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Cardiovascular Research 2000 46(2):332-345; doi:10.1016/S0008-6363(00)00031-6
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Birth associated changes in pulmonary arterial connective tissue gene expression in the normal and hypertensive lung

Rachael M Kitleya, Alison A Hislopa, Susan M Halla, Anthony J Freemontb and Sheila G Hawortha,*

aVascular Biology and Pharmacology Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK
bMusculo-Skeletal Research Group, University of Manchester, Oxford Road, Manchester, UK

* Corresponding author. Tel.: +44-171-813-84-59; fax: +44-171-813-84-59

Received 28 October 1999; accepted 19 January 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objectives: To determine the temporal and spatial expression of the connective tissue precursors, procollagen and tropoelastin mRNA in normal and pulmonary hypertensive porcine pulmonary arteries from birth onwards. Methods: Using in situ hybridisation, connective tissue gene expression for procollagen {alpha}1(I) and {alpha}1(III) and tropoelastin was studied in intrapulmonary arteries from normal piglets, 5 min–16 weeks, and from piglets made pulmonary hypertensive by exposure to hypobaric hypoxia for 3 days, from birth, 3 or 14 days of age. In addition, Type III pN—procollagen, tropoelastin and collagen I and III were studied by immunohistochemistry. Quantitative or semi-quantitative techniques were applied to both in situ and immunohistochemical studies. Results: Procollagen {alpha}1(I) and {alpha}1(III) mRNA expression increased rapidly in the media and adventitia between birth and 3 days of age (P<0.05). The increase was transient and the number of cells expressing procollagen mRNA decreased to the low newborn number after 6 days of age. Type III pN—procollagen immunostaining was greatest in newborn elastic and muscular arteries and then decreased. Collagen I and III increased mainly after 6 days of age. In animals exposed to chronic hypobaric hypoxia from birth, the increase in procollagens I and III mRNA was prevented. Exposure to hypoxia from 3 or 14 days led to little change in either gene expression or in procollagen and mature collagen from the normal. Tropoelastin gene expression was high at birth in the endothelium and media for the first 6 days, and then decreased. Normally, tropoelastin decreased in the media and increased in the adventitia after 16 days of age. Hypoxia had no effect on the mRNA but led to increased tropoelastin. Conclusion: We demonstrated marked, rapid changes in temporal and cell specific connective tissue gene expression in normal pulmonary arteries immediately after birth as the vasculature remodels. Each gene appeared to have its own timetable of expression and responded differently to hypoxia-induced hypertension.

KEYWORDS Connective tissue; Developmental biology; Gene expression; Pulmonary circulation; Remodelling


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
Connective tissue is deposited rapidly after birth in the intrapulmonary arteries of both the human and porcine lung [1–3]. In porcine vessels, the increase in deposition is not evident until about 3 weeks of age. The regulation of connective tissue deposition is not well understood. Increased connective tissue deposition may reflect increased expression and/or decreased degradation; in the pulmonary arterial system, postnatal increase is probably due to increased gene expression. We hypothesised that the increase in procollagen gene expression would occur after birth, since the pulmonary arterial wall is remodelled immediately after birth. The smooth muscle cells spread within the vessel wall, from a brick-like to a more fusiform profile, to produce a rapid increase in lumen diameter. This process is probably facilitated by the plasticity of the vessel wall which, as we have previously shown, lacks extensive distribution of collagen Type I [1–3]. In general, Type I collagen forms thick bundles of fibres and affords mechanical strength, whereas Type III forms finer, more reticulate fibres which exist as networks in tissues characterised by elasticity, such as the aorta. Collagen Types I and III can be present within the same bundle and even within the same fibre [4].

In the presence of pulmonary hypertension, the amount of connective tissue in and around the pulmonary arterial wall increases, at any age. Structural examination of the vessel wall shows a striking increase in Type I collagen within both media and adventitia [5,6]. But biochemical studies on bleomycin-induced pulmonary hypertension in adult rabbits demonstrated a commensurate increase in both Type I and Type III collagens, brought about by changes in both synthetic and degradative processes [7]. The elastin profiles forming the internal, external and medial elastic laminae of human and porcine intrapulmonary arteries normally increase in size rapidly after birth [1–3], but the increase is excessive in human and experimental neonatal pulmonary hypertension. The lobar pulmonary artery of calves exposed to hypoxia from 1 to 15 days of age showed a 4-fold increase in steady-state tropoelastin gene expression, and a 2-fold increase in collagen type I mRNA [8].

