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. 2007 Aug 16;584(Pt 2):625–635. doi: 10.1113/jphysiol.2007.138735

Thrombospondin-1 expression and localization in the developing ovine lung

Foula Sozo 1, Stuart B Hooper 1, Megan J Wallace 1
PMCID: PMC2277169  PMID: 17702817

Abstract

Fetal lung growth is critically dependent on the degree to which the lungs are expanded by liquid, although the mechanisms involved are unknown. As thrombospondin-1 (TSP-1) can regulate cell proliferation, attachment, spreading and angiogenesis, we investigated the effects of alterations in fetal lung expansion on TSP-1 expression in sheep. TSP-1 mRNA levels were investigated using Northern blot analysis and in situ hybridization, whereas the protein levels were determined by immunohistochemistry. Early growth response 1 (EGR1) mRNA levels were measured by quantitative real-time PCR. TSP-1 was expressed in type-II alveolar epithelial cells and fibroblasts and its mRNA levels increased from 100.0 ± 14.0% in control fetuses to 347.5 ± 73.6% at 36 h of increased lung expansion (P < 0.05), and were reduced to 39.4 ± 6.1% of control levels (100.0 ± 20.4%) at 20 days of decreased lung expansion (P < 0.05). The percentage of cells positive for TSP-1 mRNA increased from 1.9 ± 0.4% to 5.2 ± 0.8% at 36 h of increased fetal lung expansion (P < 0.01). The proportion of tissue stained positive for TSP-1 protein doubled at 36 h of increased lung expansion (23.3 ± 2.2%) compared to controls (11.7 ± 3.2%; P < 0.05). Conversely, at 20 days of decreased lung expansion, the percentage of tissue that stained positive for TSP-1 was halved (25.7 ± 3.2%) compared to controls (39.8 ± 3.3%; P < 0.05). The increase in TSP-1 expression may be due to increased mRNA levels of the transcription factor EGR1 at 36 h of increased lung expansion (2.7 ± 0.7-fold of control levels (1.0 ± 0.2); P < 0.05). Given the known functions of TSP-1 and its localization within the lung, we speculate that TSP-1 may have a significant role in regulating fetal lung growth.

Fetal lung growth and development is critically dependent on the degree to which the lungs are expanded by a lung liquid that is secreted across the pulmonary epithelium into the future airspaces and leaves the lungs via the trachea (Harding & Hooper, 1996). During periods of apnoea, the fetal glottis is actively constricted, which restricts lung liquid efflux (Harding et al. 1986) and promotes liquid accumulation within the future airways. As a result, an internal distending pressure of 1–2 mmHg is applied to the fetal lungs at rest (Vilos & Liggins, 1982), which maintains the lungs in a distended state. If this distending pressure is abolished, for example by draining the lungs of liquid, lung growth ceases (Alcorn et al. 1977; Moessinger et al. 1990; Hooper et al. 1993). On the other hand, if the fetal trachea is obstructed, lung liquid accumulates within the future airways, which expands the lungs further and provides a potent stimulus for fetal lung growth and maturation (Alcorn et al. 1977; Moessinger et al. 1990; Hooper et al. 1993; Nardo et al. 1998).

The lung growth response to tracheal obstruction (TO) is time-dependent, temporarily increasing lung DNA synthesis rates to ∼800% above control levels at 2 days of TO (Nardo et al. 1998). An increase in basal lung expansion induces proliferation of type-II alveolar epithelial cells (AECs), fibroblasts and endothelial cells, induces differentiation of type-II into type-I AECs, and accelerates the structural development of the terminal air sacs (Alcorn et al. 1977; Flecknoe et al. 2000; Nardo et al. 2000). However, the cellular and molecular mechanisms involved are unknown. The identification of genes that mediate expansion-induced lung growth will probably provide a critical first step in understanding the factors that regulate fetal lung development during the alveolar stage. In a recent study (Sozo et al. 2006), we identified thrombospondin-1 (TSP-1) as the most highly upregulated gene in the lung in response to 36 h of increased fetal lung expansion.

TSP-1 is a member of the thrombospondin gene family, which is subdivided into two groups depending on their structure; subgroup A consists of TSP-1 and -2, and subgroup B consists of TSP-3, -4 and -5 (Adams, 2001). TSP-1 and -2 are secreted extracellular matrix glycoproteins consisting of three disulphide-linked chains each with a molecular weight of 142 kDa (Lawler et al. 1978). All TSPs are matricellular proteins and hence do not contribute directly to the extracellular matrix (ECM) structure but can influence cell function by modulating cell–matrix interactions. TSP-1 can regulate cell proliferation (Majack et al. 1988; Bagavandoss & Wilks, 1990), cell attachment and spreading (Murphy-Ullrich & Hook, 1989; Sage & Bornstein, 1991) and angiogenesis (Good et al. 1990; Calzada et al. 2004) by its interactions with secreted proteases, cytokines and other effector proteins (Adams, 2001). Our aims were to determine how TSP-1 expression changes during normal lung development and in response to accelerated and retarded lung growth, induced by changes in fetal lung expansion. We speculate that TSP-1 is an important regulator of fetal lung growth and development, although the mechanisms by which it could exert its effects are unknown. To investigate how TSP-1 may be activated, we also examined the expression of the transcription factor early growth response 1 (EGR1), as it can modulate transcription of the TSP-1 gene (Shingu & Bornstein, 1994).

Methods

Ethical approval

All experiments involving the use of animals were approved by the Monash University Committee for Ethics in Animal Experimentation.

Experimental groups

Lung tissues were collected previously from fetal and newborn sheep during normal lung development and during periods of accelerated or retarded lung growth, induced by alterations in fetal lung expansion (Hooper et al. 1993; Keramidaris et al. 1996; Boland et al. 1997; Nardo et al. 1998; Flecknoe et al. 2003; Boland et al. 2004; Sozo et al. 2006). During normal lung development, fetal lung tissue was collected at 91 days (n= 4), 105 days (n= 5), 111 days (n= 4), 128 days (n= 5), 138 days (n= 4) and 142 days (n= 5) gestation as well as from 2-week-old lambs born at term (∼147 days; n= 5) (Flecknoe et al. 2003).

