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1 Department of Physiology, Monash University, Victoria 3800, Australia2 Cell & Matrix Biology Research Unit, Department of Pediatrics, University of Melbourne and Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, Victoria 3052, Australia3 Pulmonary and Critical Care Medicine, Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA
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(Received 8 October 2003;
accepted after revision 20 October 2003;
first published online 24 October 2003)
Corresponding author S. B. Hooper: Department of Physiology, Monash University, Victoria, 3800. Email: stuart.hooper{at}med.monash.edu.au
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Introduction |
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There is some evidence to indicate that prenatal corticosteroid treatments enhance lung development and improve survival of infants with LH. For example, antenatal dexamethasone treatment of fetal rats with a nitrofen-induced CDH enhances structural maturity of the lung (Suen et al. 1994a), resulting in increased lung compliance (Losty et al. 1995). Similarly, high doses of cortisol administered to fetal sheep with a surgically induced diaphragmatic hernia improve dynamic lung compliance, increase the protein/DNA ratio (Schnitzer et al. 1996) and enhance structural development of the lung (Hedrick et al. 1997). However, it is evident that the enhanced lung maturity may occur at the expense of lung growth (Suen et al. 1994b; IJsselstijn et al. 1997), alveolar development (Willet et al. 2001) and fetal body growth (Losty et al. 1995; IJsselstijn et al. 1997). Combined with other detrimental effects, including impaired neural development (Uno et al. 1990; Dunlop et al. 1997; Quinlivan et al. 2000), it is apparent that prenatal synthetic corticosteroid exposure may adversely affect both the fetus and the newborn and could cause some adverse effects that persist into adulthood.
Although synthetic corticosteroids can apparently enhance development of the hypoplastic fetal lung, the specific effects of corticosteroids and the mechanisms involved are largely unknown. Furthermore, it is not known whether the beneficial effects of corticosteroids on lung development can be dissociated from the detrimental effects on lung and body growth. Previous studies have indicated that cortisol infusions, designed to mimic the preparturient increase in fetal plasma cortisol concentrations without inducing labour, can enhance aspects of fetal lung development without adversely affecting fetal lung or body growth (Wallace et al. 1995). However, the specific effect of cortisol infusions, at doses that achieve increases in cortisol levels within a physiological range, on the structural development of the lung is unknown.
Our aims were to determine the biochemical and morphometric effects of physiological doses of cortisol on normal and hypoplastic lungs in fetal sheep and to investigate potential mechanisms that may mediate the cortisol-induced morphological changes in lung tissue. Severe LH in fetal sheep was induced by continuously draining the lungs of liquid, causing them to deflate (Alcorn et al. 1977; Moessinger et al. 1990; Nardo et al. 1995; Davey et al. 1999) which, in the absence of any intervention, causes lethal respiratory insufficiency after birth (Davey et al. 1999). In these fetuses, we examined the effect of a physiologically relevant cortisol infusion, which mimics the preparturient increase in fetal plasma cortisol concentrations (Wallace et al. 1995; Boland et al. 1997), on morphological aspects of fetal lung development as well as on the content and deposition of the key extracellular matrix (ECM) proteins, collagen and elastin. Cortisol-induced structural remodelling of the lung may involve altered deposition and/or metabolism of these major ECM structural proteins. Thus, we measured (1) collagen and elastin content and elastin deposition (owing to its association with septation and alveolarization) within lung parenchymal tissue and (2) the activities of the major gelatinases, MMP-2 and matrix metalloproteinase-9 (MMP-9). These gelatinases are known to be important in the degradation of a number of ECM components, including collagen.
