Umbilical cord blood–derived exosomes from healthy term pregnancies protect against hyperoxia‐induced lung injury in mice

Abstract Bronchopulmonary dysplasia (BPD) is a chronic, devastating disease primarily occurring in premature infants. To date, intervention strategies to prevent or treat BPD are limited. We aimed to determine the effects of umbilical cord blood‐derived exosomes (UCB‐EXOs) from healthy term pregnancies on hyperoxia‐induced lung injury and to identify potential targets for BPD intervention. A mouse model of hyperoxia‐induced lung injury was created by exposing neonatal mice to hyperoxia after birth until the 14th day post birth. Age‐matched neonatal mice were exposed to normoxia as the control. Hyperoxia‐induced lung injury mice were intraperitoneally injected with UCB‐EXO or vehicle daily for 3 days, starting on day 4 post birth. Human umbilical vein endothelial cells (HUVECs) were insulted with hyperoxia to establish an in vitro model of BPD to investigate angiogenesis dysfunction. Our results showed that UCB‐EXO alleviated lung injuries in hyperoxia‐insulted mice by reducing histopathological grade and collagen contents in the lung tissues. UCB‐EXO also promoted vascular growth and increased miR‐185‐5p levels in the lungs of hyperoxia‐insulted mice. Additionally, we found that UCB‐EXO elevated miR‐185‐5p levels in HUVECs. MiR‐185‐5p overexpression inhibited cell apoptosis, whereas promoted cell migration in HUVECs exposed to hyperoxia. The luciferase reporter assay results revealed that miR‐185‐5p directly targeted cyclin‐dependent kinase 6 (CDK6), which was downregulated in the lungs of hyperoxia‐insulted mice. Together, these data suggest that UCB‐EXO from healthy term pregnancies protect against hyperoxia‐induced lung injuries via promoting neonatal pulmonary angiogenesis partially by elevating miR‐185‐5p.


INTRODUCTION
Bronchopulmonary dysplasia (BPD) is a chronic disease characterized by respiratory and neurodevelopmental impairment, which mostly occurs in premature infants. 1 In general, patients with BPD require mechanical ventilation, oxygen supply, and steroid treatment to support their breathing. To date, despite the profound progress in neonatal care, there are limited strategies to prevent and treat BPD. Many novel therapeutic approaches have been proposed for BPD. For example, stem cell therapy is one of them because of several beneficial effects of stem cells, including their anti-inflammatory ability and capability to differentiate into lung cells. 2 Stem cell treatment improves pulmonary vascular growth and lung structure, [3][4][5] which is partially mediated via extracellular exosomes of mesenchymal stem cells (MSCs). 6 Thus, exosomes were emerged as a more promising therapy for BPD because cell-free exosomes have less immunogenicity and are feasible to maintain and store. 7 Exosomes are vesicles with a diameter between 30 and 150 nm and are characterized by expressing exosome markers, including tumor susceptibility gene 101 (TSG101) and CD9. Exosomes are secreted from cells after fusing with cell membrane, containing a variety of bioactive molecules (e.g., microRNAs). 8 Exosomes work as a vector that can transfer their inside contents to other cells. The cargo of exosomes is dependent on the physio-pathologic conditions. Our previous study revealed that exosomes from the umbilical cord blood of BPD preterm pregnancies impaired endothelial angiogenesis. 9 Further investigations to uncover exosomes that have protective effects on lung cells are clinically significant. Our previous study has demonstrated that exosomal miR-185-5p is decreased in umbilical cord vein blood from the BPD preterm pregnancies, leading to impaired endothelial angiogenesis. 9 In the current study, we hypothesized that umbilical cord blood exosomes from healthy term pregnancies protect against lung injury in BPD via elevating miR-185-5p. We used hyperoxia-induced lung injury mice as an in vivo model to determine if umbilical lungs of hyperoxia-insulted mice. Additionally, we found that UCB-EXO elevated miR-185-5p levels in HUVECs. MiR-185-5p overexpression inhibited cell apoptosis, whereas promoted cell migration in HUVECs exposed to hyperoxia. The luciferase reporter assay results revealed that miR-185-5p directly targeted cyclin-dependent kinase 6 (CDK6), which was downregulated in the lungs of hyperoxia-insulted mice. Together, these data suggest that UCB-EXO from healthy term pregnancies protect against hyperoxia-induced lung injuries via promoting neonatal pulmonary angiogenesis partially by elevating miR-185-5p. cord blood-derived exosomes (UCB-EXO) from healthy term pregnancies and encapsuled exosomal miR-185-5p alleviate lung injuries in BPD. In addition, to dissect cellular and molecular mechanisms underlying the regulation of these UCB-EXOs and their encapsuled miR-185-5p in human endothelial angiogenesis, hyperoxia-insulted human umbilical vein endothelial cells (HUVECs) were used. We found that UCB-EXOs from healthy term pregnancies significantly attenuated lung injuries in hyperoxia-insulted mice and upregulation of miR-185-5p protected against hyperoxia-induced injuries in HUVECs, suggesting that UCB-EXOs from healthy term pregnancies protect against hyperoxiainduced lung injuries via promoting neonatal pulmonary angiogenesis partially by elevating miR-185-5p.

