Targeting miR‐34a/Pdgfra interactions partially corrects alveologenesis in experimental bronchopulmonary dysplasia

Abstract Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth characterized by arrested lung alveolarization, which generates lungs that are incompetent for effective gas exchange. We report here deregulated expression of miR‐34a in a hyperoxia‐based mouse model of BPD, where miR‐34a expression was markedly increased in platelet‐derived growth factor receptor (PDGFR)α‐expressing myofibroblasts, a cell type critical for proper lung alveolarization. Global deletion of miR‐34a; and inducible, conditional deletion of miR‐34a in PDGFRα+ cells afforded partial protection to the developing lung against hyperoxia‐induced perturbations to lung architecture. Pdgfra mRNA was identified as the relevant miR‐34a target, and using a target site blocker in vivo, the miR‐34a/Pdgfra interaction was validated as a causal actor in arrested lung development. An antimiR directed against miR‐34a partially restored PDGFRα+ myofibroblast abundance and improved lung alveolarization in newborn mice in an experimental BPD model. We present here the first identification of a pathology‐relevant microRNA/mRNA target interaction in aberrant lung alveolarization and highlight the translational potential of targeting the miR‐34a/Pdgfra interaction to manage arrested lung development associated with preterm birth.


Introduction
Bronchopulmonary dysplasia (BPD), a serious complication of preterm birth (Jobe, 2016), is characterized by arrested alveolarization of lungs of infants, arising from oxygen toxicity and mechanical injury during oxygen supplementation to manage respiratory failure. How these insults impair lung alveolarization is unclear Morty, 2018).
How myofibroblast function is disturbed during aberrant alveolarization is not known, but a role for microRNA has been proposed, since deregulation of microRNA has been noted in clinical and experimental BPD (Nardiello & Morty, 2016), although no study has validated a causal role for any microRNA/mRNA interaction in alveolarization or BPD. We report here that the miR-34a/Pdgfra interaction is disease relevant, and can be therapeutically targeted to partially restore lung alveolarization under pathological conditions. These data highlight a new mediator, and druggable target, in arrested alveolarization associated with preterm birth.
Global loss of miR-34a partially restores lung alveolarization in experimental BPD Consistent with the arrested alveolarization that forms the hallmark of the BPD animal model, a 71% decrease in total alveoli number (Fig 2A and B;Appendix Table S1) and 10% increase in mean septal thickness (Fig 2A and C;Appendix Table S1) were noted in hyperoxia-exposed wild-type mouse lungs at P14, mimicking perturbations to lung structure noted in clinical BPD cases (Jobe, 2016;Nardiello et al, 2017b). Ablation of miR-34a (miR-34a À/À mice) partially protected against the impact of hyperoxia on alveolarization (Fig 2A; Appendix Table S1), with alveoli numbers increased by 47% ( Fig 2B); and septal thickness decreased to even thinner than that noted in healthy mice (Fig 2C), compared to wild-type hyperoxia-exposed controls. No compensatory increase in miR-34b or miR-34c levels was noted in miR-34a À/À mice (Appendix Fig S1A). In contrast, dual ablation of miR-34b/miR-34c (miR-34bc À/À mice), without a compensatory increase in miR-34a levels (Appendix Fig S1B), did not impact alveoli number during hyperoxia-driven arrest of alveolarization (Fig 2D and E; Appendix Table S2). However, protection against hyperoxia-driven septal thickening in miR-34bc À/À mice was noted (Fig 2F), perhaps related to the increased levels of the 3p strands of miR-34b and miR-34c in the lungs of hyperoxia-exposed mice (Appendix Fig S1B). These data implicate miR-34a as mediator of arrested alveolarization associated with hyperoxia, an idea reinforced by detection of miR-34a expression with a lacZ-tagged miR-34a gene-trap in the septa of developing lungs, with increased b-galactosidase staining evident after hyperoxia exposure (Fig 2G; Appendix Fig S2).