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147 days) to implant fetal and maternal vascular catheters and fetal tracheal catheters to form an exteriorized tracheal loop (Hooper et al. 1993). Fetal well-being was monitored on a daily basis by measuring fetal arterial partial pressures of O2 and CO2 (Pa, O2, Pa, CO2), pH, arterial O2 saturation (Sa,O2) and haematocrit (Hct) (ABL30, Radiometer, Denmark). A minimum of 5 days was allowed for the ewe and fetus to recover from surgery before experiments began. All experimental and surgical procedures on animals were approved by the Monash University Animal Ethics Committee. Experimental protocol
Ewes and their fetuses were divided into four groups; (1) saline-only group, in which saline was infused into the fetus for 9 days (122–131 days of gestational age [GA], n= 5); (2) cortisol-only group, in which cortisol was infused into the fetus at increasing doses (see below) for 9 days (122–131 days GA, n= 5); (3) saline & drain group, in which fetal lung liquid was drained gravimetrically into a sterile bag for 20 days (111–131 days GA) and fetuses received saline infusions as in group 1 (n= 5); and (4) a cortisol & drain group, in which lung liquid was drained gravimetrically into a sterile bag for 20 days (111–131 days GA) and fetuses received cortisol infusions as in group 2 (n= 5). In all cortisol-infused fetuses, increasing doses of cortisol (Hydrocortisone sodium succinate, Solu Cortef, Upjohn Pty Ltd, Australia) were continuously infused into the fetal jugular vein over the 9-day infusion period; 1.5 mg day-1 on 122–123 days GA, 2.5 mg day-1 on 124–125 days GA, 3.0 mg day-1 on 126–127 days GA, 3.5 mg day-1 on 128–129 days GA and 4.0 mg day-1 on 130–131 days GA. This 9-day regimen replicates the preparturient increase in fetal plasma cortisol concentrations. The cortisol infusion was prepared each day in heparinized saline and was delivered at 1.2 ml h-1; saline-infused fetuses received heparinized saline at 1.2 ml h-1. Fetal blood samples (2 ml) were collected from all fetuses every second day to measure plasma cortisol concentrations. All infusions were continued until 131 days GA, when an autopsy was performed.
Just prior to autopsy, the fetal lungs were drained of liquid via the tracheal catheter and the tracheal loop was blocked before the ewe and fetus were killed by an overdose of sodium pentobarbitone administered to the ewe (130 mg kg-1I.V.). The fetus was weighed and the fetal lungs, kidneys, heart and liver were removed and weighed. The left bronchus was ligated, the left lung removed distal to the ligature and portions frozen in liquid nitrogen then stored at –70°C for subsequent biochemical analysis. The right lung was fixed via the trachea at 20 cmH2O with 4% paraformaldehyde.
Biochemical and histological methods
Cortisol concentrations in fetal plasma were measured by an established radioimmunoassay (Bocking et al. 1986). Portions of frozen lung tissue were used to measure total lung DNA, protein, elastin and hydoxyproline contents, as previously described (Hooper et al. 1993; Keramidaris et al. 1996; Joyce et al. 2003); fetal lung tissue was dissected using a stereo-microscope to remove all major airways and blood vessels before analysis (Joyce et al. 2003). Proline residues in fibrillar collagens are abundantly hydroxylated, compared with proline residues in other proteins and therefore hydroxyproline is used as an index of tissue collagen levels.
The mRNA levels for tropoelastin were measured by Northern blot analysis using the same procedures and ovine-specific cDNA probe that have previously been described (Joyce et al. 2003). To account for minor loading differences of total RNA between lanes, the density of each tropoelastin band was expressed as a ratio of the density of the 18S ribosomal RNA band for that sample.
The right lung volume was determined using the Cavalieri method (Nardo et al. 2000). Portions of the right lung were embedded in paraffin, cut at 5 µm and stained with haematoxylin and eosin. Stereological measurements of right lung tissue fraction and volume, luminal fraction and volume, alveolar number and interalveolar wall thickness were made using standard stereological techniques and grids as previously described (Nardo et al. 2000). Lung sections were viewed using light microscopy (40x) and projected on to a screen, giving a final magnification of 720x; this was determined by simultaneous projection of a 0.1-mm graticule. In addition, paraffin-embedded sections (5 µm) of lung tissue were stained with Hart's resorcin–fuchsin stain for elastin and counterstained with tartrazine (0.25%) in saturated picric acid. Sections were viewed under the light microscope and images captured using a digital camera.