Isolation and identification of UCB-EXOs
UCB-EXOs were isolated, as previously described. 9 Briefly, umbilical cord blood of healthy term pregnancies (n = 41) was collected and kept at 4°C. Serum samples were obtained by centrifuging (1107 g at 4°C). Exosome pellets were isolated from the serum samples by a series of centrifugation and resuspended with sterile phosphate-buffered saline. Pooled UCB-EXOs were used in this study.
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 a healthy pregnancy was made by the obstetricians at the birth center (or whatever the unit), the Third Affiliated Hospital of Guangzhou Medical University, where the umbilical cords were collected. UCB-EXOs were confirmed by morphology, the size distribution, and expression of exosomal surface markers (TSG101 and CD9) using transmission electron microscope (TEM; JEOL-JEM1400; JEOL Ltd.) at an acceleration voltage of 80 kV, nanoparticle tracking analysis (NTA), and Western blot as previous described, respectively. 9

Establishing hyperoxia-induced lung injury mice model and treatments
One male and two female FVB/NJ mice (SPF grade, 8 weeks old) were purchased from Huafukang Medical Technology Co., Ltd. These mice were made breeding pairs and gave birth to neonatal mice. Neonatal mice were randomly allocated into normoxia, hyperoxia, and hyperoxia+UCB-EXO groups (5 pups in each group from 2 dams). Mice in the hyperoxia group were exposed to 75% O 2 from day 1 to day 14 post birth to generate BPD mice. The normoxia group was maintained at 21% O 2 . The hyperoxia-insulted mice in the hyperoxia+UCB-EXO group were intraperitoneally injected with UCB-EXO from healthy term pregnancies (500,000 particles in 50 μL volume, daily) for 3 consecutive days starting from day 4 post birth based on previous studies 6, 10,11 and our pilot studies. On day 14 post birth, mice in all three groups were euthanized under inhalation anesthesia with isoflurane (3%; Beijing Keyue Huacheng Technology Co., Ltd.). The lung tissues collected were fixed with 4% paraformaldehyde (Biosharp Company) or snapped frozen in liquid nitrogen for the subsequent assays.
All procedures were approved by the Institutional Animal Care and Use Committee 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 and eosin staining
Lung tissues from five mice in each group were stained with hematoxylin and eosin (H&E). The paraffin lung sections were deparaffinized and rehydrated by passing through the following solutions: xylene (I and II) for 20 min, 100% ethanol (I and II) for 5 min, 75% ethanol for 5 min, and distilled H 2 O for 5 min. The sections were stained with H&E following the H&E staining kit's instructions (Guge Biotechnology Co., Ltd.). After being dried and mounted, the sections were imaged and recorded under an optical microscope (NIKON ECLIPSE CI). Histopathological grade for the lung injury was scored in a blinded manner: no lesion (score 0), injured area less than or equal to 25% (score 1), injured area ranging from 26% to 50% (score 2), injured area ranging from 51% to 75% (score 3), and injured area greater than 75% (score 4), respectively, as previously described. 12,13

Masson trichrome staining
To determine collagen contents in lung tissues, additional tissue sections (from 5 mice in each group) were stained in Weigert's iron hematoxylin working solution (Solarbio Company) for 10 min, followed by differentiating in phosphomolybdic-phosphotungstic acid solution for 15 s, staining in Beibrich-Scarlet Acid Fuschin solution for 5 min and aniline blue solution for 5 min. After dehydrating and mounting, three images per section were captured under a microscope (NIKON ECLIPSE CI). The collagen contents (collagen volume fraction) were semiquantified using Image-Pro Plus 6.0 software.

