Introduction
Bronchopulmonary dysplasia (BPD) is a serious and long-term complication in premature infants that is caused by abnormal lung development. BPD occurs in more than 40% of preterm infants [
1,
2]. In the United States, about 15,000 new cases are diagnosed every year [
3]. BPD is closely associated with major complications, including prolonged mechanical ventilation, respiratory support and oxygen treatment, disordered angiogenesis, and a high incidence of advanced respiratory diseases [
4]. If BPD is not well treated, many complications, such as mental retardation and pulmonary hypertension (PH), occur. More than 20% of infants with moderate to severe BPD develop PH. The incidence and mortality rates of BPD patients with pH have significantly increased [
5]. Although significant advancements have been made in the fields of neonatal research and nursing [
6,
7], the incidence and mortality rates associated with respiratory diseases in preterm infants remain high owing to the development of BPD. Therefore, effective preventive therapies are urgently needed.
Many prenatal factors, including chorioamnionitis (CA) and pulmonary edema (PE), are closely associated with an increased risk of BPD, especially those related to intrauterine growth [
8,
9]. Insulin-like growth factor-1 (IGF-1), which has strong angiogenic activity, is implicated in the etiology of BPD [
10]. Zhang et al. identified IGF-1 as a potential target for BPD treatment [
11]. In addition, Han et al. investigated the relationship between serum IGF-1 levels and lung function [
12]. However, whether treatment with IGF-1 improves respiratory diseases and prevents BPD remains unclear.
Based on the vital functions of IGF-1 in lung progression and reports stating that low IGF levels after birth are associated with the further development of BPD, our study aimed to (i) illustrate whether recombinant human IGF-1 (rhIGF-1)/binding peptide 3 (BP3) could prevent the development of PH in experimental BPD and (ii) uncover the latent mechanism of rhIGF-1/BP3 in vascular growth, lung structure, and lung function to identify promising therapies for BPD treatment.
Materials and methods
Animals
Pregnant Wistar rats (6–8 weeks, 200–250 g) were provided by Hubei Laboratory Animal Research Center (Wuhan, China) and housed under standard conditions (22–25 °C, 50–60% humidity) with free food and water. The rat pups were also housed under standard conditions (22–25 °C, 50–60% humidity) with free food and water. This study was conducted in accordance with the ARRIVE guidelines. All experimental protocols were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study protocols were approved by the Ethics Committee of the First Affiliated Hospital of Bengbu Medical College.
Establishment of two models of BPD
BPD model of chorioamnionitis (CA): On day 20 of gestation, pregnant rats were randomly assigned to receive saline (control; 50 µl/sac) or LPS (10 µg/sac) stimulation [
13]. Two days after the intra-amniotic injections, the neonatal rat pups were induced with rhIGF-1/IGFBP3 (0.2 mg/Kg/d, IP injections) or saline for 2 weeks. The rat pups were then sacrificed at 14 days of age for lung tissue collection for subsequent experiments.
Hyperoxia-induced BPD group (HOX group): Newborn rats were subjected to high oxygen tension (85% oxygen) or normoxic conditions (control) from days 1 through 14 of postnatal life [
14]. Animals received rhIGF-1/BP3 (0.2 mg/Kg/d, IP injections) for 14 days. The pups were then killed at 14 days of age for lung tissue collection for subsequent experiments.
Lung function detection
According to previous study [
15], the lung function of 14-day old pups was determined using the flexiVent system (SCIREQ).
Lung wet/dry weight (W/D) ratio measurement
The lung W/D ratio was evaluated to analyze the severity of the PE. Briefly, fresh lung samples were collected from 14 day old pups, blotted with filter paper, and weighed. After drying for 48 h at 65 ℃, lung samples were weighed again.
Hematoxylin and eosin (H&E) staining
After the lung tissues were fixed with 4% paraformaldehyde for 24 h and dehydrated, slides with 5 μm paraffin sections were taken from the mid-plane of the left lower lobes of the fixed lungs. The sections were dewaxed, hydrated, incubated with hematoxylin for 5 min, and stained with eosin for 2 min, and the morphology of the lung tissue was observed using a microscope (×200 magnification).