Gelatin zymography was used to detect the presence of active and latent gelatinases in extracts of fetal lung (Heussen & Dowdle 1980). Lung samples (0.5 mg) were homogenized in 1 ml of buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, Tween-20), centrifuged at 1000 g for 10 min at 4°C, and the supernatants collected and stored frozen for analysis. The protein concentration of the lung extracts was measured and 15 µg of protein was loaded on to 7% SDS-polyacrylamide gels containing 0.5 mg ml-1 gelatin. Molecular weight markers and a positive control for gelatinases MMP-2 and MMP-9 (human fibrosarcoma cell conditioned medium; Stanton et al. 1998) were included on each gel. The gels were washed twice in 2.5% (v/v) Triton X-100 for 30 min, rinsed in water and then incubated overnight at 37°C in zymography buffer (100 mM Tris-HCl, pH 7.9, 30 mM CaCl2, 0.04% w/v sodium azide and 0.02% v/v Brij). Gelatin degrading activity was detected by staining with Coomassie blue (R-250) for 30 min and then destaining in 30% (v/v) methanol and 1% (v/v) acetic acid. The gel was incubated in 10% (v/v) glycerol overnight and dried between sheets of cellophane for storage. The dried gels were scanned, the images converted to greyscale images and the density of the lysis bands quantified using image analysis (ImageQuaNT, Molecular Dynamics). Each gel was repeated at least twice to ensure consistency of results. To demonstrate that the densities of the lysis bands were directly proportional to the amount of metalloproteinase activity, serial dilutions of lung extracts (75–2.4 µg of protein) were run on a separate gel. The densities of the lysis bands were directly proportional to the protein concentration of the sample (r= 0.999).
To confirm that the lysis bands resulted from metalloproteinase activity, separate gels were incubated in zymography buffer containing EDTA (60 mM), which blocks metalloproteinase activity. To confirm the identity of the latent and active forms of MMP-2, a lung extract was incubated at 37°C with the organomercurial activator APMA (2 mM), which caused a time-dependent activation of MMP-2.
Statistical analysis
The results are presented as mean ±S.E.M. The level of significance for all statistical analyses was P < 0.05. Differences in fetal body weights, lung and organ weights, DNA, protein, hydroxyproline and elastin contents, tropoelastin mRNA levels and right lung volumes were analysed by a one-way ANOVA. Significant differences between values were then identified with a Student–Newman–Kuels (SNK) test. Differences in fetal plasma cortisol concentrations and lung liquid drainage rates were analysed by a two-way ANOVA for repeated measures with treatment and gestational age as factors. If an interaction was identified, pairwise comparisons of data points were made using an SNK test. Differences in morphometrical measurements were analysed by nested one-way ANOVAs. When differences were found, they were identified by a least significant difference (LSD) test. Differences in gelatinase activity between samples electrophoresed on the same gel were compared using Student's unpaired t tests. For the gelatin zymography, five lung extracts from saline-only fetuses were electrophoresed on every gel, together with five lung extracts from either cortisol-only, saline & drain or cortisol & drain fetuses. This allowed comparisons to be made between each fetal group as experimental lung extracts electrophoresed on different gels were expressed as a percentage of the mean of the same control samples in each gel. Each group of lung extracts was analysed on duplicate gels. The differences between groups were then compared using a one-way ANOVA, followed by an LSD test.
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Fetal blood gases and body weights. All fetuses were considered healthy according to their arterial blood gas and acid–base status and these values were not significantly different between groups (data not shown). Similarly, fetal body weights were not different between any of the groups at the time of autopsy (Table 1).
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Fetal organ weights. Prolonged lung deflation, caused by lung liquid drainage, significantly reduced lung wet weights in saline & drain fetuses compared with saline-only fetuses. In lung-liquid-drained fetuses, the infusion of cortisol had no effect on lung wet weights, which were similar in saline & drain and cortisol & drain fetuses (Table 1). Similarly, in fetuses not exposed to prolonged lung deflation, the cortisol infusion did not significantly affect fetal lung weights.
