Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Ameliorate Airway Inflammation in a Rat Model of chronic obstructive pulmonary disease (COPD)

doi:10.21203/rs.3.rs-49230/v3

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Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Ameliorate Airway Inflammation in a Rat Model of chronic obstructive pulmonary disease (COPD)

https://doi.org/10.21203/rs.3.rs-49230/v3

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published 12 Jan, 2021

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Background: Chronic obstructive pulmonary disease (COPD) is an incurable and debilitating chronic disease characterized by progressive airflow limitation associated with abnormal levels of tissue inflammation. Therefore, stem cell-based approaches to tackle the condition are currently a focus of regenerative therapies for COPD. Extracellular vesicles (EVs) released by all cell types are crucially involved in paracrine, extracellular communication. Recent advances in the field suggest that stem cell-derived EVs possess a therapeutic potential which is comparable to the cells of their origin.

Methods: In this study, we assessed the potential anti-inflammatory effects of human umbilical cord mesenchymal stem cell (hUC-MSCs) derived EVs in a rat model of COPD. EVs were isolated from hUC-MSCs and characterized by the transmission electron microscope, western blotting, and nanoparticle tracking analysis. As a model of COPD, male Sprague Dawley rats were exposed to cigarette smoke for up to 12 weeks, followed by transplantation of hUC-MSCs or application of hUC-MSCs-derived EVs. Lung tissue was subjected to histological analysis using hematoxylin and eosin staining, alcian blue-periodic acid Schiff (AB-PAS) staining, and immunofluorescence staining. Gene expression in the lung tissue was assessed using microarray analysis. Statistical analyses were performed using GraphPad Prism 7 version 7.0 (GraphPad Software, USA). Student’s t-test was used to compare between 2 groups. Comparison among more than 2 groups was done using one-way analysis of variance (ANOVA). Data presented as median±standard deviation (SD).

Results: Both, transplantation of hUC-MSCs and application of EVs resulted in a reduction of peribronchial and perivascular inflammation, alveolar septal thickening associated with mononuclear inflammation, as well as a decreased number of goblet cells. Moreover, hUC-MSCs and EVs ameliorated the loss of alveolar septa in the emphysematous lung of COPD rats and reduced the levels of NF-κB subunit p65 in the tissue. Subsequent microarray analysis revealed that both hUC-MSCs and EVs significantly regulate multiple pathways known to be associated with COPD.

Conclusions: In conclusion, we show that hUC-MSCs-derived EVs effectively ameliorate by COPD-induced inflammation. Thus, EVs could serve as a new cell-free based therapy for the treatment of COPD.

Pathology

COPD

umbilical cord mesenchymal stem cells

extracellular vesicles

an animal model

The pathogenesis of the chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation that leads to small airway obstruction and emphysema (Li et al., 2015).Systemic analysis for Global Burden of Study 2010 demonstrated COPD to be the third leading cause of death in 2010 (Lozano et al., 2012). Eighty to 90% of all COPD cases are caused by exposure to cigarette smoke (CS) (Churg, Cosio & Wright, 2008). Inhalation of CS increases the number of neutrophils, B cells, macrophages, and CD8⁺T cells in the small airway and lungs. These cells, in turn, release multiple inflammatory cytokines, proteinases, and chemokines that together contribute to the degeneration of lung parenchyma (Shapiro, 1999; D'Agostino et al., 2010). Symptoms of COPD include chronic cough, dyspnea, and excessive production of sputum while anorexia, fatigue and weight loss may present in patients with severe COPD (Celli, MacNee & Force, 2004).

Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into osteoblasts, adipocytes and chondroblasts lineages(Fu et al., 2016; Corotchi et al., 2013). Apart from bone marrow (BM), MSCs can be isolated from various tissue including umbilical cord (UC), placenta, adipose tissue (AT), amniotic fluid, and lung tissue (Liu, Fang & Kim, 2016). However, UC represents an attractive source of MSCs as UC-MSCs is a less ethical concern like embryonic stem cells, and the isolation of UC-MSCs is non-invasive as compared to BM-MSCs. Besides, UC-MSCs has been shown to have similar efficacy in modulating the inflammation as BM-MSCs (Kagia et al., 2019). In another study, UC-MSCs depicted a greater proliferation, slower senescence rate, and greater anti-inflammatory effect as compared to BM-MSCs and AT-MSCs, suggesting that UC-MSCs might be a better alternative for stem cell-based therapy (Jin et al., 2013). Multiple pre-clinical studies suggest that MSCs have the potential to ameliorate the symptoms of many lung diseases such as pulmonary hypertension, asthma, COPD, and pulmonary fibrosis (Lee et al., 2012; Zeng et al., 2015; Gu et al., 2015; Dong et al., 2015). In the animal model of smoke-induced pulmonary emphysema, biweekly administration of adipose-derived MSCs cells decreases the level of inflammation, apoptosis, and alveolar enlargement (Schweitzer et al., 2011). The result from the first phase of clinical trials also demonstrates multiple doses of MSCs to be safe when administered in COPD patients while reducing the C-reactive protein at one month after transplantation (Weiss et al., 2013). A study in elastase-induced emphysema demonstrated that two doses of MSCs are better than single-dose MSCs. These effects are by decreasing the TNF-α, neutrophils and lymphocytes count in bronchoalveolar lavage fluid, thymus weight, the severity of hypertension, and increased elastic fibre content in the lung (Poggio et al., 2018). However, a recent study had demonstrated the efficacy of a single dose of UC-MSC in moderate-to-severe COPD patients, where the study was reported the UC-MSC was well tolerated with no clinically significant adverse effects was reported, decreased number of exacerbations in COPD Assessment Test (CAT) and mMRC scores, and the patients shown a significantly improved in terms of the quality of life (Bich et al., 2020).

Recently, an increasing number of researches have focused on studying the therapeutic effects of EVs in various diseases. EVs are small membrane vesicle of multivesicular bodies heterogeneous in size released by a variety of cell types, including MSCs. Extracellular vesicles can be found in body fluids such as milk, saliva, urine, amniotic fluid and cerebrospinal fluid. There are two commonly studied EVs which are exosomes and microvesicles. Exosomes, the size ranges from 40-100nm, are originated from the inward budding of endosome that forms multivesicular bodies (MVB) and released when the MVB fused with the cell membrane (Sarko & McKinney, 2017). While microvesicles (MV), also known as shed microvesicles size ranging from 50 to 1000nm, are formed by outward budding of the cell membrane (Rani et al., 2015). The isolation of EVs can be conducted via various methods, including differential ultracentrifugation, density-gradient separation, and immunoaffinity capture (Greening et al., 2015). The cargo of EVs is proteins, lipids, messenger ribonucleic acid (mRNA) and micro RNA (miRNA) which act as messenger molecules in intercellular communication (Ludwig & Giebel, 2012; Yu, Zhang & Li, 2014).

Studies have shown that EVs isolated from MSCs mimics the therapeutic effects of MSCs, participates in immunomodulation and regeneration in many animal models, however, MSCs depicted better effect in ameliorating the lung injury as compared to its secreted factors (Silva et al., 2019; Hayes et al., 2015). MSC-EVs have been reported to reduce the infarct size in mice model of myocardial ischemia/reperfusion injury (Lai et al., 2010). MSC-EVs were also capable of alleviating inflammation, oxidative stress, and apoptosis (Yang et al., 2015). Besides, the use of EVs has been recently suggested as a potential treatment option for COPD (Kadota et al., 2016; O’Farrell & Yang, 2019). However, to our knowledge, no attempts have yet been made to compare the impact of MSCs transplantation to EVs administration for in vivo models of COPD. In this study, we examined the effect of human umbilical cord MSCs (hUC-MSCs), and hUC-MSCs derived EVs on inflammation, airway remodelling, and emphysema in a rat model of COPD. In this study, we opted to use cigarette smoke to induce the inflammation in COPD 2 times/day, 7 days/week for 12 weeks following method from Zheng et al., (2009) with slight modification. Twelve weeks cigarette smoke exposure were chosen as our model as inflammation, increased goblet cell count, and emphysema were readily observed in this 12 weeks model (Zheng et al., 2009).

Preparation of FBSEVs deprived medium

DMEM/F12 (Thermofisher Scientific, USA) supplemented with 10% FBS (Thermofisher Scientific, USA) were subjected to ultracentrifugation at 100,000 x g at 18 hours at 4^oC by using Type 50.2Ti fixed-angle rotor, Optima L-100K Ultracentrifuge (Beckman Coulter, USA). The medium was collected and supplemented with 1% antibiotic antimycotic containing penicillin, streptomycin and amphotericin B (Thermofisher Scientific, USA), and 1% L-glutamine (Thermofisher Scientific, USA).

Cell culture, generation of conditioned media (CM) and isolation of EVs

Human umbilical cord-derived MSCs (hUC-MSCs) passage 4 was kindly provided by Cryocord Sdn Bhd (https://cryocord.com.my/). Cell preparation was conducted in the Current Good Manufacturing Practice (cGMP) accredited laboratory. The umbilical cord was shredded and enzymatically digested using collagenase (Worthington Biochem, USA) for approximately 2 hours at 37°C. The mesenchymal cells were isolated from human umbilical cord Wharton's jelly tissue by passing the tissue through a syringe and needle. hUC-MSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) - low glucose (Gibco,USA) supplemented with 10% human serum (Cryocord Sdn Bhd), and 100U/ml penicillin and 100μg/ml streptomycin and 0.25μg/ml amphotericin (Gibco, USA). The hUC-MSCs were cryopreserved using standard cryopreservation protocol until being used in the following research experiment.

hUC-MSCs were characterized using flow cytometric analysis, and multilineage differentiation capacity, according to the International Society for Cellular Therapy (ISCT) criteria for MSCs (Witwer et al., 2019). Positive cell surface markers CD90, CD105, CD73, CD166, and HLA-ABC and negative for hematopoietic markers of CD34, CD45, HLA-DR were characterized using flow cytometry analysis. Meanwhile, multilineage differentiation adipogenesis, osteogenesis, and chondrogenesis were conducted using commercially available differentiation kit.

hUC-MSC-CM were obtained from hUC-MSCs passage 5 to passage 7. The hUC-MSCs were cultured from a density of 4000 cells/cm²in complete medium, made up of DMEM/F12 (Thermofisher Scientific, USA) supplemented with 10% FBS (Thermofisher Scientific, USA), 1% antibiotic antimycotic containing penicillin, streptomycin and amphotericin B (Thermofisher Scientific, USA), 1% L-glutamine (Thermofisher Scientific, USA), and 20ng/mL basic fibroblast growth factor (bFGF) (Thermofisher Scientific, USA) and incubated at 37^oC, in humidified air with 5% CO₂. After 48 hrs of culture, media was changed to FBS-EVs deprived complete medium for the generation of hUC-MSCs conditioned media (hUC-MSC-CM). After 72 hours, hUC-MSC-CM was collected and concentrated using Amicon^® Ultra-15 Centrifugal Filter Devices (Merck Millipore, USA).