Altered lung growth was induced in chronically catheterized fetal sheep by altering the degree to which the lungs were expanded by liquid. Briefly, surgery was performed on pregnant Border Leicester × Merino ewes and their fetuses at ∼106–120 days gestational age (GA). Anaesthesia was induced by an intravenous injection of thiopental sodium (1 g) and maintained by continuous inhalation of 1–2% halothane in O2–N2O (50 : 50 v/v). Surgery was performed to implant two large-bore catheters into the mid-cervical trachea; one was directed towards the lungs and the other was directed towards the larynx (Hooper et al. 1988). These catheters were exteriorized from the ewe and connected to form a continuous tracheal loop that enabled liquid to flow to and from the lungs, via the trachea, as normal. Ewes and fetuses were allowed to recover from surgery for at least 5 days prior to the commencement of experimentation. All experiments concluded at ∼128–131 days GA, which is during the alveolar stage of lung development.

To accelerate fetal lung growth, the tracheal loop was obstructed (tracheal obstruction, TO) for a period of 36 h, 2 days, 3 days, 4 days or 10 days (n= 5 for each group) (Keramidaris et al. 1996; Boland et al. 1997; Nardo et al. 1998; Sozo et al. 2006). This prevented liquid efflux from the lungs, resulting in its accumulation within the future airways and an increase in fetal lung expansion. In a separate group of age-matched control fetuses (n= 5), the trachea was not obstructed and a normal level of lung expansion was maintained. To retard fetal lung growth, the lungs were deflated by continuously draining them of liquid via the tracheal catheter for 7 days (n= 4) (Hooper et al. 1993) or 20 days (n= 5) (Boland et al. 2004). Normal levels of lung expansion were maintained in separate groups of age-matched control fetuses (n= 5 for each).

At the conclusion of each experiment, the fetal lungs were drained of liquid and the ewe and fetus were humanely killed by an overdose of pentobarbital sodium (130 mg kg−1i.v.) administered to the ewe. The fetal lungs were removed, weighed and the left bronchus ligated, before the left and right lungs were separated. Small portions (avoiding major airways and blood vessels) of the left lung were snap-frozen in liquid nitrogen and stored at −70°C for molecular analysis. The right lung was fixed via the airways, using 4% paraformaldehyde (in 0.1 m phosphate buffer) and a distending pressure of 20 cmH2O; sections were then processed for light microscopy.

Northern blot analysis

Total RNA was extracted from lung tissue using a modified guanidine thiocyanate method. TSP-1 and EGR1 mRNA levels were quantified by Northern blot analysis and corrected for the level of 18S rRNA, as previously described (Lines et al. 1999); 40 μg of RNA per animal was used for the quantification of EGR1 mRNA levels. Specific ovine TSP-1 (nt 130–875; Accession number: DQ239623), which is 93% homologous to the bovine TSP-1 gene (Accession number: AB005287) and ovine EGR1 (Accession number: DQ239634) cDNA probes were used and labelled with 32P.

TSP-1 in situ hybridization

Randomly selected lung tissue sections (5 μm) were incubated at 60°C for ∼2 h, de-paraffinized in Histoclear, rehydrated in a series of ethanol washes, and washed in 1× phosphate buffered saline (PBS). Sections were then fixed in 4% paraformaldehyde, treated with 20 μg ml−1 proteinase K and incubated in acetylation solution (1.33% triethanolamine pH 8.0, 0.17% HCl, 0.25% acetic anhydride). Each slide was then prehybridized in 150 μl of hybridization solution (50% formamide, 10 mm Tris pH 7.5, 600 mm NaCl, 1 mm EDTA pH 8.0, 1× Denhardt's solution, 200 μg ml−1 yeast tRNA, 0.25% SDS) in a humidified chamber for 1 h at room temperature. Anti-sense and sense riboprobes were generated by RT-PCR using oligonucleotide primers (upstream 5′-CTGTCTTATCCCATCCCTTG-3′; downstream 5′-CAGTAGCTTTAAGTTTGTTAGAC-3′), which amplified a 199 bp region of the ovine TSP-1 gene (Accession number: DQ239623); this region was 100% homologous to the 3′ untranslated region of the bovine TSP-1 gene (Accession number: AB005287). Each riboprobe (80 ng) was denatured and then hybridized to the tissue sections overnight at 65°C in a humidified chamber; sections without either riboprobe served as negative controls. Following hybridization, sections were washed, treated with 20 μg ml−1 RNase A and incubated in blocking solution (20% heat-inactivated sheep serum (HISS), 2% Roche blocking reagent) for 1 h at room temperature in a humidified chamber. Slides were then incubated overnight at 4°C in blocking solution (5% HISS, 2% Roche blocking reagent), containing the antidigoxigenin antibody (0.075% anti-DIG; Roche, Switzerland). Omission of the anti-DIG antibody to one section served as a negative control. Slides were then washed and developed (BM purple substrate (Roche, Switzerland), 0.002 m levamisol, 0.1% Tween 20) in the dark at room temperature for 5 days (the solution was changed after 3 days) before counterstaining with nuclear fast red.

Sections were examined using light microscopy, and digital images were acquired and analysed using ImagePro Plus (Media Cybernetics, Silver Spring, MD, USA). At least three sections (from different regions of the lung), and at least two different fields of view per section, were analysed from each animal. For each field of view, the number of nuclei positive for TSP-1 mRNA and the total number of nuclei were counted; ∼1000 cells per animal were counted. The number of nuclei positive for TSP-1 mRNA was expressed as a percentage of the total number of nuclei counted for each animal. The analysis was performed on the alveolar region of the lung, avoiding areas containing major airways or blood vessels.

