Background
Bronchopulmonary dysplasia (BPD) is a common and debilitating complication of prematurity that occurs primarily in infants who require supplemental oxygen and mechanical ventilation [
1]. BPD affects up to 40% of infants born at less than 29 weeks gestational age [
2] and is characterized by a phenotype of impaired lung development indicated by the presence of large immature alveoli [
1,
3]. Dysregulated vascular growth also occurs in BPD and may lead to the development of pulmonary hypertension [
4,
5]. Despite numerous advances in clinical care; the incidence of BPD has remained relatively static and the identification of new therapeutic targets continues to be a priority [
6,
7].
Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine that acts as a regulator of the innate immune system, encourages cell proliferation and activates pro-angiogenic pathways [
8,
9]. When MIF was measured in the tracheal aspirates of preterm infants intubated for management of respiratory distress syndrome (RDS), infants with lower levels were found be more likely to develop BPD [
10]. This association was demonstrated again in a larger study that also confirmed that a single nucleotide polymorphism that increases expression of MIF is independently associated with reduced risk for BPD [
11]. When MIF levels were manipulated using MIF null mutant or knock out (MIFKO) and MIF overexpressing transgenic mice both extremes were associated with the development of an abnormal pulmonary phenotype [
12,
13]. These studies strongly suggest a key role for MIF in normal lung development and injury and indicate the need to more closely characterize mechanisms that control MIF expression in both physiological and pathophysiological conditions.
MicroRNA-451 (miR-451) is a 22 base pair (bp) sequence of complementary nucleotides that has been shown to bind to the 3’untranslated (UTR) region of MIF mRNA leading to inhibition of protein synthesis [
14,
15]. miR-451 has also been shown to reduce angiogenesis in colorectal cancer cells by targeting the interleukin – 6 receptor (IL-6R) [
16] and to decrease T-cell responses to infection by inhibiting the expression of the regulatory gene, myc [
17]. By inhibiting the expression of the regulatory protein Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ), miR-451 has been found to promote pro-apoptotic pathways involving the transcription factor forkhead box O3 (FOXO3) [
18,
19] and to reduce expression of pro-inflammatory cytokines in dendritic cells exposed to influenza virus [
20].
Although miR-451 mediated inhibition of MIF and other proteins promoting angiogenesis and cell division has been described in malignant cell lines, these regulatory relationships have not yet been investigated within the context of BPD. Our goal was to study the effect of hyperoxia on miR-451 expression in both murine lung endothelial cells (MLECs) and in a previously validated murine model of hyperoxia-induced BPD. MLECs were chosen for our in-vitro model as we had a particular interest in the role of miR-451 as an anti-angiogenic regulator. After establishing that miR-451 does increase significantly in both MLECs exposed to hyperoxia and in the lung tissues of BPD mice, we proceeded to evaluate the effect of a miR-451 inhibitor on the cardio-pulmonary phenotype, expression of MIF, inflammatory markers and vascular growth factors in newborn (NB) mice exposed to room air (RA) and BPD conditions.
Methods and materials
Animals
All in vivo experiments were performed using wild type (WT) C57BL/6 mice purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed at the Drexel University animal care facility. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Drexel University.
Hyperoxia exposure
Hyperoxia exposure was performed using a method previously described [
21,
22]. Briefly, NB mice were kept in an airtight Plexiglass container (55 × 40 × 50 cm) along with their mothers. A total of 32 pups, supported by 2 lactating dams were used to perform the experiments. Each litter comprising of 6 to 8 pups were randomized to either BPD or RA conditions. Animals in the BPD group were exposed to 100% oxygen from postnatal day (PN) 1 to PN4 which corresponds to the saccular stage of murine lung development. Mice were then allowed to recover in RA until PN14 when sacrifice was performed. Oxygen levels were measured continuously during the exposure period and the inside of the chamber was kept at atmospheric pressure. Lactating dams were cycled between the RA and hyperoxia groups every 24 h. Animals had free access to standard food and water and were subjected to a 12-h light-dark cycle. Survival was noted to be 100% which is consistent with previous work utilizing this experimental model of BPD [
12,
23]. There was no significant difference in body weight between the groups at the end-point of the study (PN14).
Cell culture
MLECs were purchased from Cell Biologics (Chicago, IL) and maintained in cell culture medium (Cell Biologics, Chicago, IL) as previously described [
24]. Exposure to hyperoxia was achieved by leaving the plates inside a tightly sealed modular chamber (Stem Cell Technologies, Vancouver, Canada) filled with 100% oxygen for 16 h.
