Abstract

   Bronchopulmonary dysplasia (BPD) is characterized by abnormal
   development of the blood vessels and alveoli in lungs, which largely
   occurs in premature infants. Exosomes (EXO) from very preterm infants
   (VPI) with BPD (BPD-EXO) impair angiogenic activities of human
   umbilical vein endothelial cells (HUVECs) via EXO-miRNAs cargo. This
   study aimed to determine whether and how BPD-EXO affect the development
   of BPD in a mouse model. We showed that treating BPD mice with BPD-EXO
   chronically and irreversibly aggravated lung injury. BPD-EXO
   up-regulated 139 and down-regulated 735 genes in the mouse lung tissue.
   These differentially expressed genes were enriched to the MAPK pathway
   (e.g., Fgf9 and Cacna2d3), which is critical to angiogenesis and
   vascular remodeling. BPD-EXO suppressed expression of Fgf9 and Cacna2d3
   in HUVECs and inhibited migration, tube formation, and increased cell
   apoptosis in HUVECs. These data demonstrate that BPD-EXO aggravate lung
   injury in BPD mice and impair lung angiogenesis, plausibly leading to
   adverse outcomes of VPI with BPD. These data also suggest that BPD-EXO
   could serve as promising targets for predicting and treating BPD.

   Subject terms: Molecular biology, Respiratory tract diseases

Introduction

   Nearly 15 million babies are born preterm before 37 weeks of gestation
   annually worldwide, which is a major global healthcare problem^[46]1.
   Among preterm birth, 10% with gestational age of less than 32 weeks are
   defined as very preterm infants (VPI) who are at greater risk of short-
   and long-term health problems.

   To date, the improved perinatal care has greatly increased the survival
   rate of VPI^[47]2. However, approximately 40% of VPI still develop
   bronchopulmonary dysplasia (BPD), placing a huge financial burden on
   families and healthcare as children require medical
   intervention^[48]3,[49]4. BPD can be resulted from either prenatal
   injury or repeated postnatal injuries in the developing lung. As a
   result, lung development is significantly defective, leading to
   persistent airway and pulmonary vascular dysfunction^[50]5. Despite the
   application of various means of respiratory care, such as mechanical
   ventilation and corticosteroids, BPD remains the most common and
   serious complication in VPI^[51]5. Thus, understanding the mechanisms
   underlying BPD and lung repairing will help us further improve the
   outcome of premature infants.

   The main pathological phenotype of BPD includes arrested
   alveolarization and microvascular growth^[52]5,[53]6. Increasing
   evidence supports that supplemental oxygen alone actually can cause BPD
   in animal models^[54]7. The disordered expression of angiogenic factors
   and their receptors is also associated with BPD-impaired function of
   pulmonary microvascular. Vascular endothelial growth factor-A (VEGFA)
   is one of several angiogenic factors that play a central role in the
   development and function of arteries and veins^[55]8,[56]9.
   Furthermore, the disruption of VEGFA signaling contributes to the
   pathogenesis of BPD by impairing lung vascular growth. Recently, it has
   been reported that mesenchymal stromal cells (MSC)-derived exosomes can
   regulate VEGFA and angiogenesis as a potential drug for BPD treatment,
   but lung fibroblast-derived exosomes did not show any benefit in
   hyperoxia-induced mice^[57]10,[58]11. This suggests that exosomes from
   different sources may have different effects on BPD.

   Exosomes (EXO) are small extracellular vesicles with a diameter ranging
   from 30 to 120 nm^[59]12,[60]13. Exosomes from MSC can strengthen
   endothelial cell tube formation in vitro, and vascularization and
   alveolarization in vivo^[61]14. Exosomes from endothelial progenitor
   cells can stimulate angiogenesis of pulmonary microvascular endothelial
   cells^[62]15. Exosomes from lung spheroid cells can also promote lung
   repair in pulmonary fibrosis^[63]16. An exosome-associated gene,
   fibroblast growth factor 9 (FGF9), could regulate both capillary plexus
   formation and VEGFA expression, which is required for early lung distal
   vascular development^[64]17–[65]19. We have recently reported that
   umbilical cord blood-derived EXO from VPI with BPD inhibit angiogenesis
   in vitro by miRNAs (e.g., miR-185-5p)^[66]20. However, it remains
   elusive whether BPD-EXO suppress pulmonary angiogenesis or development
   in vivo.

