Mice
Wildtype mice (males and females, CD1 background) were crossed to generate time-pregnant females. All mice received food and water ad libitum.
Hyperoxia injury (BPD mouse model)
Newborn pups were exposed to either normoxia (NOX) or hyperoxia (HOX) within 24 h after birth (P0) (Fig. 1). In experimental group 1, dams and pups were kept in NOX from P0 to P16. In experimental group 2, dams and pups were kept in NOX from P0 to P15. From P15 to P16, the dams and the pups were exposed to HOX (85% O2). In experimental group 3, newborn pups were subjected to HOX (85% O2) injury from P0 to P8 in a chamber (Proox Model 110, Biospherix). To minimize oxygen toxicity and bias, nursing dams were rotated every 24 h between NOX and HOX. Afterwards, nursing dams and pups were exposed to NOX (21% O2) from P8 to P16. From P15 to P16, the dams and the pups were re-exposed to HOX (85% O2). In experimental groups 1 and 3, lungs were harvested at P8. In all groups, lungs were harvested at P16. All dams and pups received food and water ad libitum.
Lung perfusion, isolation, tissue processing, and histology
Mice were euthanized by intraperitoneal injection of a solution made of ketamin, dormitor, heparin, and saline. After sternotomy, the lung was perfused transcardially (through the right ventricle) by using PBS (1×), then isolated and incubated for 30 min at 4 °C in COLD medium, stored at − 20 °C overnight, and finally stored at − 80 °C till further tissue processing.
For histology, the lung was flushed from the right ventricle to remove blood cells then perfused through the trachea with a pressure of 20 cm H2O with 5 ml 4% PFA. The trachea was tied off with a string, and the lung was removed and placed in 4% PFA for max. 24 h at 4 °C. Lungs were then progressively dehydrated (30, 50, 70, and 99% ethanol, 3 h each), incubated in xylole, then in paraffine overnight and finally embedded with a Leica embedding machine (EG 1150C). Paraffin blocks were kept cold, and 5 μm sections were cutted. Hematoxylin and eosin staining was performed according to protocols previously published.
Positive selection for vascular endothelial cells
Positive selection for vascular endothelial cells was performed by magnetic separation with human CD146 Microbeads (Miltenyi®, Bergisch-Gladbach, Germany) following manufacturers’ instructions with minor modifications as described previously [17].
RNA isolation
Total RNA was isolated using Trizol (Sigma-Aldrich) and isopropanol precipitation and further purification on columns. Next, RNA integrity was assayed using the Agilent Bioanalyzer 2000. Only samples with non-fragmented RNA were included.
Gene expression analyses
Gene expression analyses were performed using quantitative real-time PCR (qPCR) analyses on a Rotor-Gene 6000 (Qiagen). For PCR reactions, QuantiTect SYBR Green qPCR Master Mix (Qiagen) containing Hot Start Taq DNA polymerase and SYBR Green was used. Primers were used as described earlier [18]. The expression of gene of interest was normalized using three housekeeping genes (BACT, GAPDH, PECAM1). PCR conditions were as follows: 95 °C for 15 min, 40× [95 °C for 15 s, 60 °C for 30 s]. Melting of PCR product was done using a gradient from 55 to 95 °C rising in 0.5 °C increments.
Antibodies
Primary antibodies used in this study were (1) Rabbit anti-H2A.Zac (Diagenode pAb-173-050), (2) Rabbit anti-H3K9ac (Active Motif pAb#39137), and (3) Rabbit anti-H3K4me3 (Diagenode pAbCSP-030-050).
Chromatin purification and ChIP assays
Chromation purification and ChIP assays were performed as described previously [15]. Quantitative PCR analyses were performed using a Rotorgene 6000 (Qiagen). The relative amounts of specifically immunoprecipitated DNA were estimated as “percent of input” and quantified using individual standard curves for each amplicon. Primer pairs were used as described earlier [18].
