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Am J Physiol Lung Cell Mol Physiol 294: L3-L14, 2008. First published October 12, 2007; doi:10.1152/ajplung.00362.2007
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EDITORIAL FOCUS

Mechanical ventilation uncouples synthesis and assembly of elastin and increases apoptosis in lungs of newborn mice.

Prelude to defective alveolar septation during lung development?

Richard D. Bland, Robert Ertsey, Lucia M. Mokres, Liwen Xu, Berit E. Jacobson, Shu Jiang, Cristina M. Alvira, Marlene Rabinovitch, Eric S. Shinwell, and Anjali Dixit

Department of Pediatrics, Stanford University School of Medicine, Stanford, California

Submitted 4 September 2007 ; accepted in final form 8 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Prolonged mechanical ventilation (MV) with O2-rich gas inhibits lung growth and causes excess, disordered accumulation of lung elastin in preterm infants, often resulting in chronic lung disease (CLD). Using newborn mice, in which alveolarization occurs postnatally, we designed studies to determine how MV with either 40% O2 or air might lead to dysregulated elastin production and impaired lung septation. MV of newborn mice for 8 h with either 40% O2 or air increased lung mRNA for tropoelastin and lysyl oxidase, relative to unventilated controls, without increasing lung expression of genes that regulate elastic fiber assembly (lysyl oxidase-like-1, fibrillin-1, fibrillin-2, fibulin-5, emilin-1). Serine elastase activity in lung increased fourfold after MV with 40% O2, but not with air. We then extended MV with 40% O2 to 24 h and found that lung content of tropoelastin protein doubled, whereas lung content of elastin assembly proteins did not change (lysyl oxidases, fibrillins) or decreased (fibulin-5, emilin-1). Quantitative image analysis of lung sections showed that elastic fiber density increased by 50% after MV for 24 h, with elastin distributed throughout the walls of air spaces, rather than at septal tips, as in control lungs. Dysregulation of elastin was associated with a threefold increase in lung cell apoptosis (TUNEL and caspase-3 assays), which might account for the increased air space size previously reported in this model. Our findings of increased elastin synthesis, coupled with increased elastase activity and reduced lung abundance of proteins that regulate elastic fiber assembly, could explain altered lung elastin deposition, increased apoptosis, and defective septation, as observed in CLD.

lung growth and development; bronchopulmonary dysplasia; neonatal chronic lung disease; tropoelastin; lysyl oxidases; fibrillins; fibulin-5; emilin-1; serine elastase activity; lung cell apoptosis; alveolar septation

NEONATAL CHRONIC LUNG DISEASE (CLD), a variant of what Northway et al. (47) called bronchopulmonary dysplasia (BPD), typically develops from lung injury after premature birth and lengthy mechanical ventilation (MV) with O2-rich gas. Impaired alveolar formation and excess, disordered lung elastin are prominent histological features of this disease. How MV contributes to these structural changes and whether or not there is a link between dysregulated elastin production and defective lung growth, as described in CLD, remains unclear.

Elastin plays a pivotal role in mammalian lung development. Elastic fibers contribute to the structural integrity and distensibility of airways, alveoli, blood vessels, and extracellular matrix. Mutant mice lacking the elastin gene die soon after birth from cardiorespiratory failure associated with smooth muscle overgrowth in pulmonary arteries, defective airway branching, and lack of lung septation to form alveoli (34, 59). Mice lacking a single allele of the elastin gene exhibit increased susceptibility to smoke-induced emphysema, and mice with a two-thirds reduction in elastin protein display failed alveolar formation during early postnatal life (54).

Several groups have reported an association between excess, disordered elastin and defective development of alveoli and microvessels in lungs of premature infants who have died with CLD (16, 25, 37, 55). Urinary excretion of desmosine, a biomarker of elastin degradation, increased during the first week of MV in infants with evolving CLD (16). Breakdown of lung elastin in this disease has been attributed to inflammation associated with infection and exposure to increased O2 (15). Lungs of infants who died with CLD displayed thickened, tortuous, and irregularly distributed elastic fibers in the walls of distal air spaces, which was associated with reduced septation and fewer alveoli than in lungs of infants who died without CLD (37, 55).

In an ovine model of CLD, there were fewer alveoli and microvessels and more elastic fibers in lungs of preterm compared with term lambs that had received MV for 3 wk (1, 9, 10, 51). Abnormal abundance and distribution of elastin was particularly prominent in blunted secondary crests, where focal deposits of distally situated elastin normally define loci of future alveoli during lung development. Using lung tissue harvested from these lambs, we discovered reduced lung expression of growth factors that regulate lung septation (VEGF-A and PDGF-A and their receptors VEGF-R2 and PDGF-R{alpha}) and increased lung expression of growth factors that regulate elastin production [transforming growth factor (TGF)-{alpha} and TGF-β1] when compared with lung abundance of these proteins in unventilated control lambs that were studied at the same postconceptional age (12). In addition, there was differential expression of matrix proteins that regulate elastin synthesis and assembly and increased serine elastase activity in lung tissue of lambs with CLD. We speculated that these molecular and biochemical changes in the lungs might account for the aforementioned structural defects observed in this animal model of CLD. The complex nature of this experimental model, however, made it difficult to determine the specific role of MV, as distinguished from hyperoxia, pulmonary inflammation, recurrent infection, and marginal nutrition, in producing the pathology reported in this disease.