   Here, we verified the effect of BPD-EXO in a mouse model of
   hyperoxia-induced BPD and validated our hypothesis that BPD-EXO could
   aggravate hyperoxia-induced lung injury. At the same time, we used
   RNA-seq to find that the differentially expressed genes in the lung
   tissue of BPD mice treated with BPD-EXO were related to the MAPK
   signalling pathway, and verified that it could affect angiogenesis in
   vitro These findings are more helpful to our comprehensive
   understanding of BPD-EXO and better discovery of targets for the
   treatment of BPD.

Methods

   All experimental procedures were designed and performed following
   standard guidelines of the Third Affiliated Hospital of Guangzhou
   Medical University and the ARRIVE guidelines.

Exosome isolation and identification

   The recruitment criteria for patients with very preterm delivery
   (28–31^+6 weeks) were previously described^[67]20. All procedures were
   conducted in accordance with the Declaration of Helsinki. The umbilical
   cord blood collection protocol was approved by the Institutional Review
   Board of the Third Affiliated Hospital of Guangzhou Medical University
   (Protocol # 2021–014). All subjects gave written, informed consent
   before participating. Clinical diagnosis of healthy pregnancy was made
   by the obstetricians at the birth center (, the Third Affiliated
   Hospital of Guangzhou Medical University, where the umbilical cords
   were collected. Exosomes were obtained by ultracentrifugation of serum
   from extremely premature infants (2 ml/infant) as we described
   previously^[68]20.

   Exosome suspension (5 μl/infant mixed with 5 μl H[2]O) was dropped to
   the copper mesh for 1 min and stained with 10 μl phosphotungstic acid
   solution for 1 min. Morphology of exosomes was observed by a
   transmission electron microscope (JEOL-JEM1400, JEOL Ltd., United
   States) at an acceleration voltage of 80 kV. Exosome suspension
   (5 μl/infant mixed with 25 μl H[2]O) was used to determine the size
   distribution of exosomes by nanoparticle tracking analysis (NTA) using
   a Particle Metrix ZetaView instrument (Particle Metrix GmbH, Germany)
   with laser and video camera module (Particle Metrix GmbH, Germany).
   Exosome surface marker proteins TSG101 and Alix were determined by
   western blot.

Western blot analysis

   Exosomes were lysed with RIPA buffer (Thermo Scientific) following the
   manufacturer's instructions. Quantity was determined with the BCA kit
   (Thermo Scientific). Equal amounts of proteins were subjected to
   Western blotting analysis as described previously^[69]18. The proteins
   on membranes were probed using antibody against Alix (CST, #2171,
   1:1000) or TSG101 (Abcam, #ab125011, 1:5000). The protein signal was
   detected using Chemiluminescent ECL reagent (Forevergen, China).

Construction of a mouse model of bronchopulmonary dysplasia

   Pregnant (8-week-old) SPF FVB/NJ mice were purchased from Beijing
   Huafukang Biotechnology Co., Beijing, China. Thirty newborn mice were
   randomly divided into five groups (n = 6/group). One group was kept in
   an ambient condition (~ 21% O[2]) as the normoxia group. Other four
   groups of mice were transferred to a 75% O[2] (hyperoxia) environment
   from PND 2 to 14. After 2 days exposure to hyperoxia, mice were
   subsequently divided into 4 subgroups, one of which was used as a
   control group and was injected with only PBS, and the other three
   groups injected intraperitoneally with 16.67 μl BPD-EXO at
   concentrations of 1 × 10^4, 1 × 10^5, and 1 × 10^6 particles/μl per day
   from PND 4 to 6 for each mouse, respectively, and the total injection
   volume was 50 μl for three days of injection. Then, all 30 mice were
   sacrificed on PND14 after parturition and the lung tissues were
   dissected for pathological examination.