TSP-1 immunohistochemical analysis

Randomly selected paraffin-embedded lung tissue sections (5 μm) were incubated at 60°C for ∼2 h, de-paraffinized in xylene, rehydrated in graded ethanol washes, and washed in 1× PBS. Sections were then boiled (using a microwave) for 20 min in 0.01 m sodium citrate (pH 6.0) and washed twice in 1× PBS for 5 min. Sections were then incubated in 3% v/v hydrogen peroxide (in methanol) for 30 min at room temperature, washed in 1× PBS (three washes, 5 min each) and then incubated for 1 h in a humidified chamber with blocking buffer (25% v/v normal goat serum, 5% w/v bovine serum albumin, 0.05 m Tris-HCl pH 7.2). Mouse monoclonal anti-TSP-1 antibody (1 : 50 dilution; clone A6.1, NeoMarkers, Fremont, CA, USA) was then added to each section and incubated overnight at 4°C in a humidified chamber. The next day, sections were washed three times in 1× PBS (5 min each), incubated at room temperature in a humidified chamber with goat polyclonal antimouse biotinylated immunoglobulin (1 : 500 dilution; DakoCytomation, Denmark) for 1 h, and washed again in 1× PBS (three washes, 5 min each). Sections were then incubated with avidin-biotin complex (1 : 150 dilution in 1× PBS; Vector Laboratories, Inc, Burlingame, CA, USA) for 30 min at room temperature and washed three times in 1× PBS (5 min each). Sections were incubated with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich, Australia) for 8 min, rinsed in 1× PBS and counterstained with haematoxylin. Specificity of immunostaining for TSP-1 was confirmed by omission of the primary antibody.

Sections were examined using light microscopy, and digital images were acquired and analysed using ImagePro Plus (Media Cybernetics, Silver Spring, MD, USA). Three fields of view per section and three sections (from different regions of the lung) per animal were analysed. For each field of view, the area of tissue positively stained for TSP-1 was expressed as a percentage of the total area of tissue. This was then averaged for each animal and each experimental group. The analysis was performed on the alveolar region of the lung, avoiding areas containing major airways or blood vessels. Each treatment group with their respective control group was performed in separate immunohistochemical experiments and each run was analysed independently.

Quantitative real-time polymerase chain reaction

EGR1 mRNA levels in fetal lung tissue were measured at 36 h and 2 days of increased fetal lung expansion using quantitative real-time polymerase chain reaction (qRT-PCR). EGR1 primers (upstream 5′-AGGGTCACT-GTGGAAGGTC-3′; downstream 5′-GCAGCTGAAGTC-AAAGGAA-3′) were designed based on the ovine sequence (nt 444–532; Accession number DQ239634 (Sozo et al. 2006)). Primers for the amplification of the housekeeping gene 18S (upstream 5′-GTCTGTG-ATGCCCTTAGATGTC-3′; downstream 5′-AAGCTTA-TGACCCGCACTTAC-3′) were used to account for minor differences in the amount of cDNA template added to the reaction for each animal.

Total RNA (10 μg) was DNase-treated and reverse transcribed into cDNA (M-MLV Reverse Transcriptase, RNase H Minus, Point Mutant Kit; Promega, Madison, WI, USA). qRT-PCR was performed using a Rotor-Gene 3000 Real-Time Multiplexing System (Corbett Life Sciences, Australia) using 20 μl reactions, containing 1 μl cDNA template (50 ng μl−1 for EGR1 and 200 ng μl−1 for 18S), 1 μl of each forward and reverse primer (4 μm for EGR1 and 10 μm for 18S), 10 μl SYBR green (Platinum® SYBR® Green qPCR SuperMix-UDG; Invitrogen Life Technologies, Carlsbad, CA, USA) and 7 μl of nuclease-free water. The thermal profile used to amplify the PCR products included an initial 2 min incubation at 95°C, followed by 40 cycles of; denaturation at 95°C for 3 s, annealing at 59°C for 20 s, and elongation at 72°C for 20 s. The fluorescence readings were recorded after each 72°C step. Dissociation curves were performed after each PCR run to ensure that a single PCR product had been amplified per primer set. Each sample was measured in triplicate and a control sample, containing no template, was included in each run. The EGR1 mRNA levels of each animal were normalized to the 18S rRNA values for that animal and are expressed relative to the mean EGR1 mRNA levels in control fetuses.

Statistical analysis

Results are presented as the mean ±s.e.m. Student's unpaired t test or one-way analysis of variance (ANOVA; for >2 groups) were used to compare differences between ages or treatments. Significant differences indicated by ANOVA were subjected to the least significant difference post hoc test to detect significant differences between individual group means. The TSP-1 mRNA level data in response to sustained increases in fetal lung expansion and EGR1 mRNA level data at 2 days TO were normalized by log10 transformation. TSP-1 mRNA levels were correlated to DNA synthesis rates, which were measured by the incorporation of [3H]thymidine into DNA (Hooper et al. 1993; Keramidaris et al. 1996; Nardo et al. 1998), using the Pearson product moment correlation. A P value of < 0.05 was taken to be statistically significant.

Results

TSP-1 mRNA expression during normal lung development

TSP-1 mRNA levels remained constant during the canalicular and early alveolar stages of lung development, i.e. at 91 days, 105 days, 111 days and 128 days GA. At 138 days and 142 days GA, however, TSP-1 mRNA levels were reduced to 78.8 ± 4.4% and 78.2 ± 1.8%, respectively, of 128 days GA values (100.0 ± 3.0%; P < 0.005) (Fig. 1). By 2 weeks of postnatal age, the mRNA levels of TSP-1 had returned to the levels present during the canalicular stage of lung development.

Figure 1. TSP-1 mRNA levels during normal lung development.

Figure 1

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A, lung TSP-1 mRNA levels from control fetuses at 91 days, 105 days, 111 days, 128 days, 138 days or 142 days of gestation and control lambs at 2 weeks of postnatal age. *Values that are significantly different (P < 0.05) from values at 128 days GA. Term (∼147 days GA) is indicated by the dashed line. B, representative Northern blots containing total RNA (20 μg) from the lungs of control fetuses at various gestational ages (grey blocks) and from control lambs at 2 weeks of postnatal age (solid block). Each lane represents RNA from a different animal, and the panels show the same Northern blot hybridized with a 32P-labelled cDNA probe for ovine TSP-1 (upper panel) or 18S (lower panel). Each band that corresponded to TSP-1 on each blot was corrected for the level of 18S rRNA present in the same lane to adjust for minor differences in loading.