We therefore designed studies to test the hypothesis that prolonged cyclic stretch of the developing lung, with or without moderate hyperoxia, would adversely affect lung expression of genes and proteins that regulate alveolarization and elastin synthesis and assembly. We studied newborn mice, in which lung septation, angiogenesis, and matrix organization occur mainly after birth at term gestation (5). The initial report of this work described the effects of MV on lung expression of genes that regulate alveolar septation and the resulting structural impact of MV with 40% O2 for 24 h (11). Here we present the effects of MV for 8 h with either air or 40% O2 on lung expression of genes that regulate elastin synthesis and assembly and on lung elastase activity. In addition, we describe the impact of these changes on the amount and distribution of elastin in the lungs, and on apoptosis, which could contribute to the abnormal morphology of distal air spaces noted after MV of newborn mice with 40% O2 for 24 h (11).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Experiments

We used healthy Balb/C mice that were 2–4 days old and weighed 1.5–4 g after spontaneous vaginal delivery at term gestation. We did two sets of experiments. In the first series of studies, mouse pups received positive-pressure MV at either 60 or 180 breaths/min (bpm) with either 40% O2 or air for 8 h; unventilated littermates that spontaneously breathed either 40% O2 or air, respectively, for 8 h served as controls. In the second set of studies, 4-day-old mice had MV with 40% O2 at 180 bpm for 24 h; unventilated littermates that breathed 40% O2 for 24 h were used as controls. All surgical and animal care procedures and experimental protocols were reviewed and approved by Stanford University's Institutional Animal Care and Use Committee.

8-Hour Studies

We used six groups of mice that were 2–4 days old and weighed 2.4 ± 0.4 g. Four groups of pups (n = 8–10/group) had a sterile tracheostomy after anesthesia with intramuscular (im) ketamine [~75 µg/g body wt (BW)] and xylazine (~15 µg/g BW) in preparation for MV. Two groups of control pups (n = 8–10/group) also received ketamine-xylazine anesthesia for a small superficial neck incision before breathing either air or 40% O2 without MV. Pups that were randomly chosen to receive MV had a midline neck incision done under a dissecting microscope, followed by a tracheal slit and insertion of a custom-made polyvinyl catheter (intratracheal segment: inner diameter 0.28 mm, outer diameter 0.61 mm, length 1–2 mm; Tygon Tubing, Akron, OH) that was sutured into the trachea, as previously described (11). These pups received MV for 8 h with either air or 40% O2, delivered at a respirator rate of either 60 bpm using a custom-designed, time-cycled, pressure-limited rodent respirator (model 2000; Mallard Medical, Redding, CA) or at a rate of 180 bpm using a custom-made, piston-type small animal respirator (Microvent model 848; Hugo Sachs Elektronik, Harvard Apparatus, Holliston, MA). We used an inspired O2 concentration of 40% (FIO2 0.4) based on previous studies in which premature lambs with CLD required ~40% O2 to maintain a normal PaO2 (9). Our rationale for using one respirator rate that was slower and one that was faster than the normal breathing rate of newborn mice [110 bpm (43)] was to compare effects on the lung of different respiratory patterns incorporating different tidal volumes and inflation pressures. The ventilation circuit was configured as a loop of tubing into which a cut-off 20-gauge luer stub was inserted and connected to the tracheostomy, with a dead space volume of <5 µl. FIO2 and airway pressures were measured as previously described (11). Details of physiological measurements that were made during these studies are in the online supplement at the AJP-Lung web site.

Mice that received MV rested supine with their limbs gently taped to an underlying gauze pad, which was placed on an electric warming blanket, beneath a radiant lamp that kept ambient temperature at ~30–33°C. The pups were covered with a sheet of clear plastic to reduce heat and water losses across the skin. They were observed continuously, with close monitoring of chest movement, skin color, temperature, and response to tactile stimulation. In several studies, heart rate and systolic blood pressure were monitored at frequent intervals using a tail-cuff microsensor device (model MK-2000A; Muromachi Kikai, Tokyo, Japan) as previously described (11). Pups received additional anesthesia, 10 µg/g BW ketamine, and 2 µg/g BW xylazine, as needed for agitation, which was detected by body movement or an exaggerated response to tactile stimulation. Unventilated control pups were kept in a warm plastic chamber and breathed either air or 40% O2 that flowed into the chamber. Control and ventilated pups appeared to have milk in their stomachs at the start of studies and therefore did not receive feedings during the 8-h studies. At the end of each study, the pups received pentobarbital intraperitoneally, 100–150 µg/g BW, before the chest was opened to remove the lungs.