Pyrosequencing
DNA methylation signatures of promoter segments of NANOG, NFE2L2, and STAT3 were analyzed using a Qiagen Pyromark Q24 sequencer as previously described (Plos One Jenke et al.). Briefly, after standard sodium bisulfite conversion using the EZ DNA Methylation-Gold Kit (Zymo Research, USA) pyrosequencing methylation analysis was conducted using the PyroMark Q24 (Qiagen, Germany) according to the manufacturer’s protocol. Therefore, we designed and made use of the following oligonucleotides: 1. for NFE2L2—primer F1 (5′- gga gtt aga ggg gat agt ggt t-3′), 5′-biotinylated primer (5′-acc cca cca aat caa aac ttc ct-3′), and sequencing primer S1 (5′-agg gta aag gag gat g-3′); 2. for STAT3—primer F1 (5′-ggt gta ggg tgg ggt tat t-3′), 5′-biotinylated primer (5′-acc cta tat atc tcc tcc tat cct-3′), and sequencing primer S1 (5′-ggg tgg ggt tat ttt t-3′); 3. for NANOG (DNA methylation positive control)—primer F1 (5′-gta gga tag gaa tgg ggg ttg-3′), 5′-biotinylated primer (5′-acc tta aat tta ccc caa att cta c-3′), and sequencing primer S1 (5′-aat ggg ggt tgg gga-3′). No reliable NOS3 DNA methylation assays meeting our quality standards could be designed. The level of methylation was analyzed using PyroMark Q24 2.0.6 Software (Qiagen). Non-CpG cytosine residues and a standard fully methylated DNA (Zymo Research, USA) were used as controls to verify bisulfite conversion.
Small RNA-seq and analyses pipeline
Total RNA was purified as described above. For multiplexing, we made use of different multiplex sequencing barcodes for sequencing in a single lane as described [19]. Briefly, total RNA was separated by polyacrylamide gel electrophoresis. Gel fragments corresponding to 15 to 35 nt RNA molecules were cut, and RNA was eluted. These small RNAs were directly used for the construction of sequencing libraries in four steps: step 1: ligation of DNA oligos to the 3′-end of the RNA; step 2: ligation of RNA or, respectively, chimeric RNA/DNA oligos to the 5′-end of RNAs; step 3: cDNA library synthesis by reverse transcriptase; and step 4: amplification of the cDNA library. Subsequently, after final quality checks by microcapillary electrophoresis and qPCR, the libraries were sequenced on an Illumina Hiseq 2000 platform (single end, 50 bp). This work has benefited from the facilities and expertise of the high-throughput sequencing core facility of IMAGIF Gif-sur-Yvette (Centre de Recherche de Gif—www.imagif.cnrs.fr). The initial data analysis pipeline was as follows: CASAVA-1.8.2 was used for demultiplexing, Fastqc 0.10.1 for read quality assessment and Cutadapt-1.3 for adaptor trimming, resulting in an average sequence number for each developmental time point sample of 7.58 Mbp. File conversions, filtering, and sorting as well as mapping (Bowtie2) were done using “Galaxy” [20,21,22], a platform for data intensive biomedical research (https://usegalaxy.org/), and “Chimira” [23], respectively.
Immunofluorescence staining for eNOS
Paraffin sections were deparaffinized. Antigen retrieval was performed for 15 min in citrate buffer using a rice cooker, then slides were cooled down on ice for 20 min. After washing slides three times in TBST (TBS buffer + 0.1% Tween 20) for 5 min, slides were blocked with 3% bovine serum albumin (BSA) and 0.4% Triton X-100 [in Tris-buffered saline (TBS)] at room temperature (RT) for 1 h and then incubated with primary antibody against eNOS (ThermoFisher Scientific, PA1-037; 1:100) at 4 °C overnight. Afterwards, slides were again washed three times in TBST for 5 min, incubated with secondary antibody (Goat Anti-Rabbit Alexa 555, ThermoFisher Scientific, A-21429; 1:500) in room temperature for 1 h, and then washed three times in TBST before being mounted with Prolong Gold Anti-fade Reagent with DAPI (4,6-diamidino-2-phenylindole; ThermoFisher Scientific, P36931). Fluorescent images were acquired using Leica DM5500 B fluorescence microscope connected to Leica DFC360 FX camera.
Statistical analysis
Data were compared using Mann-Whitney U test according to normality assumptions on univariate analysis followed by Bonferroni correction for multivariate analysis. Categorical variables were compared using the Fisher exact test. Statistical analyses were performed with GraphPad Prism 5.0.
Cell culture
Human arterial endothelial cells (HAEC) were bought from PromoCell (Heidelberg, Germany) and cultivated upon manufacturer’s recommendations. For cDNA synthesis, we used 500 ng RNA per sample using the QuantiTect Reverse Transcription kit (Qiagen). DNA was isolated by phenol:chloroform:isoamylic alcohol extraction followed by precipitation with isopropanol. For in vitro experiments, HAEC (PromoCell) were cultivated upon manufacturer’s recommendations. For HDAC inhibition, HAEC were treated with 1 μM trichostatin A (TSA) for either 72 h or twice for 6 h with an 60-h interval in between. Hypoxia experiments were performed using a hypoxia incubator chamber (STEMCELL Technologies, Grenoble, France) exposing cells to a ppO2 of 0.12 bar corresponding to an oxygen fraction of 12% for 24 h. Control experiments were performed under normoxic conditions (ppO2 = 0.21 bar).