TSP-1 mRNA levels following accelerated and retarded fetal lung growth

TSP-1 mRNA levels were significantly increased to 347.5 ± 73.6% of control levels (100.0 ± 14.0%) at 36 h of increased fetal lung expansion (P < 0.05) and were reduced to 137.2 ± 18.9% of control levels at 2 days of increased fetal lung expansion; these values were not significantly different from control. TSP-1 mRNA levels in fetal lung tissue tended to be reduced to 81.0 ± 3.9% of control levels at 4 days of increased fetal lung expansion, relative to the levels at 2 days of increased fetal lung expansion (P= 0.07) but were not different from control levels. At 10 days of increased fetal lung expansion, TSP-1 mRNA levels (107.1 ± 18.9) were similar to those in control fetuses (Fig. 2). TSP-1 mRNA levels were not different from control levels (100.0 ± 14.4%) at 7 days of decreased fetal lung expansion (90.6 ± 23.8%); however, at 20 days of decreased fetal lung expansion, TSP-1 mRNA levels were significantly reduced to 39.4 ± 6.1% of control levels (100.0 ± 20.4%; P < 0.05) (Fig. 3). TSP-1 mRNA levels were positively correlated to DNA synthesis rates, previously determined by us (Hooper et al. 1993; Keramidaris et al. 1996; Nardo et al. 1998), during alterations in fetal lung expansion (y= 14.4x– 1233.4; R= 0.872, P < 0.05).

Figure 2. TSP-1 mRNA levels following accelerated fetal lung growth.

Figure 2

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A, lung TSP-1 mRNA levels from control fetuses (solid bar) and fetuses exposed to 36 h (Sozo et al. 2006) (open bar), 2 days, 4 days or 10 days of increased lung expansion (tracheal obstruction, TO; grey bars). *Values that are significantly different (P < 0.05) from control values. B, representative Northern blots containing total RNA (20 μg) from the lungs of control fetuses and from fetuses exposed to 36 h of increased lung expansion. Each lane represents RNA from a different fetus, and the panels show the same Northern blot hybridized with a 32P-labelled cDNA probe for ovine TSP-1 (upper panel) or 18S (lower panel). Each band that corresponded to TSP-1 on the blot was corrected for the level of 18S rRNA present in the same lane to adjust for minor differences in loading.

Figure 3. TSP-1 mRNA levels following retarded fetal lung growth.

Figure 3

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A, lung TSP-1 mRNA levels from control fetuses (solid bar) and fetuses exposed to 7 days (open bar) or 20 days (grey bar) of decreased lung expansion (drain). *Values that are significantly different (P < 0.05) from control values. B, representative Northern blots containing total RNA (20 μg) from the lungs of control fetuses and from fetuses exposed to 20 days of decreased lung expansion. Each lane represents RNA from a different fetus, and the panels show the same Northern blot hybridized with a 32P-labelled cDNA probe for ovine TSP-1 (upper panel) or 18S (lower panel). Each band that corresponded to TSP-1 on the blot was corrected for the level of 18S rRNA present in the same lane to adjust for minor differences in loading.

In the peri-alveolar region of the fetal lung, in both control fetuses and fetuses exposed to 36 h of increased lung expansion, TSP-1 mRNA was predominantly localized in type-II AECs and in interstitial cells, probably fibroblasts (Fig. 4). Type-II AECs were classified by their distinctive large rounded nuclei that protrude into the alveoli. TSP-1 mRNA was also present in cartilage, in the epithelium of small bronchioles, in smooth muscle cells and in cells (probably fibroblasts) present within connective tissue septa of the lung (not shown). Cells lining large blood vessels, i.e. endothelial cells, did not appear to contain TSP-1 mRNA. No staining was visible in sections hybridized with the sense riboprobe, sections without either riboprobe or sections without the anti-DIG antibody (data not shown). In fetuses exposed to 36 h of increased lung expansion, the proportion of TSP-1-labelled cells in the alveolar region of the lung was significantly greater (5.2 ± 0.8%) than in control fetuses (1.9 ± 0.4%; P < 0.01).

Figure 4. TSP-1 mRNA localization in lung tissue following accelerated fetal lung growth.

Figure 4

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High-power (bar = 10 μm) images demonstrating the cells expressing TSP-1 mRNA (purple/blue staining) in lung tissue from control fetuses (A) and fetuses exposed to 36 h of increased lung expansion (B). Note the positive staining (arrows) in interstitial cells (probably fibroblasts) of the lung parenchyma and type-II AECs.

Localization of TSP-1 in fetal lung tissue following accelerated and retarded fetal lung growth

In all control fetuses and fetuses exposed to altered lung expansion, TSP-1 was localized to the epithelium of small bronchioles, and was present in cartilage and on the luminal surface of blood vessels (Fig. 5). In the alveolar region of the fetal lung, TSP-1 was present in all cell types, especially in cells within the peri-alveolar region (probably fibroblasts). TSP-1 localized predominantly to the cell cytoplasm and interstitium in control fetuses, although a number of stained nuclei were also observed. In fetuses exposed to an increase in lung expansion, there was a greater abundance of heavily stained nuclei compared to controls, and the proportion of tissue stained positive for the TSP-1 protein in the alveolar region of the lung was doubled in fetuses exposed to 36 h (23.3 ± 2.2%) and 2 days (22.1 ± 2.3%) of increased lung expansion compared to control fetuses (11.7 ± 3.2% and 12.7 ± 1.3%, respectively; P < 0.05). The proportion of tissue stained positive for the TSP-1 protein in the alveolar region of the lung was nearly halved in fetuses exposed to 20 days of decreased lung expansion (25.7 ± 3.2%) compared to control fetuses (39.8 ± 3.3%; P < 0.05). No staining was visible in sections that were not incubated with the anti-TSP-1 antibody (Fig. 5B).