24-Hour Studies

We used two groups of mice that were 4 days old and weighed 1.9–4.5 g (3.0 ± 0.5 g, mean ± SD). One group of pups (n = 20) had a sterile tracheostomy after im anesthesia with ketamine (~75 µg/g BW) and xylazine (~15 µg/g BW), as described above, in preparation for MV with 40% O2 at 180 bpm for 24 h. In addition, a polyvinyl catheter (inner diameter 0.28 mm, outer diameter 0.61 mm, length 5 cm; Tygon Tubing) was inserted gently through the mouth into the stomach for subsequent feedings. Repeat doses of ketamine (10–20 µg/g BW) and xylazine (2–4 µg/g BW) were injected im, as needed, to prevent discomfort. During MV, pups were fed rodent milk replacer (KMR; PetAg, Hampshire, IL) ~30–40 µl every 2–3 h, which provided a daily fluid intake of ~100–120 µg/g, and a caloric intake of ~70–80 kcal/g. Care of these mice during MV, and ventilator settings applied in these 24-h studies, were similar to those used during the 8-h studies of pups that had MV at 180 bpm. Control pups (n = 16) received im ketamine-xylazine anesthesia for a superficial neck incision (sham tracheostomy), after which they breathed 40% O2 for 24 h in a warm plastic chamber, except for a 4-h period during the second 12 h in which they were kept with the dam in a larger plastic chamber with continuous flow of 40% O2, thus enabling them to feed without a change of FIO2. All pups received im antibiotics, 200 µg/g BW of ampicillin, and 4 µg/g BW of gentamicin, to reduce the risk of infection.

Postmortem Studies

RNA extraction and quantitative real-time PCR. At the end of several 8-h studies (n = 6/group), lungs were excised, frozen in liquid N2, and stored at –80°C for subsequent two-step mRNA extraction using TRIzol reagent (Invitrogen, Carlsbad, CA), and purification was with RNeasy Mini Kit columns (Qiagen, Valencia, CA), as previously described (12). Quantitative real-time PCR, using proprietary primers and probes (Taqman Gene Expression Assays; Applied Biosystems, Foster City, CA), was applied to measure lung mRNA expression of matrix proteins known to regulate elastin synthesis and assembly (tropoelastin, lysyl oxidase, lysyl oxidase-like-1, fibrillin-1, fibrillin-2, fibulin-5, and emilin-1). 18S ribosomal RNA was used as an internal control.

Protein Extraction and Western Immunoblots

At the end of several 24-h studies (4–8/group), lungs were excised, frozen in liquid N2, and stored at –80°C for later measurement of extracellular matrix proteins. Details of the protein extraction procedure were reported previously (12) and are described in the online supplement.

Immunoblot analysis was applied to measure lung proteins as follows: tropoelastin, using a 1:500 dilution of rabbit polyclonal antibody (generous gift of R. Mecham, Washington Univ., St. Louis, MO); fibrillin-1, using a 1:200 rabbit polyclonal antibody (from R. Mecham); fibrillin-2, using a 1:300 dilution of rabbit polyclonal antibody (from R. Mecham); fibulin-5, using a 1:500 dilution of rabbit polyclonal fibulin-5 antibody (from R. Mecham); lysyl oxidase-like-1, using a 1:250 dilution of rabbit polyclonal lysyl oxidase antibody and lysyl oxidase, using a 1:500 dilution of rabbit polyclonal antibody (generous gifts of K. Fong and K. Csiszar, Univ. of Hawaii, Honolulu); and emilin-1, using a 1:1,000 dilution of rabbit polyclonal antibody (generous gift of D. Forrest, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). Visualization of bound antibodies was performed using a 1:5,000 dilution of one of two secondary antibodies conjugated to horseradish peroxidase, either goat anti-rabbit IgG (sc-2301, Santa Cruz) or donkey anti-rabbit IgG (sc-2305, Santa Cruz). The membranes were stripped and reprobed with a 1:8,000 dilution of rabbit polyclonal anti-β-actin antibody (ab-8227; Abcam, Cambridge, MA) and incubated at room temperature for 1 h to provide an internal loading control.

Serine Elastase Activity

Lung tissue was frozen in liquid N2 and stored at –80°C for later measurement of serine elastase activity by a previously described method (12, 57, 64) that uses DQ-elastin substrate according to instructions provided by the manufacturer (Molecular Probes, Eugene, OR). Details of this method are included in the online supplement.

Processing of Lungs for Quantitative Histology

To obtain lungs for histopathology at the end of 24-h studies, pups were euthanized with pentobarbital, and the diaphragm was punctured via the abdomen to permit lung expansion during instillation of fixative via the tracheostomy tube. For unventilated control pups, a tracheostomy and diaphragm opening were made after death so that the lungs could be filled with fixative. Carnoy's solution was instilled to fix the lungs for assessment of elastin content and distribution. PBS-buffered 4% paraformaldehyde (PFA) solution was used to fix the lungs for immunohistochemistry (IHC). After the lungs were filled with fixative, the tracheostomy tube was connected through a plastic catheter to a column of freshly prepared fixative solution at a pressure of 20 cmH2O, which was maintained for 30 min at room temperature for fixation with Carnoy's solution, or overnight at 4°C for fixation with 4% PFA. The trachea was ligated, and the chest was opened to excise the fixed lungs, which were transferred to a vial of 70% ethanol before tissue embedding and sectioning.