We have examined the patterns of gene expression and protein deposition in porcine pulmonary arteries from birth to 16 weeks. The steady state mRNA levels of procollagen {alpha}1(I), procollagen {alpha}1(III), and tropoelastin were studied in the normal and we also examined gene expression in neonatal pulmonary hypertension caused by chronic exposure to hypobaric hypoxia. Gene expression was studied by in situ hybridisation, rather than using whole lung homogenates, in order to study spatial as well as temporal changes. Fibril-forming collagens are secreted as precursors, the procollagens, and we thus examined the expression of a precursor of type III, type III pN—procollagen, as a marker for newly-deposited collagen, by immunohistochemistry. In addition, we examined the expression of mature collagen I and III and tropoelastin after immunostaining. Porcine pulmonary arteries were studied because of the structural and haemodynamic similarities between the porcine and human circulation during early life [1–3,9].


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 Animal model
Lung tissue was obtained from healthy Large White pigs born naturally at term, and killed at 5 min (n=8), during the first 24 h (n=7), 3 days (n=9), 6 days (n=8) and 16 days (n=4) of age. Tissue was also taken from 16-week-old animals at an abattoir (n=6). All animals were killed by a lethal injection of sodium pentobarbital (100 mgkg–1). An additional 20 animals were placed in a hypobaric hypoxic chamber (50.8 kPa) for 3 days. The internal temperature was maintained at 29 °C and the air pressure maintained at 50.8 kPa. The pressure was returned to normal while cleaning the chamber and feeding the piglets, three times daily for 10–15 min. The animals also had a continuous supply of milk in the chamber. Three groups of hypoxic animals were studied; exposed to hypoxia from birth to 3 days (n=7), from 3 to 6 days (n=8), and from 14 to 17 days of age (n=5). Previous studies showed that animals treated in this manner develop pulmonary hypertension with right ventricular hypertrophy and pulmonary arterial medial hypertrophy, and those exposed from birth continue to shunt from right to left through fetal channels and have a systemic arterial oxygen saturation of 71±5% [10]. The animals received humane care in compliance with British Home Office regulations. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Department of Health and Human Services, Publ. No. (NIH) 85-23, revised 1996).

2.1.1 Study groups
The number of animals used in each study are shown in Table 1. For the in situ hybridisation studies, four experiments were carried out for procollagen {alpha}1 (III) mRNA (procollagen III) and five experiments were carried out for both procollagen {alpha}1 (I) mRNA (procollagen I) and tropoelastin mRNA. Each experiment included tissue from animals of different ages, normal and hypoxic. Animals from at least three different litters were studied at each age in the study of normal development and in each group of hypoxic animals. Immunohistochemistry experiments were carried out for type III pN—procollagen, tropoelastin, collagen I and collagen III on three or four animals in each group.


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  		Table 1 Number of animals used in each control and hypoxic group for each studya

 
2.2 Preparation of tissue
Immediately after death the heart and lungs were removed and blocks of lung tissue (approximately 2x1x0.5 cm) were cut from the hilar and peripheral regions. The tissue was fixed in 10% formaldehyde in buffered saline for at least 24 h and then processed and embedded in paraffin wax. Serial sections were cut at 7 µm for in situ hybridisation and 4 µm for immuno-histochemistry. Other blocks were embedded in OCT compound and frozen immediately in isopentane cooled by liquid nitrogen. These were stored at –80°C and 10 µm frozen sections were cut immediately before use.

2.2.1 Preparation of DNA probes
The cDNA probe for procollagen III mRNA was 295 base pairs long, cut from the plasmid (pHFS3) with Pst I [11]. The procollagen I mRNA probe was 372 base pairs long and was cut from the plasmid (pHCALLU) with Pst I and Pvu II [12]. The tropoelastin mRNA probe (T66) was 500 base pairs long and is a bovine cDNA which spans exons 6 to 15 and is a fragment of the larger clone BEL-1 [13]. The original plasmid (pGEM4Z) sample for tropoelastin was kindly provided by Dr. W.C. Parks (Washington University Medical Centre, St Louis, USA). The cDNA probes were labelled with 35S using the MegaprimeTM DNA Labelling System (Amersham), which utilises the Klenow fragment of DNA polymerase I, and then purified through SephadexTM G-50 (Pharmacia) spun columns. These protocols were carried out according to the manufacturers’ instructions.

2.2.2 In situ hybridisation
Tissue sections were dewaxed in xylene and hydrated through graded alcohols. Sections were then treated with 0.2 N HCl for 10 min, washed in 2x standard sodium citrate buffer (SSC; 1x is 150 mM sodium chloride, 15 mM sodium citrate, pH 7), incubated with 5 µgml–1 nuclease-free proteinase K (Sigma) in 0.05 M tris HCl, pH 7.4 for 1 h at 37°C and then washed in 0.2% glycine in phosphate buffered saline (PBS tablets; Sigma). Negative control sections were then washed with 0.5x SSC, incubated with 1 mgml–1 ribonuclease A (Sigma) for 1 h at 37°C and again washed in 0.5x SSC.