Figure 5. TSP-1 protein localization in lung tissue following alterations in fetal lung expansion.

Figure 5

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A and B, low-power (bar = 10 μm) images demonstrating the localization of TSP-1 protein (brown staining) in fetal lung tissue. Note the positive staining in epithelial cells lining bronchioles (EP), blood vessels (BV) and cartilage (CL) in A relative to negative control sections (B). C–F, high-power (bar = 10 μm) images demonstrating the localization of TSP-1 protein (brown staining) in lung tissue from control fetuses (C and E), fetuses exposed to 36 h of increased lung expansion (D) and fetuses exposed to 20 days of decreased lung expansion (F). Note the positive staining in cells likely to be type-I alveolar epithelial cells (I), type-II alveolar epithelial cells (II) and interstitial cells (IC; probably fibroblasts) of the lung parenchyma. Staining was absent in the negative control sections (B). The TSP-1 protein localization studies were performed for each treatment group and its control group in separate immunohistochemical runs, and each run was analysed independently.

EGR1 mRNA levels following alterations in fetal lung growth

Using real-time PCR, EGR1 mRNA levels were shown to be 2.7 ± 0.7-fold higher than control levels (1.0 ± 0.2) at 36 h of increased fetal lung expansion (P < 0.05), but were not different from control levels (1.0 ± 0.2) at 2 days of increased fetal lung expansion (1.7 ± 0.8-fold of control levels) (Fig. 6). A Northern blot analysis, using 40 μg of RNA per animal, demonstrated that EGR1 mRNA levels were not different from control levels (100.0 ± 5.6%) at 3 days (105.8 ± 9.4%) and 10 days (116.9 ± 11.1%) of increased fetal lung expansion. EGR1 mRNA levels in fetuses exposed to 20 days of decreased fetal lung expansion (111.0 ± 7.3%) were also similar to the levels in control fetuses (100.0 ± 5.6%) (data not shown).

Figure 6. EGR1 mRNA levels following accelerated fetal lung growth.

Figure 6

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Lung EGR1 mRNA levels, determined by qRT-PCR, in control fetuses (solid bars) and fetuses exposed to 36 h (open bar) or 2 days (grey bar) of increased lung expansion (tracheal obstruction, TO). *Values that are significantly different (P < 0.05) from control values.

Discussion

The expression of TSP-1 in normal lung growth and development has not previously been examined. We recently identified TSP-1 as a gene differentially expressed by increased fetal lung expansion, an intervention that accelerates fetal lung growth (Sozo et al. 2006). As TSP-1 has been suggested by others to play a role in developmental processes (O'Shea & Dixit, 1988; Corless et al. 1992; Iruela-Arispe et al. 1993; Bornstein, 2001; Wu et al. 2006), we aimed to characterize the expression and localization of TSP-1 mRNA and protein within the ovine fetal lung during the canalicular and alveolar stages of lung development. TSP-1 expression increased in response to an increase in fetal lung expansion, when the mechanisms accelerating lung cell proliferation are expected to be most active. Conversely, a decrease in fetal lung expansion, which causes lung growth to cease, resulted in a reduction in TSP-1 expression and a halving in the percentage of tissue positive for the TSP-1 protein. These data suggest that TSP-1 may play an important role in the regulation of fetal lung development.

TSP-1 and cell proliferation

It is well established that the growth response induced by an increase in fetal lung expansion involves an increase in cell proliferation (Nardo et al. 1998, 2000), differentiation of alveolar epithelial cell types (Alcorn et al. 1977; Flecknoe et al. 2000) and thinning of the peri-alveolar tissue (Alcorn et al. 1977; Nardo et al. 2000) – properties that would enhance the gas exchange potential of the lung. For example, lung DNA synthesis rates have been shown to increase to ∼800% above control levels at 2 days of increased fetal lung expansion (Nardo et al. 1998) due to the proliferation of fibroblasts, type-II AECs and endothelial cells (Nardo et al. 2000). Therefore, the alterations in TSP-1 levels just prior to this time point suggest that TSP-1 may have a role in initiating this increase in cell proliferation. Indeed, TSP-1 mRNA levels were positively correlated to DNA synthesis rates in response to alterations in fetal lung expansion.

The reported effects of TSP-1 on cell proliferation vary according to cell type and are also dependent on substratum type and protein conformation, which affects the availability of protein domains to interact with different cell-surface receptors (Bornstein, 2001). TSP-1 has been shown to stimulate the proliferation of smooth muscle cells (Majack et al. 1988; Bagavandoss & Wilks, 1990) and fibroblasts (Phan et al. 1989; Bagavandoss & Wilks, 1990). The presence and upregulation of TSP-1 mRNA and protein in fibroblasts and type-II AECs in response to an increase in lung expansion, therefore suggests that TSP-1 may induce these cells to proliferate. In contrast, TSP-1 has been shown to inhibit proliferation in endothelial cells from different sources, including endothelial cells from the pulmonary artery (Bagavandoss & Wilks, 1990), but can promote proliferation in endo-thelial cells from other sources, such as venous endo-thelial cells (Calzada et al. 2004). TSP-1 mRNA did not appear to be present in endothelial cells lining blood vessels in the lung, although the TSP-1 protein was deposited on the luminal surface of blood vessels and may therefore exert a paracrine effect on the endothelial cells. Although not studied or analysed directly, the level of TSP-1 protein on the luminal surface of blood vessels appeared to be higher in fetuses exposed to an increase in fetal lung expansion compared to control fetuses, and lower in fetuses exposed to a decrease in fetal lung expansion compared to control fetuses. Generally, TSP-1 is considered an inhibitor of angiogenesis; TSP-1 is absent where new vessels are forming but present where neovascularization is almost complete (O'Shea & Dixit, 1988; Good et al. 1990; Lane et al. 1992). The presence of TSP-1 on the luminal surface of large blood vessels within the lungs of fetuses exposed to an increase in fetal lung expansion may therefore be associated with the prevention of further proliferation and migration of endothelial cells in newly formed vessels, and thus the stabilization of the new vessel structure (Sage & Bornstein, 1991). Since endothelial cells cannot be distinguished from type-I epithelial cells at the light microscopy level, it is possible that TSP-1 is present in or beneath endothelial cells and may affect their proliferation in small capillaries. TSP-1 was also present in most chondrocytes within the lungs of all fetal sheep, supporting the finding of others that nearly all chondrocytes express TSP-1 (Iruela-Arispe et al. 1993).