Fluorescence in situ hybridization
Fluorescein amidite-labeled miR-185-5p probe was purchased from BersinBio Company. Fluorescence in situ hybridization (FISH) assay was performed following the manufacturer's instructions. After deparaffinized and hydrated, the lung sections were digested with protein K (15 μg/mL). Slides were dehydrated through an ethanol series to 100% ethanol, air-dried, hybridized overnight, and followed by 4′,6-diamidino-2-phenylindole staining to visualize the nuclei. FISH staining was recorded under a laser confocal scanning microscope (Zeiss LSM 800 with Airyscan) and miR-185-5p positive cells were counted.

Cell culture and UCB-EXO uptake measurement
To further dissect the cellular and molecular mechanisms underlying the regulation of UCB-EXO in endothelial angiogenesis, hyperoxia-insulted HUVECs were used as an in vitro model of hyperoxia-induced BPD. 14 HUVECs purchased from Cellcook were cultured as previous described. 9 After UCB-EXO treatment, cells were incubated with PKH67 green fluorescent dye (5 μg/mL; #MIDI67; Sigma) for 24 h to label UCB-EXO. Fluorescent images were captured under a laser confocal scanning microscope (Zeiss LSM 800 with Airyscan).

Cell transfection and hyperoxia insult
HUVECs were transfected with negative control mimics (NC mimics, 100 pmoL/mL) or miR-185-5p mimics (100 pmoL/mL; GenePharma Company) in the Lipofectamine 2000 reagent (Thermo Fisher Scientific) for 24 h, respectively, as we previously described. 9 Cells were exposed in hyperoxia (75% O 2 ) or normoxia (21% O 2 ) for an additional 48 h for cell apoptosis and migration assays. The NC mimics and miR-185-5p mimics sequences were reported in our previous study. 9

Reverse transcription quantitative polymerase chain reaction
After serum starvation for 6 h, HUVECs were incubated with UCB-EXO at 0, 10, 20, and 40 μg/mL for an additional 48 h to test if UCB-EXO could dose-dependently increase miR-185-5p expression in HUVECs. These concentrations of UCB-EXO used were based on our previous study. 9 Total RNA and quantitative polymerase chain reaction (qPCR) were performed as described. 9 In brief, total RNA was extracted using Trizol (MRC, TR118-500). M-MLV Reverse Transcriptase (M1705; Promega) was used to generate cDNA, followed by qPCR assay using ChamQ SYBR qPCR Master Mix (Q341-03; Vazyme). The primer sequences for VEGFA, CD31, CDK6, and GAPDH are shown in Table 1. VEGFA and CD31 were used as markers for endothelial angiogenic activities. 15 Gene expression data were normalized to U6 or GAPDH.

Cell apoptosis assay
Cell apoptosis was determined using flow cytometry. After treatment, cells were digested with EDTA free trypsin, suspended in 500 μL binding buffer, and then incubated with 5 μL Annexin V and 7 μL 7-amino-actinomycin (AAD; BD Biosciences) at RT for 15 min. Annexin V and 7-AAD signals were analyzed by flow cytometry (Accuri C6; BD Biosciences).

Transwell assay
Cell migration assay were carried out as previously described. 9 After serum starvation, HUVECs were seeded into the upper chamber of transwell for 16 h. The bottom wells were added to the complete medium with fetal bovine serum (10%) to drive cell migration. The migrated cells were fixed and stained with 1% crystal violet. Images of migrated cells were taken under a Nikon Eclipse Ti microscope (Tokyo). Migrated cells were counted using the Image J software (National Institutes of Health [NIH]).