miR-34a in PDGFRa + cells contributes to aberrant lung alveolarization
An in silico analysis identified two miR-34a-binding sites in the Pdgfra 3 0 -UTR ( Fig 3A) (Silber et al, 2012;Garofalo et al, 2013). The PDGF-AA ligand and PDGFRa are key mediators of alveolarization (Boström et al, 1996(Boström et al, , 2002, and reduced PDGFRa levels in mesenchymal cells are reported in human neonates that develop BPD (Popova et al, 2014). A synthetic miR-34a mimic reduced PDGFRa protein levels in vitro in MLg cells, a mouse lung fibroblast cell line, suggesting that a miR-34a/Pdgfra interaction occurs in mouse lung fibroblasts (Fig 3B), where increased miR-34 family microRNA transcripts ( Fig 3C) and reduced Pdgfra mRNA transcripts (Appendix Fig S3) were noted in hyperoxia-exposed MLg cells. To explore this idea in vivo, exposure of newborn mice to hyperoxia (85% O 2 ) reduced lung PDGFRa protein levels at P5 (Fig 3D), which is the peak phase of bulk alveolarization (Morrisey & Hogan, 2010;Warburton et al, 2010). Treatment of MLg cells in vitro with antimiR-34a, which neutralizes miR-34a, partially protected steady-state PDGFRa protein levels against the impact of hyperoxia exposure, while an inert ("scrambled") antimiR did not ( Fig 3E). These data support the contention that hyperoxiadriven elevations in miR-34a levels negatively regulated PDGFRa abundance. PDGFRa + cells were isolated from P5 mouse lungs by FACS (Appendix Fig S4A), where in vivo hyperoxia exposure had driven a dramatic increase in miR-34a levels in PDGFRa + cells ( Fig 3F, Appendix Fig S5), accompanied by reduced Pdgfra (Appendix Fig S4B) and Acta2 (Appendix Fig S4C) mRNA levels. The magnitude of the impact of hyperoxia on miR-34a levels in PDGFRa + cells was considerably larger than that observed in lung homogenates, highlighting the PDGFRa + cell as being particularly susceptible to hyperoxia-driven effects on miR-34a during alveologenesis.
To address miR-34a function in PDGFRa + cells, a mouse strain carrying a conditional, tamoxifen-inducible deletion of miR-34a in Pdgfra-expressing cells was generated (denoted miR-34a iDPC/iDPC ; Fig 3G) and was validated by demonstrating reduced miR-34a expression in PDGFRa + cells ( Fig 3H). Ablation of miR-34a in PDGFRa + cells protected against hyperoxia-driven arrest of alveolarization (Fig 3I; Appendix Table S3), where approximately double the number of alveoli was noted in hyperoxia-exposed mice in which miR-34a expression was blocked in PDGFRa + cells ( Fig 3J). Ablation of miR-34a expression in PDGFRa + cells did not impact hyperoxia-provoked perturbations to septal thickness (Fig 3K), which we attribute to the tamoxifen solvent, Miglyol, a complex fatty acid-derivative mixture, which we propose limited the impact of hyperoxia on septal thickening analogous to that reported for chemically related cottonseed oil (Nardiello et al, 2017b), since Miglyol alone is known to attenuate normal lung development (Fehl et al, 2019). Alternatively, it may be epithelial miR-34a that regulates septal thickening, since miR-34a regulates lung epithelial cell (notably, type II pneumocyte) apoptosis (Syed et al, 2017) in experimental BPD. These data validate a role for miR-34a in PDGFRa + cells in mediating the inhibitory effects of hyperoxia on alveolarization.  A Microarray analysis of microRNA expression changes in newborn mouse lungs exposed to 21% O 2 versus 85% O 2 , at post-natal day (P)5 and P14. Microarray data are available at the GEO database under accession number GSE89666. B Quantitative RT-PCR detection of microRNA-34a/b/c-5p family members in the lung over the course of normal (21% O 2 ) and aberrant (85% O 2 ) alveolarization. C Quantitative RT-PCR detection of microRNA-34a/b/c-3p family members in the lung over the course of normal (21% O 2 ) and aberrant (85% O 2 ) alveolarization.