Fixed lungs were embedded in paraffin for quantitative image analysis of elastin and IHC, as described below. Details of the random lung sectioning procedure were reported previously (11) and are described in the online supplement. To assess the amount and distribution of lung elastin, tissue sections were stained with Hart's elastin stain.

Lung Content and Distribution of Insoluble Elastin

The relative amount and distribution of insoluble elastin in lung was assessed by automated video thresholding of stain color (Hart's elastin stain) using the Bioquant True Color Windows Image Analysis system (R & M Biometrics, Nashville, TN) as previously described (12). Additional details of this assay are in the online supplement.

Immunohistochemical Localization of Fibulin-5 Protein

Standard IHC techniques, as previously described (12), were applied for semiquantitative assessment and localization of fibulin-5 protein in lungs of newborn mice that received MV with 40% O2 for 24 h compared with controls. Details of these methods are in the online supplement.

Assessment of Programmed Cell Death and Cell Proliferation in Lung

Apoptosis was detected and quantified with the TUNEL assay using the ApopTag In Situ Apoptosis Detection kit (Chemicon International, Temecula, CA) applied to PFA-fixed, paraffin-embedded lung tissue sections according to the manufacturer's instructions, as previously described (4). Stained cells were counted on 10 random sections of lung from each mouse without knowledge of the group of mice from which the tissue was taken. The number of stained cells was expressed as a percentage of the total number of cells counted in the 10 tissue sections.

Immunoblot analysis was used to quantify the amount of active caspase-3 protein in lung as a second and more specific way of assessing apoptosis in newborn mice. Details of this method are described in the online supplement.

Cell proliferation was detected and quantified by immunostaining for the cell cycle-specific marker, proliferating cell nuclear antigen (PCNA; clone PC-10, Dako, Carpinteria, CA), by a modification of a previously described method (7), details of which are in the online supplement.

Statistical Analysis of Data

Data in the text, table, and figures are expressed as means ± SD. To compare datasets that displayed a normal Gaussian distribution in studies that included two groups of mice (i.e., control vs. MV for 24 h), we used Student's unpaired t-test to assess for significant differences between groups. For datasets that had a skewed non-Gaussian distribution, we applied the non-parametric Mann-Whitney test to assess for significant differences. We used one-way analysis of variance and Student-Newman-Keuls multiple comparison tests to identify differences in mRNA expression in lungs of unventilated control pups that breathed either air or 40% O2 and mechanically ventilated pups that breathed either air or 40% O2. For datasets that exhibited marked differences in variability between multiple groups, we applied the non-parametric Kruskal-Wallis test with Dunn's post hoc analysis to assess for significant differences between groups (66). Statistical analysis was done using the Prism 4 software package (GraphPad, San Diego, CA). Differences were considered statistically significant if the P value was <0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Short-Term Studies: Eight Hours

Table 1 lists summary data for airway pressures that were assessed in the 8-h studies. Some of these measurements were reported previously in a paper that described the effects of MV on lung expression of genes and proteins that regulate alveolar septation in newborn mice (11). As expected, peak inflation pressure was greater when MV was delivered at 60 bpm than at 180 bpm. Mean airway pressure, however, was similar for the two respiratory patterns, as the relative duration of inspiration compared with expiration averaged 1:2.3 in the 60-bpm studies, whereas this ratio was 1:1.2 in the 180-bpm studies. Airway pressures were similar in pups that received MV with air compared with 40% O2. Positive end-expiratory pressure (PEEP) was kept close to atmospheric pressure after pilot studies showed that elevating PEEP to >1 cmH2O led to rapid onset of cyanosis, hepatomegaly, and death from circulatory failure.


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  		Table 1. Respiratory data for newborn mice that received mechanical ventilation with either air or 40% O2 for 8 h at 60 or 180 breaths/min (bpm)

 
As reported previously (11), mice that were mechanically ventilated for 8 h exhibited physiological stability, as assessed by noninvasive measurements of heart rate (523 ± 47 beats/min vs. 521 ± 42 beats/min in unventilated control pups) and systolic blood pressure (41 ± 13 mmHg vs. 47 ± 5 mmHg in unventilated controls) and by terminal measurements of heart blood pH (7.30 ± 0.12) and PCO2 (37 ± 11 mmHg). There were no significant differences in cardiovascular or respiratory gas variables between animals that breathed air compared with 40% O2 or between pups that had MV at 60 bpm compared with 180 bpm (data not shown).

Lung mRNA Expression of Elastin-Related Genes in Mice From 8-h Studies

Quantitative RT-PCR was applied to measure lung mRNA expression of several genes that are important in elastin synthesis and assembly, including tropoelastin, lysyl oxidase, lysyl oxidase-like-1, fibrillin-1, fibrillin-2, fibulin-5, and emilin-1. Expression patterns for most of these genes were virtually identical between groups that breathed 40% O2 compared with those that breathed air (Fig. 1). Lung mRNA for tropoelastin (Fig. 1A) and lysyl oxidase (Fig. 1B) increased significantly after 8 h of MV with 40% O2 or air. There was no significant change in fibrillin-1 (Fig. 1D) or fibulin-5 (Fig. 1F) mRNA after 8 h of MV with 40% O2 or air. Lung mRNA for lysyl oxidase-like-1 (Fig. 1C), fibrillin-2 (Fig. 1E), and emilin-1 (Fig. 1G) decreased after 8 h of MV with either 40% O2 or air. Except for emilin-1, mRNA expression of each elastin-related gene was similar in lungs of control pups that breathed 40% O2 compared with those that breathed air. Emilin-1 mRNA was greater in lungs of mice that breathed 40% O2 than in pups that breathed air for 8 h.


