Abstract
Transforming growth factor (TGF)-β mediates hypoxia-induced inhibition of alveolar development in the newborn lung. TGF-β is regulated primarily at the level of activation of latent TGF-β. Fibroblasts expressing Thy-1 (CD90) inhibit TGF-β activation. We hypothesized that loss of Thy-1 due to hypoxia may be a mechanism by which hypoxia increases TGF-β activation and that animals deficient in Thy-1 will simulate the effects of hypoxia on lung development. To determine if loss of Thy-1 occurred during hypoxia, non-transgenic (C57BL/6) wild-type (WT) mice exposed to hypoxia were evaluated for Thy-1 mRNA and protein. To determine if Thy-1 deficiency simulated hypoxia, WT and Thy-1 null (Thy-1−/−) mice were exposed to air or hypoxia from birth to 2 wk, the critical period of lung development, and lung histology, function, parameters related to TGF-β signaling, and extracellular matrix protein content were measured. To test if the phenotype in Thy-1−/− mice was due to excessive TGF-β signaling, measurements were also performed in Thy-1−/− mice administered TGF-β neutralizing antibody (1D11). We observed that hypoxia reduced Thy-1 mRNA and Thy-1 staining in WT mice. Thy-1−/− mice had impaired alveolarization, increased TGF-β signaling, reduced lung epithelial and endothelial cell proliferation but increased fibroblast proliferation, and increased collagen and elastin. Lung compliance was lower, and tissue but not airway resistance was higher in Thy-1−/− mice at 2 wk. Thy-1−/− mice given 1D11 had improved alveolar development and lung function. These data support the hypothesis that hypoxia, by reducing Thy-1, increases TGF-β activation, and thereby inhibits normal alveolar development.
Keywords: transforming growth factor-β, infant, CD90
exposure to chronic hypoxia inhibits lung development (8, 16). Transforming growth factor (TGF)-β signaling is dynamically regulated during alveolar development (1), and we have recently shown that TGF-β signaling plays an important role in hypoxia-induced inhibition of lung development (5). Hypoxia increases TGF-β activation and signaling, and inhibition of TGF-β signaling using a mouse model with an inducible dominant-negative TGF-β receptor attenuated the hypoxia-induced inhibition of alveolar development and abnormal vascular remodeling (5). TGF-β is regulated mainly at the level of activation of inactive latent TGF-β (2, 7), but it is not known how increased TGF-β activation occurs during hypoxia. Thy-1 (CD90) is a glycophosphatidylinositol (GPI)-linked outer membrane leaflet glycoprotein that is predominantly expressed on subsets of fibroblasts and lymphocytes, and we have previously demonstrated that presence of Thy-1 limits the ability of fibroblasts to activate latent TGF-β and express α-smooth muscle actin (αSMA) (31), whereas fibroblasts lacking Thy-1 show enhanced myofibroblastic differentiation and survival (25). We have shown that loss of fibroblast Thy-1 expression correlates with increased TGF-β activation and lung fibrogenesis in vivo (13), indicating that decreased Thy-1 expression may have adverse consequences.
We hypothesized that a loss of Thy-1 due to hypoxia may be a mechanism by which TGF-β activation is increased during hypoxic conditions and that animals deficient in Thy-1 will simulate the effects of hypoxia on lung development.
METHODS
All protocols were approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham and were consistent with the Public Health Service policy on Humane Care and Use of Laboratory Animals (Office of Laboratory Animal Welfare, Aug 2002) and the Guide for the Care and Use of Laboratory Animals (National Research Council, National Academy Press, 1996). All experiments, unless otherwise specified, were done with a minimum of six mice from at least two litters for each experimental condition.
To determine if loss of Thy-1 occurred during hypoxia, wild-type mice exposed to hypoxia were evaluated for Thy-1 mRNA expression by real-time quantitative PCR and Thy-1 protein by immunohistochemistry. To determine if animals deficient in Thy-1 simulated the effects of hypoxia on lung development, wild-type and Thy-1 null mice were exposed to air or hypoxia from birth for up to 2 wk, the critical period of lung development, and lung histology, lung function, parameters related to TGF-β signaling, and extracellular matrix protein content in the lung were measured. To test whether the phenotype in animals deficient in Thy-1 was due to excessive TGF-β signaling, measurements were also performed in animals administered neutralizing antibody to TGF-β from birth.
Animal Model
Wild-type (WT) C57BL/6 mice and Thy-1 null (Thy-1−/−) mice were exposed to normobaric 12% O2 (hypoxia) or air (normoxia) from soon after birth for 2 wk [the period of maximal alveolar development (9)] as described previously (3–6). Briefly, a Pro-Ox 110 oxygen controller (Bio-Spherix, Redfield, NY) servo-controlled the oxygen concentration to the set level by controlling the inflow of a mixture of nitrogen and oxygen gases. In addition, O2 concentration (OM-100 Oxygen Analyzer; Newport Medical Instruments, Newport Beach, CA), humidity, temperature, and barometric pressure (Fisherbrand Digital Barometer; Fisher Scientific, Pittsburgh, PA) within the chamber were monitored continuously. Carbon dioxide accumulation within the chamber was not noted.
The Thy-1−/− mouse was generated on a C57BL/6 background by insertion of a neomycin resistance gene into the third exon of the Thy-1 gene (20) and was kindly provided to Dr. Hagood by Dr. Michael Bloom (National Heart, Lung, and Blood Institute). Mice were placed in the hypoxia chamber as soon as possible after birth (<12 h of age) along with their dam. 12% O2 was used for hypoxic exposure as we have shown earlier that 10% O2, the concentration often used for chronic hypoxic exposures in adult mice, is associated with increased mortality in newborn mice (3–6). Dams exposed to 12% oxygen did well, and there was no significant change in body weight over the 2 wk of exposure (<10% variation from dams in air). A subset of litters of Thy-1−/− mice in air was also administered TGF-β neutralizing antibody (clone 1D11, MAB1835; R&D Systems, Minneapolis, MN), which neutralizes all three isoforms of TGF-β (-β1, -β2, and -β3), at a dose of 20 μg by intraperitoneal injection on postnatal days 1, 5, and 10 (∼20 μg/g body wt on postnatal day 1; 6 μg/g on day 5; and 4 μg/g on day 10). As determined in preliminary studies, this dosing resulted in a marked reduction (50–75%) of TGF-β signaling without obvious toxicity in mice over the first 14 postnatal days. Controls were given similar volumes of vehicle (saline). The antibody was not given to dams antepartum as we have previously observed that TGF-β signaling may be required for adequate milk production by dams (5). This antibody has been previously used to demonstrate that reduction of TGF-β signaling in the hyperoxia-exposed newborn mouse improves alveolar development (19).
After the completion of hypoxia or normoxia exposure, a subset of each litter was used to examine lung morphometry, and the rest of the litter had lungs removed for isolation of RNA and protein as described subsequently.