TSP-1 and ECM remodelling

An increase in fetal lung expansion induced by tracheal obstruction has also been shown to result in extracellular matrix remodelling, which includes the synthesis of extracellular matrix components such as tropoelastin and collagen (Nardo et al. 1998; Joyce et al. 2003; Sozo et al. 2006). TSP-1 is present in cultured lung fibroblasts and it induces myofibroblasts to deposit collagen in lung cell cultures (Morishima et al. 2001). In the current in vivo study, TSP-1 was also found in fibroblasts and connective tissue regions of the lung and was increased at 36 h TO, suggesting that TSP-1 may regulate the deposition of extracellular matrix components, such as collagen, at or prior to 2 days, and may also regulate remodelling of the extracellular matrix. The mechanisms are unknown but may involve the activation of transforming growth factor beta 1 (TGFβ1). TSP-1 can activate latent TGFβ1 (Schultz-Cherry et al. 1995; Ribeiro et al. 1999), which in turn can regulate remodelling of the ECM. However, we have previously shown that the bioactive TGFβ1 protein is not elevated at 2 days of increased fetal lung expansion (Wallace et al. 2006).

TSP-1 during normal lung development

During the alveolar stage of lung development, at 138 days and 142 days GA, TSP-1 mRNA levels were reduced to ∼80% of that observed in the canalicular stage of lung development; earlier periods of lung development were not studied. In mice, TSP-1 mRNA expression in the lung begins during the pseudoglandular stage of development, reaching a maximum during the early saccular stage of development and is apparent only in large bronchi (Iruela-Arispe et al. 1993). The expression of TSP-1 in the alveolar stage of lung development in mice, however, has not been studied. In the present study, TSP-1 mRNA was not only present in the cells beneath the epithelium of bronchioles, where TSP-2 is expressed in mice (Iruela-Arispe et al. 1993), but was also present in the epithelia of alveoli; TSP-3 was the only TSP expressed in distal airspaces in mice (Iruela-Arispe et al. 1993). Although it is possible that the probe we used to detect TSP-1 mRNA also detects the other ovine TSP genes, nucleotide analysis shows high homology to TSP-1 in mice and humans and lower sequence similarity to the other TSP homologues in mice and humans; the sheep sequences of TSP-2 and TSP-3 are not known. It is more likely that these differences in the distribution of TSP-1 mRNA in the lung may be due to a species difference, or it is possible that during the alveolar stage of lung development, the expression of TSP-1 changes, relative to earlier stages of lung development, and that this may correlate to differing roles TSP-1 may have during different stages of lung development. The reduction in TSP-1 mRNA levels during the alveolar stage of lung development coincides with, and may be responsible for, a decrease in the rate of lung cell proliferation that occurs at this time (Filby et al. 2006). The reduction in TSP-1 mRNA levels at this time also coincides with a period where capillaries are rapidly being formed to facilitate efficient gas exchange, suggesting that this reduction may enable endothelial cells to proliferate and migrate to form new vessels.

Regulation of TSP-1 expression

At 36 h of increased fetal lung expansion, TSP-1 mRNA levels increased to ∼3-fold of control levels. This upregulation in TSP-1 mRNA expression was accompanied by a doubling in TSP-1 protein levels at 36 h, which were sustained for at least 2 days of increased fetal lung expansion. Unfortunately, TSP-1 protein levels were unable to be studied during longer periods of increased fetal lung expansion as the fixation of that tissue was not compatible with the immunohistochemical technique used. The doubling in TSP-1 protein levels after only 36 h of increased lung expansion suggests that TSP-1 expression could be upregulated even earlier than this time point, although further studies are required to confirm this. These rapid alterations in TSP-1 levels suggest that TSP-1 is able to respond to a stimulus very quickly. Indeed, TSP-1 has previously been identified as an immediate early gene (Inuzuka et al. 1999). It is unknown, however, how TSP-1 is itself activated by an increase in fetal lung expansion. EGR1, which is also an early-response gene and a transcription factor, can positively regulate the expression of TSP-1 (Shingu & Bornstein, 1994; Moon et al. 2005). We recently identified EGR1 as a gene differentially expressed by an increase in fetal lung expansion (Sozo et al. 2006), and using qRT-PCR we have shown that EGR1 mRNA levels are increased at 36 h of increased fetal lung expansion. The TSP-1 promoter contains an EGR1 binding site and a Sp1 site that are important for TSP-1 transcription (Shingu & Bornstein, 1994), suggesting that the upregulation of EGR1 in response to an increase in fetal lung expansion could activate the TSP-1 promoter and be responsible for the increase in TSP-1 expression that occurs.

Conclusions

In summary, we have shown that TSP-1 mRNA and protein levels are increased by an increase in fetal lung expansion when cell proliferation rates are high, and reduced by a decrease in fetal lung expansion when cell proliferation rates are low. TSP-1 mRNA levels are also high during the canalicular and early alveolar stages of lung development, and decrease later in the alveolar stage of lung development, when cell proliferation rates also decrease. Given the known functions of TSP-1 as a regulator of cell proliferation, angiogenesis and extracellular matrix production and remodelling, we suggest that TSP-1 may have a significant role in regulating these processes in the fetal sheep lung. However, in order to determine the precise role of TSP-1 in lung development, further studies are needed to block TSP-1 expression in specific lung cell types where it is known to be expressed.

Acknowledgments

We are indebted to Gosia Zieba and Valerie Zahra for expert technical assistance. This work was funded by the National Health and Medical Research Council of Australia.