All sections were rinsed in PBS, post-fixed with 0.4% paraformaldehyde in PBS for 20 min at 4°C, incubated with 0.25% acetic anhydride in 0.1 M triethanolamine, pH 8, washed in water, dehydrated through graded alcohols and air dried. Sections were hybridised overnight in a humidified chamber at 37°C with 30 µl hybridisation buffer (0.1% dextran sulphate, 1 mgml–1 bovine serum albumin (BSA), 200 µgml–1 ficoll, 200 µgml–1 polyvinylpyrolidine, 0.6 M sodium chloride, 50% formamide, 200 µgml–1 salmon sperm DNA, 10 mM tris HCl pH 7.4, 500 µM EDTA pH 8, 10 µM dithiothreitol (DTT)) containing 50ngml–1 radiolabelled probe. Post-hybridisation, sections were washed successively in 4x SSC; 1 mM EDTA and 10 mM DTT in 0.5x SSC; 1 mM EDTA in 0.5x SSC and 50% formamide with 0.15 M sodium chloride 5 mM tris HCl and 0.5 M EDTA. High-stringency washes were then carried out using 0.5x SSC for a total of 20 min at 55°C (for procollagen mRNAs) or 40°C (for tropoelastin mRNA). The sections were then washed in 0.5x SSC, then in DEPC-treated water and dehydrated through alcohols and air dried.

2.2.3 Autoradiography
In order to verify that in each experiment, hybridisation had occurred evenly across each section, slides were placed under autoradiographic film (Hyperfilm MP, Amersham) in a light tight box and stored at 4°C for 5 days. Films were developed in an automated processor. The slides were then dipped in Ilford K5 photographic emulsion at 40°C, prediluted 1:1 with distilled water, and then laid flat and allowed to air dry for 1 h before being stored in a desiccated light tight box at 4°C for 7–10 days. The sections were brought to room temperature, developed in Kodak D19 developer, fixed in Amfix, and stained with haematoxylin and eosin.

2.2.4 Immunohistochemistry wax sections: type III pN—procollagenand tropoelastin
Tissue sections were dewaxed in xylene and hydrated through graded alcohols. Antigens were unmasked by autoclaving in citric acid buffer (2.1 gl–1 citric acid, pH 6). Endogenous peroxidase activity was blocked by incubation in 0.3% H2O2 in methanol for 30 min followed by serum free protein block (Dako, UK) for 1 h. Sections were incubated for 1 h at room temperature with either rabbit anti-human type III pN—procollagen antibody (1:300) (Chemicon, Ca), or anti-elastin mouse monoclonal BA-4 antibody (this antibody is specific for tropoelastin by S.D.S-PAGE, Sigma) (1:2000) in PBS containing 0.6% (BSA). The sections were washed in PBS and incubated for 1 h with secondary antibodies, either biotin conjugated swine anti-rabbit (1:200) for procollagen III or biotin conjugated rabbit anti-mouse (1:200) for elastin. After washing in PBS sections were incubated with StreptABcomplex/HRP (Dako, UK) for 30 min and staining was visualised by incubation with 3,3'-diaminobenzidine solution (Dako) for 2–15 min until a colour reaction could be seen. All sections in any one experiment were incubated for the same length of time. The reaction was stopped by immersion in distilled water. Sections were counterstained with haematoxylin to visualise the tissue morphology.

2.2.5 Immunohistochemistry frozen sections: collagen I and III
Sections were air dried at room temperature and fixed in 4% paraformaldehyde for 10 min. Endogenous peroxidase activity was blocked with 0.03% H2O2 in methanol for 30 min and antigen unmasking was carried out using 10 mM citrate buffer pH 3.0 for 30 min at 30°C. Primary antibodies were applied overnight at 4°C using collagen I polyclonal goat anti-human (1:200) (Chemicon, Ca) or collagen III polyclonal rabbit anti-human (1:100) (Chemicon, Ca). After washing in PBS the sections were incubated with either biotin conjugated rabbit anti-goat 1:200 (collagen I) or goat anti-rabbit 1:200 (collagen III). Antibody binding was visualised with diaminobenzidine as for wax sections.

Negative control slides for wax and frozen sections were incubated in PBS–0.6% BSA which was substituted for the primary antibody.

2.3 Assessment of results
2.3.1 In situ hybridisation studies
Four adjacent sections were assessed in each block of tissue, two test and two negative control sections. Pulmonary arteries were classified according to their wall structure: elastic (7–30 smooth muscle cell layers), muscular (3–6 smooth muscle layers), and small muscular (1–2 smooth muscle layers) [14]. Within each tissue section, all the elastic and muscular arteries and approximately five representative small muscular pulmonary arteries were assessed. Within each type of artery, the percentage of cells with positive signal in the endothelium, media and adventitia was estimated. The mean percentage of positive cells in each region of the vessel wall was compared in normal animals at different ages, and in normal and hypertensive animals of the same age. The values were compared using ANOVA and, where appropriate, t-tests with Bonferroni correction. P<0.05 was considered significant.

