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Mechanical ventilation (MV) of the neonatal airway alters mechanical properties and activates tissue-modeling pathways. Heat shock protein (HSP70) is a marker of tissue injury and modulates inflammation, which may influence subsequent pulmonary tissue modeling by matrix metalloproteinases (MMPs). HSP70 secretion is up regulated in MV airway tissues and associated with changes in airway elasticity and secretion of MMPs. Proximal tracheal segments were isolated in 13 newborn lambs and were either MV for 4 hr or SHAM. At baseline and hourly, tracheal segments were flushed and tracheal elasticity was determined. Tracheal wash fluid was assayed for HSP70 by ELISA and for MMPs by substrate zymography. HSP70 secretion increased from baseline to a peak at 1 hr in both groups (p<0.01), greater in the MV group (p<0.05), and returned to baseline values by 2 hr. This response was in contrast to the progressive decrease in tracheal elasticity (p<0.05). The HSP70 elevation pattern was noted in MMP-2, but beyond 1 hr, MMP-2 returned to baseline values in MV group but remained elevated in SHAM (p<0.05). HSP70 secretion is associated with the degree of biophysical tracheal injury as well as the time course of MMP-2 secretion by tracheal tissues.
Infants develop significant trauma to the large conducting airway as a result of mechanical ventilation(1). In the fetal and neonatal periods there are developmental changes in the structural and functional properties of the airways increasing bronchial distensibility (2-5). The resulting tracheal trauma is due to distending a relatively distensible airway while attempting to inflate a relatively non-compliant lung (6). The biophysical injury induced by mechanical ventilation (6-10), results in a molecular inflammatory response that contributes to biological trauma with respect to tissue proteolysis, and ultimately tissue remodeling (11-13).
In a recent model to quantify the functional impact of mechanical ventilation (MV) on the airway developed by our group, we noted that airway mechanical properties are markedly altered following only short periods of MV. Moduli of elasticity, representing dynamic airway elasticity whereby a greater value represents less elasticity, can be determined for the neonatal lamb trachea in real-time (14). Data from these studies show that over time, airways exposed to mechanical ventilation lose elasticity as compared to SHAM controls (15).
Matrix metalloproteinases (MMPs) are believed to contribute to normal tissue modeling as well as the progression of airway injury and chronic disease (11-13). MMPs contribute to the reparative process by degrading basement membrane components and modulating inflammation (16), and therefore can be a contributing factor to changes in the structure and function of ventilated airways. MMP-2 and MMP-9, specifically, are implicated in having an important role in airway development and injury (17-19), and therefore can be used as a marker of airway injury/remodeling.
Heat shock protein (HSP) 70 is secreted in response to injury by different tissues (20, 21). If induced prior to an injury, heat shock protein is believed to attenuate the degree of inflammation by stabilizing inhibitory kappa B factor (22-26). Inhibitory kappa B factor plays a major role in stabilizing nuclear factor kappa B (NFκB) activation. Upon NFκB activation, downstream genes regulating inflammatory mediators will be activated and hence, activate the inflammatory cascade (23, 27). Therefore, we reason that HSP-70 secretion may be induced during airway trauma by mechanical ventilation as a marker of injury and possible attenuate the degree of injury.
The objective of this study is to determine the relationship between ventilation induced airway injury and the secretion of HSP-70 and MMPs. We hypothesize that HSP-70 secretion will be up regulated in airway tissues secondary to mechanical ventilation and this will correspond to changes in expression of MMPs 2 and 9.
Newborn lambs between 1 and 4 days old (n = 13; 3.3 ± 1.2 d; 3.8 ± 1.2 kg) were anesthetized and instrumented as shown in Figure 1. Animals were allowed to breathe spontaneously through a lower endotracheal tube (ETT) and were not subjected to MV from the lower ETT. The experimental procedures are applied to the isolated, perfused and innervated tracheal segment via the upper ETT. The isolated tracheal segment used for analysis is the area between both ETT. This model allows the tracheal segment studied to be isolated and that the known inflammatory effects of ventilation on lung would not have a confounding influence on the airway outcomes. Following instrumentation, animals were randomized to either pressure-limited, time-cycled MV (n = 7) or SHAM (n = 6). Measurements of physiologic stability and airway functional parameters were made at baseline and hourly for 4 hrs; arterial plasma and airway wash fluid was collected at each measurement point.