Analysis of Lung Histology
Alveolar morphometry.
After the completion of hypoxia or normoxia exposure, the chest was opened, the lungs were inflation-fixed via the trachea with 10% formalin at 20 cmH2O pressure, and the right ventricle was perfused with formalin at the same pressure until the effluent was clear to flush out the blood in the pulmonary vessels. The lungs were maintained in inflation with formalin for 24 h using a silk ligature around the trachea, followed by transfer to 70% ethanol and further processing for histology. Five-micrometer sections were stained by hematoxylin and eosin for pulmonary morphometry. Each section consisted of a coronal section from apex to base of both lungs.
Alveolar development was evaluated by mean linear intercepts (MLI) (17) and radial alveolar counts (RAC) (10) on lung sections, as described earlier (5, 6). Photographs from six random ×100 lung fields were taken from each animal, with one image from the apex, middle, and base of each lung for MLI measurement, and from six ×400 lung fields from similar locations for evaluation of secondary septal crest density. Secondary septal crest density was estimated by counting of secondary septae longer than 5 μm per high power image field (50,000 μm2). The software package MetaMorph v. 6.2r4 (Universal Imaging, Downingtown, PA) was used for image analysis for lung vascular or alveolar morphometry.
Additional mice were evaluated for lung morphometry at different time points (at birth to 1 day, 2 mo, and 1 yr of age) to evaluate the temporal profile of lung development.
Electron microscopy.
Lung morphology was also qualitatively evaluated by scanning electron microscopy (scanning EM) as well as transmission electron microscopy (transmission EM) following inflation-fixation with EM-grade fixative (glutaraldehyde 6%:paraformaldehyde 2%). For scanning EM, the samples were evaluated uncoated on a Hitachi S-2460 scanning EM. For transmission EM, sections were stained with uranyl acetate and lead citrate. Higher magnifications were used as necessary to confirm identity of collagen and elastin fibers during transmission EM.
Vascular morphometry.
Vascular morphometry was assessed as described earlier (5, 6). Pulmonary arteries were defined as vessels that accompanied airways (veins are interlobular). Vessels smaller than 20 μm or larger than 150 μm (not resistance vessels) in external diameter were not analyzed. Twenty pulmonary arteries from different ×400 fields were evaluated from each section. Vessels cut transversely were measured along both axes, and the average wall thickness was obtained. Vessels cut obliquely or longitudinally were measured along the short axis. The wall thickness (%) of each artery was expressed as a percentage of the vessel diameter. The RV/LV free wall thickness ratio was determined as an index of right ventricular hypertrophy secondary to pulmonary hypertension, as described earlier (5, 6).
Immunohistochemical staining.
Antigen retrieval was performed on paraffin-embedded sections by heating in pH 6.0 citrate buffer (LabVision, Fremont, CA) for 20 min. The primary antibodies for Thy-1 (rabbit polyclonal H-110; Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Smad2 [rabbit anti-phospho Smad2 (Ser465/467) (Chemicon International, Temecula, CA)], αSMA (Sigma-Aldrich, St. Louis, MO), and Ki67 (LabVision) were used at 1:100 dilution for 30 min, and the secondary antibody and DAB staining kit were used as described in the product manual (Dako Envision+HRP-DAB; DakoCytomation, Carpinteria, CA). Nonspecific IgG and omission of primary antibody were used as controls for staining specificity. For quantitation, whole lung sections were imaged at ×400 and analyzed using the image analysis software (MetaMorph v.6.2). Six random high power (×400) fields from each of six different animals per group were evaluated. The identity of the sample was masked to observers estimating IHC staining to avoid bias. Thresholds for positive antibody staining compared with nonimmune serum controls were defined. Positive pixels were expressed as percentages of total tissue area, excluding air spaces, and as a percentage of total image area, as previously described (6).
Estimation of cell proliferation using Ki67 staining and flow cytometry for PCNA.
The number of Ki67-stained nuclei per high power image field (50,000 μm2) was determined using image analysis software, following immunohistochemical staining as described in the previous paragraph. As it was not evident if the differences in cell proliferation between WT and Thy-1−/− mice in alveolar walls and septae were due to differences in proliferation of endothelial cells, epithelial cells, or fibroblasts, we also performed flow cytometry to determine the proportions of these major lung cell types that were positive for markers of cell proliferation.
Two-day-old WT or Thy-1−/− mice in air were used for flow cytometry as cell proliferation in lungs is maximal soon after birth. Mouse pups were euthanized with isoflurane. The lungs were then removed, rinsed with PBS, minced with a scalpel, and immersed in 2 ml of collagenase (2 mg/ml, Sigma) for 1 h at 37°C. The cells were further dispersed by mixing using an 18G blunt needle attached to a 10-ml syringe and then filtered through a 30-μm nylon mesh filter (MACS Preseparation filters; Miltenyi Biotec, Auburn, CA). The filtered cell suspension was then pelleted by centrifugation and washed twice with PBS. The cells were resuspended in 250 μl of BD Cytofix/Cytoperm solution (BD Biosciences, San Jose, CA) for 20 min at 4°C, washed twice with BD Perm/Wash buffer (BD Biosciences), and finally resuspended in 50 μl of fluorochrome-conjugated antibody or isotype control at 1:50 dilution.
Cells were stained with antibodies (or their isotype control) to PCNA conjugated to phycoerythrin (Santa Cruz Biotechnology) in addition to antibodies against either 1) E-cadherin conjugated to Alexa Fluor 488 (Cell Signaling) or 2) CD31 (PECAM) conjugated to phycoerythrin-Cy7 (eBioscience, San Diego, CA) or 3) vimentin (Santa Cruz Biotechnology). Cells were incubated with the antibody for 30 min at 4°C in the dark, washed twice and resuspended, and then analyzed on a BD LSR II Analytical Flow Cytometer in the flow cytometry core facility, using the BD FACSDiva software. The percentage of cells that was positive for PCNA as well as either E-cadherin, CD31, or vimentin was analyzed.
Collagen and elastin staining.
Collagen and elastin were stained using Sirius red F3BA (picric acid Sirius red stain; Rowley Biochemical Institute, Rowley, MA) or Verhoeff's elastic tissue stain, respectively. The MetaMorph image analysis system was used to threshold collagen staining by comparison to areas with known collagen staining (perivascular collagen fibers) and absent collagen (bronchial epithelium excluding basement membrane and underlying collagen).
Analysis of RNA in Whole Lung Homogenates
Quantitative real-time RT-PCR.
Quantitative real-time PCR was performed using the Bio-Rad iCycler System as described previously (5, 6) for Thy-1, genes involved in TGF-β signaling, and extracellular matrix proteins. RNA was extracted using TRIzol from homogenized lung from mice at 14 days of age, treated with proteinase K and DNase I, and then quantified and reverse transcribed using the SYBR Green RT-PCR kit (Applied Biosystems) and a specific reverse (3′ antisense) PCR primer. cDNA was amplified by PCR in the Bio-Rad iCycler for 30–40 cycles. The mouse primer sequences are listed in Table 1.