   To determine the chronical effects of hyperoxia and BPD-EXO on lung
   development, eighteen PND1 mice were randomly divided into three groups
   (n = 6/group). Group 1 was kept under ~ 21% O[2] from PND 1 to 42 as
   the normoxia group. Groups 2 and 3 was transferred to a 75% O[2]
   (hyperoxia) environment from PND 2 to 14 and then returned to normoxia
   up to PND42. Groups 3 mice were daily injected intraperitoneally with
   16.67 μl BPD-EXO at 1 × 10^4 particles/μl per mouse from PND 4 to 6.
   Mice in all groups did not die until the 42nd day for sacrifice.

   All procedures were approved by the Institutional Animal Care and Use
   Committee of the Third Affiliated Hospital of Guangzhou Medical
   University (Protocol # 2019–188). Animals were maintained and treated
   in the Animal Centre of Guangzhou Medical University according to the
   rules of Committee on Animal Research and Ethics.

Hematoxylin & eosin staining (H&E) and Masson's trichrome staining

   The lung tissues were fixed with 10% formalin solution in 4℃ for 24 h
   and embedded in paraffin. The tissue blocks were sliced (5 μm
   thickness), dewaxed stained with H&E. The slide images were recorded
   using a Nikon Eclipse CI microscopy (Nikon, Japan). Additional serial
   sections were stained with Masson's trichrome according to routine
   procedures^[70]21 to visualize and quantify tissue collagen.

Immunohistochemistry (IHC) for VEGFA and CD31

   IHC for VEGFA and CD31 was performed on lung tissue sections as
   described^[71]20. The deparaffined sections were probed with mouse
   anti-VEGFA (Boster, BA0407, 1:200) or rabbit anti-CD31 (Abcam, ab28364,
   1:50), followed by the second antibody conjugated with Horseradish
   peroxidase (HRP) (Boster, SV0001). The staining color was developed
   using a DAB Substrate Kit (Abcam, ab64238), and counterstained with
   hematoxylin. Positive results were shown as brownish yellow. PBS
   missing the primary antibody was used as the negative staining control.
   The images were observed and recorded by a Nikon Eclipse CI microscopy
   (Nikon, Japan). Staining density was determined and quantified with
   Image J software using 20 × or 40 × images^[72]22.

Histopathology scores

   Semi-quantitative histopathology scoring criteria were described by
   Shen et al.^[73]23,[74]24. Lung injury was evaluated based on lung
   edema, infiltration of inflammatory cells, alveolar hemorrhage, hyaline
   membrane, and atelectasis. The scores were determined based on the area
   of injured: no lesion, score 0; injured area ≤ 25%, score 1; injured
   area 26–50%, score 2; injured area 51–75%, score 3; injured area > 75%,
   score 4. The scores were averaged for each animal from 10 randomly
   selected fields.

RNA-seq and bioinformatics analysis

   Total RNA was extracted from tissues using TRIzol reagent (Invitrogen,
   Carlsbad, CA) according to the manufacturer's instructions. The RNA
   concentration and purity were determined by a Nanodrop2000
   spectrophotometer (Thermo Scientific, US). The samples with OD^260:280
   around 2.0 were further measured the RNA integrity number (RIN) using
   Agilent 2100 bioanalyzer (Agilent, US). The RNA samples with RIN higher
   than 7 were enriched mRNA with magnetic beads with Oligo(dT) and
   performed the library construction using NEBNext® Ultra™ II RNA Library
   Prep Kit for Illumina (E7770, NEB, US). The libraries were qualified by
   Agilent 2100 Bioanalyzer and sequenced with 150 bp paired end on
   Illumina HiSeq2000 (Illumina, US). The resulting data were submitted to
   the Short Read Archive (NCBI) database (AccessionID: PRJNA917079).

