Dissociation between the effects of oxygen and pressure on matrix metalloproteinase-2, -7, and -9 expression in human airway epithelial cells.

We tested the hypothesis that hyperoxia or pressure exposure differentially activates expression of cytokines and/or matrix modeling proteins in human airway epithelial cells. Calu-3 epithelial cell monolayers were cultured on transwell plates with the apical surface exposed to gas. Following establishment of baseline, plates were placed in a chamber and exposed to: control (21% O (2); atm), hyperoxia (60% O (2); atm), pressure (21% O (2); 40 cm H (2)O), and combination (60% O (2); 40 cm H (2)O). At 72 hour of exposure, monolayers were assessed for integrity, viability, and expression of interleukin (IL)-6, IL-8 and matrix metalloproteinases (MMPs) -2, -7, and -9. Compared with controls, hyperoxia had lower transepithelial resistance ( P < 0.001) and greater IL-6 secretion ( P < 0.01), and pressure had lower cell viability ( P < 0.001) and greater IL-8 secretion ( P < 0.001). Hyperoxia resulted in more latent MMP-2 ( P < 0.05) and MMP-7 ( P < 0.001). Pressure was associated with a rise in MMPs independent of oxygen exposure ( P < 0.05). Hyperoxia and pressure differentially affected MMP activities in Calu-3 cells and may lead to the different functional and structural abnormalities observed in these in vitro studies.

Effects of oxygen concentration and exposure time on cultured human airway epithelial cells.

OBJECTIVES: To distinguish the direct effects of oxygen dose and exposure time on human airway epithelial cells. We hypothesized that progressive oxygen exposure would induce cell dysfunction and inflammation in a dose-dependent manner. DESIGN: Interventional laboratory study. SETTING: An academic medical research facility in the northeastern United States. SUBJECTS: Calu-3 human airway epithelial cell culture. INTERVENTIONS: Cells were cultured at a gas-liquid interface with the cells fed basolaterally with medium and grown to full confluence. The apical surfaces were then exposed to gas containing 21%, 40%, 60%, or 80% oxygen, 5% CO2, and balance nitrogen for 24 or 72 hrs. MEASUREMENTS AND MAIN RESULTS: The effects of oxygen concentration and time-induced cellular change were examined by measuring transepithelial resistance of monolayers, cell viability by trypan blue exclusion, basolateral lactate concentration, histology of monolayer cross-sections, and cytospin slides, plus interleukin (IL)-6 and IL-8 secretion in apical surface fluid. Transepithelial resistance decreased in a dose- and time-dependent manner (p < .001), whereas cell viability was reduced only at 72 hrs and in all hyperoxic groups (p < .05). IL-6 secretion was elevated in all hyperoxic groups at 24 hrs (p < .001), and both IL-6 and IL-8 levels were greater in the 40% FiO2 group compared with all other groups at 72 hrs (p < .01). CONCLUSIONS: In this model, airway epithelial cells demonstrate profound concentration and time-dependent responses to hyperoxic exposure with respect to cell physiology, viability, histology, and secretion of inflammatory mediators. This model might be a valuable tool for preliminary analysis of potentially protective therapies against hyperoxia-induced airway epithelial injury.

Growth and development in a heliox incubator environment: a long-term safety study.

BACKGROUND: Neonates exposed to mechanical ventilation may develop bronchopulmonary dysplasia (BPD). BPD neonates exhibit a 25-30% increase in energy expenditure which may decrease the rate of growth and development. Heliox has been shown to improve pulmonary function and may decrease energy expenditure. We hypothesized that heliox would provide a safe environment for sustained growth and development. OBJECTIVE: To assess the safety of the heliox environment we observed developmental milestones; recorded changes in weight, total length, limb length and head circumference; measured blood chemistries; compared primary organ and muscle weights, and analyzed muscle enzymatic activity. DESIGN/METHODS: Four-day-old rabbit pups (n = 27) were randomized into control (21% O(2); 79% N(2)) or heliox (21% O(2); 79% He) groups, then raised for 14 days at 26.7 degrees C and 50% relative humidity. Pups were euthanized on day 14, blood drawn and primary organs, diaphragm and gastrocnemius weighed and snap-frozen. RESULTS: All pups thrived in both environments, achieving expected developmental milestones. There were no physiologically significant group differences in weight, growth factors, tissue weight, blood chemistry or muscle enzyme activity. CONCLUSIONS: No observed long-term differences in growth or development. RESULTS demonstrated that long-term heliox exposure is safe in this rabbit model. These data suggest that heliox administration may provide time for pulmonary improvement in the BPD population, warranting appropriate clinical trials.

A prototype infant incubator for heliox therapy.

