidal volume of 12 ml/kg for 6 h and treated with IMD 0.025 mg/kg*h (6 h vent.+IMD) or solvent (6 h vent). NV = non ventilated mice. Leukocytes isolated from bronchoalveolar lavage fluid (BALF), lung homogenate and blood, respectively, were quantified and differentiated by flow cytometry (*p<0.05 vs. NV, **p<0.01 vs. NV, NV n = 5, 6 h vent. n = 7, 6 h vent. + IMD n = 8)
Figure 8.
VILI-induced cytokine production was not altered by IMD.
Mice were ventilated with a tidal volume of 12 ml/kg for 6 h and treated with IMD 0.025 mg/kg*h (6 h vent.+IMD) or solvent (6 h vent). NV = non-ventilated mice. A) IL-1β, IL-18, IL-6, KC, MIP-1α, MIP-2, MCP-1, IL12p40 were detemined in lung homogenate by multiplex assay technique. (*p<0.05 vs. NV, **p<0.01 vs. NV n = 8) B) IL-1β, IL-18, IL-6, KC, MIP-1α, MIP-2, MCP-1, IL12p40 were determined in plasma by multiplex assay technique. (*p<0.05 vs. NV, **p<0.01 vs. NV n = 8).