   The sequencing reads were filtered at low quality and trimmed the
   adaptors using Trim Galore software (v0.6.7)^[75]25. The resulting
   high-quality reads were aligned to the Mus musculus reference genome
   GRCm39 by Hisat2 (v2.2.1)^[76]26, and generated the gene reads count
   matrix using featureCounts^[77]27. Differential expressions between
   conditions were assessed based on the negative binomial distribution
   using DESeq2^[78]28. The differentially expressed genes (DEGs) with
   FDR ≤ 0.001 and Log2(Fold Change) ≥ 1 were used to perform GO and KEGG
   pathway enrichment analysis using clusterProfiler package (v4.0.5) in R
   (v4.0.2)^[79]29.

Quantitative reverse transcription PCR (RT-qPCR)

   Total RNA was digested to remove DNA contaminations by Turbo DNase
   (Invitrogen, US), followed by reverse transcribed using the Reverse
   Transcription System (Promega, US) according to the manufacturer’s
   instructions. Quantitative PCR was performed using SuperReal SYBR Green
   PreMix (Tiangen Biotechnology, China) in the ABI PRISM 7500 System
   (Thermo Scientific, US). Relative gene expression was evaluated using
   2^-△△Ct methods^[80]30. The housekeeping gene GAPDH was used for
   normalization. The primers used in this study were attached in Table
   [81]S2.

Cell culture

   Human umbilical vein endothelial cells (HUVECs) were purchased
   (Cellcook, Guangzhou, China). A high-glucose medium of Dulbecco's
   Modified Eagle Medium (DMEM, Gibco, US) containing 10% fetal bovine
   serum (FBS, Gibco, US) was used for maintaining HUVECs. Cells were then
   cultured at 37 ℃ with 5% CO[2]. Before treated with exosomes
   (100 μg/ml), cells were passaged and serum-starved for 16 h, followed
   by carrying out cell migration, tube formation, and cell apoptosis
   assays.

Cell migration assay

   HUVECs migration was tested by transwell chambers (8-μm pore size,
   Corning, US) based on the manufacturer’s instructions. HUVECs suspended
   in a serum-free medium (1 × 10^5 cells) was seeded to each wells of the
   upper chamber. The complete medium (600 μl/well) were added into the
   lower chambers. After 24 h culture, the migrated cells were fixed with
   4% paraformaldehyde for 15 min and stained with 1% crystal violet
   (Beyotime Biotechnology, Beijing, China) for 10 min. Cells in five
   random separate microscope fields were captured using a Nikon Eclipse
   Ti microscope (Tokyo, Japan) and counted (200 × magnification) using
   Image J software^[82]22.

Tube formation assay

   As described by the manufacturer’s instructions, 48 well plates were
   coated with Matrigel (BD Biosciences, US). HUVECs were digested and
   diluted to the concentration of 8 × 10^5 cells/ml, and then 200 μl
   cells supernatant were inoculated to Matrigel-coated 48-well plates and
   treated with exosomes. Cells were then incubated at 37 ℃ with 5% CO[2]
   Up to 8 h Three random fields were captured under a microscope (Olympus
   BX51, Japan) every two hours after treatments, and the tube formation
   was analyzed with Image J software^[83]22.

Flow cytometry analysis of cell apoptosis

   The apoptosis was measured using the Dead Cell Apoptosis Kit with
   Annexin V Alexa Fluor 488 & Propidium Iodide (PI) (Invitrogen, US).
   Briefly, after HUVECs (1 × 10^6 cells/well) were treated with exosomes
   (200 μg) for 48 h, cells were washed with PBS and suspended in
   1 × Annexin-binding buffer. Then, cells were stained with 5 μl Annexin
   V-Alexa Fluor 488 and 7 μl PI for 15 min at room temperature. The
   apoptosis was determined by flow cytometer (FACSAria III, BD, USA) and
   Flowjo software.

Statistical analysis

   All quantitative data were presented as Mean ± SD. The differences
   between two groups were analyzed with the unpaired Student’s t-test
   with p-value < 0.05 (*) or < 0.01 (**). The statistically significant
   differences between the means of three or more groups were compared
   with One-way ANOVA followed by Bonferroni’s post hoc test, and the
   different letters were used to show statistical significance
   (p-value < 0.05 (*) or < 0.01 (**)). All statistical analyses were
   carried out using SPSS 22 software.