B
Data information: For (A), a Welch's approximate t-test was employed to determine P values (n = 4 animals for each experimental group), which were corrected using the algorithm of Benjamini and Hochberg, as described in the Materials and Methods under the heading "Power and statistical analyses". For (B) and (C), data represent mean AE SD (n = 6 animals for each experimental group). P values were determined by one-way ANOVA with Tukey's post hoc modification, and all P values < 0.05 for 21% O 2 versus 85% O 2 comparisons at each developmental stage (P3, P15, and P14) are indicated.
The miR-34a/Pdfgra interaction plays a causal role in aberrant lung alveolarization MicroRNA/mRNA interactions can be interrupted using target site blocker (TSB) technology. We employed two synthetic TSBs (TSB1 and TSB2) to protect both of the miR-34a-binding sites in the Pdgfra 3 0 -UTR ( Fig 4A). Both TSBs protected PDGFRa expression from miR-34a regulation in MLg cells in vitro (upper panels, Fig 4B and C). Both TSBs exhibited specificity for the miR-34a/Pdgfra interaction, since neither TSB interfered with the impact of a synthetic miR-34a mimic on levels of c-Kit (middle panel, Fig 4B), a validated miR-34a target (Siemens et al, 2013), or of SIRT1 (middle panel, Fig 4C), another validated miR-34a target (Yamakuchi et al, 2008). A TSB cocktail of an equimolar TSB1:TSB2 mixture effectively protected PDGFRa expression from miR-34a regulation in MLg cells (Appendix Fig S6) Figure 2. miR-34a-5p functionally contributes to arrested lung alveolarization in response to hyperoxia.
A Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34a À/À mice during normal and aberrant alveolarization (scale bar, 50 lm). B Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR-34a À/À mice (34a À/À ) during normal and aberrant alveolarization. C Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR-34a À/À mice (34a À/À ) during normal and aberrant alveolarization. D Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34bc À/À mice during normal and aberrant alveolarization (scale bar, 50 lm). E Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR-34bc À/À mice (34bc À/À ) during normal and aberrant alveolarization. F Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR-34bc À/À mice (34bc À/À ) during normal and aberrant alveolarization. G Localization of miR-34a expression by b-galactosidase activity staining in the developing lungs of P14 miR-34a::lacZ +/+ mice that were undergoing normal or aberrant alveolarization (scale bar, 50 lm; larger and some additional images are presented in Appendix Fig S2).    Table S4), where increased alveoli number ( Fig 4E) and decreased mean septal thickness ( Fig 4F) were noted. Application of the TSB1,2 cocktail increased the abundance of both PDGFRa + cells ( Fig 4G; Appendix Fig S7) and PDGFRa + /aSMA + myofibroblasts ( Fig 4H) in hyperoxia-exposed mouse lungs. These data validate a causal role for the miR-34a/Pdgfra interaction in arrested lung development provoked by hyperoxia, most likely through partial restoration of PDGFRa + /SMA + myofibroblasts. MicroRNA function may be modulated in vivo using locked nucleic acid (LNA) antimiRs (Patrick et al, 2010). We theorized that dampening functional miR-34a levels in experimental BPD would improve alveolarization; therefore, an antimiR directed against miR-34a was applied therapeutically (concomitantly with hyperoxia exposure; Fig 5A), which decreased functional miR-34a levels in mouse lungs by P5 (Appendix Fig S8A), with no impact on miR-34b, and a moderate impact on miR-34c (Appendix Fig S8B and C). The effect of antimiR-34a on miR-34a was maintained up to P14 ( Fig 5B). AntimiR-34a protected alveolarization from hyperoxia (Fig 5C;  Appendix Table S5), increasing alveoli number by 40% (Fig 5D), and normalizing septal thickness ( Fig 5E). Flow cytometric quantification of PDGFRa + cells and PDGFRa + /aSMA + myofibroblasts (analysis ◀ Figure 3. miR-34a-5p acts in PDGFRa + cells to block lung alveolarization. A In silico identification of miR-34a binding sites in the Pdgfra 3 0 -UTR. B Immunoblot detection of PDGFRa levels in MLg cells after treatment with scrambled microRNA (SCR) or a miR-34a (MIM34a) mimic (n = 3 separate cell cultures for each group). C Quantitative RT-PCR detection of miR-34a/b/c-5p levels in MLg cells in vitro, maintained under 21% O 2 or 85% O 2 (n = 3 separate cell cultures for each group). D Immunoblot detection of PDGFRa levels in the lungs of mouse pups (n = 6 animals for each group) at post-natal day (P)5, during normal (21% O 2 ) and aberrant (85% O 2 ) alveolarization. E Immunoblot detection of PDGFRa levels in MLg cells in vitro, maintained under 21% O 2 or 85% O 2 , where cells had been transfected wither with a scrambled (SCR) antimiR, or an antimiR directed against miR-34a (A34a) (n = 3 separate cell cultures for each group). F Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRa + cells, sorted by FACS from the lungs of mouse pups (n = 4 animals for each group; data from an independent repetition are provided in Appendix Fig S5) at P5, maintained under 21% O 2 or 85% O 2 from birth. G Schematic illustration of the generation of a conditional, inducible deletion-ready mouse strain, where administration of tamoxifen (Tmxfn) abrogated miR-34a expression in Pdgfra-expressing cells (denoted miR-34a iDPC/iDPC ). H Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRa + cells, sorted by FACS from the lungs of either wild-type (34a wt/wt ) mouse pups, or mouse pups in which miR-34a expression in Pdgfra-expressing cells (34a iDPC/iDPC ) at P5 (n = 4 animals for each group). I Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from 34a wt/wt or 34a iDPC/iDPC mouse pups at P14 during aberrant (85% O 2 ) alveolarization, compared with 34a iDPC/iDPC during normal (21% O 2 ) alveolarization (scale bar, 50 lm). Data are representative of observations made in four other experiments. J Quantification of total number of alveoli by design-based stereology in 34a wt/wt or 34a iDPC/iDPC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group). K Quantification of mean septal thickness by design-based stereology in 34a wt/wt or 34a iDPC/iDPC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group).
Data information: For immunoblots (B, D, E), protein loading equivalence was controlled by bactin levels. (C, F, H, J, K) Data represent mean AE SD. In (C, F, and H), P values for pair-wise comparisons were calculated by unpaired Student's t-test. In (J and K), P values for selected comparisons were calculated by one-way ANOVA with Tukey's post hoc modification. Source data are available online for this figure.
▸ Figure 4. Disrupting the miR-34a/Pdgfra interaction restores myofibroblast abundance and limits hyperoxic damage to the developing alveolar architecture in mouse lungs.
A Generation of two target site blocker (TSB) locked nucleic acid sequences: TSB1 and TSB2 (in blue), for the disruption of the miR-34a/Pdgfra interaction, indicating binding sites in the Pdgfra 3 0 -UTR, Kit 3 0 -UTR, and the Sirt1 3 0 -UTR (in black), alongside the miR-34a sequence (in red). The miR-34a seed sequence, and the seedsequence binding site in the target mRNA 3 0 -UTR are indicated in bold, and brown, respectively. B Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRa and c-Kit as TSBdependent and TSB-independent target readouts, respectively. Protein loading equivalence was controlled by bactin levels. Note: PDGFRa, bactin, and c-Kit were all probed on the same membrane; hence, a single bactin immunoblot is presented. Data are representative of three experiments. C Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRa and SIRT1 as TSBdependent and TSB-independent target readouts, respectively. Protein loading equivalence was controlled by bactin levels. Note: PDGFRa, bactin, and SIRT1 were all probed on the same membrane; hence, a single bactin immunoblot is presented. Data are representative of three experiments. D Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type mouse pups at post-natal day (P)14, treated with either scrambled target site blocker (SCR) or a cocktail of both target site blockers (TSB1 and TSB2) during normal (21% O 2 ) and aberrant (85% O 2 ) alveolarization (scale bar, 50 lm). Data are representative of three or more experiments. E Quantification of total number of alveoli by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group). F Quantification of mean septal thickness by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group). G Quantitative analysis of PDGFRa + cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group). H Quantitative analysis of PDGFRa + /aSMA + cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).
Data information: Data represent mean AE SD. P values were calculated by unpaired Student's t-test. Source data are available online for this figure.
6 of 17 EMBO Molecular Medicine 11: e9448 | 2019 ª 2019 The Authors described in Appendix Fig S9A,B), revealed that the abundance of both PDGFRa + cells (Fig 5F; Appendix Fig S10A) and PDGFRa + / aSMA + myofibroblasts ( Fig 5G; Appendix Fig S10B), both of which were depleted by hyperoxia, was partially restored by antimiR-34a. However, the abundance of aSMA + cells per se was not changed (Appendix Fig S9C and D). These data imply that antimiR-34a partially restored myofibroblast numbers in injured, developing lungs (schematically presented in Fig 5H). Consistent with this idea, increased elastin foci and improved elastin fiber organization were noted in antimiR-34a-treated mice (Appendix Fig S11).