Heliox (Hx) gas has been shown to improve pulmonary function in infants, but methods for its delivery are invasive and problematic. To this end, we modified an Isolette (Hill-Rom Air-Shields) infant incubator (Hxl) to deliver Hx respiratory gas mixtures noninvasively while providing thermal stability for neonatal care in the Neonatal Intensive Care Unit (NICU). In vitro tests and in vivo animal studies were performed to compare the original design specifications and established baseline performance criteria for the Hxl design. The experimental environments at 50% and 80% relative humidity (RH) consisted of helium (He) with 21% and 50% O2 and control (C) of 21% and 50% O2 with the balance nitrogen (N). Elapsed times to steady state (SS) and recovery time back to SS (OCDss) due to opening and closing the door were recorded for each variable. All rabbits survived and appeared comfortable during all experimental conditions. These data show that the newly designed Isolette provides similar thermal, O2, CO2, and RH responses as the control incubator. Based on these positive safety/efficacy studies, study of the therapeutic impact of Hxl care on neonatal growth and development is in progress.

Physiologic implications of helium as a carrier gas for inhaled nitric oxide in a neonatal model of Bethanecol-induced bronchoconstriction.

OBJECTIVE: To compare heliox to nitrogen-oxygen (nitrox) as a carrier gas for inducible nitric oxide (iNO) in the presence of pharmacologically inhaled bronchoconstriction. We hypothesized that respiratory resistance and gas exchange would improve when iNO is delivered with heliox. DESIGN: Interventional laboratory study. SETTING: An academic medical research facility in the northeastern United States. SUBJECTS: Sedated, ventilated newborn piglets. INTERVENTIONS: Newborn piglets (n = 16; 2.3 +/- 0.1 kg) were placed on a flow-controlled ventilator and given intravenous Bethanecol (2 x 1 mg/kg followed by 1 mg/kg/hr) to induce bronchoconstriction. Piglets were randomized to heliox or nitrox (Fio2 = 0.3) and given 80 ppm iNO. MEASUREMENTS AND MAIN RESULTS: Hemodynamics, blood chemistry, and pulmonary mechanics were recorded at 30-min intervals for 2 hrs. Bethanecol dosing increased inspiratory respiratory resistance (cm H2O/L/min; p < .01) and decreased respiratory compliance (mL/cm H2O/kg; p < .01). Following carrier gas assignment, hemodynamics and respiratory compliance were similar between groups and respiratory resistance decreased (p < .01) in the heliox group. Over 2 hrs with iNO therapy, Paco2 increased (p < .01) whereas blood pH decreased (p < .01) in the heliox group. Respiratory resistance trended downward, oxygenation index improved (p < .01), and blood methemoglobin levels trended higher for nitrox compared with heliox. CONCLUSIONS: The INOvent was effective for controlling heliox delivery of iNO. Despite marked reduction in respiratory resistance with heliox gas ventilation in a neonatal model of pharmacologic bronchoconstriction, nitrox might perform better as a delivery vehicle for iNO.

Expression of matrix metalloproteinases 2, 7 and 9, and their tissue inhibitors 1 and 2, in developing rabbit tracheae.

BACKGROUND: Structural changes in the developing conducting airway impact the rigidity of the airway, altering the airway's ability to sustain its shape during ventilation. The developmental changes in airway compliance oppose the changes in compliance of the developing lung; thus the expression profiles of matrix modeling proteins likely are also opposite in these developing organs. OBJECTIVES: To determine the profiles of matrix metalloproteinases (MMPs) -2, -7, and -9 and tissue inhibitors (TIMPs) -1 and -2 in the developing trachea and test the hypothesis these profiles would contrast those previously reported for the lung. METHODS: Rabbits tracheae were harvested at 21 days of gestation, 3 and 17 days postgestation and at adulthood. Tissue homogenates were analyzed by substrate zymography for the activity of MMPs, and reverse zymography for TIMPs. Immunostainings on neonatal lamb tracheal rings were used to localize MMP-2 and 9. RESULTS: Analysis revealed an age-dependent decrease in total MMP-2 quantity and the ratio of active to latent forms. TIMP-2 shows a time-dependent increase throughout airway development. Total MMP-9 and TIMP-1 quantities were unchanged across these ages, although MMP-9 protein was found predominantly in its latent form during development and predominantly in its active form during adulthood. Respiratory epithelial cells reacted positive for both MMP-2 and 9 and trachealis muscle fibers were positive for MMP-2. No MMP-7 expression was identified in the rabbit airway. CONCLUSIONS: The opposing developmental patterns in MMP-2 expression between the airway and lung lead to speculation regarding the role of MMP-2 activity on changes in organ compliance.