All animal procedures in this study were approved by the Life Science Center Animal Care and Use Committee, Department of Biomedical Research, Nemours, and were in accordance with National Institutes of Health guidelines. Lambs were anesthetized initially with two 1 ml/kg intramuscular injections, separated by 10 min, of an anesthesia cocktail (ketamine: 23 mg/kg; azepromazine: 0.1 mg/kg; and xylazine: 0.05 mg/kg [KAX]) adapted from previously described protocols (28, 29). Prior to instrumentation, the region of the lamb's neck covering the trachea was shaved to remove the bulk of the hair, and then hair removal cream (Nair®, Carter-Horner Corp., Mississauga, ON, Canada) was applied to make the neck region hair free.
Local anesthesia (4 mg/kg 0.5% lidocaine HCl) was injected into the skin and soft tissues around caudal and rostral surgical sites as shown in Fig. 1. As previously described (14, 15) a double tracheotomy was performed at the caudal site, which was located just above the notch of the sternum where the trachea enters the thorax. At this site, the distal trachea was intubated with a 3.5 mm-internal diameter (ID) ETT (Hi-Lo™ Jet Tube: Mallincrodkt, Saint Louis, MO) to allow for continued spontaneous breathing. The distal end of the proximal trachea was cannulated with a saline filled catheter (5 French [Fr]) attached to a pressure transducer; the end of the proximal trachea was ligatured around the catheter to provide a seal to contain gas during ventilator cycles and saline during pressure-volume and ultrasound measurements. The proximal end of the proximal trachea (the isolated segment) was intubated through the rostral surgical site, just distal to the larynx, with a 3.0 mm-ID endotracheal tube, which was ligatured into the trachea to prevent gas and saline leaks.
For venous access and arterial blood sampling, respectively, 5 or 8 Fr umbilical catheters were inserted into the external jugular vein and carotid artery through the caudal surgical site. Subsequent anesthesia was maintained with intravenous infusion of KAX at 0.4 ml/kg/hr or as needed. Maintenance fluid was provided by a continuous venous infusion of 5% dextrose solution at a rate of 6 ml/kg/hr. Arterial blood pressure was monitored by attaching the arterial catheter to a standard pressure transducer via a bedside patient monitor (Model M1175A, Hewlett Packard), and electrodes were placed for ECG monitoring. Throughout the protocol, the animal's rectal temperature was monitored and maintained at 37-40°C on a radiant warmer bed (Resuscitaire®; Hill-Rom Air-Shields, Hatboro, PA). Following instrumentation, physiologic stability of the animal was confirmed by analysis of arterial blood chemistry (Stat profile®, Nova Biomedical, Waltham, MA), ECG, and blood pressure monitoring, and then the isolated tracheal segment was exposed to MV pressure cycles or randomized to SHAM.
Pressure cycles in the MV group were delivered by a neonatal ventilator (Sechrist Neonatal ventilator, Sechrist Industries, Inc., Anaheim, CA) for 4 hrs at the following settings: peak inspiratory pressure (PIP) = 35 cmH2O, positive end expiratory pressure (PEEP) = 5 cmH2O, flow rate = 10 L/min, frequency = 40 cycles/min with inspiratory time = 0.30 sec, and fraction of inspired oxygen (FIO2) = 0.21. The higher PIP was used to ensure physical injury was produced in the tracheal segment (14). The other ventilator parameters applied are used commonly in the clinical settings.