Results

Isolation and identification of exosomes from VPI with BPD

   Nanoparticles were isolated from the patients of very preterm infants
   with BPD as described^[84]20. The isolated nanoparticles were analyzed
   by transmission electron microscopy (TEM), nanoparticle tracking
   analysis (NTA), and western blot (WB). Diameters of nanoparticles
   ranged from 30 to 150 nm (Fig. [85]1A) with an oval or round
   vesicle-like structure and a negative-stain lipid bilayer membrane
   (Fig. [86]1B). The exosome-surface markers, TSG101 and Alix were both
   positively detected in the vesicles (Fig. [87]1C). All these results
   confirmed that the nanoparticles were exosomes.

Figure 1.

   [88]Figure 1
   [89]Open in a new tab

   Characterization of the isolated exosomes by NAT, TEM, and WB. (A) NAT.
   Sizes distribution of the isolated exosomes. (B) Morphology of
   negative-stained exosomes under TEM. (C) WB. Expression of exosomes
   surface markers TSG101 and Alix. n = 3.

BPD-EXO aggravate lung injury in BPD mice

   The H&E staining showed that compared with normoxia, hyperoxia induced
   BPD-like histocytology changes in the lung tissues (Fig. [90]2).
   Specifically, hyperoxia enlarged distal airspaces with fewer
   separation, reduced alveolarization, and increased alveolar
   interstitial cells in lung tissues (Fig. [91]2A). In addition,
   hyperoxia increased pulmonary fibrosis, decreased density of pulmonary
   microvessels, and dilated pulmonary capillaries based on the Masson
   staining. Compared with normoxia, hyperoxia increased (P < 0.05)
   collagen content (Fig. [92]2B). To detect the expression of VEGFA and
   CD31, the latter of which is an endothelial marker, we performed
   immunohistochemistry (IHC) in the lung tissue section. The IHC analysis
   revealed that staining areas of VEGFA and CD31 in hyperoxia group were
   decreased (P < 0.05) compared to normoxia group (Fig. [93]2C,D). These
   results demonstrate the successful establishment of the BPD animal
   model.

Figure 2.

   [94]Figure 2
   [95]Open in a new tab

   BPD-EXO aggravate lung injuries and angiogenesis impairment in BPD
   mice. (A) H&E staining. Lung tissue sections were stained with H&E and
   the histopathology scores were determined. (B) Mass staining. Lung
   tissue sections were stained with Masson and the collagen content were
   assessed. (C) Immunohistochemical staining of VEGFA (C) and CD31 (D).
   Lung tissue sections were immunostained for VEGFA and CD31.
   Representative images are shown. BPD, bronchopulmonary dysplasia;
   BPD-EXO, umbilical cord blood-derived exosomes from very preterm
   infants with BPD. PND, postnatal day; VEGFA, vascular endothelial
   growth factor A; CD31, cluster of differentiation 31. Scale
   bar = 50 μm. All data are presented as the mean ± SD. One-way ANOVA
   followed by Bonferroni’s post hoc test, and the different letters
   indicate statistical significance. n = 6. *, P < 0.05; **,. P < 0.01.

   On PND14, compared with hyperoxia, 5 × 10^7 BPD-EXO particles further
   increased (P < 0.05) pulmonary alveolar fusion and fibrosis, causing a
   significant increase in the histopathology scores (Fig. [96]2A,B).
   Compared to hyperoxia-treated group, BPD-EXO at 5 × 10^5, 5 × 10^6, and
   5 × 10^7 particles treated group also increased (P < 0.05) collagen
   content (Fig. [97]2A,B). Compared with hyperoxia, BPD-EXO at 5 × 10^5,
   5 × 10^6, and 5 × 10^7 particles decreased (P < 0.05) the VEGFA and
   CD31 staining distribution (Fig. [98]2C,D). Taken together, these data
   indicate that BPD-EXO exaggerates lung tissue injury and impairs lung
   angiogenesis in BPD mice.

