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Hyperoxia increases Sonic hedgehog (Shh) expression in neonatal rat lungs. The effect of mesenchymal stem cells (MSCs) on the hedgehog signaling pathway in hyperoxia-induced lung injury is unknown. This study evaluated whether MSCs could inhibit hedgehog signaling and improve established hyperoxia-induced lung injury in newborn rats.
Newborn rats were assigned to room air (RA) or hyperoxia (85% O2) groups from postnatal day 4–15, and some received intravenous injection of human MSCs (9 × 105 cells) in 90 μL of normal saline (NS) through the tail vein on postnatal day 15. We obtained four study groups as follows: RA + NS, RA + MSCs, O2 + NS, and O2 + MSCs. Pups from each group were sacrificed on postnatal days 15 and 29, and lungs were removed for histological and Western blot analyses.
Neonatal hyperoxia on postnatal days 4–15 reduced the body weight, increased the mean linear intercept, and decreased the vascular density of the rats, and these effects were associated with increased Shh and Smoothened (Smo) expression and decreased Patched expression. By contrast, the MSC-treated hyperoxic pups exhibited improved alveolarization, increased vascularization, and decreased Shh and Smo expression on postnatal day 29.
Human MSC treatment reversed established hyperoxia-induced lung injury in newborn rats, probably through suppression of the hedgehog pathway.
Supraphysiological oxygen is often required to treat newborns with respiratory disorders. However, supplemental oxygen administered to newborn infants with respiratory failure increases oxidant stress and leads to lung injury. The rat model is appropriate to study the effects of hyperoxia on preterm infants with respiratory distress because rats are born at the saccular stage, equivalent to a ∼30 week human gestation.
Hedgehog signaling is started by the binding of the hedgehog ligand to its transmembrane receptor Patched (Ptc), relieving suppression of the transmembrane receptor Smoothened (Smo), and initiating the signaling cascade.
In vivo studies have demonstrated that hyperoxia exposure causes increased Shh expression in neonatal rat lung, and an in vitro study discovered that hyperoxia markedly inhibited Shh expression in alveolar epithelial type II cells.
However, the effect of hyperoxia exposure on other components of the hedgehog signaling pathway in newborn rat lungs remains unknown.
Mesenchymal stem cells (MSCs) are multipotent stromal cells that have immunomodulatory, anti-inflammatory, and regenerative effects and they have been demonstrated to rescue hyperoxia-induced lung injury.
However, the effects of MSCs on the hedgehog signaling pathway in established hyperoxia-induced lung injury are largely unknown. We hypothesized that MSCs influence the hedgehog signaling pathway and ameliorate established hyperoxia-induced lung injury in newborn rats. To test this hypothesis, MSCs were intravenously administered on postnatal day 15 to newborn rats that were exposed to room air (RA) or an oxygen-enriched atmosphere on postnatal days 4–15, and the hedgehog signaling pathway and lung development were examined on postnatal day 29. The aims were to evaluate the effect of neonatal hyperoxia on hedgehog signaling pathway and the effects of MSCs on hyperoxia-induced lung injury and hedgehog pathway.
2. Materials and methods
2.1 Animal model
This study was approved by the Animal Care Use Committee of Taipei Medical University and was performed in accordance with the guidelines provided. We followed the methods of Chen et al.
to design the animal model. The first protocol was to evaluate the hyperoxia effect on the hedgehog pathway. Time-dated pregnant Sprague–Dawley rats were housed in individual cages with ad libitum access to laboratory food and water, kept on a 12:12-h light–dark cycle, and allowed to deliver vaginally at term. Within 12 h of birth, the litters were pooled and randomly redistributed to the newly delivered mothers; the pups were then randomly assigned to RA or oxygen-enriched atmosphere (85% O2) groups for postnatal days 4–15 (Supplemental Figure 1). The nursing mothers were rotated between the 85% O2 and RA groups every 24 h to prevent oxygen toxicity in the mothers and eliminate differing maternal effects between groups. Oxygen exposure was performed in a transparent 60 × 50 × 40-cm
Plexiglas chamber into which oxygen was continuously delivered at a rate of 4 L/min. Oxygen levels were monitored using a Pro:ox Model 110 monitor (NexBiOxy, Hsinchu, Taiwan). On postnatal day 15, all pups were deeply anesthetized through an overdose of isoflurane, and lung tissues were harvested at the time of sacrifice. The second protocol was to evaluate the effects of human MSCs on the hedgehog signaling pathway in established hyperoxia-induced lung injury. The remaining rats were randomly assigned to receive NS or MSCs on postnatal day 15.
2.2 Isolation of human MSCs
Human MSCs were isolated as described in a previous report.