For the generation and isolation of hUC-MSC-EVs, hUC-MSCs were similarly cultured as described above. After 48 hours, the media was changed to FBSEVs-deprived complete medium. After 72 hrs, hUC-MSC-CM was collected and subjected to differential centrifugation. First, centrifugation of hUC-MSC-CM was conducted by using Kubota 2420 Compact Tabletop Centrifuge (Kubota, Japan) at 300xg for 10 mins to remove dead cells. The supernatant was collected and centrifuged again by using Allegra X-15R Centrifuge Ultracentrifuge (Beckman Coulter, USA) at 10,000xg for 30 mins to remove debris, followed by ultracentrifugation at 100,000xg for 2 hrs to precipitate the hUC-MSC-EVs by using Type 50.2Ti fixed-angle rotor, Optima L-100K Ultracentrifuge (Beckman Coulter, USA). The supernatant was discarded, and the hUC-MSC-EVs pellet was washed by resuspending in 1xPBS then re-pelleted by ultracentrifugation for 1 hr. The hUC-MSC-EVs pellet was collected and resuspended in 150µL 1xPBS and used fresh for the treatments.

Transmission Electron Microscope

Freshly isolated hUC-MSC-EVs in 150µl of 1xPBS suspension were loaded onto carbon-coated copper grids (Ted Pella, USA) and incubated for 10 minutes. The grid was blotted with filter paper and stained with 2% Uranyl acetate (Ted Pella, USA) for 1 minute. Excessive uranyl acetate was removed, and the grid was let dry for 15 min before viewing using Energy Filter TEM Libra-120 (Carl Zeiss AG, Germany).

Nanoparticle tracking analysis

The particle size of hUC-MSC-EVs was characterized by nanoparticle tracking analysis (NTA) using a NanoSight NS300 (Malvern analytical, United Kingdom) blue laser system. hUC-MSC-EVs were diluted with 1xPBS between 1:10 and 1:20 and loaded into the laser module sample chamber. The system focuses the laser beam allowing observing and measuring small particles. Five readings were recorded for each hUC-MSC-EVs sample.

Western blot

β-actin and CD63 expression were confirmed with western blot analysis. 2mg/mL of hUC-MSC-EVs were separated by using 12% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membrane (Bio-rad). The membrane was blocked with 2% BSA for 1 hour at room temperature and incubated with primary antibodies, rabbit monoclonal antibody CD63 (Abcam, Cat. No. ab134045 ) 1:2000 dilution, and rabbit monoclonal antibody β-actin (Cell Signalling Technologies, Cat. No. 4970S) 1:5000 dilution overnight at 4^oC. The membrane was then washed with PBST and incubated with fluorescence secondary antibody goat polyclonal anti-rabbit IgG (Thermo Fisher Scientific, Cat. No. A16097) 1:10000 dilution for 1 hour at room temperature. The secondary antibodies were washed with PBST and developed using a fluorescence detection system (Licor).

Animal model of COPD

Male Sprague Dawley (SD) rats (250-350g) aged 8-9 weeks (n=36) were obtained from the Animal Research and Service Centre (ARASC), Universiti Sains Malaysia. All animal procedures were approved and performed according to the ethical standards of the Animal Ethics Committee of the Universiti Sains Malaysia [No. USM / Animal Ethics Approval/2016/(104)(812)]. The approved protocols for animal study was based on Guidelines for the Care and Use of Animals for Scientific Purposes (USM 2017) which was developed based on the Malaysian Animal Welfare Act (2015), and guidelines by the Australian Codes for the Care and Use of Animals for Scientific Purposes (8^th Edition, 2013) and the Singapore Guidelines on the Care and Use of Animals for Scientific Purposes.

The in vivo study was conducted in Good Laboratory Practice (GLP) accredited laboratory in Animal Research Facilities, Advanced Medical and Dental Institute (IPPT), Universiti Sains Malaysia. The experimental procedure was conducted as previously described by Zheng et al. (2009) with slight modifications. COPD symptoms and inflammation were established by using commercially available cigarettes, Marlboro (Philip Morris, USA) (each containing 10.0 mg of tar and 1.0 mg of nicotine). In total, 36 rats were divided into 6 groups (n=6); naïve (untreated group), CS (injury group), CSSH (2-week self-healing group), hUC-MSCs-EVs (hUC-MSCs-EVs treated group), hUC-MSCs (hUC-MSCs treated group), and hUC-MSC-CM (hUC-MSCs-conditioned media treated group). All groups except naïve were exposed to sidestream cigarette smoke for 15 minutes per session, 6 cigarettes for 2 sessions, 7 days a week, for 12 weeks in a smoking chamber. Rats were left to rest for 2 hours between each session.

Treatments were given via intratracheal delivery in 150µL vehicle (1xPBS) on day 85 post cigarette induction. Rats were anaesthetized intravenously by using ketamine (50mg/kg) xylazine (5mg/kg). hUC-MSCs (2.5x10⁶), hUC-MSC-EVs isolated from 2.5x10⁶hUC-MSCs, and hUC-MSC-CM concentrated from 2.5x10⁶hUC-MSCs were used in the experiment. Naïve and CS groups were euthanized on day 85; meanwhile, the rest of the groups were euthanized on day 99. Rats were euthanized by using intravenous injection of pentobarbital (200mg/mL) (Dolethal, Lure Cedex, France).

Peripheral blood collection

Peripheral blood (300µl) was collected from rat tail vein and placed into a 1ml EDTA tube (Greiner bio-one, Austria) and subjected to whole blood count using Cell Dyn Hematology Analyzer (Abbott, USA).

Histological assessment

Hematoxylin and eosin (H&E) staining was performed for the analysis and scoring of peribronchial and perivascular inflammation, alveolar inflammation, and emphysema. Meanwhile, alcian blue – periodic acid Schiff (AB-PAS) staining was performed for the analysis of goblet cell count. Scoring of inflammation within the airway was conducted using a semi-quantitative analysis. Slides were blindly coded before a pathologist scored the tissues. The inflammation scoring was performed using the scale of 0 to 3 based on the presence and intensity of inflammatory cell infiltration in the peribronchial and perivascular area. Two slides were analysed per animal, with a total of 5 animals per group. The score was done according to the parameters: 0, no inflammation detected; 1, occasional cuffing with inflammatory cells; 2, most bronchi and vessels are surrounded by a thin layer of inflammatory cells (1-5 cells thick), and 3, most bronchi and vessels are surrounded by a thick layer of inflammatory cells (>5 cells thick). Alveolar inflammation scoring was done by grid on tissue section photos captured by fluorescence microscopy (Olympus, Japan). One hundred points were counted on random areas on the slides. Ten areas were analysed on 2 slides per animal with a total of 5 animals per group. Goblet cells were counted using light microscopy (Olympus, Japan). Five hundred cells were counted, and the number of goblet cells was divided by total cells to get a percentage of goblet cells. One slide per animal with a total of 5 animals per group were assessed. Emphysema was evaluated by using mean linear intercept (Lm), which measures the enlargement of alveolar space. Measurement was done by using 40x objective and 10x eyepiece, and photos of the sections were taken and superimposed with 30x30µm grid. Ten pictures of 2 slides per animal with a total of 5 animals per group were captured. The number of alveolar intercepts along the gridline were counted and calculated based on the following formula as described previously (Choe et al., 2003):

Lm = NL

where;

N = number of lines across the photographed area

L = length of the line across the photographed area

m = number of intercepts

RNA extraction and microarray analysis

RNA extraction was performed on 30 mg of rat lung from Naïve, CS, hUC-MSCs, and hUC-MSC-EVs groups using the RNeasy Mini Kit (Qiagen, Germany) following the manufacturer’s instructions. The purity and concentration of RNA were measured by NanoDrop ND1000 (Thermo Fisher Scientific, US). RNA integrity was determined by Agilent RNA 6000 Nanokit (Agilent Technology, US). cDNA was synthesized and hybridized at 65^oC for 17 hours and viewed using Agilent SureScan Microarray Scanner (Agilent Technology, US). Comparison between different sample dataset was normalised and analysed using Gene Spring software. The sample datasets were subjected to t-test to identify significant changes (p<0.05) between the sample and control group. Genes with p<0.05 and fold-change >2.0 were filtered as significantly regulated. Volcano plot, heat map, principle component analysis, Venn diagram, and pathway analysis were generated using Gene Spring software. Gene Ontology analysis using Panther (www.pantherdb.org) was used to classify differential expression analysis (DEG) by its functional role. GO terms with p<0.05 was considered significantly enriched by DEG.

Immunofluorescent staining

Immunofluorescent staining was performed to study the expression of NF-κB subunit p65. Briefly, tissue sections were deparaffinized in xylene and rehydrated in graded ethanols. The tissues were blocked with 5% goat serum for 30 min and incubated with primary antibody mouse monoclonal NF-κB-P65 (F-6) (Santa Cruz Biotechnology, USA) 1:200 for 1.5 hours in room temperature. After washing with PBS, slides were incubated with secondary antibody Alexa Fluor 555 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, USA) and counterstained with DAPI 1:2000 in 1xPBS, and viewed under IX71 Fluorescence Microscope (Olympus, Japan).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 7 version 7.0 (GraphPad Software, USA). Comparison among more than 2 groups was done using one-way analysis of variance (ANOVA) with Tukey's multiple comparison test. Data presented as mean ± standard deviation (SD). Differences are considered to be statistically significant when p≤0.05, whereas p≤0.001 was considered to be highly significant.