BPD-EXO chronically aggravate lung injury in BPD mice

   After 28 days of normoxia, the hyperoxia-increased septum, enlarged
   distal cavity, reduced complexity, and increased pulmonary fibrosis
   persisted (Fig. [99]3A,B) (P < 0.05). Compared to nomoxia, hyperoxia
   decreased staining areas of VEGFA and CD31 (P < 0.05) (Fig. [100]3C,D).
   These results suggest that hyperoxia-induced BPD in neonatal mice can
   influence the lung development late in life. To determine the chronic
   effects of BPD-EXO on PBD mice, exposure of hyperoxia-treated mice from
   PND 4 to 6 to BPD-EXO (5 × 10^5 particles) increased pulmonary damage
   and fibrosis (Fig. [101]3A,B). Compared with hyperoxia, BPD-EXO at
   5 × 10^5 particles decreased (P < 0.05) the VEGFA and CD31 staining
   distribution (Fig. [102]3C,D). From the above results, we confirmed
   that BPD-EXO has an irreversible aggravating effect on lung injuries of
   hyperoxia-induced rats and the adverse effects can last up to the
   sexually mature stage.

Figure 3.

   [103]Figure 3
   [104]Open in a new tab

   Re-normoxia does not to reverse BPD-EXO- aggravated pulmonary damage.
   One-day-old mice were exposed to hyperoxia for 14 days then return to
   normoxia for 28 days. The lung tissue sections were stained with H&E
   (A) and Masson (B) to quantify histopathology scores (A) and collagen
   content (B), respectively. Additional tissue sections were
   immunostained for VEGFA (C) and CD31 (D) to quantify the expression.
   Representative images are shown. PND, postnatal day; VEGFA, vascular
   endothelial growth factor A; CD31, cluster of differentiation 31. Scale
   bar, 50 μm. All data are presented as the mean ± SD. n = 6. One-way
   ANOVA followed by Bonferroni’s post hoc test, and the different letters
   were used to show statistical significance (*P < 0.05; **P < 0.01).

BPD-EXO alter gene expression in lung tissues of BPD mice

   More than 196 million reads were trimmed adaptor and passed the quality
   filter. Clean reads were aligned to the mouse genome (GRCm39/mm39)
   using HISAT2^[105]26, with a mean alignment rate of more than 96.0%
   (Table [106]S1). A total of 874 BPD-EXO-induced DEGs were identified,
   of which 139 were up-regulated and 735 were down-regulated in the
   BPD-EXO-treated lung tissues (Fig. [107]4A).

Figure 4.

   [108]Figure 4
   [109]Open in a new tab

   Differential gene expression profile, gene ontology, and pathway
   enrichment analysis for lung tissues from BPD mice treated with BPD-EXO
   or vehicle. (A) The volcano plot of the differential gene expression
   profile was determined by RNA-seq. (B) The gene ontology (GO)
   enrichment analysis of the biological process. (C) KEGG pathway
   enrichment analysis identified the top 15 pathways affected by BPD-EXO.

   GO analysis revealed that these DEGs were significantly enriched in
   biological processes closely related to vascular formation, remodeling,
   and development, such as regulation of cellular response to vascular
   endothelial growth factor stimulus, vascular process in circulatory
   system, and angiogenesis (Fig. [110]4B). KEGG pathway enrichment
   analysis found that the DEGs were mainly enriched in signaling pathways
   involved in the function of angiogenesis and vascular remodeling,
   including peroxisome proliferator-activated receptors (PPAR) signaling
   pathway, vascular smooth muscle contraction, focal adhesion, tight
   junction, mitogen-activated protein kinase (MAPK) signaling pathway,
   Ras signaling pathway, AMP-activated protein kinase (AMPK) signaling
   pathway, etc. (Fig. [111]4C).