For the small muscular arteries hybridised for procollagen III mRNA a more detailed assessment of silver grain density was made. The number of silver grains was determined per unit area (1000 µm2) of media and of adventitia in six pulmonary arteries from each case. For each vessel, the value of positive signal was obtained by subtracting the mean grain density of the control sections from that of the test section. Within each experiment, the six grain density values from each normal animal were compared at different ages and values from normal and hypertensive animals of the same age were also compared. Paired values from each of the four experiments were analysed using the non-parametric Sign Test (Campbell RC, Statistics for biologists, 1989, Cambridge University Press, p. 75). This test determines whether there are differences between paired sets of values, but is not influenced by the size of any differences which may be present.

2.3.2 Immunostaining
The amount and the distribution of immunostaining were assessed in the elastic, muscular and small muscular pulmonary arteries of each case, using a semi-quantitative grading system for the endothelium, media and adventitia; ‘0’ indicated absent staining, ‘+’ indicated weak staining, ‘++’ indicated strong staining, and ‘+++’ indicated very strong staining.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Distribution of procollagen III, I and tropoelastin mRNA
At all ages, the signal of 35S cDNA detected on autoradiographic film showed that procollagen III mRNA, procollagen I mRNA and tropoelastin mRNA were present in all those major lung structures which had a high connective tissue content. The two procollagen mRNAs had high levels of signal in the pleura, septa and connective tissue surrounding airways and blood vessels [Fig. 1(a) and (c)]. For tropoelastin mRNA, the level of signal was high in the vessel walls only [Fig. 1(d)]. The control sections pretreated with RNAse demonstrated a low level of non-specific signal over the entire tissue section [Fig. 1(b)]. On the tissue sections, the signal detected by the photographic emulsion demonstrated that both procollagen I and III mRNAs were present in the media and adventitia but not on the endothelium (Figs. 2 and 3Go). The signal was greater in the adventitia than media. For procollagen III, the signal was evenly distributed throughout the media but that for procollagen I was concentrated towards the lumen during the first 6 days of life. Tropoelastin mRNA was detected in the endothelium, media and adventitia, particularly in the endothelium and media (Fig. 4). The expression was greater in the inner than the outer part of the adventitia. The distribution of the mRNAs did not change with age, but the density of binding changed in the normal with age, and in tissues from pulmonary hypertensive animals when compared with age-matched controls.


Figure 1
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  		Fig. 1 Autoradiographs of lung sections from a 3-day-old piglet after in situ hybridisation. (a) Test section for procollagen III showing high level of signal in connective tissue septa, pleura and around airways and blood vessels. (b) Negative adjacent section for the same probe showing low level of non-specific binding over all structures. (c) Test section for procollagen I at greater magnification showing specific binding in walls of pulmonary arteries, veins and airways. (d) Test section for tropoelastin showing binding in pulmonary artery and vein wall but not in the airway. For (a) and (b), bar represents 500 µm. For (c) and (d), bar represents 250 µm. a, pulmonary artery; br, bronchus; v, pulmonary vein.

 

Figure 2
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  		Fig. 2 Photomicrographs of muscular pulmonary arteries after in situ hybridisation for procollagen III. (a) Newborn test section. Silver grains are located over the media (med) and adventitia (adv) but not endothelium (end). (b) Newborn negative control section. (c) 3-day-old test section with similar distribution but greater density in comparison with normal newborn. (d) 3-day hypoxic animal with similar distribution and grain density to newborn. Bar represents 20 µm.

 

Figure 3
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  		Fig. 3 Photomicrograph of muscular pulmonary arteries after in situ hybridisation for procollagen I. (a) Newborn test section. Grain density is greater over adventitia (adv) than the media (med). Density is greater at the luminal side of the media. (b) Newborn negative control section. (c) 3-day-old test section with greater grain density over media and adventitia than newborn. (d) 3-day hypoxic animal with grain density reduced in comparison with control 3-day old. Bar represents 20 µm.

 

Figure 4
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  		Fig. 4 Photomicrograph of muscular pulmonary arteries after in situ hybridisation for tropoelastin. (a) Newborn test section. Silver grains dense over media (med) and endothelium (end) with fewer grains over adventitia (adv). (b) Newborn negative control section. (c) 3-day control, similar to newborn. (d) 3-day hypoxic animal with greater number of silver grains over the adventitia than 3-day control. Bar represents 20 µm.