References

  1. Adams JC. Thrombospondins: Multifunctional regulators of cell interactions. Annu Rev Cell Dev Biol. 2001;17:25–51. doi: 10.1146/annurev.cellbio.17.1.25. [DOI] [PubMed] [Google Scholar]
  2. Alcorn D, Adamson TM, Lambert TF, Maloney JE, Ritchie BC, Robinson PM. Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat. 1977;123:649–660. [PMC free article] [PubMed] [Google Scholar]
  3. Bagavandoss P, Wilks JW. Specific-inhibition of endothelial-cell proliferation by thrombospondin. Biochem Biophys Res Comm. 1990;170:867–872. doi: 10.1016/0006-291x(90)92171-u. [DOI] [PubMed] [Google Scholar]
  4. Boland R, Joyce BJ, Wallace MJ, Stanton H, Fosang AJ, Pierce RA, Harding R, Hooper SB. Cortisol enhances structural maturation of the hypoplastic fetal lung in sheep. J Physiol. 2004;554:505–517. doi: 10.1113/jphysiol.2003.055111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boland RE, Nardo L, Hooper SB. Cortisol pretreatment enhances the lung growth response to tracheal obstruction in fetal sheep. Am J Physiol Lung Cell Mol Physiol. 1997;273:L1126–L1131. doi: 10.1152/ajplung.1997.273.6.L1126. [DOI] [PubMed] [Google Scholar]
  6. Bornstein P. Thrombospondins as matricellular modulators of cell function. J Clin Invest. 2001;107:929–934. doi: 10.1172/JCI12749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Calzada MJ, Zhou L, Sipes JM, Zhang J, Krutzsch HC, Iruela-Arispe ML, Annis DS, Mosher DF, Roberts DD. α4β1 integrin mediates selective endothelial cell responses to thrombospondins 1 and 2 in vitro and modulates angiogenesis in vivo. Circ Res. 2004;94:462–470. doi: 10.1161/01.RES.0000115555.05668.93. [DOI] [PubMed] [Google Scholar]
  8. Corless CL, Mendoza A, Collins T, Lawler J. Colocalization of thrombospondin and syndecan during murine development. Dev Dynamics. 1992;193:346–358. doi: 10.1002/aja.1001930408. [DOI] [PubMed] [Google Scholar]
  9. Filby CE, Hooper SB, Sozo F, Zahra VA, Flecknoe SJ, Wallace MJ. VDUP1: a potential mediator of expansion-induced lung growth and epithelial cell differentiation in the ovine fetus. Am J Physiol Lung Cell Mol Physiol. 2006;290:L250–L258. doi: 10.1152/ajplung.00244.2005. [DOI] [PubMed] [Google Scholar]
  10. Flecknoe S, Harding R, Maritz G, Hooper SB. Increased lung expansion alters the proportions of type I and type II alveolar epithelial cells in fetal sheep. Am J Physiol Lung Cell Mol Physiol. 2000;278:L1180–L1185. doi: 10.1152/ajplung.2000.278.6.L1180. [DOI] [PubMed] [Google Scholar]
  11. Flecknoe SJ, Wallace MJ, Cock ML, Harding R, Hooper SB. Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep. Am J Physiol Lung Cell Mol Physiol. 2003;285:L664–L670. doi: 10.1152/ajplung.00306.2002. [DOI] [PubMed] [Google Scholar]
  12. Good DJ, Polverini PJ, Rastinejad F, Lebeau MM, Lemons RS, Frazier WA, Bouck NP. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc Nat Acad Sci U S A. 1990;87:6624–6628. doi: 10.1073/pnas.87.17.6624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harding R, Bocking AD, Sigger JN. Influence of upper respiratory tract on liquid flow to and from fetal lungs. J Appl Physiol. 1986;61:68–74. doi: 10.1152/jappl.1986.61.1.68. [DOI] [PubMed] [Google Scholar]
  14. Harding R, Hooper SB. Regulation of lung expansion and lung growth before birth. J Appl Physiol. 1996;81:209–224. doi: 10.1152/jappl.1996.81.1.209. [DOI] [PubMed] [Google Scholar]
  15. Hooper SB, Dickson KA, Harding R. Lung liquid secretion, flow and volume in response to moderate asphyxia in fetal sheep. J Dev Physiol. 1988;10:473–485. [PubMed] [Google Scholar]
  16. Hooper SB, Han VKM, Harding R. Changes in lung expansion alter pulmonary DNA synthesis and IGF-II gene expression in fetal sheep. Am J Physiol Lung Cell Mol Physiol. 1993;265:L403–L409. doi: 10.1152/ajplung.1993.265.4.L403. [DOI] [PubMed] [Google Scholar]
  17. Inuzuka H, Nanbu-Wakao R, Masuho Y, Muramatsu M, Tojo H, Wakao H. Differential regulation of immediate early gene expression in preadipocyte cells through multiple signaling pathways. Biochem Biophys Res Comm. 1999;265:664–668. doi: 10.1006/bbrc.1999.1734. [DOI] [PubMed] [Google Scholar]
  18. Iruela-Arispe ML, Liska DJ, Sage EH, Bornstein P. Differential expression of thrombospondin 1, 2, and 3 during murine development. Dev Dyn. 1993;197:40–56. doi: 10.1002/aja.1001970105. [DOI] [PubMed] [Google Scholar]
  19. Joyce BJ, Wallace MJ, Pierce RA, Harding R, Hooper SB. Sustained changes in lung expansion alter tropoelastin mRNA levels and elastin content in fetal sheep lungs. Am J Physiol Lung Cell Mol Physiol. 2003;284:L643–L649. doi: 10.1152/ajplung.00090.2002. [DOI] [PubMed] [Google Scholar]
  20. Keramidaris E, Hooper SB, Harding R. Effect of gestational age on the increase in fetal lung growth following tracheal obstruction. Exp Lung Res. 1996;22:283–298. doi: 10.3109/01902149609031776. [DOI] [PubMed] [Google Scholar]
  21. Lane TF, Iruela-Arispe ML, Sage EH. Regulation of gene-expression by SPARC during angiogenesis in vitro – changes in fibronectin, thrombospondin-1, and plasminogen-activator inhibitor-1. J Biol Chem. 1992;267:16736–16745. [PubMed] [Google Scholar]