   Among these DEGs identified, the top 15 enriched signaling pathways,
   PPAR and MAPK pathways are related to angiogenesis and vascular
   remodeling^[112]31,[113]32. The PPAR signaling pathway was enriched
   with the genes of Rxrg, Fabp3, and Adipoq genes, and the MAPK pathway
   was enriched with genes of Cacng1, Cacng6, Egf, Cacna2d3, and Fgf9. We
   verified the expression of these genes in lung samples of BPD mice and
   BPD-Exon-treated BPD mice by RT-qPCR. In line with the transcription
   results, RT-qPCR results showed that Fabp3, Fgf9, and Cacna2d3 were
   significantly down-regulated in the BPD-EXO treated BPD mice
   (Fig. [114]5).

Figure 5.

   [115]Figure 5
   [116]Open in a new tab

   Validation of DEGs expression by RT-qPCR. Differential expression of
   genes in PPAR signaling pathway (Rxrg, Fabp3, Adipoq) and MAPK pathway
   (Cacng1, Cacng6, Egf, Cacna2d3, Fgf9) between pulmonary samples
   collected from BPD mice and BPD-EXO treated BPD mice. n = 4. *P < 0.05.

BPD-EXO inhibit endothelial cell angiogenesis, and promote endothelial cell
apoptosis by regulating the MAPK signaling pathway in HUVECs

   Compared with the negative control, BPD-EXO significantly inhibited
   (P < 0.05) endothelial cell migration and tube formation
   (Fig. [117]6A,B). Compared with vehicle control, BPD-EXO reduced
   (P < 0.01) mRNA expression of CD31 and VEGFA (Fig. [118]6C). BPD-EXO
   also increased (P < 0.05) the number of apoptotic cells in BPD-EXO
   group compared with the vehicle control group) (Fig. [119]6D).

Figure 6.

   [120]Figure 6
   [121]Open in a new tab

   Effects of BPD-EXO on endothelial angiogenesis and apoptosis. HUVECs
   were treated with BPD-exosomes (100 μg/ml) or vehicle control for 48 h
   under 21% O[2] conditions. (A) Migrated cells were quantified by
   transwell assay. (B) Tube formation was quantified by measuring tubes
   per well using ImageJ software. (C) Expression of CD31 and VEGFA were
   quantified by RT-qPCR. (D) Cell apoptosis was determined using flow
   cytometry. Scale bar = 30 μm. n = 3. **P < 0.01.

   We further verify the effects of BPD-EXO on mRNA expression of Fabp3,
   Fgf9, and Cacna2d3 in HUVECs. The RT-qPCR results showed that BPD-EXO
   significantly decreased expression of Fgf9 and Cacna2d3, but not Fabp3
   (Fig. [122]7), which was in line with the results in pulmonary tissues
   (Fig. [123]5). As Fgf9 and Cacna2d3 both belong to the MAPK signaling
   pathway, it suggests that BPD-EXO may impair endothelial functions by
   dysregulating the MAPK signaling pathway.

Figure 7.

   [124]Figure 7
   [125]Open in a new tab

   BPD-EXO suppress gene expression involved in the MAPK pathway in
   HUVECs. Cells were treated with BPD-EXO or vehicle control for 48 h.
   Expressions of Fabp3 (A), Fgf9 (B), and Cacna2d3 (C) were determined by
   RT-qPCR. n = 3. **P < 0.01.

Discussion

   In the present study, we have demonstrated: 1) BPD-EXO aggravate lung
   injury and impair lung angiogenesis in a hyperoxia mouse model; 2) we
   have further shown that the adverse effect of BPD-EXO on lung tissue
   and angiogenesis in neonate can persist in adult mice; 3) many
   BPD-EXO-induced DEGs and pathways may contribute to these adverse
   effects on lung tissues and vascular growth; and 4) BPD-EXO inhibit
   angiogenesis in vitro in association with decreased fgf9 and Cacna2d3
   in HUVECs. These data suggest that BPD-EXOs may contribute to the
   development of BPD and BPD-related lung disease, and BPD-EXO and their
   downstream genes and pathways could serve as the predictive biomarkers
   and therapeutic targets for VPI with BPD.