To further explore the role of hyperoxia and miR-34a on PDGFRa + cell abundance, apoptosis was assessed in PDGFRa + cells from hyperoxia-treated mouse pups by flow cytometry (Appendix Fig  S12A), where increased apoptosis of PDGFRa + cells was noted at P5    (Appendix Fig S12B), but not at P14 (Appendix Fig S12C). Furthermore, the density of PDGFRa on PDGFRa + cells was reduced by hyperoxia (Appendix Fig S12D), suggesting abundance of PDGFRa in PDGFRa + cells from mouse lungs exposed to hyperoxia (proposed here to be attributable to increased miR-34a levels within those cells). Flow cytometry did not permit an S-phase analysis of cell proliferation due to too few cells per run, as evident in the histogram in Appendix Fig S13, which has insufficiently developed G0/G1 and G2/M peaks. However, immunofluorescence staining of lung cryosections of P5 mice (Fig 6) revealed fewer proliferating PDGFRa + cells in the lungs of hyperoxia-exposed mice at P5 versus normoxiaexposed mice. Together, these data indicate that hyperoxia does reduce PDGFRa + cell abundance and proliferation in vivo in mice. In vitro, primary mouse lung fibroblasts exhibited reduced PDGFRa levels after hyperoxia exposure (Appendix Fig S14A), and increased miR-34a levels after application of a miR-34a mimic (Appendix Fig  S14B), consistent with what was noted in MLg cells in vitro (Fig 3E). A miR-34a mimic had a moderate impact on baseline proliferation (Appendix Fig S14C) and no impact on baseline apoptosis (Appendix Fig S14D) in primary mouse lung fibroblasts in vitro.
Collectively, these data indicate that hyperoxia can decrease proliferation ( Fig 6B) and increase apoptosis (Appendix Fig S12B  and C) of PDGFRa + cells in vivo in developing mouse lungs. Additionally, hyperoxia drives increased levels of miR-34a in PDGFRa + cells that are resident in the developing mouse lung (Fig 3F), which in turn decreases the abundance of PDGFRa in affected cells (Appendix Fig S12D; ostensibly, PDGFRa + myofibroblasts in the developing septa). We propose that this results in defective elastin production and remodeling (one of the functions of myofibroblasts during alveolarization), which in turn impairs secondary septation, leading to alveolar simplification characteristic of BPD (Fig 5H). By neutralizing miR-34a (Fig 5) or disrupting the miR-34a/Pdgfra interaction (Fig 4), the abundance of PDGFRa + myofibroblasts was partially restored, leading to partial correction of this alveolarization defect. This is noteworthy given the recent first-in-man report using an antimiR to manage hepatitis C infection, by targeting miR-122 (Janssen et al, 2013). We further propose that interventions to block miR-34a function or the miR-34a/Pdgfra interaction are candidates for translational development.
A role for miR-34a in septal thickening as well?
BPD is also characterized by septal thickening (Jobe, 2016). As a secondary observation in this study, we demonstrate here that miR-34a/b/c impacted septal thinning during alveolarization. Thickened septa arose in this model from multicellular stacking of cells, which revert to the normally observed single cell layer after antimiR-34a treatment (Fig 7). Almost all septal cells stained for aquaporin 5 (Aqp5), a type I pneumocyte marker-a cell type that exhibits tremendous plasticity during alveologenesis (Yang et al, 2016)-in both thickened and restored (thinner) septa (Fig 7). In the background of hyperoxia, antimiR-34a treatment did not impact the number or apoptosis (Appendix Fig S15A-G) of type I pneumocytes; or whole-lung gene expression assessed by mRNA microarray at P5 and P14 (Appendix Table S6; GEO accession number GSE89730; validated in Appendix Fig S16). Thus, antimiR-34a most likely affected gene expression in a rare cell population, such as PDGFRa + myofibroblasts, and not broadly throughout the alveolar epithelium, composed largely of type I pneumocytes. We suggest that changes in septal complexity arose not from loss or gain of epithelial cells, but rather from the spatial organization of the type I pneumocytes, that is directed by PDGFRa + myofibroblasts. This may be related to the production of extracellular matrix (ECM) by PDGFRa + myofibroblasts, where perhaps ECM laid down and remodeled during alveologenesis provides migration cues to epithelial cells organize themselves within the newly-generated septa. Such cues might possibly include receptor-mediated interactions between the epithelial cells and the ECM, or matrikine gradients, the latter having been recently implicated in epithelial remodeling in asthma (Patel et al, 2018). This general idea is consistent with the observation that PDGFRa + lung fibroblasts decline in number during septal thinning (McGowan & McCoy, 2011) and is in-line with current thinking that epithelial-mesenchymal interactions drive lung development (Hogan et al, 2014).
To date, pivotal roles for microRNA processing by Dicer (Harris et al, 2006) and Argonaute (Lü et al, 2005) in lung branching suggested microRNA control of early (embryonic) lung development (Metzger et al, 2008), where functional roles for the miR-17 family have been demonstrated (Carraro et al, 2014). In contrast, in late ◀ Figure 5. Antagonizing miR-34a function partially restores proper lung alveolarization.
A Schematic illustration of the antimiR administration protocol. B Quantitative RT-PCR detection of miR-34a-5p levels in wild-type mouse pups at post-natal day (P)14 that had been treated either with a scrambled antimiR (S), or antimiR-34a (A34a), during normal (21% O 2 ) and aberrant (85% O 2 ) alveolarization (n = 4 animals for each group). C Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (scale bar, 50 lm). Data are representative of three or more experiments. D Quantification of total number of alveoli by design-based stereology in wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant lung alveolarization (n = 5 animals for each group). E Quantification of mean septal thickness by design-based stereology in wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group). F Quantitative analysis of PDGFRa + cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group). G Quantitative analysis of PDGFRa + /aSMA + cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group). H Schematic illustration of the role and translational scope of the miR-34a/Pdgfra interaction during arrested lung alveolarization. Hyperoxia drives miR-34a expression in myofibroblasts, downregulating PDGFRa expression and reducing PDGFRa + cell abundance, causing the perturbed elastin fiber production and blunted alveolarization seen in bronchopulmonary dysplasia (BPD). The effects of hyperoxia are attenuated when miR-34a function is blocked with an antimiR (A34a) or when the miR-34a/Pdgfra interaction is disturbed with a target site blocker (TSB).  A Mice expressing nuclear-localized GFP under the control of the Pdgfra promoter were maintained under normoxic (21% O 2 ) or hyperoxic (85% O 2 ) conditions, and lungs were harvested, processed, and immunostained for Ki67 to determine proliferation status. DAPI staining revealed nuclei of all cells present in the section. Lowmagnification images from individual channels are presented to the right of the merged (large) image first row of images. The area demarcated by the white box in the merge image of the first row is magnified in the second and third rows to allow for visualization of greater magnification of the demarcated region of the merged image, as well as visualization of a single Ki67 + , GFP + cell (white arrowhead) in all three channels separately. Scale bar: 100 lm. B The number of PDGFRa + cells in four microscopic fields was assessed for co-staining with an anti-Ki67 antibody to reveal proliferating cells. P values were calculated by unpaired Student's t-test (n = 4 fields for each group, trends are representative of those observed in two other experiments). Data represent mean AE SD. C The Ki67 staining and GFP fluorescence was controlled for by examining lungs from wild-type mice that were treated with an isotype-matched control IgG used for the Ki67 staining experiments. Sections were examined for GFP fluorescence as well as in the red channel used to detect the Ki67 staining. Scale bar: 100 lm. DAPI, 4 0 ,6-diamidino-2-phenylindole; GFP, green fluorescent protein.  The impact of administration of scrambled antimiR (S) or an antimiR directed against miR-34a (A34a) on the abundance of type I alveolar epithelial cells (marked by aquaporin 5, Aqp5) and type II alveolar epithelial cells (marked by pro-surfactant protein C, Sftpc) was assessed in 3-lm sections of paraffin-embedded lung tissue from P5 mice undergoing normal (21% O 2 ) or aberrant (85% O 2 ) lung alveolarization. DAPI, 4 0 ,6-diamidino-2-phenylindole. In the DAPI images, white lines delineate tissue from airspaces, and in the 85% O 2 groups demarcate septa. Antibody specificity was validated by rabbit IgG isotype control primary antibodies. The control experiments for the Aqp5 and Sftpc staining runs are illustrated here. Scale bars, 50 lm.
ª 2019 The Authors EMBO Molecular Medicine 11: e9448 | 2019 model of human disease. Notably, translation of these findings into the use of antimiR treatment of mice in the BPD model documented marked benefit in this preclinical model. We, therefore, highlight a potentially druggable pathway to manage arrested alveolarization following preterm birth.