Masson staining
The 5 μm lung paraffin sections were dewaxed, hydrated, and stained using the Masson Staining Kit (G1006, Servicebio) according to the manufacturer’s protocol. The collagen fibers were stained blue, and the muscle fibers and red blood cells were stained red, indicating the content of collagen fibers and the degree of fibrosis in the tissue (×200 magnification).
Evaluation of vascular density
Upper and lower lobe lung Sect. (5-µm-thick) were dissected, deparaffinized, rehydrated, and stained with von Willebrand factor (vWF; 1:100; Santa Cruz Biotechnology) and DAPI (D8417-1MG; Sigma). Subsequently, the number of vWF-positive vessels in each high-energy field (HPF) was observed using a blind method (×200 magnification).
Immunofluorescence
Upper and lower lobe lung Sect. (5-µm-thick) were obtained and incubated with N-cad (1:200, 22018-1-AP, PTG), SP-C (1:100, DF6647, affinity), E-cad (1:200, 20874-1-AP, PTG), FSP1 (1:200, 20886-1-AP, PTG), and vim (1:100, 10366-1-AP, PTG) antibodies overnight at 4 °C. The next day, the sections were incubated with the secondary antibody for 2 h. Finally, fluorescence signals were visualized using a fluorescence microscope (×400 magnification).
Right ventricular hypertrophy (RVH) evaluation
The right ventricle (RV) and left ventricle plus the ventricular septum (LV + S) were dissected and weighed. The RV/LV + S and RV/body weight ratios were then measured to analyze RVH.
Evaluation of lung alveolarization
Slides with 5-µm paraffin sections were stained with H&E. Radial alveolar count (RAC) was used to evaluate alveolarization as previously described [
16,
17].
qRT-PCR analysis
Total RNA from lung tissues exposed to rhIGF-1/BP3 was harvested (EP013, ELK Biotechnology) and reverse-transcribed (Eq. 002, ELK Biotechnology). The relative mRNA levels of IGF-1 were determined using QuFast SYBR Green PCR Master Mix (Eq. 001, ELK Biotechnology) on StepOne™ Real-Time PCR (Life technologies). Target gene levels were analyzed using 2−ΔΔCt method.
Western blot analysis
Proteins were collected from the lung samples for western blot analysis of IGF-1 and eNOS using RIPA buffer (ASPEN, AS1004). Proteins were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, IPVH00010). The membranes were blocked with 5% nonfat milk and incubated with primary and secondary antibodies. Finally, the results were analyzed using ECL detection system reagents (ASPEN, AS1059) according to the manufacturer’s protocol.
Statistical analysis
Statistical analyses were performed using the GraphPad Prism 8.0 software. Results are expressed as the mean ± SD from three independent measurements and analyzed using one-way analysis of variance (ANOVA) or Student’s t-test. Statistical significance was set at *P < 0.05 and **P < 0.01.
Discussion
BPD, a chronic lung disease caused by abnormal lung development, is common in premature infants and is characterized by the destruction of pulmonary angiogenesis [
18,
19]. Approximately 30% of infants will develop PH, which is one of the major causes of the high incidence rate and mortality in BPD. The pathophysiology of PH in patients with BPD includes pulmonary vascular reduction, endothelial cell dysfunction, increased vascular tension, and altered vascular reactivity [
20]. Therapeutic therapies for endothelial cell function and survival may help develop new methods for the prevention of BPD. Papagianis et al. have confirmed the potential anti-inflammatory therapies for BPD [
6], and Tin et al. have reported potential drug therapies for BPD [
21]. However, the specific mechanism by which placental dysfunction affects lung disease progression and leads to BPD remains unclear.