Characterization of hUC-MSCs

Mesenchymal stem cells were isolated from human umbilical cord blood were subjected to immunocytochemistry and differentiation analysis. hUC-MSCs were positive for CD73, CD90, CD105, and CD166, and negative for CD34, CD45, CD31, and HLA DR DP DQ (Table 1). Differentiation analysis showed the ability of MSCs to differentiate into adipocyte evidenced by lipid droplet formation, osteocyte evidenced by calcification formation, and chondrocyte evidenced by cell-matrix formation – Fig 1.

Table 1: Expression analysis of hUC-MSCs surface marker

Surface marker	Expression (%)
CD73	92.4
CD90	93.1
CD105	84.1
CD45	0.2
CD34	0.0
CD31	0.2
CD166	63.1
HLA-ABC	60.9
HLA DR DP DQ	0.0

Characterization of hUC-MSCs-EVs

hUC-MSC-EVs were isolated by differential centrifugation to remove cell debris and apoptotic bodies. hUC-MSC-EVs pallet suspended in 1xPBS was characterized based on morphology, size distribution and protein marker expression. Energy filtered transmission electron microscopy examination showed hUC-MSC-EVs were rounded in shape with the average size of 200nm (Fig. 2A). Western Blot analysis revealed the presence of the specific exosome marker CD63 at 30-65kDa and β-actin at 42kDa (Fig. 2B). Nanoparticle tracking analysis of hUC-MSC-EVs showed an average diameter of 153nm (Fig. 2C). Table 2 shows the mean, mode, SD and range of three hUC-MSC-EVs samples used in NTA.

Table 2: Analysis of hUC-MSC-EVs size distribution

Sample	Mean (nm)	Mode (nm)	SD (nm)	Range (nm)
1	141.2	115.0	51.4	36-737
2	156.5	116.9	68.4	64-795
3	163.0	123.1	68.3	25-740

hUC-MSC-EVs decreased lymphocyte count in peripheral blood

To study the effect of hUC-MSC-EVs on the circulating immune cells, peripheral blood was collected and subjected to full blood count. Figure 3 (A) depicted the white blood cell counts of peripheral blood. The following graphs show the differential cell counts of (B) neutrophils (C) lymphocytes (D) monocytes (E) eosinophils (D) basophils in peripheral blood. CS exposure for 12 weeks observed a non-significant increase in white blood count (WBC) count with no reduction seen following a 2 weeks self-healing rest period without exposure to CS (CSSH). Treatment with hUC-MSC-EVs and hUC-MSC-CM did not reduce WBC counts, however, a non-significant decrease was seen in response to hUC-MSCs (Figure 3 (A). Notably, CS significantly increased the percentage of lymphocytes compared to the naïve group with no observed mitigation following 2 weeks of self-healing (CSSH). A significant decrease in the percentage of lymphocytes was seen in response to treatment with whole-cell hUC-MSCs (*p<0.05), whereas a slight non-significant decrease in response to hUC-MSCs-EVs and hUC-MSC-CM Figure 3 (C)

hUC-MSC-EVs alleviates airway inflammation.

The analysis on histological scoring was conducted on the CS effects on the inflammation in rat airway and lung parenchyma. Figure 4 (A) showed the histological image of peribronchial, (B) histological image of parenchyma, (C) Semi-quantitative histological scoring and analysis of airway inflammation, (D) semi-quantitative histological scoring of lung parenchymal inflammation (D). The results showed an increase in inflammation scores in response to CS (Fig 4A-B). The accumulation of immune cells significantly increased in the lung parenchyma. Meanwhile, 2 weeks of self-healing (CSSH) did not reduce the inflammation. However, there was a significant reduction of inflammation scores observed in the parenchyma following treatment with hUC-MSC-EVs, whole-cell hUC-MSCs, and hUC-MSC-CM (**p<0.001, ***p<0.0001) – Fig 4C-D.

hUC-MSC-EVs reduce the infiltration of the immune cells in the lung

The accumulation of immune cells (neutrophils, eosinophils, lymphocytes and macrophages) in the lung is a key marker for the development of chronic inflammation in COPD. Figure 5 showed semi-quantitative histological scoring and analysis of (A) Neutrophils (B) Eosinophils (C) Lymphocytes (D) Macrophages in the lung. Our result showed that CS caused an influx of these immune cells into the lung (Fig. 4), predominantly neutrophils and macrophages, while lymphocytes and eosinophils remained present at low levels. Two weeks of self-healing (CSSH) failed to reduce the infiltration of all cell types examined. Notably, administration of hUC-MSC-EVs, hUC-MSCs, and hUC-MSC-CM was significantly reduced the immune cells influx as compared to the CS group.

hUC-MSC-EVs, hUC-MSCs, and hUC-MSC-CM decreased mucus production

To assess mucus overproduction, the semi-quantitative histological analysis was conducted to count the incidence of goblet cells between groups (figure 6). Histological sections of bronchi were stained with AB-PAS where the cell nucleus was stained blue, while goblet cells stained magenta (Fig 6A). Statistical analysis shows that treatment of CS groups with MSCs had significantly reduced the number of goblet cells (p<0.05) as compared to CS and self-healing (CSSH), and far better than groups received treatments with hUCMSC-EVs and hUCMSC-CM (Fig 6B).

hUC-MSC-EVs decreased emphysema

To study the effect of CS and treatment intervention on emphysema, the mean linear intercept of alveolar pores were measured on H&E histological slides (Fig. 7A). Quantitative analysis showed that 12 weeks of CS exposure caused alveolar destruction with a significant increase in the mean linear intercept of alveolar pores (Fig 7B), whilst 2 weeks of self-healing failed to mitigate these effects (CSSH). However, a significant (*p<0.05) reduction in the mean linear intercept of the alveolar pores and restoration of tissue was observed following treatment with hUC-MSC-EVs. Meanwhile, a non-significant reduction was observed in hUC-MSCs, and hUC-MSC-CM.

hUC-MSC-EVs decreased the levels of p65 in lung tissue

p65 is a subunit of the prototypic, pro-inflammatory transcription factor NF-κB. Translocation of p65 to the nucleus of a cell is indicative of the cells pro-inflammatory response. To study the translocation of p65 into the nuclei of cells, IHC stained and imaged lung tissue sections was quantified following CS and treatment intervention. A significant increase in the percentage of p65 positive cells was observed in the CS group. Following two weeks of self-healing, a significant reduction of p65 was observed. Treatment with hUC-MSC-EVs, hUC-MSCs and hUC-MSC-CM further reduced the p65 expression in CS-exposed lung – Fig 8.

CS, hUC-MSC-EVs and hUC-MSCs alter the gene expression

Our microarray analysis aimed to determine the pathways and the differential gene expressions altered in CS-induced inflammation lung and the treatment group (hUC-MSC-EVs and hUC-MSCs). Differentially expressed genes (DEG) which has been upregulated or downregulated more than the two-fold difference (p<0.05) are considered significant for further investigation to understand the biological, cellular and molecular functions. CS exposure was shown to lead to a total of 17689 DEG, while treatment with HUC-MSC-EVs and hUC-MSCs are shown to have led to 15160 and 23485 DEG, respectively (Fig 9A). Heatmap shows different regulation of DEG from CS group as compared to Naïve, hUC-MSC-EVs, and hUC-MSCs groups (Fig 9B). PCA plot shows a cluster of samples (n=2) in CS, hUC-MSC-EVs, and hUC-MSCs groups, but high variation was observed in the Naïve group (Fig 9C). Volcano plot shows DEG in CS, hUC-MSC-EVs, and hUC-MSCs groups (Fig 9D). Venn diagram shows overlapping DEG among CS, hUC-MSC-EVs, and hUC-MSCs (Fig 9E). 9888 DEG were overlapped in 3 groups, 1610 DEG were overlapped between CS and hUC-MSC-EVs, 4597 DEG were overlapped between CS and hUC-MSCs, and 1976 DEG were overlapped between hUC-MSCs-EVs and hUC-MSCs.

Gene Ontology analysis

GO Slim analysis of the biological process was performed on the DEG results presented in the tables below. The enriched GO terms were identified in CS (Suppl 1 Table A) hUC-MSCs (Suppl 1 Table B) and hUC-MSC-EVs (Suppl 1 Table C) groups. The GO terms for the CS group are related to the regulation of the cellular process, regulation of catalytic activity, regulation of signalling, and regulation of the metabolic process. While GO terms for hUC-MSC-EVs are related to the regulation of catalytic activity, movement of a cell or subcellular component, regulation of signalling, regulation of cell communication, cellular protein modification process, and regulation of RNA metabolic process. GO terms for hUC-MSCs are related to chemical synaptic transmission, sensory perception of the chemical stimulus, G-protein coupled receptor signalling pathway, regulation of signalling, regulation of cell communication, ion transport, regulation of biological quality, developmental process, and ribosome biogenesis.

Pathway analysis

The selection of regulated pathways related to COPD was determined based on the significant value of p<0.05. Thirty-eight pathways were significantly regulated in response to CS. Whereas, following hUC-MSC-EVs treatment, 58 pathways were significantly regulated, and only 17 pathways were significantly regulated following treatment with UCMSCs. The notable pathways which were highly regulated in the CS and hUC-MSC-EVs groups include; TGF-β receptor signalling pathway, IL-4 signalling pathway, and TNF-alpha NF-kB signalling pathway. Meanwhile, pathways which were highly regulated in response to the UCMSCs group include; TNF-alpha NF-kB signalling pathway, senescence and autophagy pathway, and IL-9 pathways. All the significantly regulated pathways are shown in Suppl 2.