Regulatory authority compliance and legal approvals
Animal experiments reported in this study were approved by the Regierungspräsidium Darmstadt, under approval numbers B2/277, B2/1002, and B2/1060.

Mice
Wild-type Mus musculus C57Bl/6J mice were obtained from The Jackson Laboratory. The generation and characterization of a tamoxifeninducible Pdgfra-Cre driver mouse strain [Tg(Pdgfra-cre/ERT2)1Wdr; MGI:3832569] referred to herein as Pdgfra-CreER T2 , on a C57BL/6J background has been described previously (Rivers et al, 2008;Ntokou et al, 2015). A lacZ-tagged miR-34a gene-trap strain (Mir34a tm1.1Lhe ; MGI:5308792) (Choi et al, 2011), herein referred to as miR-34a::lacZ or miR-34a À/À (on a C57BL/6J background) was always employed in the homozygous state and was used interchangeably as a miR-34a global knockout and a miR-34 lacZ reporter, was obtained from the Jackson Laboratory. A miR-34bc global knockout strain (Mir-c21 tm1.1Lhe ; MGI:5308794) (Concepcion et al, 2012) on a C57BL/6J background was always employed in a homozygous state, is referred to herein as miR-34bc À/À , and was obtained from the Jackson Laboratory. A mouse strain expressing a human histone 2B-enhanced green fluorescent protein fusion protein under the control of the Pdgfra promoter (B6.129S4-Pdgfra tm11(EGFP)Sor /J; MGI:3766768) (Hamilton et al, 2003) was obtained from the Jackson Laboratory, and was always employed in the heterozygous state, and allowed the detection of Pdgfra-expressing cells through nuclear-localized GFP fluorescence. A strain carrying a floxed miR-34a allele (Mir34a tm1.2Aven ; MGI: 5320795) (Concepcion et al, 2012) on a C57BL/6J background was always employed in the homozygous state, is referred to herein as miR-34a fl/fl , and was obtained from the Jackson laboratory. A conditional, inducible deletion-ready strain, where administration of tamoxifen can abrogate miR-34a expression exclusively in Pdgfra-expressing cells (denoted miR-34a iDPC/iDPC ), was created by crossing the Pdgfra-CreER T2 driver strain with a miR-34a fl/fl strain. This strain was always employed on a C57BL/6J background with the Pdgfra-CreER T2 allele in the heterozygous state, and the floxed miR-34a allele in the homozygous state. Studies employing the miR-34a iDPC/iDPC strain were controlled for with a strain heterozygous for Pdgfra-CreER T2 but carrying two wild-type miR-34a alleles. For induction of tamoxifen-responsive genes, a protocol has been developed and validated that allows for the tamoxifen treatment of newborn pups under hyperoxic conditions, where tamoxifen is poorly tolerated (Ruiz-Camp et al, 2017): Newborn pups received a single intraperitoneal injection on the day of birth [post-natal day(P)1)] of 0.2 mg tamoxifen/pup in 10 ll Miglyol 812. All mice were maintained on a 1320 formula maintenance diet for rats and mice (Altromin), available ad libitum together with drinking water, with as 12 h:12 h day/night cycle.