Table 1.
Mouse primer sequences for real-time quantitative PCR
| Primer Name | Sequence |
|---|---|
| 18S-forward | GTC TGC CCT ATC AAC TTT CG |
| 18S-reverse | ATG TGG TAG CCG TTT CTC A |
| Collagen a1 forward | CCA AGG GTA ACA GCG GTG AA |
| Collagen a1 reverse | CCT CGT TTT CCT TCT TCT CCG |
| Collagen a2 forward | TGT TGG CCC ATC TGG TAA AGA |
| Collagen a2 reverse | CAG GGA ATC CGA TGT TGC C |
| Collagen 3a1 forward | TCA AGT CTG GAG TGG GAG G |
| Collagen 3a1 reverse | TCC AGG ATG TCC AGA AGA ACC A |
| Id-1 forward | GCC CCA GAA CCG CAA AGT GA |
| Id-1 reverse | AAC CCC CTC CCC AAA GTC TCT G |
| PAI-1 forward | TCA TCA ATG ACT GGG TGG AA |
| PAI-1 reverse | GCC AGG GTT GCA CTA AAC AT |
| TGF-β1 forward | GCC CTG GAT ACC AAC TAT TGC TT |
| TGF-β1 reverse | AGT TGG CAT GGT AGC CCT TG |
| Thy-1 forward | GCC TGA CCC GAG AGA AGA AGA AG |
| Thy-1 reverse | TGG TGG TGA AGT TCG CTA GAG TAA G |
| Tropoelastin forward | TGG TAT TGG TGG CAT CGG |
| Tropoelastin reverse | CCT TGG CTT TGA CTC CTG TG |
| Lysyl oxidase forward | GTG ACA TTC GCT ACA CAG GAC ATC |
| Lysyl oxidase reverse | CCA AAC ACC AGG TAC GGC TTT ATC |
Analysis for Proteins in Whole Lung Homogenates
Newborn mouse lungs were homogenized in 1 ml of a tissue protein extraction reagent (T-PER; Pierce Biotechnology, Rockford, IL) with added protease inhibitor cocktail (Sigma-Aldrich) and centrifuged at 7,000 g for 5 min, and the supernatant was frozen at −80°C until analysis. Protein measurements were normalized to total protein as measured using the Bio-Rad Bradford Protein Assay (Bio-Rad).
ELISA.
All samples were analyzed as a single batch for TGF-β1 by ELISA as described in the manufacturer's protocol (MB100B, R&D Systems). The range of measurement of this ELISA was 5–2,000 pg/ml, with good intra-assay and interassay precision. Active and total TGF-β1 were measured separately by ELISA. Total (latent + active) TGF-β1 was measured by the addition of hydrochloric acid to activate latent TGF-β1, followed by neutralization with sodium hydroxide, as described in the product manual. This TGF-β1 ELISA does not recognize the latent form of TGF-β1 (unless activated as described above), TGF-β2, TGF-β3, or any of the bone morphogenic proteins.
SEAP assay.
Samples were assayed for bioactive TGF-β using a highly sensitive bioassay developed by Dr. Tony Wyss-Coray (27). Embryonic fibroblasts from TGFb1−/− mice were stably transfected with a reporter plasmid consisting of TGF-β-responsive Smad-binding elements coupled to a secreted alkaline phosphatase reporter gene (SBE-SEAP) (27). These fibroblasts (MFB-F11 clone) show more than a 1,000-fold induction with 1 ng/ml TGF-β1 and detect as little as 1 pg/ml TGF-β1 (27). The MFB-F11 cells were kindly provided to us by Dr. Tony Wyss-Coray (27). MFB-F11 cells are induced by all three isoforms of TGF-β (unlike the ELISA, which responds only to TGF-β1) and do not show induction to related family members activin, nodal, BMP-2, or BMP-6. The bioassay was done as described in the original report (27), with SEAP activity measured using Great EscAPe SEAP Reporter system (BD Biosciences).
Western blot.
Western blots were done as previously described (5). Ten micrograms of protein per lane were fractionated by 10% Tris-glycine SDS-PAGE electrophoresis, followed by transfer to a PVDF membrane (Millipore). Western blot analysis for pSmad2, Smad2, and β-tubulin was done using specific primary antibodies (developed in rabbit, reactive against mouse) for pSmad2 (Cell Signaling), Smad 2 (Cell Signaling), or β-tubulin (Santa Cruz) at 1:1,000–2,000 dilution for 2 h at room temperature (pSmad2, Smad2) or overnight at 4°C (β-tubulin). The secondary antibody was a goat anti-rabbit secondary antibody (Sigma) used at 1:10,000 dilution for 1 h at room temperature. Immunoreactive bands were visualized by treatment with Immun-Star Western blotting detection reagents (Bio-Rad) according to the manufacturer's instructions. Densitometry was done using a Kodak Gel Logic 100 digital imaging system (Carestream Health) and Kodak Molecular Imaging Software (v.4.5.1, Carestream Health).
Extracellular matrix protein and matrix metalloproteinase-2 and -9 assessment.
Collagen and elastin were measured in homogenized lung extracts as an estimate of total lung collagen or elastin, respectively. Collagen was measured using the Sircol soluble collagen assay (Biocolor, Newtonabbey, Northern Ireland) as described previously (4). The dye reagent (Sirius red in picric acid) binds specifically to the [Gly-X-Y]n helical structure found in all collagens. Elastin was measured using the Fastin assay (Biocolor) as described in the manufacturer's protocol. This is a quantitative dye-binding method for elastins extracted from biological materials, using the dye label 5,10,15,20-tetraphenyl-21,23-porphine tetra-sulfonate.
MMP-2 and -9 were estimated by gelatin zymography as previously described (6) in homogenized lung extracts.
Analysis of Lung Function
Pulmonary function was measured in WT and Thy-1−/− mice (maintained in air) at 14 days of age and at 4 mo of age. Mice were anesthetized with ketamine/xylazine, and tracheostomy was performed using either a 24G Angiocath for 14-day mice or an 18G blunt needle for 4-mo-old mice. The flexiVent apparatus (SCIREQ, Montreal, Canada) equipped with a Module 1 (suitable for mice as small as 10 g) was used to perform measurement maneuvers, including perturbations (predefined pressure of volume waveforms) such as forced oscillations, via the tracheostomy using room air in the closed-chest animal. The delivered tidal volume was 6 ml/kg, with a respiratory rate of 150/min. Measurements made included total resistance (R; which encompasses Rn, G, and chest wall resistance; chest wall resistance in the mouse is essentially zero), compliance (C), elastance (E), input impedance (Z), airway resistance (Rn or Raw; Newtonian resistance, which is primarily the resistance of the central or upper airways), tissue damping (G), tissue elastance (H), quasistatic compliance (Cst), and hysteresivity (Eta; the ratio of G and H) (Rn, G, and H were calculated by fitting the constant phase model to input impedance). Calibration of the flexiVent was done using the tracheal cannula to be used before each experiment. Lung volumes were measured by volume displacement after completion of the flexiVent measurements. The mice were euthanized at the conclusion of the procedure.