Figure 1
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  		Fig. 1. Lung mRNA expression of genes that regulate elastin synthesis and assembly. Tropoelastin (A), lysyl oxidase (B), lysyl oxidase-like-1 (LOX-L1; C), fibrillin-1 (D), fibrillin-2 (E), fibulin-5 (F), and emilin-1 (G) all expressed relative to 18S rRNA in 2- to 4-day-old mice (n = 6–10 pups/group) that received mechanical ventilation (MV) with either 40% O2 or air at 60 or 180 bpm for 8 h compared with unventilated control pups that breathed either 40% O2 or air for 8 h. Values are means and SD. *Significant difference compared with the relevant control group, P < 0.05. {dagger}Significant difference between air-breathing control group and 40% O2-breathing control group, P < 0.05.

 
Serine Elastase Activity in Lungs of Mice From 8-h Studies

As elastase activity is increased in the lungs of mechanically ventilated preterm infants and lambs with evolving CLD (12, 41, 49), we measured serine elastase activity in the lungs of newborn mice after 8 h of MV at 180 bpm with either air or 40% O2 compared with unventilated control pups that spontaneously breathed either air or 40% O2 for 8 h. Although there was not a significant increase in elastase activity measured in the lungs of mice that received MV with air, there was a fourfold increase of serine elastase activity in lungs of mice that received MV with 40% O2 for 8 h (Fig. 2).


Figure 2
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  		Fig. 2. Serine elastase activity measured in lungs of 3- to 4-day-old mice (n = 4 pups/group) that received MV with either 40% O2 or air for 8 h compared with control pups that spontaneously breathed either air or 40% O2. For this series of studies, we used pups that received MV at 180 bpm whose 8-h survival was nearly 100% (compared with ~60% for pups that had MV at 60 bpm). Results (means and SD) are expressed relative to the unventilated control group that breathed either 40% O2 or air. *Significant difference compared with the relevant control group, P < 0.05.

 
Long-Term Studies: Twenty-Four Hours

Lung content of proteins that impact elastic fiber formation. As MV at 60 and 180 bpm for 8 h had virtually identical effects on lung mRNA expression of elastin-related genes, and 8-h survival was nearly 100% when MV was applied at 180 bpm, compared with ~60% for MV at 60 bpm, we used mice that had MV for 24 h at 180 bpm to assess lung content of elastin-related proteins. Because serine elastase activity increased only after MV with 40% O2, and not with air, these 24-h studies were done with pups that breathed 40% O2, with or without MV.

The effects of prolonged MV with O2-rich gas on lung content of proteins involved in elastin synthesis and assembly were variable. MV with 40% O2 for 24 h doubled the amount of tropoelastin protein in lung (Fig. 3A) without increasing lysyl oxidase or lysyl oxidase-like-1 proteins (Fig. 3B). Lung content of fibrillin-1 and fibrillin-2 proteins also did not change after MV with 40% O2 for 24 h (Fig. 3C), whereas lung content of fibulin-5 protein, which has been shown to play a critical role in assembly of lung elastin (45, 62), decreased in MV pups compared with controls (Fig. 3D). IHC confirmed the reduction of fibulin-5 protein in lung that was associated with prolonged MV (Fig. 4). Lung content of emilin-1, a matrix protein that spans the interface between amorphous elastin and microfibrils in arteries and in developing lung, where it binds elastin and fibulin-5 (13, 14, 65), was reduced in mice after 24 h of MV with 40% O2 (Fig. 3D).


Figure 3
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  		Fig. 3. Abundance of elastin-related proteins measured by immunoblot relative to β-actin protein in lungs of 5-day-old mice (n = 4–8 pups/group) that received MV with 40% O2 at 180 bpm for 24 h compared with unventilated controls that breathed 40% O2 for 24 h. Measured proteins: tropoelastin (A), lysyl oxidase and lysyl oxidase-like-1 (B), fibrillin-1 and fibrillin-2 (C), and fibulin-5 and emilin-1 (D). Values are means and SD. *Significant difference compared with the control group, P < 0.05.

 

Figure 4
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  		Fig. 4. Immunohistochemical (brown) staining for fibulin-5 protein (arrows) in sections of lung obtained from 5-day-old mice after breathing 40% O2 for 24 h either without MV (control, left) or with MV at 180 bpm (right). Decreased stain in the walls of distal air spaces after MV verifies that lung expression of fibulin-5, which has a pivotal role in elastic fiber assembly, was reduced after exposure to prolonged cyclic stretch with O2-rich gas.