Gene expression

We look for the highest frequency of genes that are regulated in pathways in injury (CS) and treatment (hUC-MSCs and hUC-MSC-EVs) groups. Table 3 shows 10 genes with the highest frequency in CS. NFKB1 and Mapk1 are expressed in 11 and 12 pathways respectively, followed by Jun and Map2k1, which are regulated in 10 pathways. Table 4 shows 8 genes with the highest frequency in the hUC-MSC-EVs group. Akt1 is expressed in 22 pathways, meanwhile, Mapk1, NFKB1, Map2k1 are regulated in 18 and 15 pathways respectively. Table 5 shows 8 genes with the highest frequency in UCMSCs group. Akt1 and Mapk1 are regulated in 6 pathways, while Map2k1 and TGFB1 are regulated in 5 and 4 pathways respectively.

Table 3: Genes with the highest frequency in the CS group.

Genes	P value	FC	Frequency
Nfkb1	0.0015	6.0905	11
Mapk1 (ERK2)	0.002193779	7.168577	12
Jun	0.041863125	-3.24888	11
Map2k1 (MEK1)	0.0097	3.2654	10
Mapk9	0.0026	-4.401	8
Crebbp (CBP)	0.0089	-4.07	9
Prkcz	0.0098	3.6678	8
p65	0.0017	6.3732	8
Grb2	0.0235	-2.237	7
Src	0.0115	-2.948	7

Table 4: Genes with the highest frequency in the hUC-MSCs-EVs group.

Gene	P value	FC	Frequency
Akt1	0.00172	-4.53957	22
Nfkb1	0.003537	-4.44685	15
Map2k1	0.003896	-3.49505	15
Pik3r1	0.002295	5.379887	14
Mapk1	0.010161	-3.689022	18
Grb2	0.003507	-3.40208	12
Prkcz	0.006365	-3.68258	10
p65	0.001438	-5.04044	10

Table 5: Genes with the highest frequency in the hUC-MSCs group.

Gene	P value	FC	Frequency
Tgfb1	0.024022	-5.81517	4
Akt1	0.004107	-6.88884	6
Mapk1	0.001274641	-8.08983	4
Pik3r1	0.001114	9.6399	4
Map2k3	0.002436	-4.57343	4
Mapk3	0.01819	2.465821	4
Mapk8	0.006342	-3.81553	4
Map2k1	3.85E-04	-9.33631	5

Our study aimed to determine the effects of hUC-MSC-EVs in comparison to hUC-MSCs for the treatment of COPD. The therapeutic potential of MSCs and MSCs derived secreted factors have been widely demonstrated in various diseases, including rheumatoid arthritis, asthma, and Crohn’s disease (Gonzalez-Rey et al., 2010; Song et al., 2015; Panes et al., 2016). In COPD, MSCs capabilities to mitigate inflammation has been tested in the preclinical and clinical setting around the world (Weiss et al., 2013; Liu, Fang & Kim, 2016; Bich et al., 2020). However, little is known about the effect of extracellular vesicles isolated from MSCs for the treatment of inflammation in COPD. hUC-MSCs used in this study were positive for CD73, CD90, CD105, and CD166, and negative for CD34, CD45, CD31, and HLA DR DP DQ as previously described by (Witwer et al., 2019).

Meanwhile, differentiation analysis showed the ability of hUC-MSCs to differentiate into adipocyte, osteocyte, and chondrocyte. hUC-MSC-EVs isolated from hUC-MSCs showed a rounded morphology with the average of 153nm in diameter, and protein analysis showed a positive marker for CD63 exosomal marker. Following 12 weeks of CS exposure, the evidence of accumulation of inflammatory cell infiltrated in peribronchial and perivascular tissues as well as the parenchyma, goblet cell hyperplasia, expression of p65, and the development of emphysema was consistent to that of previously published studies (Nie et al., 2012; Zhang et al., 2014) indicating the development of COPD by CS inhalation. Two weeks of self-healing has significantly reduced the expression of p65, but did not reduce the inflammation and remodelling the destruction of alveolar in the lung. The treatment of hUC-MSC-EVs, hUC-MSCs, as well as hUC-MSC-CM, were significantly reversed the effect of sidestream CS on lung inflammation, expression of p65, and emphysema. Our study on microarray also revealed that CS was significantly regulated pathways related to COPD and upregulated genes related to inflammation including NFKB1, p65, and protein kinase Cζ (PRKCZ), whilst treatment with hUC-MSC-EVs and hUC-MSCs were observed to reverse these CS-induced gene expression effects.

Cigarette smoke is the leading risk factor of COPD, with over 80% of all COPD cases attributed to cigarette smoking. Therefore, cigarette smoke is widely employed by the researchers to develop the in vivo COPD model over other inducers such as biomass fuel, lipopolysaccharide, and elastase (Borzone et al., 2007; Al Faraj et al., 2014; He et al., 2017; Ghorani et al., 2017). For the establishment of COPD model in animal, the cigarette smoke was exposed to the animals for 6 months period in order to exhibit the severe injury in the lung (Huh et al., 2011; Kim et al., 2016). However, there are studies which employed 12 weeks cigarette smoke exposure demonstrated characteristic of COPD including inflammation, airway remodelling, fibrosis, goblet cell hyperplasia, and emphysema (Gu et al., 2015; He et al., 2015). This method is more feasible for in vivo study as compared to 6 months period, which is time-consuming. Our study is in agreement with the previous studies that showed 12 weeks of cigarette smoke exposure is sufficient to induce characteristics similar to COPD in SD rats. Importantly, our method of CS exposure for 2 times/day, 7 days/week for 12 weeks exposure induced the emphysema in rat lung, a characteristic of the chronic model of COPD (Leberl, Kratzer & Taraseviciene-Stewart, 2013). It should be noted that animal models do not fully mimic human condition, and regardless types of animal used. The duration of cigarette smoke exposure, the severity of the injury are only equivalent to the Global Initiative for Obstructive Lung Disease (GOLD) stage I or II diseases (Fricker et al., 2014).

COPD is characterized by airway and parenchymal inflammation that leads to mucus overproduction and emphysema, although these characteristic may not present in all patients, as the emphysematous lung only occurs in 20% of all COPD patients (Churg, Cosio & Wright, 2008; Akram et al., 2012). Nevertheless, in the animal model, the presence of emphysema is one of the important characteristics to confirm the development of COPD (de Oliveira, 2016). On the other hand, mucus overproduction is considered challenging to reproduce in the rat model due to the low number of goblet cells in the bronchi (Churg, Cosio & Wright, 2008). Our study using CS exposure for 12 weeks in SD rats successfully developed characteristic of COPD as we can observe the increased influx of immune cells indicating the development of inflammation in the lung, increased goblet cells count which shows increase mucus production, and increased mean linear intercept which shows the development of emphysema.

Airway inflammation begins with the disruption of the airway and vascular function, allowing infiltration of immune cells in the lung (Schweitzer et al., 2011; Presson Jr et al., 2011). In the acute phase of CS exposure that lasts until the second week, increased of neutrophils was observed. After the second week, macrophage begins to increase, and neutrophils start to decrease but not fully resolve, indicating that chronic inflammation began to develop (Stevenson et al., 2007). In our study, the increased in neutrophils, eosinophils, lymphocytes, and macrophages counts were observed, however, neutrophils and macrophages are the predominant immune cells infiltrating the lung. Our results also showed that immune cells accumulation was observed more prominently in the alveolar area rather than the peribronchial and perivascular area, which destroy the alveolar wall leading to the emphysematous lung.

The accumulation of immune cells in alveolar walls are prerequisite for the development of emphysema. Neutrophils elastase (NE) was reported to induce the epithelial apoptosis and emphysema, meanwhile excessive MMP-9 released by macrophage can result in permanent alveolar destruction (Atkinson et al., 2011; Hou et al., 2014). Shapiro et al. (2003) was demonstrated that crosstalk between these two cells is crucial in the development of emphysema. The presence of neutrophils is essential as neutrophils release NE that is required to recruit more neutrophils and monocytes into the lung. The study was also reported that mice deficient of NE (NE^-/^-) had shown significantly protected from the development of emphysema. Shapiro and colleagues further proved that the synergistic effects of neutrophil and macrophage are required to enhance the potency of both cells. The absence of NE causes the tissue inhibitors of metalloproteinases (TIMPs) to inhibit the action of macrophage elastase. Likewise, the absence of macrophage elastase caused an increased in α-1 anti-trypsin, a major inhibitor of NE. Thus, the presence of both neutrophils and macrophages are an important factor in the development of emphysema (Shapiro et al., 2003).

CS exposure also causes mucus overproduction, although the symptoms may not present in all COPD patients (Burgel & Martin, 2010). The mechanism by which CS-induced the overproduction of mucus occurs through activation of TNF-α converting enzyme (TACE) which cleaved pro-TNF-α to release TNF-α that activates epidermal growth factor receptor (EGFR) which result in mucin production (Shao, Nakanaga & Nadel, 2004). The accumulation of neutrophils in the lung during CS exposure may also exacerbate the mucus overproduction as neutrophils are also in part responsible for the impaired mucociliary clearance, increased goblet cells count, and excessive mucus production. NE released by neutrophils increased the expression of MUC5AC by enhancing the mRNA stability via reactive oxygen species mechanism (Arai et al., 2010; Fischer & Voynow, 2002). Besides, activation of TNF-α and subsequent activation epidermal growth factor pathway can also stimulate NE to induce the expression of MUC5AC (Kohri, Ueki & Nadel, 2002).