In brief, the stem cells were isolated from placental-derived tissues and characterized by analyzing the expression of CD markers (CD44, CD73, CD90, and CD105) and the cell surface receptor HLA-DR by using flow cytometry (BD Stemflow hMSC Analysis Kit; BD Biosciences, Franklin Lakes, NJ, USA). We used placenta-derived MSCs because placental tissue was easily obtained as medical waste and Talwadekar et al. demonstrated that MSCs obtained from placenta exhibit better immunomodulatory efficacy compared with MSCs obtained from umbilical cord.
Normal saline (NS; 90 μL) with or without human MSCs (9 × 105 cells) was intravenously administered into a tail vein using a 33-gauge needle with an insulin syringe on postnatal day 15. After the procedure, the animals were allowed to recover from the anesthesia and were returned to the mothers. We formed four study groups as follows: RA + NS, RA + MSCs (9 × 105 cells), O2 + NS, and O2 + MSCs (9 × 105 cells). Pups from each group were deeply anesthetized with an overdose of isoflurane on postnatal day 29, and their body and lung weights were recorded. Immediately after death, the left lung was ligated and the right lung was fixed through tracheal instillation of 10% buffered formalin at a pressure of 25 cm H2O for 10 min.
2.4 Western blot analysis
The lung tissues were homogenized in 0.6 mL of ice-cold lysis buffer containing 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.01 M deoxycholic acid, and a complete protease cocktail inhibitor (s8830, Sigma–Aldrich, St Louis, MO, USA). The samples were then centrifuged at 13,000 rpm for 20 min at 4 °C, and the supernatants were aliquoted and stored at −20 °C. The proteins (30 μg) were resolved using 8%–10% SDS–polyacrylamide gel electrophoresis under reduced conditions and were electroblotted on an Immobilon-P polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA, USA). After they had been blocked with 5% nonfat milk, the membranes were incubated with anti-Ptc/PTCH1 (Abcam, Cambridge, MA, USA), anti-Ptc (Abcam), anti-Smo (, E−5) and anti-Shh (E−1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies at 4 °C overnight. Subsequently, the membranes were incubated for 1 h with horseradish-peroxidase-conjugated secondary antibody at room temperature. Signals were visualized using enhanced chemiluminescence reagents according to the manufacturer's protocol. Mouse anti-β-actin mAb (C4, Santa Cruz Biotechnology) was used as an internal control. The densitometry unit of the protein expression in RA-exposed lungs was assigned as 1 after it had been normalized to β-actin.
2.5 Histology and morphological analysis
The lung lobes were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The tissue was then dehydrated in alcohol, cleared in xylene, and embedded in paraffin. Sections 5-μm thick were cut for further processing. After deparaffinization and rehydration, the lung sections were stained with hematoxylin and eosin (H&E) for general morphological observation and morphometric analysis. The mean linear intercept (MLI), an indicator of mean alveolar diameter, was assessed in 10 nonoverlapping fields. In brief, 10 nonoverlapping fields of every section were randomly selected, and each alveolar wall was counted as two crossings, with the crossings first counted in the horizontal direction and then in the vertical direction by turning the ocular 90°. To obtain the MLI, the average of two numbers per field was used in the following equation: Lm = (0.57/average intercepts) × 1000 (μm). Microvessel density was determined by counting the vessels with positive von Willebrand factor (vWF) staining in an unbiased manner for a minimum of four random lung fields at 400 × magnification.
After routine deparaffinization, heat-induced epitope retrieval was performed by immersing slides in 0.01 M sodium citrate buffer (pH 6.0). To block endogenous peroxidase activity and nonspecific antibody binding, the sections were first preincubated in 0.1 M phosphate-buffered saline containing 10% normal goat serum and 0.3% H2O2 for 1 h at room temperature, before being incubated with rabbit polyclonal anti-vWF and anti-Ptc/PTCH1 (1:100 for anti-vWF and 1:200 for anti-Ptc/PTCH1; Abcam), anti-Smo (E−5), and anti-Shh (E−1) (1:50; Santa Cruz Biotechnology) antibodies as primary antibodies for 20 h at 4 °C. The sections were then treated for 1 h at 37 °C with biotinylated goat antirabbit IgG or goat antimouse IgG (1:200; Jackson ImmunoResearch Laboratories Inc., PA, USA), before undergoing a reaction with the reagents from an avidin-biotin complex kit (Vector, CA, USA). The brown reaction products were visualized using a diaminobenzidine substrate kit (Vector) as per the manufacturer's recommendations. All immunostained sections were viewed and photographed using an Olympus BX43 microscope.