 
Lung Elastin Accumulation After Prolonged MV

Figure 5 shows representative images of tissue sections that were stained to detect elastin in lungs of mice that breathed 40% O2 for 24 h with or without MV. Elastin was expressed mainly at the tips of alveolar septa in lungs of unventilated control pups, whereas elastic fibers were prominent throughout the walls of distal respiratory units in the lungs of animals that had received MV with O2-rich gas for 24 h. Quantitative image analysis demonstrated that elastin accumulation in the lungs was ~50% greater in pups that received MV compared with unventilated controls (elastin fiber density, as % lung parenchyma: MV = 10.1 ± 2.3%, n = 6; control = 6.7 ± 0.8%, n = 6; significant difference, P < 0.05).


Figure 5
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  		Fig. 5. Images of Hart's-stained lung tissue taken from 5-day-old mice after breathing 40% O2 for 24 h, either with (right) or without (left) MV at 180 bpm. Lung elastin accumulation (arrows, pointing to dark elastic fiber staining) was distinctly greater in the mice that received MV: quantitative image analysis showed that elastin density, expressed as a percentage of lung parenchyma, averaged 10.1 ± 2.3% in the group that received MV compared with 6.7 ± 0.8% in the unventilated control group (significant difference, P < 0.05). Note that elastin is expressed mainly at the tips of septa in the unventilated control lung, whereas elastic fibers are prominent throughout the walls of distal air spaces in the lung that was exposed to MV for 24 h.

 
Lung Cell Apoptosis and Proliferation Assays

We previously reported that MV of newborn mice with 40% O2 for 24 h nearly doubled air space area without altering total lung volume, findings that are indicative of diminished lung septation (11). To determine if these structural changes in the lung that occurred after lengthy MV might reflect differences in lung cell apoptosis or proliferation resulting from cyclic lung stretch, we assessed TUNEL staining in lung sections, some of which were obtained from the earlier study (11), to detect apoptosis, and PCNA staining of cells in sections of lung taken from pups that breathed 40% O2 for 24 h with or without MV. We also measured active caspase-3 protein in lung tissue obtained from pups that had MV for 24 h compared with unventilated control pups that breathed 40% O2 for 24 h. Apoptosis was greater in the group that received MV compared with controls (Fig. 6, A and B, and Fig. 7), whereas cell proliferation was not significantly different between the two groups of mice (Fig. 6A).


Figure 6
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  		Fig. 6. A: TUNEL stain assay for apoptosis in lung tissue sections showed significantly more apoptotic cells in lungs of newborn mice (n = 15) that received MV with 40% O2 for 24 h compared with control pups (n = 11) that breathed 40% O2 without MV for 24 h. Proliferating cell nuclear antigen (PCNA) staining for proliferating cell nuclei did not show a significant difference between pups that received MV with 40% O2 for 24 h (n = 16) compared with unventilated control pups (n = 7). Some tissue sections used for these assays came from an earlier study (11), which included newborn mice that had MV at either 60 or 180 bpm for 24 h. B: lung abundance of active caspase-3 protein, relative to β-actin, in newborn mice that had MV with 40% O2 at 180 bpm for 24 h compared with unventilated controls that spontaneously breathed 40% O2 for 24 h. Values are means and SD. *Significant difference compared with unventilated control group, P < 0.05.

 

Figure 7
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  		Fig. 7. TUNEL staining of lung tissue sections showed that most of the apoptotic cells (arrows) were located in the walls of distal air spaces. Staining was most apparent in epithelial cells, with some staining of what appeared to be pulmonary macrophages within the air spaces.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Neonatal Mouse Model To Study the Impact of MV on Lung Development

This report on MV of newborn mice for up to 24 h establishes a new experimental model that can be used to help define mechanisms of disordered alveolar septation and angiogenesis observed in neonatal CLD. Here we focus specifically on the putative link between prolonged cyclic stretch of the lung during development and abnormal elastin deposition in the walls of distal air spaces.

Gene Regulation of Elastin Synthesis and Assembly in the Developing Lung

Elastin plays a critical role in the formation of alveoli and blood vessels during lung development (38). Elastin-null mice do not survive beyond the immediate postnatal period because of cardiovascular and respiratory failure associated with overgrowth of arterial smooth muscle, defective airway branching, and lack of alveolar septation (34, 59). Mutations in several genes that are known to affect elastin synthesis and assembly, including fibulin-5, fibrillin-1, lysyl oxidase, and lysyl oxidase-like-1 yield offspring that exhibit abnormal lung development, often leading to pulmonary pathology that resembles emphysema in those mice that survive beyond the perinatal period (27, 35, 36, 45, 46, 62).