In-vitro inhibition of miR-451
When MLECs had reached a confluence of approximately 70%, they were transferred to an antibiotic free growth medium (Cell Biologics, Chicago, IL) and transfected with 50 nM miR-451 inhibitor (catalog ID: IH-310630-07, Dharmacon, Lafayette, CO) using the Lipofectamine 3000 kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA). Dosage of the inhibitor was determined based on the results of previous work published by our group [
24,
25]. Cells were then re-plated and incubated at 37 °C for 24 h prior to exposure to either RA or hyperoxia.
In-vivo inhibition of miR-451
A subgroup of mice in both the RA (
n = 7) and BPD (
n = 8) groups were treated intranasally with 20 μM of an oligonucleotide based miR-451 inhibitor (Qiagen, Valencia, CA) on PN2 and PN4. Dosage of the inhibitor was determined based on the results of previous work performed in our laboratory [
23,
25].
Bronchoalveolar lavage (BAL)
BAL specimens were obtained, and cell count analysis performed as previously described [
21,
24]. Total protein content of BAL fluid was evaluated using the Pierce™ BSA Assay Kit (Thermo Fisher Scientific, Waltham, MA) as previously described [
24,
26].
Real-time reverse transcription PCR
RNA was extracted from both murine lung tissue and MLECs using the miRNeasy mini kit (Qiagen, Valencia, CA). Real-time reverse transcription PCR was performed as previously described [
23]. miScript primer assay IDs MIMAT0001632 and MS00033740 were used for miR-451 and RNU6 respectively.
Histological analysis
Lung tissues obtained from NB mice underwent a standardized inflation protocol (25 cm H2O) and were fixed in 4% paraformaldehyde. Specimens were then embedded in paraffin and 5 μm sections were obtained prior to staining with hematoxylin and eosin (H&E). This preparation was performed at the Department of Pathology Core Facility (Drexel University College of Medicine). Two random sections of both heart and lung tissues were obtained per animal with 6–8 animals represented in each experimental group.
Lung morphometric analysis
All images for morphometric analysis were captured on an Olympus IX70 with DP73 camera attachment. At least 4 to 7 low power (magnification × 20) images were acquired for each animal with care taken to avoid capturing vessels and large airspaces. Alveolar size was estimated by measuring the mean chord length of the airspace automatically using ImageJ as previously described [
23]. Four – 7 separate readouts were obtained for each animal. This software was also used to measure septal thickness. Measurements of radial alveolar counts were obtained as previously described [
27].
Measurement of BPD induced right ventricular hypertrophy (RVH)
Quantification of right ventricular (RV) wall thickness, left ventricular (LV) wall thickness and interventricular septal (IVS) thickness was performed by examining H&E stained specimens under 40x magnification using Cell Sens Olympus software. These measurements were then used to calculate the ratio of RV/(LV + IVS).
Elastin staining
Elastin staining was performed on 5 μm thick lung paraffin sections using the Modified Verhoff’s elastin staining protocol of Percival and Radi [
28]. The arterial thickness was measured by drawing an arbitrary line on the arterial wall using Olympus Cell Sens software (version 7).
Immunostaining
Immunostaining was done following the methodology as previously described [
23]. Briefly, 5um paraffin slides were dewaxed and dehydrated through a series of graded alcohol, followed by antigen retrieval in citrate buffer (pH 6) and incubation with vWF (DAKO, 1:100, Germany) at 4 °C, overnight. The following day, slides were washed 5 times with 1X PBS, 5 min each wash and incubated with the appropriate secondary antibody at room temperature for 2 h, washed again with 1X PBS for 5 times, 5 min each wash and mounted with vectashield 4′,6-diamidino-2-phenylindole (DAPI) (Vector laboratories, CA).
For imaging, the slides were manually counted under high power field for the number of vessels to cover the entire lung. Five – 6 animals were taken for each group. Photomicrographs were taken at 10X and 40X magnifications, and intensity adjusted with Adobe Photoshop 13.
Western blotting
Detection of MIF, angiopoietin (Ang)1, Ang 2, tyrosine kinase with immunoglobulin and epidermal growth factor homology domains receptor 2 (Tie2), Vascular Endothelial Growth Factor-A (VEGF-A), interleukin (IL)-6, IL-1β, YWHAZ and FOXO3 was performed using vinculin and β-actin as loading controls from lung tissue and MLEC lysates using Western blot as previously described [
25].