Statistical Analysis
Results were expressed as means + SE. Normalization to the air group was required for the mRNA analysis, to calculate fold-changes in mRNA expression. The average of the measurements (mRNA for gene of interest/18S RNA) in the WT air group at 14 days was considered as = 1.0, and all other measurements were expressed relative to the expression in the WT air group. Other measurements (e.g., protein as measured by ELISA, Western blots, Sircol, Fastin) are shown as measured in absolute amounts (mg/mg protein or as a ratio to internal standard). Data were analyzed by two-way ANOVA using SigmaStat (v. 3.11; Systat Software, San Jose, CA) to test for separate and combined effects of genotype and hypoxia on measurements. Multiple comparison testing (Student-Newman-Keuls) was performed if statistical significance (P < 0.05) was noted by two-way ANOVA.
RESULTS
Thy-1−/− mice appeared phenotypically similar to WT mice of comparable age and were of similar size. Survival of newborn Thy-1−/− mice in air was comparable to WT mice, but was decreased during hypoxia (10–15% mortality of WT pups exposed to either air or hypoxia and air-exposed Thy-1−/− pups vs. 40–60% mortality in hypoxia-exposed Thy-1−/− pups). The mortality in hypoxia-exposed Thy-1−/− pups was not seen soon after birth, but peaked at 5–10 days of age. Hypoxia led to a similar magnitude of growth retardation in both WT and Thy-1−/− mice (30–40% smaller at 14 days than air-exposed pups of the same genotype).
Thy-1 Decreases With Hypoxia
qPCR of lung homogenates and immunohistochemical staining of lung sections showed that hypoxia reduced Thy-1 mRNA and protein expression, respectively, in WT mice (Fig. 1). By immunohistochemical staining at 14 days of age, Thy-1 was noted at alveolar septal tips as well as on alveolar walls, and was not seen in vascular endothelium or tunica media.
Fig. 1.
Thy-1 decreases with hypoxia. A: mRNA for Thy-1 by competitive real-time PCR in homogenized lungs from mice exposed to air or hypoxia from birth for up to 14 days (n = 6 mice/group; means ± SE, *P < 0.05 vs. air at same time point). B–H: immunohistochemistry for Thy-1 on mouse lung sections at 14 days of age. Quantitation of Thy-1 immunohistochemical staining is shown as a percentage of total image area (B) and as a percentage of tissue area (C). Representative photomicrographs are shown in D–H. No primary antibody (D), no secondary antibody (E), and Thy-1−/− mouse (F) are negative controls. Thy-1 staining was increased in wild-type (WT) mice exposed to air (G) compared with WT mice exposed to hypoxia (H) (×400; calibration bars = 50 μm).
Thy-1−/− Mice in Air and Hypoxia-Exposed WT Mice Have Increased TGF-β Signaling
TGF-β1 mRNA expression was comparable in WT mice exposed to air (WT-air) and Thy-1−/− mice exposed to air (Thy-1−/−-air) mice, and hypoxia increased TGF-β1 mRNA to a similar extent in both genotypes (WT and Thy-1−/−) (Fig. 2). WT mice exposed to hypoxia (WT-hypoxia), as well as Thy-1−/− mice in either air (Thy-1−/−-air) or hypoxia (Thy-1−/−-hypoxia), all had similar increases in active TGF-β1 protein in lung homogenates by ELISA compared with WT-air mice (Fig. 2). Hypoxia did not increase total TGF-β1 protein within a genotype (WT or Thy-1−/−), but Thy-1−/−-air and Thy-1−/−-hypoxia mice had increased total TGF-β1 compared with corresponding WT mice (Fig. 2). Thy-1−/−-air mice administered the 1D11 antibody (Thy-1−/−-air-1D11) had increased TGF-β1 mRNA expression compared with Thy-1−/−-air mice and active and total TGF-β1 protein not statistically different from the other groups (Fig. 2). Results by the SEAP assay were similar to those by ELISA (data not shown).
Fig. 2.
TGF-β signaling is increased in WT-hypoxia and in Thy-1−/−-air and Thy-1−/−-hypoxia mice and is reduced by 1D11 (TGF-β neutralizing antibody) in Thy-1−/−-air mice at 14 days of age. Measurement of mRNA for TGF-β1 (A) by competitive real-time PCR, active TGF-β1 (B), and total (latent + active) TGF-β1 (C) by ELISA in homogenized lungs. D–H: representative photomicrographs of mouse lung sections stained for phospho-Smad2. Nuclear staining for phospho-Smad2 is increased in WT-hypoxia and Thy-1−/−-air and Thy-1−/−-hypoxia and reduced in Thy-1−/−-air-1D11 mice (×400). PA, pulmonary artery; Br, bronchus; calibration bars = 50 μm. I: pSmad2/Smad2 ratio in Western blots quantitated by densitometry (n = 6 mice/group; means ± SE, *P < 0.05 vs. corresponding air controls, †P < 0.05 vs. corresponding WT, ‡P < 0.05 vs. corresponding vehicle).
Phospho-Smad2 by immunohistochemical staining and by Western blot was increased in WT-hypoxia mice as well as Thy-1−/−-air mice, providing additional evidence for increased TGF-β signaling during hypoxia in WT mice, and in Thy-1−/− mice even without hypoxia exposure. Thy-1−/−-hypoxia mice had a further increase in pSmad2 compared with Thy-1−/−-air mice (Fig. 2). Thy-1−/−-air-1D11 mice had pSmad2 much lower than Thy-1−/−-air mice and similar to WT-air mice (Fig. 2).
Confirmation for increased TGF-β signaling in hypoxia and during Thy-1 absence was provided by qPCR for PAI-1, which revealed increased PAI-1 in WT-hypoxia compared with WT-air, and increased PAI-1 in Thy-1−/−-air compared with WT-air, and a further increase in Thy-1−/−-hypoxia (Fig. 3). Thy-1−/−-air-1D11 mice had lower PAI-1 compared with Thy-1−/−-air mice. Id-1 [a marker of bone morphogenic protein (BMP) signaling that is normally reciprocally regulated with regard to PAI-1 in chronic hypoxia, i.e., decreasing with hypoxia while PAI-1 increases with hypoxia (11)] decreased in hypoxia-exposed compared with air-exposed animals, and genotype (WT or Thy-1−/−) did not influence Id-1 expression (Fig. 3). Thy-1−/−-air-1D11 mice had higher Id-1 compared with Thy-1−/−-air mice.
Fig. 3.