Adjusted for body weight (g kg-1), the wet weights of the fetal liver and kidney were similar in all treatment groups, whereas the wet weight of the fetal heart was reduced in the cortisol-only group compared with the saline & drain and cortisol & drain groups of fetuses (Table 1).
Fetal lung DNA and protein content. Prolonged lung deflation significantly increased the DNA concentration of fetal lung tissue in both cortisol-infused and saline-infused fetuses, and the infusion of cortisol had no additional effect. The pulmonary DNA concentration was similar in cortisol & drain fetuses compared with saline & drain fetuses and was similar in cortisol-only fetuses compared with saline-only fetuses (Table 2). Although lung liquid drainage significantly reduced total lung DNA content, the infusion of cortisol had no additional effect. The total DNA content was similar in cortisol & drain fetuses compared with saline & drain fetuses and in cortisol-only fetuses compared with saline-only fetuses (Table 2). Similarly, prolonged lung deflation significantly reduced total lung protein content and the infusion of cortisol did not significantly affect this decrease; total lung protein content was similar in cortisol & drain fetuses compared with saline & drain fetuses and in cortisol-only compared with saline-only fetuses (Table 2).
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Fetal lung tropoelastin mRNA levels and elastin content. Prolonged lung deflation reduced tropoelastin mRNA levels in fetal lung tissue from 38.9 ± 5.7 in saline-only fetuses to 26.4 ± 1.4 in saline & drain fetuses (Fig. 3), although this reduction just failed to reach statistical significance (P= 0.06). The infusion of cortisol significantly increased tropoelastin mRNA levels in fetal lung tissue to 75.4 ± 10.4 in cortisol-only fetuses compared with saline-only fetuses (38.9 ± 5.7). Although lung tropoelastin mRNA levels tended to be greater in cortisol & drain fetuses (32.7 ± 3.5) compared with saline & drain fetuses (26.4 ± 1.4), the difference was not significant.
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Lung morphometry
Percentage lung luminal and tissue space. In saline-infused fetuses, prolonged lung deflation significantly decreased the percentage of potential airspace within the fetal lung and increased the percentage of space occupied by lung tissue (Table 3). The infusion of cortisol significantly increased the percentage of potential airspace and reduced the percentage of tissue space in cortisol-only compared with saline-only fetuses and in cortisol & drain fetuses compared with saline & drain fetuses. As a result, the percentages of tissue space and potential airspace in cortisol & drain fetuses were similar to those in saline-only fetuses. In spite of the increase in percentage of potential airspace, total right lung volume and the luminal volume of the right lung (adjusted for fetal body weight) were significantly reduced by lung liquid drainage and the infusion of cortisol did not alter this (Table 3).
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Prolonged lung deflation significantly increased the interalveolar wall thickness from 12.6 ± 2.1 µm in saline-only fetuses to 21.5 ± 3.7 µm in saline & drain fetuses (Fig. 4). Cortisol infusions tended to decrease the interalveolar wall thickness in cortisol-only fetuses (9.4 ± 1.4 µm) compared with saline-only fetuses (12.6 ± 2.1 µm) as well as in cortisol & drain fetuses (16.0 ± 2.5 µm) compared with saline & drain fetuses (21.5 ± 3.7 µm). However, these reductions in interalveolar wall thickness were not quite significant (Fig. 4).
Elastin deposition within the alveolar parenchyma. In saline-only fetuses, elastin staining was focused at the tips of secondary septal crests, indicating the formation of numerous secondary septal crests at this stage of gestation in fetal sheep (Fig. 5). By contrast, in saline & drain fetuses, focal aggregates of elastin were irregularly spaced along the saccule walls; most elastin fibres appeared to be located along the saccule wall and were not associated with the formation of secondary septal crests (Fig. 5). This is indicative of abnormal secondary septal crest formation and alveolarization. Elastin deposition in blood vessels and airways, however, did not appear to be altered (data not shown). In cortisol-only fetuses, elastin deposition in alveolar parenchymal tissue was very similar to that observed in saline-only fetuses. Similarly, the infusion of cortisol into cortisol & drain fetuses did not significantly alter the deposition of elastin in the alveolar parenchyma compared with saline & drain fetuses (Fig. 5). Thus, despite the increase in alveolar number observed in cortisol & drain fetuses compared with saline & drain fetuses, the deposition of elastin was still abnormal, predominantly occurring as aggregates within the saccule walls.