The primary antibodies used were MIF (Abcam, Cambridge, UK, 1:400), Vinculin (Santa-Cruz Biotechnology, Dallas, TX; 1:10,000), β-actin (Cell Signaling Technology, Danvers, MA), Ang1, (Sigma-Aldrich, St. Louis, MO, 1:500), Ang2 (Sigma-Aldrich, St. Louis, MO, 1:500), Tie2 (Santa-Cruz Biotechnology, Dallas, TX; 1:200), VEGF-A (Abcam, Cambridge, UK, 1:200), IL-6 (Santa-Cruz Biotechnology, Dallas, TX; 1:500), IL-1β (Cell Signaling Technology, Danvers, MA), FOXO3 (Cell Signaling Technology, Danvers, MA; 1:1000), YWHAZ, (Santa-Cruz Biotechnology, Dallas, TX; 1:800).
ImageJ software was used to analyze expression of target proteins relative to either vinculin or β-actin loading controls.
Statistical analysis
Previous work has shown that mean chord length, as a measure of alveolarization is abnormal in > 99% of WT BPD mice lungs. We expected that treatment with a miR-451 inhibitor would yield an 80% improvement in mean chord length. Hence, with an alpha = 0.005, beta = 0.2, and a power of 80%, we calculated a need for n = 6 in each group. All statistical analysis was performed using Graph Pad Prism Version 7 (GraphPad software, San Diego, CA). Values are expressed as mean +/− SEM. Groups were compared using the Student’s two-tailed unpaired T-test or one-way analysis of variance (ANOVA) with Tukey’s post-hoc test to correct for multiple comparisons, where appropriate. A p value of < 0.05 was considered statistically significant.
Discussion
During normal lung development angiogenesis is coordinated precisely, leading to a balance in factors that promote expansion with those that promote stability of the endothelial barrier [
30,
31]. Normal alveolar development has been shown to be dependent on angiogenesis [
4,
32,
33] and dysregulated vascular growth is a well-established feature of the BPD phenotype [
1,
4]. miR-451 is a miRNA that has been shown to inhibit the expression of pro-angiogenic and anti-apoptotic mediators such as MIF, IL-6R and YWHAZ in a variety of different malignant cell types and tumors [
14,
16,
18,
34,
35]. In tumors, reduced expression of miR-451 and increased expression of MIF is associated with increased likelihood of progression [
15,
36]; however, in preterm infants, increased expression of MIF is linked to a reduced risk for BPD [
10,
11]. Studies published by our group utilizing murine models of neonatal hyperoxia induced lung injury (HALI) and BPD have strongly suggested that the protective effect of MIF is mediated through regulation of the Ang-Tie2 axis [
12,
13] and promotion of angiogenesis through upregulation of VEGF-A [
37,
38]. Studies evaluating both removal of MIF and elevation of expression to supraphysiological levels have shown that both extremes are associated with the development of a pulmonary phenotype similar to that seen in hyperoxia induced BPD [
12,
13]. These findings led to the consideration of potential mechanisms that regulate MIF expression in the developing lung in both physiological and pathophysiological conditions.
The goal of this study was to evaluate miR-451 as a potential contributor to the pathogenesis of BPD and to investigate the possibility that MIF signaling pathways in the developing lung could be subject to miRNA mediated regulation. We chose to evaluate miR-451 expression using both an in-vitro model of MLECs exposed to hyperoxia and an in-vivo murine model of severe hyperoxia induced BPD. Endothelial cells were selected due to our interest in the effect of miR-451 on angiogenesis. We have demonstrated that miR-451 is upregulated in both MLECs exposed to hyperoxia and in lung tissues of NB mice exposed to 100% O
2 during the critical saccular phase of lung development. This rise in miR-451 expression in response to hyperoxia coincides with the decrease in MIF expression previously reported by our group using the same experimental model of hyperoxia induced BPD [
12] and is consistent with published findings that miR-451 is one of several miRNAs differentially regulated in a murine model of hyperoxia-induced BPD [
39].
Treatment of NB mice with a miR-451 inhibitor was found to be associated with mitigation of the BPD phenotype indicated by reduced mean chord length, reduced septal thickness and increased mean radial alveolar counts relative to WT BPD mice. Treatment with a miR-451 inhibitor was also associated with a significant reduction in RVH similar to that noted in MIF overexpressing transgenic mice exposed to a model of HALI [
13]. Inhibition of miR-451 was also shown to partially preserve vascular growth in BPD mice and reduce vascular remodeling indicating that the rise in miR-451 noted in MLECs and murine lung tissues may contribute to the dysregulated vascular growth that is part of the BPD phenotype.