At baseline and each subsequent measurement period, the isolated tracheal segment was flushed with normal saline at room temperature, and 5 ml was collected to be assayed for HSP-70 and MMPs. The collected tracheal wash fluid was centrifuged at 5°C and 18,000 × g for 20 min to remove debris before storage at -70°C. While saline filled, the tracheal segment was assessed for the static pressure-volume relationship and static/dynamic phase ultrasound imaging. Pressure-volume measurements and ultrasound imaging were performed as described in a previous paper by our group (14, 15). A high frequency ultrasound system, Vevo 770 High-Resolution Imaging System (VisualSonics, Toronto, Canada), was used to measure tracheal wall thickness and resting segment volumes. Tracheal wall thickness was measured for both posterior and anterior walls, representing the muscle and cartilaginous walls, respectively. Resting segment volumes were derived by multiplying cross-sectional area by the segment length. After each measurement set, the saline in the tracheal segment was flushed out with air so that the airway would be gas filled throughout the duration of the protocol.
Following the 4 hr measurements, an IV bolus of 1 ml/kg KAX was administered to achieve deep sedation before the isolated tracheal segment was harvested. At the time of harvest, the isolated tracheal segment length was measured as the distance between ligatures while the segment was still situated in vivo. Euthanasia was then achieved by thoracotomy and pulmonary exsanguination. In total, each animal was under sedation for approximately 5 hrs.
Methodology and results for in vivo static and dynamic elasticity from these studies was previously reported (14, 15). Briefly, static airway pressure-volume relationships were constructed by first filling the isolated tracheal segment with normal saline and allowing the pressure within to equilibrate with the ambient condition. A two-way stopcock was attached to the distal catheter and 0.2 ml aliquots of saline was injected into the closed tracheal segments in 15 sec intervals until a pressure ≤ 80 cmH2O was achieved, the saline was then withdrawn in the same manner. The resultant pressure profiles associated with the step volume increments were recorded and injected volume units were normalized to resting segment volume. Bulk modulus (K) was calculated as the slope of the pressure-volume curves obtained at each interval.
In ventilated animals, the relationship between tangential wall stress and strain of the trachea was determined during dynamic ventilator pressure loading. Bulk and elastic moduli, represent the amount of force (stress) to achieve a degree of deformation (strain). The trachea normally has some degree of elasticity, but our data demonstrate that over time more force is needed to distend the airway. In this regard, the airways are losing elasticity in response to ventilator stress whereby the enlarging internal diameter is associated with the breakdown of elastic components and airway distention is accomplished through stretch of the non-elastic structural components. Bulk modulus measures the volumetric adjustments of the trachea resulting from changes in the airway elastance, measured globally. The Young's modulus derivation accounts for dynamic tissue properties such as inertia and viscoelastic resistance.
Ultrasound imaging provided real time cross-sectional images at the exact moments where pressure waves peaked, and at end expiratory pressure. Stress-strain relationships were constructed where wall stress was a function of ventilatory pressure, luminal diameter and wall thickness, and strain was the associated change in diameter. Young's modulus was adapted to the trachea model as the slope of the stress-strain relationship at each interval.
Substrate zymography was used to quantify the relative abundance of both the physiologically active and latent forms of MMP-2 and MMP-9. Prior to analysis, lung tissue was homogenized in a lysis buffer containing a non-metalloprotease inhibitor cocktail (Sigma, St. Louis, MO). Loading buffer was added directly to tracheal wash samples, and equal volumes of each sample were loaded into gels for electrophoresis. Protein was separated in 10% acrylamide gels containing 1 mg/ml gelatin (Sigma, St. Louis, MO) according to previously described methods (11, 29). Following electrophoresis, SDS was eluted by washing the gels in 2.5% Triton-X-100; then the gels were incubated for 18 hrs at 37°C in incubation buffer containing 50mM Tris-HCl, pH 7.5, 5 mM calcium chloride, and 0.01 mM zinc chloride. Gels were subsequently stained with Coomassie brilliant blue R250 and scanned to a digital image for analysis.
Densitometric analysis of the images was performed using Scion Image software (Scion Corp., Frederick, MD) to determine pixel values for the expression of each sample. Relative quantification in arbitrary units (au) of all MMP bands was accomplished by running a standard curve of a common sample on all gels. The total quantity of each MMP was derived by adding the raw pixel values for the active and latent bands from each lane before extrapolation of the au value from the standard curve. A ratio of the quantity of active (A) to latent (L) forms for each relevant MMP (A:L ratio) was calculated for each sample as the ratio of raw pixel values.