Based on earlier studies that showed abnormal accumulation of elastin in lungs of preterm lambs with CLD, in which there was discordant expression of elastin-related genes (12, 51), we tested the hypothesis that MV of newborn mice with O2-rich gas would induce changes in lung expression of genes that affect elastin synthesis and assembly, which in turn might lead to abnormal elastin deposition, and perhaps contribute to impaired lung septation. The specific way that abnormal elastin production contributes to failed alveolar and lung vascular formation in CLD is unclear, but there is much evidence that cyclic stretch can induce tropoelastin gene expression in the developing lung, which in turn may yield increased elastin accumulation (22, 28, 44). It was therefore not surprising that lung expression of tropoelastin mRNA increased after 8 h of MV with either air or 40% O2 and that an extended period of MV with O2-rich gas led to an increase in lung elastin protein. Less predictable were the discordant expression patterns of genes and proteins that impact elastin assembly in lungs of MV mice compared with controls. It is notable that lung expression of genes that regulate elastin assembly either did not change (fibrillin-1 and fibulin-5) or decreased (lysyl oxidase-like-1, fibrillin-2, and emilin-1) after MV for 8 h and that lung content of two key elastin assembly proteins, fibulin-5 and emilin-1, decreased after MV for 24 h, as tropoelastin mRNA and elastin fiber density increased in the lungs of these newborn mice. These findings offer a plausible explanation for the abnormal abundance and distribution of elastin in the lungs after prolonged MV with 40% O2. Deficiencies of fibulin-5, lysyl oxidase, and lysyl oxidase-like-1 result in abnormal elastin assembly associated with defective lung septation and emphysema in mutant mice (27, 35, 36, 45, 62). Mice that are deficient in emilin-1, which is expressed in lung during fetal development (13), exhibit structural defects of elastic fibers in aorta and skin (65). Thus, the observed reduction in fibulin-5 and emilin-1 proteins in lungs of newborn mice after lengthy MV with O2-rich gas may have contributed to the air space enlargement that was apparent after 24 h of MV.

Serine Elastase Activity in Lungs of Mice After 8 h of MV

Several studies have demonstrated inflammation and increased proteolytic activity in lungs of infants with evolving CLD (21, 41, 49, 58) and in animal models of this disease (3, 18, 63). In previous studies of preterm lambs that received MV with O2-rich gas for 3 wk, serine elastase activity increased during the initial phase of CLD, and this was associated with subsequent accumulation of excess, disordered lung elastin (12). We therefore were not surprised to find that serine elastase activity increased on average by about 400% in newborn mice after 8 h of MV with O2-rich gas. It is noteworthy, however, that there was little or no inflammation noted in the lungs of these mice after MV with either air or 40% O2 for up to 24 h (11). This suggests that increased lung elastase activity in the mice may have come from lung parenchymal cells, perhaps smooth muscle cells or fibroblasts, for which there is evidence of inducible serine elastase activity (31, 32, 48, 53, 56, 60, 67). The association of increased proteolytic activity and later accumulation of widely dispersed elastic fibers in the lung begs the question: does early degradation of elastin-related proteins contribute to impaired tissue remodeling and disordered elastin deposition?

Apoptosis and Failure of Lung Septation

There are several possible mechanisms by which MV might have induced apoptosis in the lungs of our newborn mice. Conditions that may increase lung cell apoptosis include prolonged exposure to cyclic stretch, moderate hyperoxia, and the direct or indirect effects of impaired elastin assembly and heightened elastin degradation. In vitro studies of both fetal and adult rat type II lung epithelial cells exposed to mechanical stretch for up to 24 h showed a significant increase in apoptosis (24, 33). A recent report showed that angiotensin converting enzyme inhibitors prevented stretch-induced apoptosis of adult rat lung epithelial cells through a mechanism that involves bradykinin-mediated stimulation of the phosphoinositol 3-OH-kinase-Akt-Bcl-2/Bcl-XL pathway, which helps to regulate cell survival (23). The increased apoptosis reported in cultured fetal rat lung epithelial cells after 20% cyclic stretch for 24 h was attributed to release of the proinflammatory cytokine IL-8 and reduced production of the anti-inflammatory cytokine IL-10 (33). Another report described increased apoptosis and reduced cell proliferation of cultured fetal rat lung fibroblasts in response to cyclic mechanical stretch for up to 24 h (52). These ex vivo studies need to be tested in living animals to determine if this putative mechanism of stretch-induced apoptosis applies to the mammalian lung exposed to prolonged positive-pressure MV.

Several studies have shown that hyperoxia can induce apoptosis of lung epithelial cells, which in turn may contribute to the impaired alveolar septation observed after prolonged breathing of >80% O2 in newborn mice (8, 39, 40). A recent report indicated that lung epithelial cell apoptosis in newborn mice, induced by continuous exposure to 85% O2 for 28 days, may be mediated through TGF-β and bone morphogenic protein signaling, resulting in arrested lung growth and pathology similar to BPD (2). In our studies, however, O2 exposure was limited to 40% for 24 h, during which MV led to a threefold increase in apoptosis and a corresponding increase in air space size, consistent with impaired septation.

As prolonged MV with O2-rich gas can stimulate release of TGF-β, as described in a recent report of dysregulated elastin production in preterm lambs with CLD (12), it is possible that activation of TGF-β during lengthy MV of newborn mice could have contributed to the observed increase in lung cell apoptosis and abnormal elastin deposition. Such a mechanism has been invoked to explain the pulmonary emphysema and defective elastin assembly observed in fibrillin-1 null mice, in which there is dysregulated activation and signaling of TGF-β, resulting in apoptosis and impaired alveolar septation (46).