Regulation of the balance of vascular growth factors in the developing lung is known to be critical in the creation of the extensive network of blood vessels necessary to support alveolarization [
30,
31]. The Ang-Tie2 axis has been shown to play an essential role in maintaining both vascular homeostasis and expansion during lung development [
31,
40]. As previous work published by our group has indicated that the known miR-451 target MIF acts as a regulator of Ang signaling, we proceeded to evaluate the effect of a miR-451 inhibitor on the expression of MIF, Ang1, Ang2 and the Ang receptor Tie2. MIF expression did not decrease in MLECs exposed to hyperoxia; however, administration of a miR-451 inhibitor was associated with increased expression of MIF. This finding differs from results shown in murine BPD lung specimens where MIF decreases in response to hyperoxia and expression is preserved following treatment with a miR-451 inhibitor. Discrepancies in the expression of both Ang1, Ang2 and the Ang receptor, Tie2 were also noted between in-vitro and in-vivo models. Ang1 acts via the Tie2 receptor to increase expression of vascular adhesion molecules, inhibit apoptosis and promote stability of the endothelial cell barrier [
31,
41]. Ang2 competes with Ang1 for Tie2 binding sites and has the opposite effect; loosening connections between endothelial cells in order to promote branching [
31,
40]. Although no changes in Ang1 expression were noted in MLECs exposed to hyperoxia, Ang1 expression was noted to be significantly decreased in WT BPD mice relative to the WT RA group. This decrease in Ang1 expression is consistent with the pattern noted in both experimental models of BPD [
12] and in preterm infants who go on to have adverse pulmonary outcomes [
42,
43]. Administration of a miR-451 inhibitor to NB mice was associated with a significant increase in Ang1 expression that was partially maintained following exposure to hyperoxia. No change was noted in the expression of Ang1 in MLECs either following exposure to hyperoxia or to a miR-451 inhibitor. Ang2 expression was found to increase in both MLECs exposed to hyperoxia and in the lungs of BPD mice. This result is consistent with previous reports in the literature that identify Ang2 as a pathogenic regulator in the response to hyperoxia [
29,
44] and a potential biomarker denoting increased risk for BPD [
45,
46]. miR-451 inhibitor administration was associated with a decrease in Ang2 in MLECs exposed to hyperoxia; however, Ang2 expression was significantly increased in the lungs of both RA and BPD mice who were treated with the antagomir. Administration of the miR-451 inhibitor was also associated with increased expression of the Ang receptor Tie2 in murine lung tissues. No changes in Tie2 expression noted in MLECs. Previous work has demonstrated that pulmonary vascular development is driven by interactions between alveolar epithelial and endothelial cells [
47] and it is possible that the different expression patterns noted in the in-vivo model could have occurred due to the influence of other pulmonary cell types. MIF is constitutively expressed in nearly every cell type [
8] and it is also possible that other pulmonary cell types are making a greater contribution to MIF expression during normoxic conditions. Interactions between different cell types occurring in-vivo could also explain the differences noted in the expression of the Ang proteins and their receptor Tie2.
miR-451 has been shown to inhibit angiogenesis through targeting MIF, calcium-binding protein 39 (CAB39) and IL-6R mediated pathways that influence expression of VEGF-A [
16,
35,
48]. In our study, administration of a miR-451 inhibitor was also associated with increased expression of VEGF-A. VEGF expression has been shown to be decreased in both experimental models of BPD and in preterm infants who develop adverse pulmonary outcomes [
32,
49‐
51] and gene therapy with VEGF has been shown to improve alveolarization in a rat model of hyperoxic lung injury [
32]. The pro-apoptotic activity of Ang2 is also known to be reduced in the presence of increased levels of VEGF [
52] which may explain in part why treatment with a miR-451 was associated with both increased expression of Ang2 and mitigation of the BPD phenotype. The increase in Ang1 expression associated with administration of a miR-451 inhibitor could also potentially balance the deleterious effects of Ang2. The Ang1:Ang2 ratio which is known to decreased in the presence of hyperoxia [
12,
25] was significantly improved in the lungs of BPD mice treated with a miR-451 inhibitor. Increased expression of Tie2 noted following miR-451 inhibitor administration might also contribute to the relative improvements in vascular growth and alveolarization seen in the BPD group.