MSCs has been actively investigated as a potential therapy for COPD. Clinical studies measuring C-reactive protein in COPD patient revealed the benefit of MSCs administration in mitigating the inflammation (Hayes et al., 2020). In the animal model, MSCs alleviates the inflammation by reducing the alveolar macrophage, while at the same time promoting the expression of the anti-inflammatory cytokine, IL-10 in macrophages (Gu et al., 2015). MSCs also reduced the neutrophil infiltration regardless of the route of administration (Antunes et al., 2014). This therapeutic effects of MSCs are governed by the released of paracrine factors, including growth factor, cytokine, and EVs rather than cell-to-cell contact (Fontaine et al., 2016). Recently, research begins to unravel the therapeutic effects of MSCs derived EVs and better understand the mechanism behind this ability. Several studies have shown anti-inflammatory effects of MSCs derived EVs in mitigating the inflammation similar to MSCs. Maremanda et a., (2019) study the effect of MSC, MSC-exosomes, and combination of MSC + MSC-exosomes in acute CS exposure in mice model. The group measured the total cell count, and differential cells count in BAL fluid. The treatment of MSC, MSC-exosomes, and combination MSC + MSC-exosomes decreased total cell count, macrophages, neutrophils, and CD4⁺ T cells count. However, the group did not measure the accumulation of immune cells in the peribronchial and parenchyma area (Maremanda, Sundar & Rahman, 2019). Apart from CS-induced inflammation, MSC-exosomes also have been shown to modulate the differentiation, activation, and proliferation of T cells in vitro (Blazquez et al., 2014). Reduced number of eosinophils, lymphocytes, and airway remodelling were observed in the animal model of asthma when treated with adipose tissue MSC-EVs (de Castro et al., 2017). In the rat model of hepatic ischemia-reperfusion injury, hUC-MSC-EVs inhibited the activity of the neutrophils by attenuation of respiratory burst and oxidative stress, thus reducing the apoptosis of hepatocytes (Yao et al., 2019). Also, MSC-EVs attenuated the pro-inflammatory cytokines such as IL-17, TNF-α, RANTES, MIP1α, MCP-1, CXCL1, HMGB1, while enhancing the production of IL-10, PGE2, and KGF (Stone et al., 2017). In agreement with the previous studies, our study demonstrated that hUC-MSC-EVs possess anti-inflammatory similar to its cell counterpart, hUC-MSCs. The treatment with hUC-MSC-EVs significantly reduced immune cells accumulation in the lung, especially neutrophils accumulation, reduced emphysema, reduced protein expression of p65, and downregulated DEG related to COPD.

To date, there are no treatment options available to regenerate the lung damage in emphysema. However, stem cell-based therapy demonstrates a promising regenerative capability to restore the function of the damaged lung. MSCs and MSC-CM are shown to restore the lung function by mitigating the apoptosis in the emphysematous lung (Huh et al., 2011). This anti-apoptosis effect is in part is mediated by vascular endothelial growth factor (VEGF) and VEGF receptor (Guan et al., 2013). Besides, MSCs reduced expression of cyclooxygenase-2 in alveolar macrophage, thereby mitigating the emphysema in a rat model of COPD (Gu et al., 2015).

On the other hand, relatively few studies were conducted to decipher the effects of MSCs derived EVs in the emphysematous lung. The study by Kim and colleague (2017) comparing the regenerative effects of nanovesicles generated from adipose stem cells (ASC) and ASC derived exosomes in the elastase-induced emphysematous lung. The result showed that nanovesicles significantly reduced the emphysema via its cargo content, FGF2, while no significant reduction of emphysema was observed in ASC derived exosome (Kim et al., 2017). In a study examining the effect of MSC-exosome on bronchopulmonary dysplasia, a chronic lung disease in the preterm infant, characterized by restricted lung growth, subdued alveolar and blood vessel development, and impaired pulmonary function, MSC-exosome are shown to reduce mean linear intercept, while increasing the lung alveolarization, through alteration of macrophages pro-inflammatory M1 phenotype into anti-inflammatory M2 phenotype (Willis et al., 2018). Our result provides the evidence of hUC-MSC-EVs ability to reduce emphysema in CS-induced COPD in a rat model. Considering the importance of neutrophils and macrophages accumulation in the pathogenesis of emphysematous lung, a significant reduction in the accumulation of neutrophils when treated with hUC-MSC-EVs and hUC-MSCs in our study, in part might explain the reduction of emphysema. Decreased in macrophages accumulation were also observed when treated with hUC-MSC-EVs, hUC-MSCs, and hUC-MSC-CM, although the reduction was not significantly different from the injury group. Recent studies also reported that MSCs derived microvesicles reduced the influx of neutrophils through the effects of KGF (Zhu et al., 2014). However, macrophages are shown to play an essential role in MSCs anti-inflammatory effects by changing from M1 to M2 phenotype which produces IL-10 that involve in the reduction of inflammation when treated with MSCs and MSC-EVs (Etzrodt et al., 2012; Gu et al., 2015; Sicco et al., 2017). Although the accumulation of macrophages is prerequisite for emphysema, however, in allergic asthma, depletion of alveolar macrophage reversed the immunosuppressive effect of MSCs in which the production of IL-10 was dependent on the presence of alveolar macrophage (Mathias et al., 2013). The macrophages role might explain why macrophages in our study did not significantly reduce as it aids in MSCs anti-inflammatory response.

To date, relatively few studies examining the effect of MSCs in reducing the mucus overproduction. Although there are reports stated that mucus could be mitigated with the administration of MSCs, in-depth analysis on the mechanism involves remaining unknown (Lee et al., 2011; Mohammadian et al., 2016). Besides, there is no report on the ability of MSC-EVs to reduce mucus overproduction. Our study showed a significant reduction of goblet cells count in hUC-MSCs. Reduction of goblet cells can be observed in hUC-MSC-EVs and hUC-MSC-CM, however, the reduction was not significant. The extracellular environment can alter the MSCs fate and the paracrine factors released by the MSCs (Sullivan et al., 2014). Thus, the hUC-MSCs transplanted into the lung will be influenced by the lung microenvironment, and the paracrine factors that are being released by the transplanted MSCs will be different from the hUC-MSC-EVs and hUC-MSC-CM collected from the hUC-MSCs grown in the flask, thereby will affect the lung differently. This effect can be observed in the various pathways that hUC-MSCs and hUC-MSC-EVs regulated in our study. We also speculate that hUC-MSC-EVs, and hUC-MSC-CM can affect the lung tissues faster than hUC-MSCs, as hUC-MSCs will also need to establish cell-to-cell contact, and the lung microenvironment will also have to communicate with hUC-MSCs, in order for hUC-MSCs to produce an effect. Meanwhile, MSC-EVs and paracrine factors in MSC-CM can readily be taken up by the cells in the lung due to its small size (De Jong et al., 2014). Hence, we observed more reduction of goblet cells count in MSC-EVs and MSC-CM treatment groups as compared to MSCs alone.

Our microarray analysis aimed to determine the pathways associated with COPD and gene expression profile in our COPD model. We also seek to understand how the treatment with hUC-MSC-EVs and hUC-MSCs can change the gene expression profile and pathways in COPD model. Our on DEG analysis of microarray data revealed the importance of p50, p65, and PRKCZ in our animal model. 12 weeks CS exposure significantly upregulated p50, p65, and PRKCZ and the treatment with hUC-MSC-EVs were significantly downregulated the expression of these genes. Immunohistochemistry staining on p65 confirms the significant upregulation of p65 protein in the CS group, and significant downregulation of p65 when treated with hUC-MSC-EVs, hUC-MSCs, and hUC-MSC-CM. Our study was also revealed that p50, p65, and PRKCZ involved in many pathway regulations that include TNF-α NF-κβ signalling pathway, IL-2 signalling pathway, oxidative stress, estrogen signalling pathway, and IL-4 signalling pathway.

The expression of PRKCZ and NF-κβ play a vital role in inflammation and thus, the pathogenesis of COPD. PRKCZ is upstream of NF-κβ, phosphorylating p65 at serine 311 to promote the acetylation of Lysine 310, thus activating the κβ transcription (Diaz‐Meco & Moscat, 2012). Mice deficient of PRKCZ was found to reduce myeloperoxidase and influx of neutrophils, and reduced pro-inflammatory cytokines such as IL-13, IL-17, IL-18, IL-1β, TNF-α, MCP-1, MIP-2, and IFN-γ, while the use of PRKCZ inhibitors blocked the activation of NF-κβ by TNF-α, thus reducing the pro-inflammatory IL-8 expression (Yao et al., 2010; Aveleira et al., 2010). Meanwhile, NF-κβ composed of five members, NF-κβ1 (p50), NF-κβ2 (p52), RelA (p65), RelB, and c-Rel, that regulate a multitude of genes involved in inflammatory responses (Liu et al., 2017). Among all heterodimers of NF-κβ, p50/p65 heterodimer represents the most abundant NF-κβ activated by the canonical pathway (Giridharan & Srinivasan, 2018).

Cigarette smoke activates NF-κβ within one hour of exposure to the lung thus causing inflammatory reactions which increase white blood cell count, lymphocyte count, and granulocyte count (Churg et al., 2003; Flouris et al., 2012). Data from the pre-clinical study showed 4 weeks of CS exposure significantly increased p65 and Iκβα in mice lung as compared to control group (Yu et al., 2018). NF-κβ is also required by IL-1β and IL-17A to induce the expression of MUC5B in bronchial epithelial cells that cause goblet hyperplasia in COPD (Fujisawa et al., 2011). Besides, various studies demonstrated the upregulation of p65 and p50 expression in COPD patients (Di Stefano et al., 2002; Caramori et al., 2003; Tan et al., 2016; Zhou et al., 2018). Microarray study conducted by Yang et al., (2013) revealed the vital role of p50 in regulating many pathways of COPD including toll-like receptor signalling pathway, cytokine-cytokine receptor interactions, chemokine signalling pathway, and apoptosis (Yang et al., 2013).

Our study revealed the downregulation of PRKCZ, p65, and p50 expression when treated with hUC-MSC-EVs. p50 regulated 18 pathways in the hUC-MSC-EVs group, while PRKCZ and p65 regulated 10 pathways suggesting the vital role of the NF-κβ pathway in hUC-MSC-EVs therapeutic effects in our model. Downregulation NF-κβ subunit by hUC-MSC-EVs can affect multiple pathways in our model, thus reducing the inflammation. MSC-EVs has been shown to decrease the expression of NF-κβ in an in vitro model of cystic fibrosis and experimental colitis (Yang et al., 2015; Zulueta et al., 2018). MSC-exosomes also interfered with TLR-4 signalling of BV2-microglia, prevented the degradation of NFκβ inhibitor, Iκβα, and phosphorylation of MAPK family protein in response to LPS stimulation (Thomi et al., 2019). However, much is still unknown about how MSC-EVs regulates the NF-κβ pathway. In our study, we did not elucidate the cargo content of hUC-MSC-EVs that is responsible for anti-inflammatory effects on CS-induced lung inflammation. Nevertheless, the study demonstrated that micro-RNA content of MSCs derived exosome could reduce p50 NF-κβ pathway in macrophage, thus preventing the Toll-like receptor-induced macrophage activation (Phinney et al., 2015). In addition, CCR2 in MSCs derived exosomes abolished the ability of CCL2 to induce p65 phosphorylation in macrophages (Shen et al., 2016). Meanwhile, knockdown of GPX-1 in human MSCs, reverse the effect of MSCs derived exosomes in reducing the phosphorylation of p65 (Yan et al., 2017). These results proved that multiple cargo contents of MSC-EVs play a vital role in mediating the inflammation.