Dual luciferase gene reporter assays
Plasmids (pmirGLO-CDK6-wild type [WT]-3′UTR and pmirGLO-CDK6-mutant [MUT]-3′UTR) were constructed by GenePharma Company. Cells were grown overnight to 70% of confluence and then were replaced with serum-free culture medium. Plasmid mixtures were prepared (0.2 μg/ well WT or MUT plasmid mixed with 0.6 μg/well miRNA-185-5p specific miRNA mimics or negative control). Lipofectamine 2000 transfection reagent (Life Technologies) was used to transfect cells with plasmid mixtures following the manufacturer's instructions. Forty-eight h after transfection, the protein in each well was extracted using 100 μL passive lysis buffer and detected for luciferase activity using Dual-Luciferase Reporter System (Promega) by a platereading luminometer (TECAN Spark10M) according to the manufacturer's instructions. The relative luciferase activity (a fold change of the control group) was expressed.

Statistical analysis
Data were expressed as means ± SD. Statistical analyses were carried out with SPSS 22 software. The unpaired Student's t-test or one-way analysis of variance followed by Bonferroni's post hoc test were used. Any p < 0.05 was considered statistically significant unless stated otherwise.

UCB-EXO of healthy term pregnancies prevent lung injuries in hyperoxia-insulted mice
Isolated vesicles were first characterized. These vesicles in the TEM images appeared as a cup-shape morphology (Figure 1a), a typical feature of exosomes. Over 99.77% isolated vesicles had a diameter ranging from 30 to 150 nm (Figure 1b). The NTA results are consistent among samples of UCB-EXO from all healthy pregnancies. Exosome-surface markers, TSG101 and CD9, were positively expressed in the vesicles (Figure 1c). These results confirmed that these vesicles were exosomes. H&E staining results showed that, as compared with normoxia, hyperoxia increased (p < 0.05, n = 5 mice/ group) the histopathological grade of lung tissues by 2.67 folds (Figure 1d,e). Masson staining also showed that hyperoxia elevated (p < 0.05, n = 5 mice/group) extracellular matrix deposition in the alveolar wall by 25-folds (Figure 1f,g). These data demonstrate the successful establishment of the hyperoxia-induced lung injury mice model. More importantly, UCB-EXO treatments completely blocked (p < 0.05, n = 5 mice/group) the hyperoxia-induced increases in the histopathological grade score and the collagen content (Figure 1e,g), suggesting that UCB-EXO of healthy term pregnancies prevent the development of hyperoxia-induced lung injury in mice.

UCB-EXO of healthy term pregnancies alleviate the disrupted angiogenesis of lung tissues in hyperoxia-insulted mice
To evaluate the development of pulmonary vessels, immunostaining for CD31 and VEGFA was performed in lung tissues as abnormal pulmonary vascular development is one hallmark of BPD. 16 As shown in Figure 2, CD31 and VEGFA immunoreactivities in lung tissues were significantly (p < 0.05, n = 5 mice/group) reduced in the hyperoxia group compared with the normoxia group, whereas UCB-EXO treatment slightly increased CD31 levels but restored VEGFA (p < 0.05, n = 5 mice/group) levels in the BPD group to that in the normoxia group. These results suggest that UCB-EXO of healthy term pregnancies inhibit vascular injury in lungs of the hyperoxia-induced lung injury mice model via increasing VEGFA expression.

UCB-EXO of healthy term pregnancies increase miR-185-5p positive cells in lung tissues of hyperoxia-insulted mice
Our previous study showed that miR-185-5p expression was robustly decreased in UCB-EXO from BPD preterm pregnancies, which contributes to defects in endothelial angiogenesis. 9 In this study, FISH assay revealed that hyperoxia decreased (p < 0.05, n = 5 mice/group) miR-185-5p positive cells in the lungs by 3.1-folds compared with normoxia ( Figure 3). In contrast, compared with hyperoxia alone, UCB-EXO increased (p < 0.05, n = 5 mice/ group) the miR-185-5p positive cells in the hyperoxia group (Figure 3). These results indicate that UCB-EXO of healthy term pregnancies deliver miR-185-5p to the lungs of hyperoxia-insulted mice through intraperitoneal injection.
To further confirm that CDK6 is a direct target of miR-185-5p, we used the luciferase reporters that have either the WT or the MUT 3′-UTR sequence of the CDK6 gene. The luciferase reporter assay results showed that transfecting miR-185-5p mimics and plasmids containing the