 
3.2 Microscopic assessment of in situ hybridisation
3.2.1 Procollagen III mRNA
Quantitative studies confirm that at all ages the percentage of positive cells in the adventitia was generally higher than that in the media in all types of pulmonary artery and the difference was statistically significant in both elastic and muscular arteries (Fig. 5). The percentage of positive cells in the adventitia was lower in the small muscular pulmonary arteries than in muscular and elastic pulmonary arteries at all ages, significantly at 3, 6 and 16 days.


Figure 5
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  		Fig. 5 Percentage of cells positive for procollagen III in the media and adventitia of large and small muscular arteries from control (solid) and hypoxic (open) animals. NB, newborn; d, day; wk, week. Comparisons using t-test after ANOVA.

 
Between birth and 3 days of age the mean percentage of procollagen III mRNA positive cells per unit area appeared to increase in both the media and adventitia of muscular and small muscular arteries (Fig. 5). A similar pattern was seen in the elastic arteries (data not shown). This increase was transient and by 16 days of age, the mean percentage of positive cells had returned to the lower newborn level. Using the more detailed assessment of grain density per unit area (Fig. 6), procollagen III mRNA expression was significantly higher in both the media and adventitia of small pulmonary arteries (50–250 µm diameter) at 3 days of age than in the newborn and 6 day, 16 day and 16 week animals (P<0.05 for each comparison). In an additional experiment (n=7), the grain density was found to be significantly higher in both the media and adventitia at 19–24 h than at 0–8 h (P<0.05), and was similar to that at 3 days of age.


Figure 6
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  		Fig. 6 Number of silver grains in small muscular pulmonary arteries counted per unit area of media and adventitia after in situ hybridisation for procollagen III showing results for one experiment. Each point is the count from one vessel. Control (solid) and hypoxic (Hyp) (open) animals. Comparison using non-parametric Sign Test.

 
Following exposure to chronic hypobaric hypoxia from birth for 3 days, the normal postnatal increase in the number of cells per unit area positive for procollagen III mRNA did not take place (Figs. 2 and 5Go). In the small muscular pulmonary arteries the grain density per unit area was lower than in the 3-day-old control animals (P<0.05) (Fig. 6). Conversely, in animals exposed to a hypoxic environment from 3 to 6 days of age, the mean percentage of positive procollagen III mRNA in the muscular pulmonary arteries was increased compared to the normal at 6 days of age in both the media (from 13% to 47%) and adventitia (from 70% to 95%). For the small pulmonary arteries, the grain density per unit area at 6 days was also greater than normal in both the media and adventitia (P<0.05). Hypoxic exposure from 14 to 17 days had no effect on mRNA expression.

3.2.2 Procollagen I mRNA
Quantitative studies confirm that at all ages the percentage of positive cells was higher in the adventitia than in the media in all types of pulmonary artery (Figs. 3 and 7Go). In both structures there were fewer positive cells in the small muscular pulmonary arteries than in the large muscular (Fig. 7) and elastic pulmonary arteries (data not shown). The mean percentage of positive procollagen I mRNA labelled cells appeared to increase between birth and 3 days of age in both media and adventitia of muscular and small muscular arteries. The difference was statistically significant in the media of one vessel type and the adventitia of the other (Fig. 7). By 16 weeks of age the percentage of positive cells had decreased in both media and adventitia of all arteries and the values were similar to those seen in the newborn.


Figure 7
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  		Fig. 7 Percentage of cells positive for procollagen I in the media and adventitia of large and small muscular arteries from control (solid) and hypoxic (open) animals. Values compared by t-test after ANOVA.

 
Following exposure to hypoxia from birth the increase in expression normally seen in the media of the muscular pulmonary arteries between birth and 3 days of age did not occur (Figs. 3 and 7Go). Exposure from 3 or 14 days had no effect on the mRNA expression.

3.2.3 Tropoelastin mRNA
Tropoelastin mRNA was located on endothelial cells, medial smooth muscle cells and adventitial fibroblasts (Fig. 4). At nearly all ages the percentage of positive cells was generally greater in the muscular than in the small muscular pulmonary arteries and in both types of artery it was usually greater in the endothelium and media than in the adventitia, particularly during the first week of life (Fig. 8).


Figure 8
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  		Fig. 8 Percentage of cells positive for tropoelastin in the endothelium, media and adventitia of large and small muscular arteries from control (solid) and hypoxic (open) animals.