The hyperoxia-based mouse model of bronchopulmonary dysplasia
Bronchopulmonary dysplasia was modeled in mice in a protocol well established in our laboratory (Nardiello et al, 2017b) where newborn mouse pups, randomized to litters of equal numbers of pups per nursing dam, are exposed to 85% O 2 from P1 to P14, while control mouse pups with normal lung development are exposed in parallel to 21% O 2 . In the case of tamoxifen-induced gene expression where mice received tamoxifen on P1, hyperoxia exposure was initiated on P2. Both male and female animals were used, since no sex bias has been noted in studies on perturbations to lung development of C57Bl/6J mice in response to hyperoxia (Nardiello et al, 2017b). Nursing dams were rotated between normoxia and hyperoxia at 24-h intervals, to limit oxygen toxicity. At either P3 (prior to bulk lung alveolarization), at P5 (the peak period of bulk lung alveolarization), or at P14 (after completing of the bulk alveolarization phase), mice were killed by pentobarbital overdose (500 mg/kg, intraperitoneal) and lungs were removed en bloc for further analysis. The investigators were not blinded to group allocation, but were blinded to outcome assessment.

Microarray analyses
For an unbiased analysis of microRNA expression over the course of normal and aberrant lung development, microRNA was isolated with a miRNeasy Mini kit (Qiagen), and microRNA expression was assessed using an Agilent-035430 mouse miRNA array platform (miRBase release 17 miRNA ID version; Mouse_8x60K-v17). For an unbiased analysis of mRNA expression over the course of normal and aberrant lung development after antimiR administration, mRNA was isolated with a peqGOLD total RNA kit (Peqlab), and mRNA expression was assessed using an Agilent-028005 SurePrint G3 Mouse GE 8 × 60K Microarray platform. Microarray analyses were undertaken by IMGM Laboratories (Munich).

Stereological analysis of lung structure
Lung structure was assessed by design-based stereology with systemic uniform random sampling, on mouse lungs that were pressure fixed at 20 cm H 2 O, and treated with arsenic, osmium and uranium, and embedded in plastic (Technovit 7100) resin, sectioned at 2 lm, stained with Richardson's stain, and image captured in a Nanozoomer-XR C12000 (Hamamatsu), exactly as described previously (Madurga et al, 2014;Mi zíková et al, 2015;Nardiello et al, 2017b). Lung volume was determined by the Cavalieri principle (Madurga et al, 2014). Stereological analyses were undertaken using the NewCast PLUS version VIS4.5.3. computer-assisted stereology system (Visiopharm) and facilitated the determination of inter alia total number of alveoli in the lung, the mean septal thickness, and total gas-exchange surface area.
In situ b-galactosidase activity detection Cryosections (

Cell isolation and cell culture
Primary mouse lung fibroblasts were isolated from C57Bl/6J mice. Briefly, lungs were instilled with approximately 500 ll of preheated (37°C) collagenase type I (2 mg/ml; Sigma-Aldrich), and subsequently excised en bloc from adult female C57BL/6J mice that were killed by isoflurane inhalation. Lungs were placed in 50-ml Falcon tubes containing 25 ml of preheated (37°C) collagenase type I and incubated on a Unimax1010 orbital rotator with gentle agitation at 70 r.p.m, for 1 h at 37°C. Lungs were minced with sterile scissors, and the tissue suspension was dispersed by repeated gentle passage through a 20G syringe needle. The cell suspension was then passed through a 40-lm filter into a new 50-ml Falcon tube. The cell suspension was centrifuged at 120 × g for 8 min at 4°C. The supernatant was discarded, and the cell pellet was resuspended in 5 ml of pre-warmed (37°C) high-glucose DMEM containing 10% (v/v) FCS, 100 U/ml penicillin (ThermoFisher), 100 lg/ml streptomycin (ThermoFisher), and seeded into a T-75 cell culture flask (1 flask per lung) and passaged in low-glucose DMEM containing 10% (v/v) FCS, 100 U/ml penicillin (ThermoFisher), 100 lg/ml streptomycin (ThermoFisher). Primary mouse lung fibroblasts were employed throughout this study, with the exception of in vitro hyperoxia exposure and when Lipofectamine â 2000 was used as transfection reagent. In the latter two cases, the MLg mouse lung fibroblast cell line (ATCC â CCL-206 TM ) was employed, and was obtained from the American Type Culture Collection, and maintained in EMEM supplemented with 10% (v/v) FBS. Cultures of primary cells and cell lines were routinely (monthly) screened for mycoplasma contamination.

MicroRNA mimic, antimiR, and target site blocker interventions in vitro and in vivo
A synthetic scrambled miR mimic and a miR-34a mimic (catalog numbers SI03650318 and MSY0000542, respectively; Qiagen) were transfected into primary mouse lung fibroblasts with HiPerFect (Qiagen) or MLg cells with Lipofectamine â 2000 or 3000, as per manufacturer's instructions. Locked nucleic acid (LNA) oligonucleotides (purchased from Exiqon) included a scrambled (inert) sequence (5 0 -ACGTCTATACGCCCA-3 0 ); an antimiR directed against miR-34a (5 0 -AGCTAAGACACTGCC-3 0 ) and miRCURY LNA TM microRNA Target Site Blockers (herein referred to as target site blockers) directed to target the interaction between the two miR-34a binding sites in the mouse Pdgfra 3 0 -UTR and miR-34a: 5 0 -TTGGCAGTATTCTCCA-3 0 (TSB1) and 5 0 -AGGCAGTGATACAGCT-3 0 (TSB2) (see Fig 3A). In vitro, synthetic oligonucleotides were transfected into MLg cells with Lipofectamine â 2000. When combined, synthetic microRNA mimics and LNA target site blockers were applied together as a cocktail, at a final cumulative concentration of 160 nM (Fig 4B). In vivo, both target site blockers (applied as a cocktail of a 1:1 mixture of TSB1 and TSB2) and a scrambled or miR-34a-specific antimiR were all applied by intraperitoneal injection at a dose of 10 mg/kg at P1 and P3, in ddH 2 O.