DISCUSSION
The present study has revealed, for the first time, that UCB-EXO from healthy term pregnancies alleviate lung injury in hyperoxia-insulted neonatal mice in association with increases in their encapsuled miR-185-5p. We have further shown that UCB-EXO from healthy term pregnancies elevate the level of miR-185-5p in HUVECs, which inhibits cell apoptosis and promotes cell migration in HUVECs exposed to hyperoxia. We have also demonstrated that miR-185-5p directly targets CDK6, suppressing CDK6 expression in HUVECs. Thus, UCB-EXO from healthy term pregnancies might protect against hyperoxiainduced lung injury and prevent BPD development via the miR-185-5p/CDK6 cascade. Exosomes or extracellular vesicles from a variety of resources have promising therapeutic effects on experimental hyperoxia-induced lung injury models. For example, extracellular vesicles from MSCs have been shown to reduce lung injury in a mouse or rat model of hyperoxia-induced lung injury. [17][18][19][20] Exosomes derived from early gestational MSCs also inhibit the BPD development in neonatal mice. 10 In contrast, exosomes derived from activated polymorphonuclear leukocyte of patients with BPD could induce BPD-like injuries in neonatal mice. 21 Moreover, our previous study has also revealed that UCB-EXOs from BPD preterm pregnancies impair angiogenic ability in endothelial cells. 9 These data clearly indicate that exosome function is highly depending on the sources of exosomes. The current study provides experimental evidence that UCB-EXO from healthy term pregnancies prevent lung injuries in hyperoxia-insulted mice, suggesting that these UCB-EXO might be a potential candidate for the prevention of BPD.
Our current findings show that miR-185-5p in UCB-EXO from healthy term pregnancies might contribute to UCB-EXO-improved pulmonary development (indexed by decreased histopathological grade and collagen content) and pulmonary vascular growth (indexed by increased CD31 and VEGFA levels) in hyperoxia-induced lung injury mice. This is not surprising because a variety of miRNAs are known to affect the BPD development in rodent models. In particular, it has been shown that miR-421 negatively regulate the BPD development in hyperoxia-insulted mice 22 ; whereas overexpression of miR-101-3p inhibits lung injuries in BPD mice. 23 In addition, both miR-451 and miR-203a-3p actively participate in the BPD development in mice 24 and rats, 25 partially via regulating vascular growth and function increase. We have previously revealed that miR-185-5p, which is lower in UCB-EXO from BPD versus non-BPD preterm pregnancies, promote endothelial angiogenesis in vitro. 9 The current finding of low miR-185-5p levels in lung tissues of hyperoxia-insulted mice is consistent with our previous report in UCB-EXO from BPD preterm pregnancies. 9 Together with the current observation that miR-185-5p also inhibited apoptosis and enhanced migration in endothelial cells, these findings demonstrate that miR-185-5p encapsuled in UCB-EXO from healthy term pregnancies plays an important role in the lung development in BPD. Most importantly, these data suggest the potential of miR-185-5p as a target for impaired lung angiogenesis in BPD.
Many genes have been identified as the target of miR-185-5p including VEGFA, 26 cyclin D2, 27 and cathepsin K. 28 Our previous study has also predicted that CDK6 and DNA methyltransferase 1 (DNMT1) are targets of miR-185-5p. 9 Here, we have further shown that miR-185-5p and CDK6 levels are inversely correlated on in lung tissues of hyperoxia-insulted mice, suggesting a negative regulation of miR-185-5p in CDK6. This is supported by the current finding that CDK6 expression is significantly inhibited by UCB-EXO in mouse lungs and by miR-185-5p overexpression in hyperoxia-exposed HUVECs. These observations indicate that CDK6 is indeed a direct target of miR-185-5p, which is consistent with the current data from the luciferase reporter assay. The role of CDK6 in regulating angiogenesis during BPD development remains elusive. However, as CDK6 inhibition suppresses the angiogenic activity of HUVECs, 29 further investigations are needed to confirm if CDK6 plays an essential role in miR-185-5p-regulated lung development in BPD.
In conclusion, this study demonstrates that UCB-EXOs from healthy term pregnancies protect against the lung injuries in hyperoxia-insulted mice possibly through promoting endothelial functions by the miR-185-5p cascade. Future investigations are needed to further validate and confirm if miR185-5p can serve as a promising target for BPD intervention.