Our study had successfully isolated the hUC-MSC-EVs from hUC-MSCs. Twelve weeks of CS exposure induced the inflammation, increased goblet cells count, and emphysema in the rat model. The treatment with hUC-MSC-EVs, hUC-MSCs, and hUC-MSC-CM decreased the inflammation in the lung, decreased the goblet cells, and destruction of the lung in a rat model of COPD similar to hUC-MSCs. hUC-MSC-EVs reduced the inflammation in part by expression of PRKCZ, and NF-κβ subunits p65, and p50, which regulates many genes responsible for innate and adaptive immune response. Confirmation study using immunofluorescence on p65 showed a similar result as microarray analysis of DEG. Taken together, there are still limited data demonstrating the regenerative and the anti-inflammatory effects of MSC-EVs to mitigate the inflammation in COPD. More studies should be conducted to decipher the anti-inflammatory effects of MSC-EVs as a whole, as well as exosomes, and microvesicles as different particle might exhibit different therapeutic effects. Determination of cargo content of MSCEVs responsible for the anti-inflammatory effects and the mechanism of action of the cargo content of MSC-EVs can provide a clear with the ways toward the goal of using hUC-MSCs as a new treatment for COPD.

COPD Chronic obstructive pulmonary disease

EVs Extracellular vesicles

hUC-MSCs human umbilical cord mesenchymal stem cell

AB-PAS alcian blue-periodic acid Schiff

ANOVA one-way analysis of variance

SD standard deviation

CS cigarette smoke

MSCs Mesenchymal stem cells

BM bone marrow

UC umbilical cord

AT adipose tissue

MVB multivesicular bodies

mRNA messenger ribonucleic acid

miRNA micro ribonucleic acid

IL interleukin

iNOS inducible nitric oxide synthase

MDA malondialdehyde

MPO myeloperoxidase

SOD superoxide dismutase

GSH glutathione

FBS fetal bovine serum

DMEM Dulbecco’s modified Eagle’s medium

ISCT International Society for Cellular Therapy

CD cluster of differentiation

HLA human leukocyte antigen

NTA nanoparticle tracking analysis

PVDF polyvinylidene difluoride

H&E Hematoxylin and eosin

Lm mean linear intercept

WBC white blood count

K/uL cubic per microliter

DEG Differentially expressed genes

NF-kB nuclear factor kB

GO gene ontology

TGF-β transforming growth factor-β

TNF-alpha tumor necrosis factor-alpha

Map2k1 dual specificity mitogen-activated protein kinase kinase 1

Akt1 RAC-alpha serine/threonine-protein kinase

MAPK mitogen-activated protein kinase

Crebbp creb binding protein

Prkcz protein kinase C zeta

Grb2 growth factor receptor bound protein 2

Src Proto-oncogene tyrosine-protein kinase

Pik3r1 Phosphatidylinositol 3-kinase regulatory subunit alpha

Map2k3 Mitogen-Activated Protein Kinase Kinase 3

Ethical approval

This experimental procedure involving animals was approved by the Animal Ethics Committee of the Universiti Sains Malaysia (No. USM / Animal Ethics Approval / 2016 / (104) (812)). All procedures were followed the Universiti Sains Malaysia’s safety policies. Tissue culture was carried out in compliance with regulations for containment class II pathogens.

Consent for publication

Not applicable

Availability of data and material
Not applicable

Funding
The study was supported by the Universiti Sains Malaysia (USM) Research University Grant (1001/CIPPT/8012203).

Authors' contributions

NR – Designed the experiment, performed experiments, analysed and interpreted the data, prepared draft of the manuscript, finalized the manuscript. NZ – Performed microarray experiments, analysed the data, revised and finalized the manuscript. DW – Guided NR in preparing EVs and characteried the EVs, analysed and interpreted the data, revised and finalized the manuscript. JS – Guided NR in preparing EVs and characteried the EVs, analysed and interpreted the data, revised and finalized the manuscript. MM – Guided NZ in preparing samples for microarray and run the microarray experiment, analysed and interpreted the data, revised and finalized the manuscript. HK – Guided NZ in preparing samples for microarray and run the microarray experiment, analysed and interpreted the data, revised and finalized the manuscript. SAMI – Guided NR analysed the histopathological slides, analysed and interpreted the data, revised and finalized the manuscript. GKKS - Guided NR analysed the histopathological slides, analysed and interpreted the data, revised and finalized the manuscript. KYT – Guided NR in stem cell culture, characterized the MSCs, analysed and interpreted the data, revised and finalized the manuscript. GCO – Guided NR in stem cell culture, characterized the MSCs, analysed and interpreted the data, revised and finalized the manuscript. BHY – Designed the experiment, guided and supervised NR performed experiments, analysed and interpreted the data, the PI for the research funding, revised and finalized the manuscript

Acknowledgements

The author would like to thank the staff in Dr Darius Widera’s lab for helping in hUC-MSCs-EVs characterisation and western blot analysis, staff in Dr Mitsuru Morimoto’s lab for helping with microarray analysis, and the staff in Regenerative Medicine Lab, and staff in Animal Research Facilities (ARF) Advanced Medical and Dental Institute (IPPT) for helping the study.

Competing interests

The authors declare no other financial involvement with any organization or entity with financial interest and/or conflict with matters discussed in the manuscript apart from those disclosed.

Akram, K. M., Samad, S., Spiteri, M. & Forsyth, N. R. (2012). Mesenchymal stem cell therapy and lung diseases. Mesenchymal Stem Cells-Basics and Clinical Application II. Springer.

Al Faraj, A., Shaik, A. S., Afzal, S., Al Sayed, B. & Halwani, R. (2014). MR imaging and targeting of a specific alveolar macrophage subpopulation in LPS-induced COPD animal model using antibody-conjugated magnetic nanoparticles. International journal of nanomedicine, 9, p. 1491.

Antunes, M. A., Abreu, S. C., Cruz, F. F., Teixeira, A. C., Lopes-Pacheco, M., Bandeira, E., Olsen, P. C., Diaz, B. L., Takyia, C. M. & Freitas, I. P. (2014). Effects of different mesenchymal stromal cell sources and delivery routes in experimental emphysema. Respiratory Research, 15(1), p. 118.

Arai, N., Kondo, M., Izumo, T., Tamaoki, J. & Nagai, A. (2010). Inhibition of neutrophil elastase-induced goblet cell metaplasia by tiotropium in mice. European Respiratory Journal, 35(5), p. 1164-1171.

Atkinson, J. J., Lutey, B. A., Suzuki, Y., Toennies, H. M., Kelley, D. G., Kobayashi, D. K., Ijem, W. G., Deslee, G., Moore, C. H. & Jacobs, M. E. (2011). The role of matrix metalloproteinase-9 in cigarette smoke–induced emphysema. American journal of respiratory and critical care medicine, 183(7), p. 876-884.

Aveleira, C. A., Lin, C.-M., Abcouwer, S. F., Ambrósio, A. F. & Antonetti, D. A. (2010). TNF-α signals through PKCζ/NF-κB to alter the tight junction complex and increase retinal endothelial cell permeability. Diabetes, 59(11), p. 2872-2882.

Bich, P. L. T., Thi, H. N., Chau, H. D. N., Van, T. P., Do, Q., Khac, H. D., Le Van, D., Huy, L. N., Cong, K. M. & Ba, T. T. (2020). Allogeneic umbilical cord-derived mesenchymal stem cell transplantation for treating chronic obstructive pulmonary disease: a pilot clinical study. Stem Cell Research & Therapy, 11(1), p. 1-14.

Blazquez, R., Sanchez-Margallo, F. M., de la Rosa, O., Dalemans, W., Alvarez, V., Tarazona, R. & Casado, J. G. (2014). Immunomodulatory Potential of Human Adipose Mesenchymal Stem Cells Derived Exosomes on in vitro Stimulated T Cells. Front Immunol, 5, p. 556.

Borzone, G., Liberona, L., Olmos, P., Sáez, C., Meneses, M., Reyes, T., Moreno, R. & Lisboa, C. (2007). Rat and hamster species differences in susceptibility to elastase-induced pulmonary emphysema relate to differences in elastase inhibitory capacity. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 293(3), p. R1342-R1349.

Burgel, P. & Martin, C. (2010). Mucus hypersecretion in COPD: should we only rely on symptoms? : Eur Respiratory Soc.

Caramori, G., Romagnoli, M., Casolari, P., Bellettato, C., Casoni, G., Boschetto, P., Chung, K. F., Barnes, P., Adcock, I. & Ciaccia, A. (2003). Nuclear localisation of p65 in sputum macrophages but not in sputum neutrophils during COPD exacerbations. Thorax, 58(4), p. 348-351.

Celli, B. R., MacNee, W. & Force, A. E. T. (2004). Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. Eur Respir J, 23(6), p. 932-46.

Churg, A., Cosio, M. & Wright, J. L. (2008). Mechanisms of cigarette smoke-induced COPD: insights from animal models. Am J Physiol Lung Cell Mol Physiol, 294(4), p. L612-31.

Churg, A., Wang, R. D., Tai, H., Wang, X., Xie, C., Dai, J., Shapiro, S. D. & Wright, J. L. (2003). Macrophage metalloelastase mediates acute cigarette smoke–induced inflammation via tumor necrosis factor-α release. American journal of respiratory and critical care medicine, 167(8), p. 1083-1089.