Flow cytometry and FACS
All flow cytometry protocols and gating strategies are indicated in the relevant supplementary figures in the Appendix. Antibody conjugates, dilutions, and commercial sources are detailed in Appendix Table S9. Flow cytometry was performed to estimate apoptosis (by annexin V staining) and to quantify cell populations in developing mouse lungs, using the antibodies listed in Appendix Table S9. Flow cytometry and FACS were performed with an LSR Fortressa or an FACSAria III cell sorter, respectively, operated with DIVA software (BD Bioscience). Single-cell suspensions were prepared from mouse pup lungs by instilling approximately 300 ll of Dispase (50 U/ml; BD Bioscience) followed by incubation for 30 min at 37°C. Lungs were dissociated in a gentleMACS TM Dissociator (Miltenyi) in 5 ml (per lung) DMEM supplemented with 10% (v/v) FCS, 100 U/ml penicillin (ThermoFisher), 100 lg/ml streptomycin (ThermoFisher), and 320 U/ml bovine pancreatic DNAse 1 (Serva). To remove cell debris and blood clots, whole-lung cell suspensions were filtered through 100 lm and 40-lm filters. After centrifugation at 266 × g for 10 min at 4°C, cell pellets were resuspended in Flow Cytometry Staining Buffer (eBioscience; 00-4222-26), blocked with 1:100 Mouse BD Fc Block TM (BD Biosciences), and incubated with the appropriate primary antibodies or isotype controls diluted in Flow Cytometry Staining Buffer for 20 min at 4°C in the dark. After washing with Flow Cytometry Staining Buffer, whole-lung cell suspensions were incubated with secondary antibodies diluted in Flow Cytometry Staining Buffer for 20 min at 4°C in the dark. In the case of intracellular staining for myofibroblasts, cells were fixed in 0.15% (m/v) paraformaldehyde in PBS for 10 min at 4°C and permeabilized with 0.2% (m/v) saponin (Calbiochem) diluted in PBS for 15 min at 4°C prior to addition of antibodies. For assessment of epithelial cell apoptosis, stainings were carried out on fresh non-permeabilized cells, where cells were washed and resuspended in annexin V buffer (BD Biosciences) prior to annexin V-Alexa Fluor 647 conjugate (Thermo-Fisher A23204; 1:100) incubation in annexin V buffer for 20 min at 4°C in the dark. Where a fluorophore-conjugated secondary antibody was not employed, cells that had been labeled with an unconjugated primary antibody were treated with a biotin-conjugated secondary antibody, followed by a Streptavidin-phycoerythrin conjugate (Biolegend 405204; 1:300). For S-phase analysis, live-cell determinations were made by incubation of cell suspensions Hoechst 33342 (Sigma B2261, 5 lg/ml) in PBS for 45 min at 37°C in the dark. For assessment of PDGFRa + cell apoptosis, live cell single-cell suspensions were prepared as described above, up to and including the step employing Mouse BD Fc Block TM , after apoptosis was detected in cell suspensions with an Annexin V kit (Biolegend,640906). For isolation of PDGFRa + cells by cell sorting, the anti-PDGFRa-APC conjugate was coupled to microbeads using 30 ll of anti-APC MicroBeads  for 20 min at 4°C in the dark, and separated in a AutoMacs separator (Miltenyi) prior to cell sorting. The RNAqueous-Micro kit (Thermo Fisher) was employed to isolate mRNA from PDGFRa + cells, which are present in low number. To exclude dead cells and debris, 1 ll of 5 mg/ml DAPI or 5 ll of 50 lg/ml 7-ADD (Biolegend) was pipetted into the wholelung cell suspensions just before the cell analysis.
For the assessment of cell proliferation by immunofluorescence, cryosections were prepared from P5 mouse lungs, where lungs were exposed by midline thoracotomy, perfused transcardially with 1× PBS, and inflated with 1:1 PBS: Tissue-Tek â O.C.T. (Sakura, 4583), removed en bloc from the thorax, and frozen at À20°C. Frozen tissue was sectioned at 10 lm with a cryostat; sections were mounted on glass slides and stored at À20°C before fixation. Frozen sections were fixed with cold (À20°C) 4% (m/v) paraformaldehyde (15 min, room temperature) and blocked with normal goat serum diluted 1:1 with 3% (m/v) BSA dissolved in 1× PBS containing 0.3% (v/v) Triton X-100 (1 h at room temperature). Sections were permeabilized with 1% (m/v) saponin in 1× PBS (20 min at room temperature). Ki67 was detected using an anti-Ki67 primary antibody (Appendix Table S9), and an Alexa Fluor 647-conjugated goat anti-rat (Invitrogen, A21247; 1:500) secondary antibody followed by incubation in 4 0 ,6-diamidino-2-phenylindole (DAPI; 1:1,000 dilution of a 1 mg/ml stock solution in PBS). Z-stack images of the sections were acquired using a Zeiss LSM710 Laser Scanning Confocal Microscope. For enumeration of Ki67 + cells, the number of Ki67 + cells and GFP + cells was assessed in a total of 500 DAPI + cells, per microscopic field; and four fields were assessed per experimental condition.