HSP-70 levels in isolated tracheal segment tissue homogenates and airway wash aliquots were measured using a commercially available HSP70 ELISA Kit (StressXpress®, Assay Designs Corporation). Airway wash fluid aliquots were lyophilized and reconstituted to 10x concentrates for HSP-70 detection. All standards and samples were assayed in duplicates and all samples were diluted to fall within the detection range of this assay. The assay's sensitivity is 500 pg/ml. Inter-assay and intra-assay coefficients of variance is <10%. HSP-70 samples from tracheal tissue were standardized to total protein concentration and samples from airway wash fluid were standardized to surface area of the isolated tracheal segment.
All time-dependent parameters were analyzed using a general linear model analysis of variance (ANOVA) with Bonferroni post hoc tests where applicable. With the exception of elastic modulus, time-dependent parameters were analyzed for main group effects and group by time interaction using two-way ANOVA. All time-dependent parameters were evaluated for group dependent time effects using one-way ANOVA. Tissue content of HSP-70 and MMPs (only at 4 hr time point) were analyzed by t-test. Significance was accepted at p ≤ 0.05.
As previously reported, animals tolerated the protocol with normal physiology, and there were no group differences in vital signs or physiologic parameters (15). All animals displayed a modest respiratory acidosis that was constant throughout the protocol. Hemoglobin saturation remained above 80% in all animals and heart rate remained constant.
Tracheal tissues was harvested at the end of the 4-hour ventilation and homogenized to analyze for presence of HSP-70 as well as MMP-2 and 9. HSP-70 was detected in both SHAM and ventilated tracheal tissue but no difference was found between these two groups (Figure 2A). MMP-2 and 9 were detected in tracheal tissues in both their latent and active forms. Latent MMP-2 was significantly more abundant in the ventilated trachea (p < 0.05; Figure 2B). There were no differences in the other parameters. Data for all MMP parameters are presented in Table 1.
HSP-70 concentration was detected in concentrated airway wash fluid from both groups at baseline and each subsequent time point. In each group, HSP-70 secretion was elevated at 1 hr (Figure 3), with a greater elevation in the ventilated group as determined by a significant interaction between group and time (p < 0.05). HSP-70 secretion then returned to baseline values by 2 hrs.
Both MMP-2 and 9 were found predominantly in their latent form. Active, latent and total MMP-2 secretion (Figure 4) was elevated at 1hr. Beyond 1 hr, total and latent MMP-2 secretion (Figure 4A, C) returned to baseline values in the ventilated group only, resulting in a difference from SHAM values (p < 0.05) that remained elevated. Total and latent MMP-9 (Figure 5A, C) increased over time in the SHAM group only to produce a main effect for group (p < 0.05) by 4 hrs. There were no group differences in active MMP-2 or MMP9.
Data for the bulk and Young's moduli of elasticity are summarized in figure 6. Analysis of bulk modulus corrected for percent change from baseline (Figure 6A) revealed significant effects for group and time. By 4 hrs, bulk modulus values for ventilated tracheae were greater than SHAM tracheae (p < 0.01) and greater than baseline values (p < 0.05). Bulk modulus remained unchanged in the SHAM animals. Analysis of elastic modulus (Figure 6B), representing the dynamic stress-strain relationships in the ventilated animals only, revealed a time effect (p < 0.05) where values were greater by 4 hrs. These data were discussed in detail in by our group in prior papers (14, 15).
In this study, we evaluated the relationship between the time course of airway injury resulting from typical mechanical ventilator pressure waveforms and trauma response / tissue modeling proteins. We have shown that HSP-70 and MMP 2 & 9 are produced by tracheal tissue and secreted into the airway lumen as a result of mechanical distention of the airway. Furthermore, HSP-70 and MMP-2 secretion peaked early (within 1 hr) in the time course of the initiation of pressure exposure. In this regard, HSP-70 secretion profile was associated with the secretion profile for MMP-2, and thus may be related to the tissue remodeling response secondary to airway trauma.