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Prolonged lung deflation also significantly increased latent MMP-2 levels from 100.0 ± 4.4% in saline-only fetuses to 136.2 ± 5.0% in saline & drain fetuses (Fig. 7). However, the infusion of cortisol did not significantly affect latent MMP-2 levels, as values were similar in cortisol-only (98.5 ± 4.2%) and saline-only (100.0 ± 4.4%) fetuses as well as in cortisol & drain (146.1 ± 6.7%) and saline & drain (136.2 ± 5.0%) fetuses (Fig. 7).
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The doses of cortisol infused were similar to those used in previous studies (Wallace et al. 1995; Boland et al. 1997) and caused fetal plasma cortisol concentrations to increase gradually over the infusion period, mimicking the preparturient increase that occurs in sheep. However, as plasma cortisol concentrations must increase above 40–50 ng ml-1 for 3 days to induce normal parturition in sheep (Elsner et al. 1980; Nathanielsz et al. 1982), this dose was insufficient to induce preterm labour. By contrast, plasma cortisol concentrations remained at pre-infusion levels in saline-infused fetuses, which is consistent with previous studies demonstrating that the preparturient increase in fetal plasma cortisol concentrations does not begin until 135 days of gestation in sheep (Magyar et al. 1980).
A 20-day period of lung liquid drainage caused marked reductions (60%) in fetal lung DNA, protein, elastin and hydroxyproline contents resulting in severe LH and marked structural abnormalities, which are similar to those reported by others (Alcorn et al. 1977; Moessinger et al. 1990; Nardo et al. 1995; Davey et al. 1999). Previous studies have shown that prolonged lung liquid drainage causes lung growth and development to cease (Alcorn et al. 1977; Nardo et al. 1995) and the resulting LH renders the lungs incapable of sustaining independent life after birth (Davey et al. 1999). The degree of reduction in fetal lung weight was also similar to the reductions caused by both nitrofen-induced (Sluiter et al. 1992; Losty et al. 1996; IJsselstijn et al. 1997) and surgically created (Hassett et al. 1995; Lipsett et al. 2000), diaphragmatic hernias in rats and sheep, as well as the reductions observed in human fetuses with CDH.
Previous studies examining the effects of corticosteroids on lung development in fetuses with LH induced by a CDH have shown that corticosteroid treatment decreases fetal body weights (Losty et al. 1995; IJsselstijn et al. 1997) and further reduces lung weights (Suen et al. 1994a; IJsselstijn et al. 1997). Similarly, reductions in birth weight (Reinisch et al. 1978; Jobe et al. 1998) and lung weights (Schellenberg et al. 1987; Adamson & King 1988) have been reported following corticosteroid treatment in fetuses without LH, as well as in human clinical trials. By contrast, we have shown that cortisol, infused at doses (1.5–4.0 mg day-1) that increase circulating cortisol concentrations within a physiological range, do not affect fetal lung or body growth. These findings are consistent with previous studies showing that prenatal exposure to corticosteroids need not affect fetal lung growth (Liggins et al. 1988; Wallace et al. 1995; Boland et al. 1997). It is now apparent that the route of delivery (maternal versus fetal), number of doses, duration of treatment, as well as the dose and type of corticosteroid administered are important factors in determining whether fetal growth restriction occurs (Newnham et al. 1999; French et al. 1999). It is important to note that synthetic corticosteroids, which are commonly used antenatally, have a bioactivity that is 30 times greater than cortisol. Thus, in view of our findings, it is evident that although high-dose synthetic corticosteroids may adversely affect fetal lung and body growth, physiological doses of cortisol can induce lung maturation without inducing these adverse affects.