2.7 Statistical analysis
All data are presented as means ± standard deviation (SD). Statistical analyses were performed using two-way analysis of variance with Bonferroni post hoc test for multiple group comparisons. The survival rate was evaluated using the Kaplan–Meier method, and the log-rank test was used for intergroup comparisons. Differences were considered statistically significant at p < 0.05.
Eight dams gave birth to a total of 80 pups; 40 pups each were randomly distributed to the RA and 85% O2 groups. On postnatal day 15, ten rat pups each from the RA and 85% O2 groups were sacrificed, and the rest were randomly assigned to receive NS or human MSCs.
3.1 Body weight, lung weight, and lung-to-body weight ratio on postnatal day 15
The rats reared in 85% O2 exhibited significantly lower body and lung weights and a significantly higher lung-to-body weight ratio on postnatal day 15 than those reared in RA (Table 1).
Table 1Body weight, lung weight, and lung-to-body weight ratio of rats on postnatal day 15.
3.2 Hyperoxia from postnatal days 4–15 reduced alveolarization and vascularization
Representative lung sections stained with H&E and vWF on postnatal day 15 are displayed in Fig. 1. The lungs of rats reared in 85% O2 during postnatal days 4–15 contained large thin-walled air spaces and exhibited a significantly higher MLI than those of the rats reared in RA (Fig. 1A and B). The lungs of the hyperoxic rats also exhibited significantly lower vascular density than those of the RA-reared rats (Fig. 1C and D).
3.3 Hyperoxia from postnatal days 4–15 activated the hedgehog signaling pathway
The immunoreactivity of Shh was primarily detected in the epithelial cells of bronchioles, endothelial cells of blood vessels, and interstitum of alveolar walls (Fig. 1E). The immunoreactivity of Ptc was detected in the interstitum of alveolar walls and the endothelial cells of blood vessels. The immunoreactivity of Smo presented in the endothelial cells of blood vessels and epithelial cells of alveoli. The rats reared in 85% O2 on postnatal days 4–15 exhibited significantly higher Shh and Smo and significantly lower Ptc protein expression than those reared in RA.
3.4 Effect of intravenous MSCs administration on survival rate after lung injury
The rats reared in RA and treated with NS or MSCs on postnatal day 15 were all alive on postnatal day 29 (Supplemental Figure 2). Of the rats in the 85% O2 group, one receiving NS died on postnatal day 16, and one receiving MSCs died on postnatal day 17. On postnatal day 29, the survival rates between the rats treated with NS or MSCs were comparable.
3.5 Body weight, lung weight, and lung-to-body weight ratio on postnatal day 29
The rats reared in 85% O2 and treated with NS or MSCs on postnatal day 15 had significantly lower body and lung weights on postnatal day 29 than those reared in RA and treated with NS or MSCs (Table 2). Treatment with MSCs did not significantly influence body or lung weight. The lung-to-body weight ratio was comparable between the rats treated with NS and those treated with MSCs.
Table 2Body weight, lung weight, and lung-to-body weight ratio of rats on postnatal day 29.
Lung-to-body weight ratio
RA + NS
87.5 ± 6.5
0.60 ± 0.07
0.68 ± 0.05
RA + MSCs (9 × 105 cells)
83.1 ± 5.4
0.64 ± 0.04
0.78 ± 0.05
O2 + NS
68.2 ± 5.4∗∗
0.59 ± 0.05
0.86 ± 0.07∗∗
O2 + MSCs (9 × 105 cells)
73.2 ± 5.6∗∗
0.63 ± 0.08
0.85 ± 0.06∗∗
Values represent means ± SD.
∗∗p < 0.01 compared with RA + NS and RA + MSCs (9 × 105 cells) group.
3.6 Intravenous administration of MSCs improved alveolarization and vascularization in rats with lung injury
Representative lung sections stained with H&E and vWF on postnatal day 29 are presented in Fig. 2A and C, respectively. The rats exposed to 85% O2 from postnatal days 4–15 and treated with NS on postnatal day 29 exhibited a significantly higher MLI and lower vascular density than the rats exposed to RA and treated with NS or MSC (Fig. 2B and D). Treatment with human MSCs significantly improved the hyperoxia-induced alteration of the MLI and vascular density to normoxic levels.
3.7 Effect of intravenous MSCs administration on the hedgehog signaling pathway in rats with lung injury
Representative immunohistochemistry, Western blots, and quantitative data of Shh, Ptc, and Smo protein expression in the 29-day-old rats are presented in Fig. 3. Shh immunoreactivity was primarily detected in the epithelial cells of bronchioles, endothelial cells of blood vessels, and interstitum of alveolar walls (Fig. 3A). The expression pattern of Smo was detected in the epithelial cells of alveoli and bronchioles and endothelial cells of blood vessels. Ptc immunoreactivity was mainly detected in the endothelial cells of blood vessels and interstitum of alveolar walls. The rats exposed to 85% O2 and treated with NS exhibited more intense Shh and Smo immunoreactivity and less intense Ptc immunoreactivity than did the RA-exposed rats treated with NS. MSC treatment significantly hindered the hyperoxia-induced increase in Shh and Smo immunoreactivity and protein level and increased the Ptc protein level (Fig. 3B).