Downstream markers of TGF-β signaling are increased in WT-hypoxia and in Thy-1−/−-air and Thy-1−/−-hypoxia mice and are reduced by 1D11 (TGF-β neutralizing antibody) in Thy-1−/−-air mice at 14 days of age. Measurement of mRNA for PAI-1 (A) and Id-1 (B) by competitive real-time PCR indicates hypoxia and Thy-1 absence increases PAI-1 and 1D11 reduces PAI-1, with reciprocal changes in Id-1. C–G: representative photomicrographs of mouse lung sections stained for αSMA. Cytoplasmic staining for αSMA is increased in WT-hypoxia (D) and Thy-1−/−-air (E) and Thy-1−/−-hypoxia (F) compared with WT-air mice (C), primarily in alveolar septal tips (arrows), and reduced in Thy-1−/−-air-1D11 mice (G; ×400; calibration bars = 50 μm). I: αSMA /β-tubulin ratio in Western blots quantitated by densitometry (n = 6 mice/group; means ± SE, *P < 0.05 vs. corresponding air controls, †P < 0.05 vs. corresponding WT, ‡P < 0.05 vs. corresponding vehicle).
WT mice exposed to hypoxia (WT-hypoxia), as well as Thy-1−/− mice in either air (Thy-1−/−-air) or hypoxia (Thy-1−/−-hypoxia), all had increased αSMA compared with WT-air (Fig. 3). Thy-1−/−-air-1D11 mice had lower αSMA compared with Thy-1−/−-air mice (Fig. 3).
Thy-1−/− Mice Have Impaired Lung Development, Which Is Prevented By Inhibition of TGF-β Signaling
Air-exposed Thy-1−/− mice had similar RAC compared with WT mice at birth, but lower RAC at 2 wk and 2 mo of age, indicating a delay in alveolar development (Fig. 4). By 1 year of age, the differences in RAC between Thy-1−/− mice and WT mice had diminished and were not statistically significant (Fig. 4). At 2 wk of age, increases in MLI indicating larger alveoli were noted in Thy-1−/−-air mice, confirming impaired alveolar development (Fig. 4). Thy-1−/−-air-1D11 mice had improved alveolar septation, as indicated by radial alveolar counts similar to WT-air mice, and well above Thy-1−/−-air mice (Fig. 4). Secondary septal crest density measurements showed that WT-hypoxia mice had lower crest density compared with WT-air mice. Thy-1−/−-air mice had crest density comparable to WT-hypoxia mice, and Thy-1−/−-hypoxia mice had a further reduction in crest density. Thy-1−/−-air-1D11 mice had crest density similar to WT-air mice (Fig. 4).
Fig. 4.
Alveolar development in WT-air, WT-hypoxia, Thy-1−/−-air, Thy-1−/−-hypoxia, and Thy-1−/−-air-1D11 mouse pups at 14 days of age. A–E: representative photomicrographs of hematoxylin & eosin-stained sections of lungs from WT (A and B) and Thy-1−/− (C–E) mouse pups after 14 days of air (A, C, E) or hypoxia (B and D) exposure (×100; calibration bars = 250 μm). Alveolar size is larger with fewer septations in WT-hypoxia (B), Thy-1−/−-air (C), and Thy-1−/−-hypoxia (D) compared with WT-air mice (A). Thy-1−/−-air-1D11 mice (E) have alveolar size and septation similar to WT-air mice (A). Radial alveolar counts at 1 day, 14 days, 2 mo, and 1 yr of age in WT-air and Thy-1−/−-air mice (F) are similar at birth but lower in Thy-1−/− mice by 14 days. Alveolarization as estimated by radial alveolar count (G), mean linear intercept (H), and secondary septal crest density (I) displays impairment in alveolarization during hypoxia and Thy-1 absence at 14 days of age (n = 6 mice/group; means ± SE, *P < 0.05 vs. corresponding air controls, †P < 0.05 vs. corresponding WT, ‡P < 0.05 vs. corresponding vehicle).
Analysis of cell proliferation revealed that Thy-1−/−-air mice had fewer Ki67(+) cells compared with WT-air and WT-hypoxia mice, and Thy-1−/−-hypoxia mice had a further reduction in Ki67(+) cells (Fig. 5). WT-hypoxia mice had Ki67 labeling ∼20% less than in WT-air mice. Thy-1−/−-air-1D11 mice had Ki67 labeling similar to WT-air mice. Resistance pulmonary arteries and bronchi were similar in Ki67 labeling in WT-air and Thy-1−/−-air mice (data not shown). Flow cytometry of dispersed cells from WT-air and Thy-1−/−-air mice indicated that WT mice have more epithelial cell (E-cadherin + cells) and endothelial cell (CD31+ cells) proliferation compared with Thy-1−/− mice, but Thy-1−/− mice have more proliferation in fibroblasts (increased PCNA+ in vimentin+ but not in CD31+ cells). Smooth muscle cells and leukocytes are also vimentin+ but constitute only a small fraction of cells in newborn mouse lungs.
Fig. 5.
Cell proliferation in WT-air, WT-hypoxia, Thy-1−/−-air, Thy-1−/−-hypoxia, and Thy-1−/−-air-1D11 mouse pup lungs. A–E: representative photomicrographs of Ki67-stained sections of lungs from WT (A and B) and Thy-1−/− (C–E) mouse pups after 14 days of air (A, C, E) or hypoxia (B and D) exposure (×400; calibration bars = 50 μm). F: quantitation of Ki67 staining in alveoli, expressed as number of Ki67(+) nuclei per 50,000 μm2 of lung cross-sectional area. G: quantitation of PCNA(+) cells as a percentage of E-cadherin(+), CD31(+), and vimentin(+) cells in dispersed mouse lung cells by flow cytometry (n = 6 mice/group; means ± SE, *P < 0.05 vs. corresponding air controls, †P < 0.05 vs. corresponding WT, ‡P < 0.05 vs. corresponding vehicle).
Scanning EM done on 2-wk-old Thy-1−/− and WT mice, exposed to air or hypoxia from birth, confirmed that alveolar size was qualitatively larger in WT-hypoxia mice compared with WT-air mice, and that Thy-1−/−-air had larger alveoli, comparable to WT-hypoxia mice, and larger than those of WT-air mice. Alveolar ultrastructure, including pores of Kohn by SE, was qualitatively similar in both genotypes (WT or Thy-1−/−) and conditions (air and hypoxia) (data not shown).
Transmission EM was done primarily to evaluate structural extracellular matrix proteins such as collagen and elastin in the developing alveolar septae. Both collagen and elastin fibrils were at similar locations in the developing alveolar septae (Fig. 6), but elastin in Thy-1−/− mice, whether in air or hypoxia, was denser (Fig. 6).
Fig. 6.