The drainage of lung liquid for 20 days increased the percentage tissue space (by 70%) and reduced the percentage of potential airspace (by 22%) in the lungs of saline-infused fetuses compared with saline-only fetuses; this resulted from both thicker interalveolar walls and reduced alveolar diameters (Fig. 3). A reduction in tissue space and increase in potential airspace characterizes the structural maturation of the lung over the last third of gestation, such that the percentage of potential airspace increases from 20% at 90 days of gestation to 75–80% near term (145 days of gestation) in fetal sheep (Alcorn et al. 1981). However, after 20 days of lung liquid drainage (at 131 days of gestation) the percentage of potential airspace was 60% in saline & drain fetuses, which equates to a value expected at 110 days of gestation in control fetal sheep (Alcorn et al. 1981). As this was the age at which lung liquid drainage began, it appears that liquid drainage not only causes lung growth to cease (Nardo et al. 1995), but it also causes structural development of the lungs to cease. This arrest of structural development was associated with a marked reduction in alveolar number, indicating that the process of alveolarization was also inhibited. This is consistent with our finding that prolonged lung deflation reduces the expression of tropoelastin in lung tissue and markedly alters the deposition of elastin in the alveolar region of fetal sheep, whereby it is no longer focused at the tips of secondary septal crests (Joyce et al. 2003). Instead, aggregates of elastin form at irregular intervals within saccule walls, further indicating that the process of alveolarization is severely restricted. The hypoplastic lungs of fetuses with a CDH have a similar degree of structural immaturity, as indicated by reduced radial alveolar counts, reduced surface area, increased tissue space and reduced potential airspace (Losty et al. 1996).
Although the cortisol infusion did not affect fetal lung growth, it markedly improved structural maturation of the lung in both fetuses with hypoplastic lungs and fetuses with normal sized lungs. In particular, cortisol decreased the percentage tissue space and increased the percentage airspace in cortisol & drain fetuses compared with saline & drain fetuses, such that values in cortisol & drain fetuses were not different from those in saline-only fetuses (controls). The cortisol-induced changes were most probably the result of a decrease in interalveolar wall thickness and an increase in alveolar diameter. Cortisol infusions had a similar effect on lung structure in fetuses not exposed to lung liquid drainage. Although previous studies have shown similar effects of corticosteroids on normally grown fetal lungs, the dose of corticosteroid used, for example 17 mg day-1 (Boshier et al. 1989; Kendall et al. 1990), was much greater than that used in the present study (1–4 mg day-1). High doses of synthetic corticosteroids have also been administered to fetuses with hypoplastic lungs (Schnitzer et al. 1996; Hedrick et al. 1997) that were, in total, an order of magnitude greater than doses used in the present study. Despite the differences in dose and bioactivity of the corticosteroid used, we achieved alterations in lung structure in fetuses with hypoplastic lungs that were similar to those achieved previously with higher doses of synthetic corticosteroids (Schnitzer et al. 1996; Hedrick et al. 1997). This suggests that increases in fetal cortisol, within a physiological range, can deliver the same beneficial effects on lung structural maturity in the absence of any detrimental effects on lung and body growth.
The reduction in alveolar number (90%) caused by prolonged lung deflation was substantially greater than the change in any other parameter measured; in comparison, lung weights and total DNA content were reduced by 50% and 30%, respectively. The infusion of cortisol alone increased alveolar number by 40% in normal fetuses (cortisol-only) compared with saline-only fetuses, and by 50% in fetuses with LH (cortisol & drain) compared with saline & drain fetuses; cortisol also increased tropoelastin expression in cortisol-only fetuses compared with saline-only fetuses. These findings are contrary to previous studies that have shown that higher doses of synthetic corticosteroids inhibit alveolarization during both fetal (Willet et al. 2001) and postnatal development (Tschanz et al. 2002). However, despite the 50% increase in alveolar number induced by cortisol, the absolute number of alveoli was still severely reduced in cortisol-infused fetuses with LH (cortisol & drain) compared with control (saline-only) fetuses. Furthermore, cortisol infusions did not correct the abnormal deposition of elastin within the alveolar parenchyma, indicating that secondary septal crest formation was still abnormal in cortisol & drain fetuses. Thus, although cortisol can enhance alveolarization and increase tropoelastin expression in normal fetuses, lung deflation has such a profound impact on secondary septal crest formation (as indicated by elastin deposition) and alveolarization that physiological doses of cortisol cannot reverse it.