 
The temporal pattern of tropoelastin gene expression in the endothelium and media was not like that of the procollagens. In the endothelium and media, the percentage of cells expressing tropoelastin mRNA was high at birth, remained high during the first 6 days of life and appeared to decrease to a lower level at 16 days–16 weeks of age (Fig. 8). By contrast, the mean percentage of positive cells labelled for tropoelastin mRNA in the adventitia was low at birth, increased by 3 days of age and remained at the high level throughout the 16-week study period (Fig. 8). There was considerable case-to-case variation and the findings were not statistically significant, but the pattern of change with age was clear. There was no change in expression of tropoelastin mRNA as a result of exposure to hypoxia for 3 days.

3.3 Assessment of immunostaining
3.3.1 Type III pN—procollagen
At all ages, type III pN—procollagen staining was seen over the pleura, septa, the connective tissue surrounding the airways and blood vessels and in the media of elastic and muscular pulmonary arteries (Fig. 9). In the vessel walls a network of type III procollagen fibres was seen associated with the medial elastic laminae, particularly in the subendothelium. Little or no staining was seen on smooth muscle cells (Fig. 9). The amount of immunostaining decreased with decrease in size of artery. The adventitia showed stronger staining at all ages. Considering the amount and intensity of staining in relation to age (Table 2), immunostaining was greatest in newborn elastic and muscular arteries and decreased with age, particularly in the subendothelium and media. Conversely, immunostaining increased in the subendothelium and media of small arteries during the first 6 days of life. Following exposure to hypoxia from birth to 3 days of age staining was similar to that normally seen at birth and greater than in normal 3-day control animals — a maintenance of the normal newborn condition (Table 2). Exposure to hypoxia in the ranges 3–6 and 14–17 days of age did not alter procollagen III immunostaining.


Figure 9
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  		Fig. 9 Upper panel: Photomicrographs of muscular pulmonary arteries stained with antibody for Type III pN—procollagen, counter-stained with haematoxylin. Positive staining is greater in the newborn than at 16 days particularly in the subendothelial region. It is found close to the elastic laminae of the media and is dense in the adventitia. The negative control section has no primary antibody. Bar represents 20 µm. end, endothelium; med, media; adv, adventitia. Middle panel: Photomicrographs of muscular pulmonary arteries immunostained for collagen III. Staining is low in the newborn adventitia and media; by 6 days staining is greater, particularly in the subendothelium (*). At 16 days staining is uniform across the media. Bar represents 25 µm. Bottom panel: Photomicrographs of muscular pulmonary arteries immunostained for collagen I. Staining is greater in the adventitia than the media and increases with age. Bar represents 25 µm.

 

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  		Table 2 Summary of staining appearance for type III pN—procollagen in elastic, muscular and small muscular pulmonary arteries from control and hypoxic animals (n=3–5 in each group)a

 
3.3.2 Collagen III
Immunostaining of mature collagen III showed a similar distribution in the lung to the procollagen III. However, at birth there was little mature collagen III in either the media or adventitia of the elastic and large muscular arteries (+) (Fig. 9). By 6 days of age this had increased both in the adventitia (+++) and the media where there was a gradient from the inner subendothelial layer (+++) to the outer layers (++) (Fig. 9), similar to that seen in the procollagen at birth. By 16 days and 16 weeks there was even expression throughout the wall (+++). Conversely, in the small arteries at birth there was mature collagen in the media (++) with less in the adventitia (+). This did not change with age, and hypoxic exposure had no effect on the collagen staining.

3.3.3 Collagen I
Immunostaining for collagen I was seen at all ages in the pleura, septa and around the airways and blood vessels. In all arteries staining in the media was almost undetectable at birth but increased with age to (+) at 6 days and (++) at 16 weeks (Fig. 9). The adventitia of large arteries stained strongly (+++) at all ages. However, in the adventitia of small arteries there was less at birth (++) but this increased (+++) by 3 days of age. An increase in mRNA for collagen I was seen at this age. The normal small increase in collagen was not seen in those animals hypoxic from birth.

3.3.4 Tropoelastin
In our sections tropoelastin only partially co-localised with elastic laminae as stained by Millers elastic stain (Fig. 10). In the elastic and large muscular arteries at birth there was strong tropoelastin reactivity (+++) closely associated with the mid and outer elastic laminae of the media, but the two innermost elastic laminae were only weakly positive (+) (Fig. 10). The adventitia also stained weakly (+). A similar appearance was seen up to 16 days of age. In the 16-week-old animals medial staining (++) was patchy and diffuse although still associated with the elastic laminae and immuno reactivity was now apparent close to the internal elastic lamina (Fig. 10). The density of adventitial staining had increased (++). In the small muscular arteries at all ages tropoelastin was predominantly associated with the external elastic lamina (+) (Fig. 10). Adventitial staining was weak except in the adult (++). In animals exposed to hypoxia from birth to 3 days, tropoelastin expression was greater in the media and adventitia than age-matched controls (+++) in both large and small arteries. The increase was most noticeable in the small arteries (Fig. 10).