  22. Lawler JW, Slayter HS, Coligan JE. Isolation and characterization of a high molecular-weight glycoprotein from human-blood platelets. J Biol Chem. 1978;253:8609–8616. [PubMed] [Google Scholar]
  23. Lines A, Nardo L, Phillips ID, Possmayer F, Hooper SB. Alterations in lung expansion affect surfactant protein A, B and C mRNA levels in fetal sheep. Am J Physiol Lung Cell Mol Physiol. 1999;276:L239–L245. doi: 10.1152/ajplung.1999.276.2.L239. [DOI] [PubMed] [Google Scholar]
  24. Majack RA, Goodman LV, Dixit VM. Cell-surface thrombospondin is functionally essential for vascular smooth-muscle cell-proliferation. J Cell Biol. 1988;106:415–422. doi: 10.1083/jcb.106.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moessinger AC, Harding R, Adamson TM, Singh M, Kiu GT. Role of lung fluid volume in growth and maturation of the fetal sheep lung. J Clin Invest. 1990;86:1270–1277. doi: 10.1172/JCI114834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moon Y, Bottone FG, McEntee MF, Eling TE. Suppression of tumor cell invasion by cyclooxygenase inhibitors is mediated by thrombospondin-1 via the early growth response gene Egr-1. Mol Cancer Ther. 2005;4:1551–1558. doi: 10.1158/1535-7163.MCT-05-0213. [DOI] [PubMed] [Google Scholar]
  27. Morishima Y, Nomura A, Uchida Y, Noguchi Y, Sakamoto T, Ishii Y, Goto Y, Masuyama K, Zhang MJ, Hirano K, Mochizuki M, Ohtsuka M, Sekizawa K. Triggering the induction of myofibroblast and fibrogenesis by airway epithelial shedding. Am J Respir Cell Mol Biol. 2001;24:1–11. doi: 10.1165/ajrcmb.24.1.4040. [DOI] [PubMed] [Google Scholar]
  28. Murphy-Ullrich JE, Hook M. Thrombospondin modulates focal adhesions in endothelial cells. J Cell Biol. 1989;109:1309–1319. doi: 10.1083/jcb.109.3.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nardo L, Hooper SB, Harding R. Stimulation of lung growth by tracheal obstruction in fetal sheep: relation to luminal pressure and lung liquid. Pediatr Res. 1998;43:184–190. doi: 10.1203/00006450-199802000-00005. [DOI] [PubMed] [Google Scholar]
  30. Nardo L, Maritz G, Harding R, Hooper SB. Changes in lung structure and cellular division induced by tracheal obstruction in fetal sheep. Exp Lung Res. 2000;26:105–119. doi: 10.1080/019021400269907. [DOI] [PubMed] [Google Scholar]
  31. O'Shea KS, Dixit VM. Unique distribution of the extracellular matrix component thrombospondin in the developing mouse embryo. J Cell Biol. 1988;107:2737–2748. doi: 10.1083/jcb.107.6.2737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Phan SH, Dillon RG, McGarry BM, Dixit VM. Stimulation of fibroblast proliferation by thrombospondin. Biochem Biophys Res Comm. 1989;163:56–63. doi: 10.1016/0006-291x(89)92098-6. [DOI] [PubMed] [Google Scholar]
  33. Ribeiro SMF, Poczatek M, Schultz-Cherry S, Villain M, Murphy-Ullrich JE. The activation sequence of thrombospondin-1 interacts with the latency-associated peptide to regulate activation of latent transforming growth factor-beta. J Biol Chem. 1999;274:13586–13593. doi: 10.1074/jbc.274.19.13586. [DOI] [PubMed] [Google Scholar]
  34. Sage EH, Bornstein P. Extracellular proteins that modulate cell–matrix interactions – SPARC, tenascin, and thrombospondin. J Biol Chem. 1991;266:14831–14834. [PubMed] [Google Scholar]
  35. Schultz-Cherry S, Chen H, Mosher DF, Misenheimer TM, Krutzsch HC, Roberts DD, Murphy-Ullrich JE. Regulation of transforming growth factor-beta activation by discrete sequences of thrombospondin 1. J Biol Chem. 1995;270:7304–7310. doi: 10.1074/jbc.270.13.7304. [DOI] [PubMed] [Google Scholar]
  36. Shingu T, Bornstein P. Overlapping Egr-1 and Sp1 sites function in the regulation of transcription of the mouse thrombospondin-1 gene. J Biol Chem. 1994;269:32551–32557. [PubMed] [Google Scholar]
  37. Sozo F, Wallace MJ, Zahra VA, Filby CE, Hooper SB. Gene expression profiling during increased fetal lung expansion identifies genes likely to regulate development of the distal airways. Physiol Genomics. 2006;24:105–113. doi: 10.1152/physiolgenomics.00148.2005. [DOI] [PubMed] [Google Scholar]
  38. Vilos GA, Liggins GC. Intrathoracic pressures in fetal sheep. J Dev Physiol. 1982;4:247–256. [PubMed] [Google Scholar]
  39. Wallace MJ, Thiel AM, Lines AM, Polglase GR, Sozo F, Hooper SB. Role of platelet-derived growth factor-B, vascular endothelial growth factor, insulin-like growth factor-II, mitogen-activated protein kinase and transforming growth factor-beta 1 in expansion-induced lung growth in fetal sheep. Reprod Fertil Dev. 2006;18:655–665. doi: 10.1071/rd05163. [DOI] [PubMed] [Google Scholar]
  40. Wu Z, Wang S, Sorenson CM, Sheibani N. Attenuation of retinal vascular development and neovascularization in transgenic mice over-expressing thrombospondin-1 in the lens. Dev Dyn. 2006;235:1908–1920. doi: 10.1002/dvdy.20837. [DOI] [PubMed] [Google Scholar]

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