Corotchi, M. C., Popa, M. A., Remes, A., Sima, L. E., Gussi, I. & Plesu, M. L. (2013). Isolation method and xeno-free culture conditions influence multipotent differentiation capacity of human Wharton’s jelly-derived mesenchymal stem cells. Stem cell research & therapy, 4(4), p. 1-16.

D'Agostino, B., Sullo, N., Siniscalco, D., De Angelis, A. & Rossi, F. (2010). Mesenchymal stem cell therapy for the treatment of chronic obstructive pulmonary disease. Expert Opin Biol Ther, 10(5), p. 681-7.

de Castro, L. L., Xisto, D. G., Kitoko, J. Z., Cruz, F. F., Olsen, P. C., Redondo, P. A. G., Ferreira, T. P. T., Weiss, D. J., Martins, M. A. & Morales, M. M. (2017). Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma. Stem cell research & therapy, 8(1), p. 151.

De Jong, O. G., Van Balkom, B. W., Schiffelers, R. M., Bouten, C. V. & Verhaar, M. C. (2014). Extracellular vesicles: potential roles in regenerative medicine. Front Immunol, 5, p. 608.

de Oliveira, M. V. (2016). Animal Models of Chronic Obstructive Pulmonary Disease Exacerbations: A Review of the Current Status. Journal of Biomedical Sciencies, 05(01), p.

Di Stefano, A., Caramori, G., Oates, T., Capelli, A., Lusuardi, M., Gnemmi, I., Ioli, F., Chung, K. F., Donner, C. F., Barnes, P. J. & Adcock, I. M. (2002). Increased expression of nuclear factor- B in bronchial biopsies from smokers and patients with COPD. European Respiratory Journal, 20(3), p. 556-563.

Diaz‐Meco, M. T. & Moscat, J. (2012). The atypical PKCs in inflammation: NF‐κB and beyond. Immunological reviews, 246(1), p. 154-167.

Dong, L. H., Jiang, Y. Y., Liu, Y. J., Cui, S., Xia, C. C., Qu, C., Jiang, X., Qu, Y. Q., Chang, P. Y. & Liu, F. (2015). The anti-fibrotic effects of mesenchymal stem cells on irradiated lungs via stimulating endogenous secretion of HGF and PGE2. Sci Rep, 5, p. 8713.

Etzrodt, M., Cortez-Retamozo, V., Newton, A., Zhao, J., Ng, A., Wildgruber, M., Romero, P., Wurdinger, T., Xavier, R., Geissmann, F., Meylan, E., Nahrendorf, M., Swirski, F. K., Baltimore, D., Weissleder, R. & Pittet, M. J. (2012). Regulation of monocyte functional heterogeneity by miR-146a and Relb. Cell Rep, 1(4), p. 317-24.

Fischer, B. M. & Voynow, J. A. (2002). Neutrophil elastase induces MUC 5AC gene expression in airway epithelium via a pathway involving reactive oxygen species. American journal of respiratory cell and molecular biology, 26(4), p. 447-452.

Flouris, A. D., Poulianiti, K. P., Chorti, M. S., Jamurtas, A. Z., Kouretas, D., Owolabi, E. O., Tzatzarakis, M. N., Tsatsakis, A. M. & Koutedakis, Y. (2012). Acute effects of electronic and tobacco cigarette smoking on complete blood count. Food and chemical toxicology, 50(10), p. 3600-3603.

Fontaine, M. J., Shih, H., Schäfer, R. & Pittenger, M. F. (2016). Unraveling the mesenchymal stromal cells' paracrine immunomodulatory effects. Transfusion medicine reviews, 30(1), p. 37-43.

Fu, W., Xie, X., Li, Q., Chen, G., Zhang, C., Tang, X. & Li, J. (2016). Isolation, characterization, and multipotent differentiation of mesenchymal stem cells derived from meniscal debris. Stem cells international, 2016, p.

Fujisawa, T., Chang, M. M.-J., Velichko, S., Thai, P., Hung, L.-Y., Huang, F., Phuong, N., Chen, Y. & Wu, R. (2011). NF-κB mediates IL-1β–and IL-17A–induced MUC5B expression in airway epithelial cells. American journal of respiratory cell and molecular biology, 45(2), p. 246-252.

Ghorani, V., Boskabady, M. H., Khazdair, M. R. & Kianmeher, M. (2017). Experimental animal models for COPD: a methodological review. Tob Induc Dis, 15, p. 25.

Giridharan, S. & Srinivasan, M. (2018). Mechanisms of NF-κB p65 and strategies for therapeutic manipulation. Journal of inflammation research, 11, p. 407.

Gonzalez-Rey, E., Gonzalez, M. A., Varela, N., O'Valle, F., Hernandez-Cortes, P., Rico, L., Buscher, D. & Delgado, M. (2010). Human adipose-derived mesenchymal stem cells reduce inflammatory and T cell responses and induce regulatory T cells in vitro in rheumatoid arthritis. Ann Rheum Dis, 69(1), p. 241-8.

Greening, D. W., Xu, R., Ji, H., Tauro, B. J. & Simpson, R. J. (2015). A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods. Methods Mol Biol, 1295, p. 179-209.

Gu, W., Song, L., Li, X. M., Wang, D., Guo, X. J. & Xu, W. G. (2015). Mesenchymal stem cells alleviate airway inflammation and emphysema in COPD through down-regulation of cyclooxygenase-2 via p38 and ERK MAPK pathways. Sci Rep, 5, p. 8733.

Guan, X. J., Song, L., Han, F. F., Cui, Z. L., Chen, X., Guo, X. J. & Xu, W. G. (2013). Mesenchymal stem cells protect cigarette smoke-damaged lung and pulmonary function partly via VEGF-VEGF receptors. J Cell Biochem, 114(2), p. 323-35.

Hayes, J., Schuster, M., Grossman, F., Rutman, O. & Itescu, S. (2020). Mesenchymal stem cell therapy improves pulmonary function and exercise tolerance in patients with chronic obstructive pulmonary disease (copd) and high baseline inflammation. Cytotherapy, 22(5), p. S188-S189.

Hayes, M., Curley, G. F., Masterson, C., Devaney, J., O’Toole, D. & Laffey, J. G. (2015). Mesenchymal stromal cells are more effective than the MSC secretome in diminishing injury and enhancing recovery following ventilator-induced lung injury. Intensive care medicine experimental, 3(1), p. 1-14.

He, F., Liao, B., Pu, J., Li, C., Zheng, M., Huang, L., Zhou, Y., Zhao, D., Li, B. & Ran, P. (2017). Exposure to ambient particulate matter induced COPD in a rat model and a description of the underlying mechanism. Scientific reports, 7, p. 45666.

He, Z. H., Chen, P., Chen, Y., He, S. D., Ye, J. R., Zhang, H. L. & Cao, J. (2015). Comparison between cigarette smoke-induced emphysema and cigarette smoke extract-induced emphysema. Tob Induc Dis, 13(1), p. 6.

Hou, H.-H., Cheng, S.-L., Chung, K.-P., Wei, S.-C., Tsao, P.-N., Lu, H.-H., Wang, H.-C. & Yu, C.-J. (2014). PlGF mediates neutrophil elastase-induced airway epithelial cell apoptosis and emphysema. Respiratory research, 15(1), p. 106.

Huh, J. W., Kim, S. Y., Lee, J. H., Lee, J. S., Van Ta, Q., Kim, M., Oh, Y. M., Lee, Y. S. & Lee, S. D. (2011). Bone marrow cells repair cigarette smoke-induced emphysema in rats. Am J Physiol Lung Cell Mol Physiol, 301(3), p. L255-66.

Jin, H. J., Bae, Y. K., Kim, M., Kwon, S.-J., Jeon, H. B., Choi, S. J., Kim, S. W., Yang, Y. S., Oh, W. & Chang, J. W. (2013). Comparative analysis of human mesenchymal stem cells from bone marrow, adipose tissue, and umbilical cord blood as sources of cell therapy. International journal of molecular sciences, 14(9), p. 17986-18001.

Kadota, T., Fujita, Y., Yoshioka, Y., Araya, J., Kuwano, K. & Ochiya, T. (2016). Extracellular vesicles in chronic obstructive pulmonary disease. International journal of molecular sciences, 17(11), p. 1801.

Kagia, A., Tzetis, M., Kanavakis, E., Perrea, D., Sfougataki, I., Mertzanian, A., Varela, I., Dimopoulou, A., Karagiannidou, A. & Goussetis, E. (2019). Therapeutic effects of mesenchymal stem cells derived from bone marrow, umbilical cord blood, and pluripotent stem cells in a mouse model of chemically induced inflammatory bowel disease. Inflammation, 42(5), p. 1730-1740.

Kim, Y.-S., Kim, J.-Y., Cho, R., Shin, D.-M., Lee, S. W. & Oh, Y.-M. (2017). Adipose stem cell-derived nanovesicles inhibit emphysema primarily via an FGF2-dependent pathway. Experimental & molecular medicine, 49(1), p. e284-e284.

Kim, Y. S., Kokturk, N., Kim, J. Y., Lee, S. W., Lim, J., Choi, S. J., Oh, W. & Oh, Y. M. (2016). Gene Profiles in a Smoke-Induced COPD Mouse Lung Model Following Treatment with Mesenchymal Stem Cells. Mol Cells, 39(10), p. 728-733.

Kohri, K., Ueki, I. F. & Nadel, J. A. (2002). Neutrophil elastase induces mucin production by ligand-dependent epidermal growth factor receptor activation. American Journal of Physiology-Lung Cellular and Molecular Physiology, 283(3), p. L531-L540.

Lai, R. C., Arslan, F., Lee, M. M., Sze, N. S., Choo, A., Chen, T. S., Salto-Tellez, M., Timmers, L., Lee, C. N., El Oakley, R. M., Pasterkamp, G., de Kleijn, D. P. & Lim, S. K. (2010). Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res, 4(3), p. 214-22.

Leberl, M., Kratzer, A. & Taraseviciene-Stewart, L. (2013). Tobacco smoke induced COPD/emphysema in the animal model-are we all on the same page? Front Physiol, 4, p. 91.