Assessment of apoptosis and cell proliferation in vitro
Primary mouse lung fibroblasts were seeded at 4,000 cells (in 100 ll) per well of a 96-well tissue culture plate (Greiner, 655180, for proliferation; Greiner, 655098, for apoptosis), incubated overnight, and starved in serum-free OptimMEM (Gibco, 31985-062) for 1 h.
For assessment of proliferation, cells were transfected either with a scrambled microRNA mimic or a miR-34a-5p mimic (80 nM final concentration, as described above), and proliferation was monitored by BrdU incorporation using a colorimetric Cell Proliferation ELISA kit (Roche, 11647229001) after a 1-h serum starvation period in DMEM GlutaMAX (Gibco, 21885-025), followed by 24 h in DMEM GlutaMAX supplemented with 10% (v/v) FCS and 1% Penicillin-Streptomycin solution. Signal was allowed to develop over 5-30 min, as was read in an Infinite M200 Pro spectrophotometer (Tecan).
For assessment of apoptosis, cells were transfected with a scrambled microRNA mimic or a miR-34a-5p mimic as described for proliferation, above, and caspase 3 and caspase 7 activity was detected as a surrogate for apoptosis, using a Caspase-Glo â 3/7 Assay System (Promega, G8091) after 24 h. For a positive control, medium was supplemented with staurosporine (Cayman Chemical, 62996-74-1; 0.5 lM) for the last 6 h of the 24-h period. Luminescence was determined for 60 min, in an Infinite M200 Pro luminometer (Tecan).

Power and statistical analyses
A prospective power analysis was undertaken for all animal studies, to assess the sample size required. Samples sizes were calculated using G*Power 3.1.9.2 (Faul et al, 2007). For changes in microRNA and mRNA expression assessed by real-time RT-PCR in mouse lung homogenates, a DCt of |0.5| was considered relevant, resulting in an effect size of d = 2.70 (using miR-34a-5p expression as reference values), where d is Cohen's effect size, and using a = 0.5 (where a is the Type I error), and a power (1-b) of 0.8, where b is the type II error; required a sample size of n = 4 animals per group. For cells sorted by FACS from mouse lungs and processed for microRNA or mRNA analyses, where a pronounced change in gene expression was anticipated, a DCt of |1.0| was considered relevant, resulting in an effect size of d = 2.79 (using miR-34a-5p levels in FACS-sorted PDGFRa + cells as reference values), and using a = 0.5 and a power (1-b) of 0.8, required a sample size of n = 4 animals per group. For changes in rare cell populations in single-cell suspensions from whole lungs of mice, assessed by flow cytometry, a doubling of the cell population (100% increase) was considered relevant, resulting in an effect size of d = 2.88 (using PDGFRa + cell abundance in whole-lung suspensions as reference values), and using a = 0.5 and a power (1-b) of 0.8, required a sample size of n = 4 animals per group. Assessment of lung structure included two parameters (total number of alveoli in the lung and mean septal thickness), both assessed by design-based stereology in the same lungs from the same animals. For assessment of total number of alveoli, a 50% increase in the total number of alveoli was considered relevant, resulting in an effect size of d = 4.13 (using the hyperoxia-treated wild-type mouse lungs as reference values), and using a = 0.5 and a power (1-b) of 0.8, required a sample size of n = 3 animals per group. For assessment of mean septal thickness, an increase of 2 lm in mean septal thickness was considered relevant, resulting in an effect size of d = 11.11 (using the hyperoxia-treated wild-type mouse lungs as reference values), and using a = 0.5 and a power (1-b) of 0.8, required a sample size of n = 2 animals per group. Since both the total number of alveoli in the lung and the mean septal thickness are measured in the same animals, a sample size of n = 3 animals per group was required, which was extended to four animals per group, in the event of an outlier arising from technical issues related to tissue processing during embedding for stereological analysis.
Data are presented as mean AE SD. Differences between groups were evaluated by one-way ANOVA with Tukey's post hoc test for multiple (more than two) comparisons, while two-group comparisons were performed with an unpaired Student's t-test. All statistical analyses were performed with GraphPad Prism 6.0. For microarray studies, a Welch's approximate t-test was used to determine P values which were corrected using the algorithm of Benjamini and Hochberg, to generate the corrected P-value, P(corr) (Benjamini & Hochberg, 1995). The presence of statistical outliers was tested by Grubbs' test, and no outliers were found. In general, data sets were too small to test normal distribution, and normality was assumed.

Data Availability
Microarray data comparing microRNA steady-state levels in lungs of mouse pups exposed to 21% O 2 versus 85% O 2 are available at the GEO database under accession number GSE89666 (https://www. ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE89666).
Expanded View for this article is available online. The paper explained Problem Bronchopulmonary dysplasia (BPD) is a common and severe complication of preterm birth, where the lungs of preterm born infants do not properly develop. Notably, the formation of the alveoli-the principal gas-exchange units of the lung-is stunted, which has important consequences for the long-term respiratory health of BPD survivors. While oxygen support of infants with acute respiratory failure causes BPD, the disease mechanisms that underlie the stunted lung development are unknown.

Results
Our report identifies the interaction between microRNA-34a and the mRNA encoding platelet-derived growth factor receptor (PDGFR)a as a disease-relevant interaction in stunted lung developed associated with BPD that was experimentally modeled in mice. Our report also documents that this interaction is "druggable", and can be manipulated in vivo to protect the development of alveoli from the damaging effects of oxygen support.

Impact
Our report highlights a new pathological pathway that can also be pharmacologically targeted to attenuate experimental disease pathology. Targeting this specific microRNA-34a/mRNA interaction may form the basis of a new approach to the medical management of BPD.