Transmission electron micrographs of alveolar septal tips from WT and Thy-1−/− mice exposed to either air or hypoxia at 14 days of age. Collagen (Col) and elastin (EL) are at similar locations, but elastin in Thy-1−/−-air (C) and Thy-1−/−-hypoxia (D) mice is denser than in WT mice in air (A) or hypoxia (B) (×6,500; calibration bars = 500 nm).
Vascular morphometry and RV/LV thickness ratios indicated that Thy-1−/−-air mice were comparable to WT-air mice (% wall thickness:WT-air:13.33 ± 0.5; Thy-1−/−-air:11.7 ± 0.3, P NS, not significant; RV/LV:WT-air:0.36 ± 0.02; Thy-1−/−-air:0.34 ± 0.03; P NS). Hypoxia increased RV/LV thickness in both WT-hypoxia and Thy-1−/−-hypoxia mice, and the changes in RV/LV thickness were comparable in both genotypes (RV/LV:WT-hypoxia:0.50 ± 0.04; Thy-1−/−-hypoxia:0.50 ± 0.05; P NS), indicating that the loss of Thy-1 did not significantly affect normal postnatal pulmonary vascular remodeling or right ventricular remodeling in response to chronic hypoxia.
Thy-1−/− Mice Have Increased Lung Collagen, Which Is Reduced By Inhibition of TGF-β Signaling
The Sircol assay for whole lung soluble collagen indicated that Thy-1−/−-air mice had more collagen compared with WT-air mice. In addition, WT-hypoxia mice had increased collagen compared with WT-air mice, and Thy-1−/−-air and WT-hypoxia mice had similar amounts of collagen (Fig. 7). Thy-1−/−-hypoxia mice had collagen amounts similar to Thy-1−/−-air and WT-hypoxia mice (Fig. 7). Thy-1−/−-air-1D11 mice had lower collagen compared with Thy-1−/−-air mice and were not different from WT-air mice. Picrosirius red-stained lung sections showed collagen staining consistent with the Sircol quantitation, with collagen primarily in alveolar septal tips and alveolar walls (Fig. 7). Perivascular collagen was increased by hypoxia but did not differ by genotype (data not shown). Collagen mRNA expression was increased by hypoxia, but not by Thy-1 absence (Fig. 7), indicating that increased collagen in Thy-1 absence was not due to increased transcription. WT-air mice and Thy-1−/−-air mice had similar total MMP-2 and MMP-9 concentrations by zymography (data not shown), indicating that increased collagen is probably not associated with reductions in these MMPs.
Fig. 7.
Lung collagen is increased in WT-hypoxia and in Thy-1−/−-air and Thy-1−/−-hypoxia mice compared with WT-air mice and is reduced by 1D11 in Thy-1−/−-air mice at 14 days of age. A–E: representative photomicrographs of picrosirius-red stained sections of lungs from WT (A and B) and Thy-1−/− (C–E) mouse pups after 14 days of air (A, C, E) or hypoxia (B and D) exposure, with collagen staining (highlighted) primarily in alveolar septal tips (arrows) and alveolar walls (×100; calibration bars = 250 μm). Measurement of soluble collagen in lung homogenates by the Sircol assay (F) shows collagen is increased in WT-hypoxia, Thy-1−/−-air, and Thy-1−/−-hypoxia compared with WT-air mice, and Thy-1−/−-air-1D11 mice have collagen similar to WT-air mice. Measurement of mRNA for collagen isoforms (G–I) by qPCR indicates hypoxia increases collagen synthesis without significant interaction by genotype (n = 6 mice/group; means ± SE, *P < 0.05 vs. corresponding air controls, †P < 0.05 vs. corresponding WT, ‡P < 0.05 vs. corresponding vehicle).
Thy-1−/− Mice Have Increased Lung Elastin, Which Is Reduced By Inhibition of TGF-β Signaling
The Fastin assay for whole lung soluble elastin showed that Thy-1−/−-air mice had more elastin compared with WT-air mice. WT-hypoxia mice had increased elastin compared with WT-air mice and comparable to Thy-1−/−-air mice (Fig. 8). Thy-1−/−-hypoxia mice had more elastin than Thy-1−/−-air or WT-hypoxia mice (Fig. 8), indicating synergistic effects of hypoxia and Thy-1 absence on lung elastin content. Thy-1−/−-air-1D11 mice had lower elastin compared with Thy-1−/−-air mice and were not different from WT-air mice. Verhoeff's elastin-stained lung sections showed elastin staining consistent with the elastin quantitation, with elastin primarily in alveolar septal tips (Fig. 7). Tropoelastin mRNA was increased in WT-hypoxia compared with WT-air, with a trend toward increase in Thy-1−/−-air and Thy-1−/−-hypoxia mice compared with WT-air (0.05 < P < 0.2).
Fig. 8.
Lung elastin is increased in WT-hypoxia and in Thy-1−/−-air and Thy-1−/−-hypoxia mice compared with WT-air mice and is reduced by 1D11 (TGF-β neutralizing antibody) in Thy-1−/−-air mice at 14 days of age. A–E: representative photomicrographs of elastin-stained sections of lungs from WT (A and B) and Thy-1−/− (C–E) mouse pups after 14 days of air (A, C, E) or hypoxia (B and D) exposure, with elastin staining primarily in alveolar septal tips (arrows) (×100; calibration bars = 250 μm). Measurement of soluble elastin in lung homogenates by the Fastin assay (F) shows elastin is increased in WT-hypoxia, Thy-1−/−-air, and Thy-1−/−-hypoxia compared with WT-air mice, and Thy-1−/−-air-1D11 mice have elastin similar to WT-air mice. Measurement of mRNA for tropoelastin (G) and lysyl oxidase (H) by qPCR indicates hypoxia increases tropoelastin mRNA in WT mice, with a trend toward increase in Thy-1−/−-air and Thy-1−/−-hypoxia mice compared with WT-air (0.05 < P < 0.2), and no significant changes in lysyl oxidase (n = 6 mice/group; means ± SE, *P < 0.05 vs. corresponding air controls, †P < 0.05 vs. corresponding WT, ‡P < 0.05 vs. corresponding vehicle).
Thy-1−/− Mice Have Increased Lung Damping (Tissue Resistance) at 2 Wk of Age, Which Is Reduced By Inhibition of TGF-β Signaling
Thy-1−/−-air mice had reduced lung compliance and increased total lung resistance compared with WT-air mice at 2 wk of age. The increase in lung resistance was due to an increase in lung damping (tissue resistance), as airway resistance was similar in both Thy-1−/−-air and WT-air mice (Fig. 9). Thy-1−/−-air-1D11 mice had lower total lung resistance to WT-air levels due to a reduction in tissue resistance compared with Thy-1−/−-air at 2 wk of age, although lung compliance did not improve (Fig. 9). Changes in elastance and tissue elastance were opposite to those seen in lung compliance, with increased elastance in Thy-1−/−-air and Thy-1−/−-air-1D11 mice compared with WT-air mice. Lung volumes/body weight were not different (data not shown). Changes in lung compliance and airway resistance when adjusted for lung volume were similar to unadjusted changes in these variables [compliance (ml/cmH2O) per ml lung volume:WT-air:0.016 ± 0.001, Thy-1−/−-air:0.010 ± 0.001, Thy-1−/−-air-1D11:0.011 ± 0.001, P < 0.01 for WT-air vs. other two groups; airway resistance (cmH2O·s/ml) per ml lung volume:WT-air:0.99 ± 0.22, Thy-1−/−-air:0.87 ± 0.17, Thy-1−/−-air-1D11:1.14 ± 0.15, P = 0.69]. Hysteresivity (Eta) was unchanged, ranging from 0.13 to 0.15 in all groups.