BPD associated PAH is characterized by various forms of vascular remodeling, and elastin being a major component of blood vessels is easily visualized using Verhoeff’s stain. In the present study, we found that the arterial walls were thickened and dense with abundant deposition of collagen following exposure to hyperoxia; however, treatment with the miR-451 inhibitor was associated with a significant decrease in arterial wall thickness and a reduction in the deposition of collagen. These findings indicate remodeling of the large vessels towards a state of normalcy in the presence of a miR-451 inhibitor. Taken together with the significant reduction in RVH and relative preservation of in vascular density in BPD mice treated with the miR-451 inhibitor these findings suggest that increased expression of miR-451 in the presence of hyperoxia may contribute to the dysregulation of vascularization that is part of the BPD pulmonary phenotype.
In addition to anti-angiogenic and pro-apoptotic activities, miR-451 has also been shown to downregulate the inflammatory responses in a variety of different experimental models [
20,
53,
54]. In our model of hyperoxia induced BPD, treatment with a miR-451 inhibitor was also associated with several indications of an increased inflammatory response including elevation of BAL total neutrophil count, elevation in BAL total protein and increased expression of pro-inflammatory cytokines IL-6 and IL-1β. MIF has been shown to promote arrest and transmigration of neutrophils across the endothelial cell barrier through interactions with the chemokine receptors CXCR2 and CXCR4 [
55,
56]. Therefore, upregulation of MIF expression in the presence of a miR-451 inhibitor could account for the increased numbers of neutrophils present in the BAL specimens. miR-451 has also been shown to reduce production of pro-inflammatory cytokines in murine dendritic cells exposed to influenza virus by inhibiting the expression of the regulatory protein YWHAZ [
20]. YWHAZ acts as an inhibitor of the zinc finger protein ZFP36 and the inhibitory transcription factor FOXO3. Inhibition of miR-451 in this model was associated with increased expression of YWHAZ, decreased nuclear expression of FOXO3 and increased production of IL-6, among other cytokines [
20]. In the current study we did note that IL-6 and IL-1β expression was not only significantly increased in the WT BPD group but also in both groups of miR-451 inhibitor treated mice. We did note changes in the expression of FOXO3 with significantly reduced expression of the phosphorylated nuclear form in the miR-451 inhibitor treated BPD mice when compared to the WT BPD mice. This appears to be occurring independently of YWHAZ. The inferences we can draw from these findings are limited as western blotting was performed on whole lung tissue lysates rather than discrete nuclear and cytoplasmic fractions; however, regulation of FOXO3 by miR-451 may be an area for future investigation in this BPD model.
An excessive inflammatory response has been established as an important contributor to the pathogenesis of BPD [
57,
58]; however, the impact of pro-inflammatory cytokines on the NB lung has been shown to be dependent on the timing and duration of exposure [
59,
60]. IL-6 in addition to having pro-inflammatory activities, also plays an important role in anti-inflammatory and regenerative processes [
61]. When NB IL-6 transgenic mice were studied in a model of hyperoxia-induced lung injury, absence of IL-6 was found to be associated with higher levels of pro-inflammatory cytokines and increased apoptosis in comparison to WT controls [
62]. IL-6 is also known to act via hypoxia-inducible factor (HIF)-1α to increase VEGF expression [
63] and this process has been shown to be inhibited by miR-451 mediated regulation of IL-6R expression in colorectal cancer cells [
16]. It is therefore possible to explain why disruption of miR-451 mediated inhibition of both inflammation and angiogenesis could result in relative improvements in lung morphometry and vascular growth in murine lungs exposed to hyperoxia.
Our study does have several limitations. Although the in-vivo model we used correlates with the pathological phenotype and long term consequences of neonatal lung disease [
22,
59,
64] these experiments cannot fully replicate the combination of factors that contribute to the development of BPD. The decision to randomize animals from the same litter to the same experimental group is another limitation of our study. As the sample size of the mice dams used for these experiments was low, we cannot exclude the possibility of the influence of maternal genetic variation on the differences we noted. As miR-451 acts on multiple targets, at this stage we are not yet able to ascribe the benefits of miR-451 inhibition exclusively to upregulation of a specific target or pathway. Although comparison of our results with those obtained from the study of MIF over-expressing transgenic mice (MIFTG) [
12,
13] lead us to speculate that MIF is involved, a relationship between miR-451 and MIF within the context of BPD can only be suggested, as modulation of this signaling pathway by other molecules is probably playing a role. Although the relative improvements seen in alveolarization and angiogenesis are impressive, more work needs to be performed to define the specific pathways that contribute to these findings.
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