One of the mechanisms by which prolonged lung deflation and cortisol may induce changes in lung structure is via alterations in ECM components, particularly collagen and elastin. Although total hydroxyproline content of the lungs was reduced by prolonged lung deflation, the concentration per gram of lung tissue was increased by over 50%, which is reflected by an increase in the hydroxyproline-to-protein ratio, at least in cortisol & drain fetuses compared with cortisol-only fetuses. The increased hydroxyproline concentration, combined with impaired elastin deposition, would be expected to alter the mechanical properties of the tissue, thereby contributing to a lower compliance in these lungs relative to their size; similar observations have been made in hypoplastic lungs induced by CDH (Hassett et al. 1995). However, prolonged lung deflation also increased the level of active and latent MMP-2 levels, which is a gelatinase known to be important in the degradation of a number of ECM components, including collagen. Despite this increase in active MMP-2 levels, the concentration of hydroxyproline in lung tissue from fetuses exposed to lung liquid drainage was elevated. Furthermore, cortisol had no effect on hydroxyproline levels in either group of cortisol-infused fetuses, despite a reduction in active MMP-2 levels in cortisol & drain fetuses.
In most cell types, MMP-2 is constitutively expressed and does not respond to stimulation by growth factors or cytokines (Alexander & Werb 1991). Thus, the mechanism for the increase in active and latent MMP-2 levels in saline & drain fetuses is unknown. It is possible that this increase may relate to an increase in the proportion of type-II alveolar epithelial cells (AECs) associated with the reduction in lung expansion (Alcorn et al. 1977; Flecknoe et al. 2002). As MMP-2 has been localized to type II AECs in the fetal lung (Fukuda et al. 2000), an increase in the proportion of type II cells in response to prolonged lung deflation would be expected to increase MMP-2 levels, although an effect of mechanical stress on lung fibroblasts cannot be excluded (Tomasek et al. 1997). The levels of active (but not latent) MMP-2 in lung tissue were significantly reduced in cortisol & drain fetuses compared with saline & drain fetuses. As a result, when expressed as a proportion of total (active plus latent) MMP-2 levels, active MMP-2 levels were significantly reduced from 33.3 ± 0.9% to 24.4 ± 1.3% by the cortisol infusion in fetuses exposed to prolonged lung deflation. One effect of such a decrease in active MMP-2 levels could be reduced proteolysis of ECM molecules.
MMP-2 activation is unique, compared with other MMPs, as it involves both MT1-MMP (a membrane-bound MMP) and TIMP-2 (Murphy et al. 1999). Thus, the increase in both latent and active MMP-2 in response to prolonged lung deflation may be due to an increase in MMP-2 expression, whereas the reduction in active MMP-2 levels in response to cortisol may be mediated via effects on MT1-MMP or TIMP-2 levels. It has been suggested that MT1-MMP may be regulated by corticosteroids as well as by a number of cytokines and growth factors (Lohi et al. 1996). However, although gelatin zymography can detect the presence/absence of gelatinases, it does not predict gelatinase activity; MMP/TIMP complexes are disrupted during zymography, allowing MMP-2 to migrate free of TIMPs in the gel.
In summary, we have shown that relatively low, physiological doses of cortisol, which are insufficient to induce labour in sheep, enhanced structural development of the hypoplastic fetal lung without adversely affecting either fetal lung or body growth. However, the enhanced structural development was predominantly restricted to reductions in alveolar parenchymal tissue volumes with little or no effect on septal crest formation and subdivision of the terminal air sacs. Furthermore, as we could detect no change in tissue collagen concentration following cortisol treatment in fetuses with severe LH, it is likely that the cortisol-induced changes in lung structure may involve remodelling of other matrix molecules, including the interstitial proteoglycans. The increase in active and latent MMP-2 levels in hypoplastic fetal lungs indicates that proteolysis of matrix molecules may be elevated in these lungs and that an effect of cortisol may involve a reduction in the activity of this enzyme.
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