Increased elastase activity associated with MV also might cause lung cell apoptosis, as there is abundant evidence that elastin degradation, both in human emphysema and in murine models of emphysema, releases elastin fragments that can induce apoptosis and lead to air space enlargement (6, 20, 26). Reports that serine elastase inhibitors, including {alpha}1-antitrypsin, prevent lung cell apoptosis (50) and help to ameliorate cigarette smoke-induced emphysema in mice (17, 61) support the notion that increased elastase activity may have contributed to the lung cell apoptosis and air space enlargement noted in newborn mice after MV with 40% O2 for 24 h.

Working Model of Dysregulated Synthesis and Assembly of Lung Elastin From MV of Newborn Mice

Figure 8 shows schematically how elastic fiber synthesis and assembly is thought to occur in the lung and the putative effects that our reported lung changes (defined in red) of gene and protein expression and elastase activity might have on elastic fiber formation in newborn mice that receive MV with O2-rich gas. Tropoelastin is secreted from lung myofibroblasts onto microfibril scaffolds that attach to the cell through the interaction of fibrillins, microfibril-associated glycoproteins, and integrin receptors on the cell membrane. Fibulin-5 and emilin-1 act as bridging molecules, helping to attach tropoelastin to the cell and adjacent microfibril by interacting with matrix proteins and integrins on the plasma membrane. Lysyl oxidase-like-1, which is secreted by fibroblasts and smooth muscle cells, helps bind tropoelastin and matrix proteins to the cell and also contributes to elastic fiber homeostasis, converting tropoelastin into a lysyl-deaminated form that enables covalent crosslinking of tropoelastin to form mature elastic fibers (19, 35). Lysyl oxidase, an extracellular copper-dependent enzyme that is secreted by fibroblasts and smooth muscle cells, oxidizes lysine residues in both elastin and collagen and thereby creates covalent cross-linkages that stabilize these fibrous proteins, which provide resilience and distensibility to the lung's respiratory units and blood vessels (29, 36).


Figure 8
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  		Fig. 8. Working model of excess, disordered elastin production in lungs of newborn mice that had MV with O2-rich gas for 24 h. This figure, redrawn and modified from Midwood and Schwarzbauer (42), depicts how elastic fiber synthesis and assembly is thought to occur in the lung during development and the putative effects on the formation of elastic fibers in lung that might ensue from changes in gene and protein expression and elastase activity seen in newborn mice exposed to lengthy MV. Tropoelastin is secreted from myofibroblasts and deposited on microfibril scaffolds that adhere to the cell surface through tight interaction of fibrillins, microfibril-associated glycoproteins, and integrin receptors on the cell membrane. Fibulin-5 and emilin-1 are bridging molecules that interact with matrix proteins and integrin receptors to help tether tropoelastin to the cell surface. Lysyl oxidase-like-1 and lysyl oxidase are enzymes that induce oxidative deamination of lysine residues contained in tropoelastin, thereby initiating covalent cross-linkages that are essential for producing stable, insoluble elastic fibers. Our working model suggests that prolonged cyclic stretch of the developing lung stimulates release of tropoelastin from myofibroblasts without a corresponding increase in matrix proteins that are critical for elastic fiber assembly. Increased tropoelastin production, without a corresponding increase in fibulin-5 and emilin-1, coupled with increased lung elastase activity induced by MV, could contribute to excess accumulation of poorly organized elastic fibers and associated failure of alveolar septation.

 
In our working model, tropoelastin secretion increases in lung myofibroblasts exposed to cyclic stretch, without a corresponding increase of matrix proteins that regulate elastin assembly. Lung content of two such proteins, fibulin-5 and emilin-1, decreases during lengthy MV. Because molecular interactions of tropoelastin and several extracellular matrix proteins, including fibulin-5, lysyl oxidase, and lysyl oxidase-like-1, are essential for normal elastic fiber formation (30, 35), discordant lung expression of these closely linked proteins could result in defective elastin assembly. These findings, coupled with increased elastolytic activity that occurs during MV with O2-rich gas, might yield increased production of poorly organized elastic fibers that contribute to the abnormal lung structure and function seen in neonatal CLD. Validation of this model awaits further inquiry on how mechanical forces applied to the lung, with or without hyperoxia or inflammation, may disrupt the tightly coordinated patterns of myofibroblast migration and elastin synthesis and assembly that are essential for normal lung septation and growth.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-62512 (R. D. Bland) and HL-56401, Specialized Center of Research Project V (R. D. Bland), and the Vera Moulton Wall Cardiopulmonary Research Center, Stanford University.


   ACKNOWLEDGMENTS
 
We thank Dr. Robert Mecham (Washington Univ., St. Louis, MO) for generously providing antibodies to tropoelastin, fibrillin-1, fibrillin-2, and fibulin-5; Dr. Douglas Forrest (National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases) for providing the emilin-1 antibody; and Drs. Katalin Csiszar and Keith S. K. Fong (Univ. of Hawaii, Honolulu) for providing antibodies to lysyl oxidase and lysyl oxidase-like 1.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. D. Bland, Stanford Univ. School of Medicine, CCSR Bldg., Rm. 1225, 269 Campus Drive, Stanford, CA 94305-5162 (e-mail: rbland{at}stanford.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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