HSP-70 appears to be a sensitive marker of injury in the airway as even mild trauma from instrumentation and measurements in this study may have resulted in a degree of HSP-70 secretion in our sham animals. In order to obtain pressure volume data to calculate elastic properties, the sham airways were exposed to static stretch at each hourly interval. It is noteworthy, that this static stretch most likely resulted in HSP-70 secretion from the sham airways despite no change in mechanical properties or secretion of measured proteins. The higher PIP used in this study ensures physical injury was produced in the trachea and allows correlation with HSP-70 as a marker of injury. Our group plans to use lower and more clinically relevant PIP in future studies. A crucial point is the rapid time course of HSP-70 secretion, i.e., within the first hour of trauma to the airway tissues. Reports of the time course of HSP-70 kinetics associated with injury to other tissues indicate that HSP-70 typically does not appear until after a few hours (30). In the current study, this early HSP-70 appearance is associated with MMP-2 secretion, which also peaked within the first hour of injury and is attenuated following the spike in HSP-70. It is difficult to interpret the rapid elevation in HSP-70. This could represent an earlier spike (prior to the 1st hour) in HSP-70 (not measured in our study) and therefore, may represent an acute response to the physical stress experienced by tracheal tissue in response to mechanical ventilation. The fall in HSP-70 after the 1st hour would also support that HSP-70 is secreted or produced early and acutely in response to injury. Although we did not note a difference in tracheal tissue content of HSP-70 between ventilated animals and SHAM when harvested at the 4 hr interval, we hypothesize that if tracheal tissue was harvested and analyzed within the 1st hour of mechanical ventilation, tissue content of HSP-70 between the groups would have been different and may correspond with tracheal aspirate HSP-70 secretions pattern. A limitation of this study is the single time duration (4 hr), which the animals were exposed to mechanical ventilation. Proteases like MMPs 2 & 9 can degrade and cleave components of extracellular matrix in response to various factors such as physical stress (31). The cleaved matrix fragments and non-matrix substances can act as inflammatory mediators contributing to further inflammation and tissue remodeling (32). MMPs 2 & 9 have been shown to play a role in lung branching and alveoralization via interaction between MMP and inflammatory mediators (17, 18). Therefore, it is speculated that MMPs may play a role in airway tissue remodeling after physical stress.
HSP-70 is also known to function as a molecular chaperone activated by cellular stress, which helps to attenuate inflammation by stabilizing inhibitory kappa B (IκB). This prevents activation of NFκB and subsequent downstream genes for inflammatory mediators (26). Heat shock proteins interact with the MAP (Mitogen-activated protein) kinase pathways by a balanced inhibition of both extracellular signal-regulated kinase (ERK) and Jun N-terminal kinase (JNK). Transcription of HSPs in turn is regulated by MAP kinase. Both may be important factors affecting balance between cell death and survival programs (33, 34). The role of MMPs in tissue remodeling and therefore, cell death and survival, after physical stress may be associated with the MAP kinase pathways as well. Unfortunately, the available NFkB assays were not compatible with ovine tissues and therefore, we were unable to evaluate the interaction between HSP-70 and NFkB.
The interactions between HSP-70 and MMPs, and their relationship to airway trauma, have not been fully elucidated. Our data indicates that HSP-70 is secreted by tracheal tissues in response to injury and may play a role in regulating the response to airway injury. The significant and early response of trauma-related and tissue-modeling proteins associated with mechanical ventilator induced injury of the upper airway has important clinical and further research implications. Ongoing and future studies are aimed at further evaluating the interaction between trauma-response mediators, tissue proliferation pathways and airway remodeling.
We would like to acknowledge Barbara E. Gray, BA, CPM, Administrative Manager, Nemours Research Lung Center, for her contribution to the research effort.
This work was performed at the Alfred I. duPont Hospital for Children of the Nemours Foundation.
Financial Support: This study was funded through Nemours, an NIH COBRE grant 1 P20 RR020173-01 (The Center for Pediatric Research).
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