Fig. 9.
Lung function measurements in WT-air, Thy-1−/−-air-vehicle, and Thy-1−/−-air-1D11 mice at 14 days of age. Lung dynamic compliance (A) was reduced, and elastance (C) was increased in Thy-1−/−-air-vehicle and Thy-1−/−-air-1D11 mice compared with WT-air mice. Total lung resistance (B) and tissue damping (E) but not airway resistance (D) were increased in Thy-1−/−-air-vehicle compared with WT-air and Thy-1−/−-air-1D11 mice. Changes in tissue elastance (F) were similar to changes in total elastance (C) (n = 4–6 mice/group; means ± SE, *P < 0.05 vs. WT-air).
However, by 4 mo of age, these changes in lung function in Thy-1−/−-air mice had disappeared and in fact were partly reversed, with the adult Thy-1−/− mice having lower resistance (both airway and tissue resistance) (adult Thy-1−/− mice vs. adult WT mice:compliance:0.035 ± 0.01 vs. 0.024 ± 0.03 ml/cmH2O, P = 0.10; total resistance:0.64 ± 0.06 vs. 0.84 + 0.03 cmH2O·s/ml, P < 0.05; airway resistance:0.15 ± 0.01 vs. 0.22 ± 0.01 cmH2O·s/ml, P < 0.01; tissue damping:7.12 ± 0.7 vs. 9.4 ± 0.39 cmH2O/ml, P < 0.05).
DISCUSSION
The present study is the first to test the effects of hypoxia on Thy-1 in the newborn lung and to determine the effects of Thy-1 deficiency on lung development. Previous in vivo studies by us (5) and other investigators (29) of the functional role of TGF-β in normal lung development and in the response to hypoxia have determined that excessive TGF-β signaling contributes to the hypoxia-induced inhibition of lung development and abnormal vascular remodeling. As TGF-β is primarily regulated by activation, it is probable that regulators of TGF-β activation may play a key role in initiating the response of the lung to hypoxia. We have previously shown that fibroblasts expressing Thy-1 on their cell surface inhibit TGF-β activation and are less fibrogenic (31). Therefore, a natural consequence of a decrease in Thy-1 would be increased TGF-β activation. Our results demonstrating that inhibition of TGF-β signaling in Thy-1−/− mice improves alveolar development and lung function reveal that excessive TGF-β signaling may contribute to the lung phenotype in Thy-1−/− mice. Our study therefore indicates that a hypoxia-mediated reduction in Thy-1 may lead to excessive TGF-β activation and downstream signaling, resulting in inhibition of lung development and abnormal lung function.
Our study using WT and Thy-1−/− mice in the newborn period is a comprehensive evaluation of the role of Thy-1 in lung development and function. It is important to note that changes in lung histology and function due to lack of Thy-1 were predominantly observed in the developing lung, and the lack of persistent effects may be due to compensatory phenomena in other signaling pathways. Our study focused on Thy-1 in the lung during hypoxia, but other stimuli such as inflammatory cytokines (IL-1, TNFα) and fibrogenic stimuli (e.g., intratracheal bleomycin) are also known to decrease Thy-1 surface expression, probably due to increased Thy-1 shedding (13). It is hence likely that a reduction in Thy-1 may contribute to increased TGF-β activation and impaired lung development in other models of newborn lung pathology.
The observation that Thy-1 is a regulator of TGF-β activation and is involved in lung development in the newborn mouse is clinically very relevant as TGF-β is considered a mediator of bronchopulmonary dysplasia (BPD) in preterm infants (15, 28). Mouse lung development in the first two postnatal weeks parallels lung development in humans from week 24 of gestation through the first 2 years of age (9) as alveolar development is postnatal in mice, whereas it begins in utero and continues postnatally in humans (32). TGF-β overexpression in newborn animal models leads to pathology similar to BPD (12, 30). Vicencio et al. (29) have demonstrated inhibition of alveolar development and increased TGF-β in bronchoalveolar lavage fluid in rats exposed to hypoxia during the first 2 wk of life but not adulthood. We have recently shown chronic hypoxia leads to increased TGF-β activity and that inhibition of increased TGF-β signaling attenuates hypoxia-induced abnormal vascular remodeling and impaired alveolar development (5).
The reduction in Thy-1 with hypoxia may be due to multiple mechanisms. One mechanism may be reduction of Thy-1 mRNA expression, as we have observed in this study. It is also possible that hypoxia provides selection pressure for fibroblasts lacking Thy-1, such that hypoxia is associated with a relative increase in Thy-1(−) fibroblast proliferation. This is consistent with data indicating that Thy-1(−) fibroblasts have reduced apoptosis compared with Thy-1(+) fibroblasts and that transfection of Thy-1 into Thy-1(−) fibroblasts reduces cell survival (25). Thy-1 is shed from fibroblasts on exposure to cytokines such as IL-1 and TNFα (13), and these fibroblasts then transiently acquire the Thy-1(−) fibroblast phenotype and are able to activate TGF-β (13). A similar process may occur with exposure to hypoxia, and studies are underway to determine the effects of oxygen tension on Thy-1 shedding. Additional studies are in progress evaluating the time course of Thy-1 expression and localization during embryonic and fetal periods (in the relatively hypoxic in utero environment) and postnatal lung development.
It is important to recognize that while Thy-1 is an inhibitor of TGF-β activation, the loss of Thy-1 does not activate TGF-β by itself, but permits existing stimuli (e.g., thrombospondin-1, αvβ6-integrin, reactive oxygen species) to activate TGF-β. The unresponsiveness of Thy-1(+) cells is not due to defective TGF-β signaling because both Thy-1(+) and Thy-1(−) fibroblasts respond to exogenous active TGF-β. Rather, Thy-1(+) cells fail to activate latent TGF-β in response to fibrogenic stimuli, whereas Thy-1(−) fibroblasts do so (31). As seen in our data, the concentration of active TGF-β1 in lung homogenates is 5–10 pg/mg protein compared with total TGF-β1 of 750–1,000 pg/mg protein, indicating that only ∼1% of the available TGF-β1 is normally activated. Therefore, even small changes in the regulation of TGF-β activation may have major effects. Many molecules such as thrombospondin-1 (18), the αvβ6- and αvβ8-integrins (26), proteases (14), as well as physical stimuli (e.g., redox status) are known to increase TGF-β activation, but Thy-1, as one of the few negative regulators, may also play a critical role in this process.