Figure 10
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  		Fig. 10 Photomicrographs of muscular pulmonary arteries immunostained with anti-elastin mouse monoclonal BA-4 antibody (tropoelastin) and Millers elastic stain counterstained with van Gieson stain (EVG). In the newborn the internal elastic lamina stains with EVG but not with tropoelastin. The adventitia has less stain than the media. At 16 weeks the media has less staining than in the newborn but it is associated with the internal elastic lamina. The adventitial staining is greater. In smaller muscular pre-acinar arteries from a normal 3-day-old (3dN) traces of tropoelastin are seen associated with the internal and external elastic laminae (arrowed) of the media (M). In the 3-day hypoxic (3dH) staining is increased particularly in the external elastic lamina. Bar represents 25 µm. E, endothelium; A, adventitia.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
In the present study, by using quantitative techniques, we showed that both procollagen I and III gene expression increased rapidly between birth and 3 days and the increase in procollagen III mRNA was evident by 19–24 h in small muscular arteries. For both genes, the increase in expression was transient, falling after 6 days of age. By using in situ hybridisation rather than biochemical techniques we were able to localise gene expression to specific regions of the vessel wall, in specific segments of the pulmonary arterial tree. During the first week of life the procollagen I mRNA and the type III pN procollagen was concentrated at the adlumenal border of the media. This indication of preferential collagen synthesis is compatible with the synthetic phenotype of the inner smooth muscle cells seen on ultrastructural examination at birth [1,15]. However, little mature collagen I was seen until after the first week, although there was more collagen III in the subendothelium by 3–6 days.

Relatively little is known about the regulation of the procollagen genes and why birth should be followed by a sudden increase in procollagen gene expression is not clear. But the abrupt increase in mechanical stretch which occurs when the lung expands and the vessels become distended with blood, and the shear stress imposed on the endothelial cells as blood flow increases, may both be important. In vitro studies on cultured fibroblasts show an increase in procollagen III mRNA level and in collagen III synthesis after 24 h of cyclical stretch [16] and a later increase in Type 1 collagen synthesis [17]. In endothelial cells, shear stress activates a shear stress response element in several growth factor genes [18]. A complex relationship exists between stretch and up-regulation of growth factor and connective tissue gene expression. High lung inflation increases wall stress in rabbit pulmonary capillaries, causes an increase in expression of procollagen III mRNA, basic fibroblast growth factor, and transforming growth factor β1(TGFβ1) [19]. Balloon angioplasty of rabbit atherogenic vessels increased the gene expression of procollagens I and III, and TGFβ, and there were direct correlations between TGFβ1 and both Type I and Type III procollagen mRNA expressions [20].

Once translation of the mRNAs encoding the pre-procollagens has taken place, intracellular post-translational modifications result in the formation of soluble triple-helical procollagen molecules, the precursors of the fibrillar collagens [4]. Using semi-quantitative analysis, we found that there was more type III pN—procollagen in the elastic and large muscular arteries at birth and during the first 3 days of life than subsequently. This higher level of procollagen III could be attributed to stretch of the vessel wall at birth. Mechanical stretch increases the synthesis of total protein, collagen and elastin within 4 h in isolated adult rat pulmonary arteries [21]. The procollagen III antibodies stained predominantly a network of small fibres in the subendothelium, the ablumenal side of each medial elastic lamina and the inner adventitia, adjacent to the external elastic lamina. Previous ultrastructural studies showed that between birth and 4 days of age there is a rapid increase in the amount of banded collagens, that is of collagens Type I and III, in the subendothelium and media [1–3]. In the present study mature collagen III antibody staining showed an increase by 6 days, though collagen I appeared more slowly. Considered together, these observations suggest that birth is followed by secretion, assembly and cross-linking of fibrils, while an upregulation of procollagen gene expression ensures its replacement and the maintenance of a potentially high level of connective tissue formation during early postnatal life [1,15].

In normal porcine pulmonary arteries, tropoelastin mRNA expression on the media and on the endothelium was high at birth, unlike the procollagen gene expression. The high, early, time-limited tropoelastin gene expression we found on the endothelial and smooth muscle cells has been reported by others in rat pulmonary arterial endothelial and smooth muscle cells [22]. We also found high tropoelastin protein expression in the newborn and immature porcine lungs which decreased by 16 weeks. This is presumably responsible for the marked, rapid increase in thickness and continuity of the medial elastic laminae which occurs during the first 3 weeks of life [1,3,15]. But in the adventitia, tropoelastin mRNA expression was low at birth, increased by 3 days of age, and remained high throughout the 16-week study period. Tropoelastin and elastin increased in the adventitia after 16 days of age. This later increase in tropoelastin gene expression on the porcine adventitial fibroblasts is reminiscent of another study in which Bruce [23] showed that in the rat lung, expression peaked at 4 days of life in vascular smooth muscle cells and on the 11th day in interstitial fibroblasts as alveolar development occurred. Moreover, regulation of gene expression appeared to differ in the two cell types in vitro, in that exposure to insulin-like growth factor 1 increased tropoelastin mRNA in aortic smooth muscle cells, but not lung fibroblasts taken from 2 to 3-day-old rats [24]. Also TGFβ increased tropoelastin steady-state mRNA in lung fibroblasts but not in aortic smooth muscle cells [25]. Regulation of tropoelastin gene expression probably changes with age. It appears to be regulated principally at the transcriptional level in neonatal rat lung fibroblasts but is also controlled by additional post-transcriptional mechanisms in the adult cell [26,27].