Our in vivo model revealed that neonatal hyperoxia from postnasal days 4–15 arrested alveolarization and reduced the amount of angiogenesis. This hyperoxia-induced lung injury was associated with increased Shh and Smo expression and decreased Ptc expression. Intravenous administration of MSCs on postnatal day 15 resulted in improved alveolarization, increased amount of angiogenesis, decreased Shh and Smo expression, and increased Ptc expression on postnatal day 29. These results suggest that in newborn rats, hyperoxia activated the hedgehog signaling pathway and human MSC therapy reversed hyperoxia-induced lung injury through suppression of the hedgehog pathway.
In this study, we used 85% O2 exposure of rat pups from postnatal days 4–15 because murine alveolar development begins on postnatal day 4 and saccular division is complete by postnatal day 14.
Additionally, rats exposed to 85% O2 from postnatal days 4–14 exhibit larger and more simplified alveolar airspaces than those with 4-day excessive oxygen exposure before postnatal day 4 and after postnatal day 9.
Our study demonstrated that rats reared in 85% O2 from postnatal day 4 had significantly lower body and lung weights on postnatal day 15 than those reared in RA. The lung-to-body weight ratio was significantly higher in the rats reared in 85% O2 than in those reared in RA. These results indicate that hyperoxia exhibited a significant negative effect on body weight gain. The rats reared in 85% O2 and treated with NS or MSCs exhibited significantly lower body weight on postnatal day 29 than those reared in RA and treated with NS or MSCs. Treatment with MSCs did not influence body weight or lung-to-body weight ratio.
Preclinical studies have demonstrated the therapeutic effects of intratracheal MSCs transplantation on established neonatal hyperoxia-induced lung injury.
The intravenous route was not evaluated for established hyperoxia-induced lung injury in neonatal animals. Thus, we evaluated the therapeutic effects of human MSC by intravenous administration in this study.
The hedgehog pathway regulates morphogenesis of the lung and other organs.
The role of hedgehog signaling in the progress of lung development after hyperoxia is unknown. In this study, we discovered that hyperoxia on postnatal days 4–15 increased Shh and Smo expression, and these effects persisted on postnatal day 29 after return to RA from postnatal day 15. These results demonstrate that neonatal hyperoxia activates the hedgehog signaling pathway during hyperoxia and RA recovery and confirm that this pathway plays a crucial role in lung development through the hedgehog expression pattern in parallel to alveolarization and angiogenesis.
Hedgehog signaling commences when a hedgehog ligand binds to its transmembrane receptor Ptc, relieving suppression of the transmembrane receptor Smo and initiating the signaling cascade.
Inhibition of hedgehog signaling during early postnatal lung development causes airspace enlargement in mice, and upregulation of Shh signaling pathways was demonstrated in patients with progressive idiopathic pulmonary fibrosis.
Our study demonstrated that neonatal hyperoxia on postnatal days 4–15 arrested lung development and activated the hedgehog signaling pathway in rat lungs which induced airspace enlargement in newborn. Intravenous administration of human MSCs on postnatal day 15 improved alveolarization and increased the amount of angiogenesis. These effects are associated with the inhibition of the hedgehog signaling pathway. These findings suggest that precise regulation of the hedgehog signaling pathway is vital to postnatal lung development.
Our results suggest that human MSCs reverse established hyperoxia-induced lung injury in newborn rats, probably through suppression of hedgehog signaling. Identification of strategies to correct alterations in the hedgehog signaling pathway could eventually facilitate the development of novel therapies for hyperoxia-induced lung injury or neonatal lung diseases such as BPD. Further in vivo and in vitro studies are needed to evaluate the effects of MSCs in Shh gene knockout mice and on cell cultures treated with anti-hedgehog blocking monoclonal antibodies.
Declaration of Competing Interest
This study was supported by the Ministry of Science and Technology in Taiwan ( 107-2314-B-038-060-MY2 ). The authors are grateful to the Meridigen Biotech Co., Ltd. Taipei, Taiwan for assistance in harvesting and preparing the MSCs used in this study.
Appendix A. Supplementary data
Animal models of bronchopulmonary dysplasia. The term rat models.
Am J Physiol Lung Cell Mol Physiol.2014; 307: L948-L958