In vitro investigations have revealed that developing endothelial cells are vital for the regulation of epithelial growth by producing key factors, including hepatocyte growth factor and IGF-1 [
22]. IGF-1 plays an important role in fetal growth and organogenesis by regulating cell growth, maturation, and differentiation. 21. Schmidt et al. confirmed that IGF-1 treatment improves outcomes in rats with cancer cachexia [
23]. Seedorf et al. reported that rhIGF-1/BP3 injections significantly reduced BPD and increased IGF-1 levels in preterm infants [
13]. Based on these findings, we hypothesized that the early restoration of IGF-1 levels may play a protective role against severe retinopathy in premature infants. Therefore, this study aimed to evaluate whether postnatal treatment with rhIGF-1/BP3 could prevent PH in BPD.
Several factors are involved in the pathogenesis of BPD, including hyperoxia and mechanical ventilation [
24,
25]. In our study, we generated two models of BPD to investigate the underlying mechanism of rhIGF-1/BP3 in the progression of BPD: one model was associated with CA induced by intra-amniotic fluid and exposure to LPS, and the other was exposed to postnatal hyperoxia. The newborn rats were then injected with rhIGF-1/BP3 (0.2 mg/Kg/d) or saline. Previous studies have suggested that exposure to hyperoxia may damage the alveolarization and pulmonary vascularization in preterm infants [
26]. First, we evaluated the degree of lung histopathology and pulmonary fibrosis using H&E and Masson staining of the lower lobe of the left lung. Our findings are in accordance with other reports suggesting that rhIGF-1/BP3 improves LPS-induced changes in lung histology, pulmonary fibrosis, and increased collagen volume in infant lungs. RAC was used to assess the effect of rhIGF-1/BP3 on lung tissue injury in rats with BPD. We found that RAC was inhibited, and similar findings were obtained in the high oxygen-treated groups, suggesting that rhIGF-1/BP3 treatment improves distal lung structure in BPD rats.
Adverse prenatal elements such as chorioamnionitis, PE, or postpartum damage may result in pulmonary vascular disease, which not only causes PH but also affects distal lung growth [
27]. Therefore, we determined the effects of rhIGF-1/BP3 on the vessel density in infant rats. Our findings revealed that LPS treatment or high-oxygen stimulation decreased vessel density; however, rhIGF-1/BP3 stimulation restored vascular density to normal levels. It should be mentioned that we measured the vascular density through vWF staining, and as it is the localization of extracellular matrix and secreted proteins, there will definitely be non-specific staining. During the analysis process, we can only try to narrow the selection range and avoid including too many non-specific staining in the statistical range. This was a limitation of the current study. We also assessed the effects of rhIGF-1/BP3 on right ventricular hypertrophy and pulmonary function by determining RVH, lung resistance, and lung compliance. Our findings show that rhIGF-1/BP3 therapy preserves the lung structure and function and prevents RVH in rat pups. Therefore, in the two models of BPD, rhIGF-1/BP3 treatment played a significant role in normalizing lung function and preventing RVH in infant rats, which further supports previous research that has identified IGF-1 as a critical regulator of lung development.
To further explain the mechanisms related to rhIGF-1/BP3 in the lung structure of these models, we detected the expression of IGF-1 or eNOS in the lungs using western blotting. We observed reduced IGF-1 mRNA level and protein expression and reduced eNOS protein levels after LPS or hyperoxia exposure. Moreover, we assessed the alveolar epithelial and mesenchymal cell markers in the lungs of BPD rats. Immunofluorescence revealed that LPS induction or hyperoxia exposure reduced the number of alveolar epithelial cells and promoted the EMT of airway epithelial cells, whereas we found the opposite results in the rhIGF-1/BP3 treated groups. However, this study did not elucidate the specific molecular regulatory mechanism of rhIGF-1/BP3 in regulating EMT, which is another limitation of this study.
In conclusion, our results revealed that rhIGF-1/BP3 treatment accelerated lung angiogenesis, improved lung structure and function, and prevented the development of RVH in two experimental BPD models. Our study suggests a promising therapy for BPD in preterm infants. Following these achievements in BPD research, additional antenatal models that result in impaired lung development need to be further investigated.
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