For both procollagen and tropoelastin, gene expression had diminished before the connective tissue had been deposited. We found that procollagen I and III gene expression returned to the lower newborn level between 6 and 16 days of life, while much of the increase in protein deposition was seen after the first week. Previous studies had indicated a marked increase in protein content after 3 weeks of age [1,15]. A comparable time lag between procollagen I and III gene expression and protein deposition has been demonstrated in experimental balloon angioplasty, when the procollagen genes were transcriptionally activated early, after 2–7 days while collagen deposition increased between 7 and 30 days after injury [28]. Similarly we found that tropoelastin gene expression was already high at birth in the endothelium and media, and decreased between 6 days and 16 weeks of age, as did tropoelastin immunostaining. But our previous ultrastructural studies show a marked increase in medial elastin deposition later, after 3 weeks of age [1]. In summary, the evidence suggests that postnatal connective tissue deposition is governed at least as much by post-translational events as by gene expression.

In babies with persistent pulmonary hypertension of the newborn, the pulmonary arterial pressure is usually elevated from birth, and because hypoxia is a relatively common cause of persistent pulmonary hypertension, we exposed the piglets to hypobaric hypoxia from the moment of birth. This intervention prevented the normal postnatal increase in procollagen I and III gene expression. This may be due to a failure to increase blood flow due to the hypoxic vasoconstriction and shunting of blood away from the lungs through the ductus arteriosus and foramen ovale causing a reduced shear stress response. The high level of procollagen III immunostaining seen in the newborn was maintained after 3 days of hypoxic exposure suggesting enhanced synthesis but there was no apparent effect on mature collagen III deposition, and collagen I deposition was reduced for age. These features all suggest maintenance of the fetal state. By contrast, tropoelastin gene expression remained high at 3 days in the smooth muscle cells, as in normal animals, but there was an increase in tropoelastin deposition suggesting that the high expression of mRNA allowed for rapid translation to protein. In calves exposed to hypoxia from 1 day to 15 days of age, there was a 2-fold increase in both steady-state collagen type I mRNA, and collagen synthesis, and a 4-fold increase in steady-state elastin mRNA levels and elastin synthesis [8]. Differences between this and our study are likely to be due to the increased duration of hypoxic exposure and probably also to species variation. In the porcine lung, exposure to hypoxia from 3 to 6 days elicited a smaller response than exposure from birth. Procollagen III gene expression increased, but procollagen I and tropoelastin gene expression remained normal. Procollagen I mRNA might have increased had exposure been continued for a longer period of time because expression of procollagen III precedes that of procollagen I during early postnatal development [15]. Interestingly, exposing newborn rats to hyperoxia inhibited the normal postnatal increase in tropoelastin gene expression associated with alveolarisation of the newborn lung but the time of onset of hyperoxia influenced the gene expression [26], as we found for the procollagens.

In conclusion, we have demonstrated marked, rapid changes in temporal and cell-specific connective tissue gene expression occurring in normal pulmonary arteries immediately after birth, as the pulmonary vasculature remodels to extrauterine life and pulmonary vascular resistance falls. Each gene appeared to have its own timetable. The findings indicate differences in regulatory mechanisms for procollagen I and procollagen III and tropoelastin in endothelial, smooth muscle cells and fibroblasts in vivo, as indicated by the in vitro studies of other investigators. Connective tissue genes responded differently to hypoxia-induced pulmonary hypertension and the response of each gene depended upon the age at which the animals were exposed to hypoxia. One might hypothesise that birth increases the mechanical stretch and shear stress imposed on the vessel wall, leading to changes in cell shape and connective tissue gene expression. This leads to later deposition of collagens and elastin to stabilise and reinforce the remodelled pulmonary artery in its new postnatal environment, whether normal or hypoxic.

Time for primary review 28 days.


   Acknowledgements
 
This work was funded by the British Heart Foundation. The authors are grateful to Prof. C. Kielty for her helpful criticism of the manuscript.


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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