The observation that Thy-1 absence is associated with impaired lung alveolar development and lung function is a novel finding. This observation is consistent with the data that TGF-β overexpression in newborn animal models leads to pathology similar to BPD (12, 30) and that Thy-1 absence would increase TGF-β activation (31). Our data indicate that WT mice have more epithelial cell and endothelial cell proliferation compared with Thy-1−/− mice, but Thy-1−/− mice have more proliferation in fibroblasts. This is consistent with the concept that increased TGF-β activity increases fibroblast proliferation and reduces epithelial and endothelial cell proliferation, and the increase in fibroblast proliferation may increase extracellular matrix protein production and alter lung function, while the reduction in alveolar epithelial and endothelial cell proliferation would lead to inhibition of septation. αSMA staining is normally seen at the tip of the developing septal crests, the smooth muscle layer of resistance pulmonary arteries, and as a thin layer surrounding major bronchi. We noted that WT mice in hypoxia and Thy-1−/− mice (whether in air or hypoxia) had fewer septal crests, but an increase in αSMA was seen within the interstitium and alveolar walls, as well as in the septal crests that remained. These observations suggest that hypoxia and Thy-1 absence lead to abnormal (dysplastic) αSMA accumulation. Elastin, which is another important constituent of the alveolar septal crest, was also increased with hypoxia and Thy-1 absence and appeared denser on electron microscopy. Abnormalities in elastin homeostasis or structure may potentially affect alveolar septal crest development. As the effects of Thy-1 absence on lung structure and function change over time, it is possible that other compensatory mechanisms affecting extracellular matrix proteins are involved. We evaluated two gelatinases (MMP-2 and MMP-9), but many other proteases and elastases are also involved in matrix protein turnover and may be altered with hypoxia or absence of Thy-1.
The observation that Thy-1−/− mice have increased lung collagen and elastin and that this increase is mediated through enhanced TGF-β signaling is consistent with our previous demonstration that attenuation of TGF-β signaling in the chronic hypoxia model reduces lung collagen and elastin (5). The finding of increased lung tissue resistance that is abrogated by inhibition of TGF-β signaling suggests that the increase in extracellular matrix proteins such as collagen and elastin may be responsible for the abnormalities in lung function. However, lung compliance did not improve significantly in Thy-1−/− mice in response to inhibition of TGF-β signaling, indicating that other mechanisms may be contributing to the abnormal lung function. It is important to remember that while Thy-1 absence is associated with evidence of increased TGF-β activity and downstream signaling, the presence of Thy-1 also regulates non-TGF-β-mediated effects such as cell motility, adhesion, and other aspects of the fibroblast phenotype (23, 24). One of our intriguing observations is that the lung function abnormalities in the Thy-1−/− mice spontaneously reverse in the adult mouse, indicating that lung structure and function do not remain static after the period of rapid lung development in the first two postnatal weeks.
It is likely that the increased mortality in the Thy-1−/−-hypoxia mice and the lack of any additional impairment in their lung development compared with Thy-1−/−-air mice is an indication that additional impairment in alveolarization is not compatible with survival in this model. We have previously noted that TGF-β signaling attenuates hypoxia-induced pulmonary arterial remodeling (5), but no pulmonary arterial remodeling or right ventricular hypertrophy was noted in mice lacking Thy-1. We postulate that this may be because Thy-1 is normally expressed in the lung mainly in fibroblasts and not in resistance blood vessels. This is consistent with our finding that Thy-1−/−-air mice have similar pulmonary arterial wall thickness and RV/LV ratios to WT-air mice, indicating that abnormal pulmonary vascular remodeling and pulmonary hypertension are not seen with Thy-1 deficiency under normoxic conditions. Other stimuli of vascular TGF-β activation during hypoxia, such as via thrombospondin-1 (21, 22), may be responsible for the vascular changes.
Administration of the neutralizing antibody 1D11 resulted in a marked reduction in pSmad2 immunohistochemical staining as well as pSmad2/Smad2 by Western blot, and this reduction in TGF-β signaling is also accompanied by reductions in PAI-1 mRNA and increased Id-1 mRNA, further confirming that downstream markers of TGF-β signaling are also reduced. However, this reduction in TGF-β signaling was accompanied by an increase in TGF-β1 mRNA, and no change in active TGF-β1 protein, leading us to speculate that there may be compensatory pathways that increase TGF-β synthesis in response to a reduction in downstream signaling, and that active TGF-β1 bound to 1D11 and functionally inactive may still be measured by ELISA. Therefore, although the neutralizing antibody is effective, additional research into compensatory pathways is required, and monitoring of dosing adequacy in vivo requires measurement of downstream markers such as PAI-1.
A limitation of our study is that the Thy-1−/− mouse is not a conditional knockout, and expression of Thy-1 could not be precisely regulated or targeted in the developing lung. However, these results generate hypotheses that can be tested using transgenic models with targeting to lung. Future studies evaluating overexpression of Thy-1 in the lung are required to test whether preventing reductions in Thy-1 in the lung will attenuate the effects of hypoxia. Other limitations of the newborn mouse model are that interspecies differences limit extrapolation of observations to human infants, and the fact that chronic hypoxia, while a good reproducible model in animals, is not the main etiological factor underlying BPD or PPHN in human infants. The chronic hypoxia model may more accurately simulate the impairment of lung development seen with chronic intrauterine hypoxemia and growth restriction.
Our findings indicate that Thy-1 is an important regulator of TGF-β signaling in the newborn lung and suggest that preserving or increasing Thy-1 expression may be a possible therapeutic strategy in attenuating the abnormal lung development observed in BPD. Thy-1 may prove to be a critical regulator of TGF-β signaling in other neonatal disorders that may be characterized by excessive TGF-β activation (e.g., hyperoxic lung injury, necrotizing enterocolitis, posthemorrhagic neonatal hydrocephalus, etc.).
GRANTS
This work was supported in part by National Institutes of Health Grants R01-HL-092906, K08-HD-046513, HL-044195, HL-082818, HL-50147, HL-007457, and HL-56046, a CCRI grant, American Thoracic Society/Pulmonary Hypertension Association Grant ATS PH-06-006, Research Facilities Improvement Program Grant C06 RR 15490, and American Heart Association Grant 0455197B.
Acknowledgments
We acknowledge the assistance of Dr. Trenton Schoeb, Dr. David Kelly, Dr. Ona M. Faye-Petersen, and Melissa M. Chimento for assistance with the transmission electron microscopy, and Vista Engineering in Birmingham for assistance with the scanning electron microscopy. This work was presented in part at the American Thoracic Society Meeting, San Diego, CA, May 19–24, 2006.
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