   Our previous study showed that BPD-EXO impairs angiogenesis in
   HUVECs^[126]20, but whether BPD-EXO has such an effect in vivo needs
   further verification. Gareth R et al. found that exosomes derived from
   human MSC medium supernatant can alleviate hyperoxia-induced lung
   tissue damage in neonatal mice^[127]33. Liping Teng et al. also
   reported that exosomes secreted by umbilical cord blood-derived MSCs
   can promote the proliferation HUVECs and angiogenesis^[128]34. The
   exosomes secreted by these umbilical cord blood MSCs can regulate
   related diseases through miRNA, regulate proliferation and
   migration-related TGF-β signaling pathways^[129]35, and also regulate
   inflammation-related GSK3β/Wnt/β^[130]36 and IL-6/STAT3
   pathway^[131]37. Umbilical cord blood exosomes are primarily protective
   in lung disease^[132]38. Another study of ours also showed that
   exosomes derived from healthy term umbilical cord blood can alleviate
   hyperoxia-induced lung injury^[133]39. But in the present study, we
   found that treatment with cord blood exosomes from BPD aggravated
   hyperoxia-induced lung injury. This discrepancy may be due to the
   heterogeneity of exosomes from different sources or severities of the
   same disease may exhibit different actions^[134]12,[135]40. For
   example, it is possible that only umbilical cord blood EXO from VPI but
   not other preterm infants with BPDs can cause the aggravating of
   hyperoxia-induced lung injury.

   In the current study, we used two mouse models of hyperoxia injury to
   evaluate the effect of BPD-EXO on hyperoxia-induced lung injury,
   short-term hyperoxia-induced and long-term hyperoxia-induced. Mice's
   lung development is largely completed by 2–3 weeks after birth^[136]41.
   Five days after birth, the lungs of the mice were still saccular, and
   complete alveoli were not formed until the 30th-day^[137]42,[138]43.
   Therefore, it is best to choose a short-term model to study the damage
   of hyperoxia to alveoli, that is, induce hyperoxia for 14 days.
   Hyperoxia-induced injury is associated with long-term pulmonary
   complications such as secondary pulmonary hypertension (PH)^[139]33. We
   chose to continue to observe the 42nd day after hyperoxia induction to
   evaluate mice's lung injury after hyperoxia induction in adulthood. Our
   results indicated that hyperoxia-induced damage was more severe in
   BPD-EXO-treated mice, regardless of the early model or the long-term
   model. However, further experiments are needed to verify whether
   BPD-EXO treatment is more serious for hyperoxia-induced early lung
   development damage or longer-term lung damage.

   The current observation that BPD-EXO down-regulated Fgf9 and Cacna2d3
   gene expression both in vitro and in vivo is not surprising since both
   of these two genes are enriched in the MAPK signaling pathway and are
   closely related to angiogenesis^[140]44. Particularly, Fgf9 could
   regulate both capillary plexus formation and VEGFA expression in
   endothelial cells, which is required for early lung distal vascular
   development^[141]17–[142]19. Cacna2d3 regulates cell apoptosis and cell
   replication through the MAPK pathway and PI3K/AKT pathway, both of
   which are associated with angiogenesis^[143]45. The human and mouse
   FGF9 3’ UTR are highly conserved and are similarly regulated by miR-182
   and miR-328. Interestingly, both miR-183 and miR-328 were up-regulated
   EXO-miRNA in the BPD-EXO^[144]20,[145]46,[146]47. Cacna2d3 are target
   genes of miR-27a which was the up-regulated EXO-miRNA in the BPD
   samples^[147]20,[148]48. These findings suggest that EXO could be used
   as treatment options for BPD, since their involvements in the
   pathogenesis of BPD. Preterm infant-derived exosomes are a possible
   cause of BPD progression and regulating molecule cargo of EXO might
   rescue BPD-impaired fetal angiogenesis and promote lung development.
   Future investigations are needed to be performed on the role of
   specific molecules in exosomes on BPD progression and to provide more
   clinical and research evidence for innovative diagnosis tools and
   therapy methods for VPI with BPD.

Supplementary Information

   [149]Supplementary Information 1.^ (54.6KB, pdf)
   [150]Supplementary Information 2.^ (9.6KB, xlsx)
   [151]Supplementary Information 3.^ (9.9KB, xlsx)

Acknowledgements