Lee, C., Mitsialis, S. A., Aslam, M., Vitali, S. H., Vergadi, E., Konstantinou, G., Sdrimas, K., Fernandez-Gonzalez, A. & Kourembanas, S. (2012). Exosomes mediate the cytoprotective action of mesenchymal stromal cells on hypoxia-induced pulmonary hypertension. Circulation, 126(22), p. 2601-11.

Lee, S.-H., Jang, A.-S., Kwon, J.-H., Park, S.-K., Won, J.-H. & Park, C.-S. (2011). Mesenchymal stem cell transfer suppresses airway remodeling in a toluene diisocyanate-induced murine asthma model. Allergy, asthma & immunology research, 3(3), p. 205-211.

Li, X., Wang, J., Cao, J., Ma, L. & Xu, J. (2015). Immunoregulation of Bone Marrow-Derived Mesenchymal Stem Cells on the Chronic Cigarette Smoking-Induced Lung Inflammation in Rats. Biomed Res Int, 2015, p. 932923.

Liu, T., Zhang, L., Joo, D. & Sun, S.-C. (2017). NF-κB signaling in inflammation. Signal transduction and targeted therapy, 2(1), p. 1-9.

Liu, X., Fang, Q. & Kim, H. (2016). Preclinical studies of mesenchymal stem cell (MSC) administration in chronic obstructive pulmonary disease (COPD): a systematic review and meta-analysis. PLoS One, 11(6), p.

Lozano, R., Naghavi, M., Foreman, K., Lim, S., Shibuya, K., Aboyans, V., Abraham, J., Adair, T., Aggarwal, R. & Ahn, S. Y. (2012). Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. The lancet, 380(9859), p. 2095-2128.

Ludwig, A. K. & Giebel, B. (2012). Exosomes: small vesicles participating in intercellular communication. Int J Biochem Cell Biol, 44(1), p. 11-5.

Marciniuk, D., Ferkol, T., Nana, A., de Oca, M. M., Rabe, K., Billo, N. & Zar, H. (2014). Respiratory diseases in the world. Realities of today—opportunities for tomorrow. African Journal of Respiratory Medicine Vol, 9(1), p.

Maremanda, K. P., Sundar, I. K. & Rahman, I. (2019). Protective role of mesenchymal stem cells and mesenchymal stem cell-derived exosomes in cigarette smoke-induced mitochondrial dysfunction in mice. Toxicology and Applied Pharmacology, 385, p. 114788.

Mathias, L. J., Khong, S. M., Spyroglou, L., Payne, N. L., Siatskas, C., Thorburn, A. N., Boyd, R. L. & Heng, T. S. (2013). Alveolar macrophages are critical for the inhibition of allergic asthma by mesenchymal stromal cells. J Immunol, 191(12), p. 5914-24.

Mohammadian, M., Boskabady, M. H., Kashani, I. R., Jahromi, G. P., Omidi, A., Nejad, A. K., Khamse, S. & Sadeghipour, H. R. (2016). Effect of bone marrow derived mesenchymal stem cells on lung pathology and inflammation in ovalbumin-induced asthma in mouse. Iranian journal of basic medical sciences, 19(1), p. 55.

Nie, Y.-C., Wu, H., Li, P.-B., Luo, Y.-L., Zhang, C.-C., Shen, J.-G. & Su, W.-W. (2012). Characteristic comparison of three rat models induced by cigarette smoke or combined with LPS: to establish a suitable model for study of airway mucus hypersecretion in chronic obstructive pulmonary disease. Pulmonary pharmacology & therapeutics, 25(5), p. 349-356.

O’Farrell, H. E. & Yang, I. A. (2019). Extracellular vesicles in chronic obstructive pulmonary disease (COPD). Journal of thoracic disease, 11(Suppl 17), p. S2141.

Panes, J., Garcia-Olmo, D., Van Assche, G., Colombel, J. F., Reinisch, W., Baumgart, D. C., Dignass, A., Nachury, M., Ferrante, M., Kazemi-Shirazi, L., Grimaud, J. C., de la Portilla, F., Goldin, E., Richard, M. P., Leselbaum, A., Danese, S. & Collaborators, A. C. S. G. (2016). Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn's disease: a phase 3 randomised, double-blind controlled trial. Lancet, 388(10051), p. 1281-90.

Phinney, D. G., Di Giuseppe, M., Njah, J., Sala, E., Shiva, S., St Croix, C. M., Stolz, D. B., Watkins, S. C., Di, Y. P. & Leikauf, G. D. (2015). Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs. Nature communications, 6, p. 8472.

Poggio, H. A., Antunes, M. A., Rocha, N. N., Kitoko, J. Z., Morales, M. M., Olsen, P. C., Lopes-Pacheco, M., Cruz, F. F. & Rocco, P. R. (2018). Impact of one versus two doses of mesenchymal stromal cells on lung and cardiovascular repair in experimental emphysema. Stem cell research & therapy, 9(1), p. 296.

Presson Jr, R. G., Brown, M. B., Fisher, A. J., Sandoval, R. M., Dunn, K. W., Lorenz, K. S., Delp, E. J., Salama, P., Molitoris, B. A. & Petrache, I. (2011). Two-photon imaging within the murine thorax without respiratory and cardiac motion artifact. The American journal of pathology, 179(1), p. 75-82.

Rani, S., Ryan, A. E., Griffin, M. D. & Ritter, T. (2015). Mesenchymal Stem Cell-derived Extracellular Vesicles: Toward Cell-free Therapeutic Applications. Mol Ther, 23(5), p. 812-823.

Sarko, D. K. & McKinney, C. E. (2017). Exosomes: Origins and Therapeutic Potential for Neurodegenerative Disease. Front Neurosci, 11, p. 82.

Schweitzer, K. S., Hatoum, H., Brown, M. B., Gupta, M., Justice, M. J., Beteck, B., Van Demark, M., Gu, Y., Presson Jr, R. G. & Hubbard, W. C. (2011). Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides. American Journal of Physiology-Lung Cellular and Molecular Physiology, 301(6), p. L836-L846.

Shao, M. X., Nakanaga, T. & Nadel, J. A. (2004). Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-α-converting enzyme in human airway epithelial (NCI-H292) cells. American Journal of Physiology-Lung Cellular and Molecular Physiology, 287(2), p. L420-L427.

Shapiro, S. D. (1999). The macrophage in chronic obstructive pulmonary disease. Am J Respir Crit Care Med, 160(5 Pt 2), p. S29-32.

Shapiro, S. D., Goldstein, N. M., Houghton, A. M., Kobayashi, D. K., Kelley, D. & Belaaouaj, A. (2003). Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice. The American journal of pathology, 163(6), p. 2329-2335.

Shen, B., Liu, J., Zhang, F., Wang, Y., Qin, Y., Zhou, Z., Qiu, J. & Fan, Y. (2016). CCR2 positive exosome released by mesenchymal stem cells suppresses macrophage functions and alleviates ischemia/reperfusion-induced renal injury. Stem cells international, 2016, p.

Sicco, C. L., Reverberi, D., Balbi, C., Ulivi, V., Principi, E., Pascucci, L., Becherini, P., Bosco, M. C., Varesio, L. & Franzin, C. (2017). Mesenchymal stem cell‐derived extracellular vesicles as mediators of anti‐inflammatory effects: Endorsement of macrophage polarization. Stem cells translational medicine, 6(3), p. 1018-1028.

Silva, J. D., de Castro, L. L., Braga, C. L., Oliveira, G. P., Trivelin, S. A., Barbosa-Junior, C. M., Morales, M. M., Dos Santos, C. C., Weiss, D. J. & Lopes-Pacheco, M. (2019). Mesenchymal stromal cells are more effective than their extracellular vesicles at reducing lung injury regardless of acute respiratory distress syndrome etiology. Stem cells international, 2019, p.

Song, X., Xie, S., Lu, K. & Wang, C. (2015). Mesenchymal stem cells alleviate experimental asthma by inducing polarization of alveolar macrophages. Inflammation, 38(2), p. 485-92.

Stevenson, C. S., Docx, C., Webster, R., Battram, C., Hynx, D., Giddings, J., Cooper, P. R., Chakravarty, P., Rahman, I., Marwick, J. A., Kirkham, P. A., Charman, C., Richardson, D. L., Nirmala, N. R., Whittaker, P. & Butler, K. (2007). Comprehensive gene expression profiling of rat lung reveals distinct acute and chronic responses to cigarette smoke inhalation. Am J Physiol Lung Cell Mol Physiol, 293(5), p. L1183-93.

Stone, M. L., Zhao, Y., Smith, J. R., Weiss, M. L., Kron, I. L., Laubach, V. E. & Sharma, A. K. (2017). Mesenchymal stromal cell-derived extracellular vesicles attenuate lung ischemia-reperfusion injury and enhance reconditioning of donor lungs after circulatory death. Respiratory research, 18(1), p. 212.

Sullivan, K. E., Quinn, K. P., Tang, K. M., Georgakoudi, I. & Black, L. D. (2014). Extracellular matrix remodeling following myocardial infarction influences the therapeutic potential of mesenchymal stem cells. Stem cell research & therapy, 5(1), p. 14.

Tan, C., Xuan, L., Cao, S., Yu, G., Hou, Q. & Wang, H. (2016). Decreased histone deacetylase 2 (HDAC2) in peripheral blood monocytes (PBMCs) of COPD patients. PLoS One, 11(1), p.

Thomi, G., Surbek, D., Haesler, V., Joerger-Messerli, M. & Schoeberlein, A. (2019). Exosomes derived from umbilical cord mesenchymal stem cells reduce microglia-mediated neuroinflammation in perinatal brain injury. Stem Cell Res Ther, 10(1), p. 105.

Weiss, D. J., Casaburi, R., Flannery, R., LeRoux-Williams, M. & Tashkin, D. P. (2013). A placebo-controlled, randomized trial of mesenchymal stem cells in COPD. Chest, 143(6), p. 1590-1598.

Willis, G. R., Fernandez-Gonzalez, A., Anastas, J., Vitali, S. H., Liu, X., Ericsson, M., Kwong, A., Mitsialis, S. A. & Kourembanas, S. (2018). Mesenchymal stromal cell exosomes ameliorate experimental bronchopulmonary dysplasia and restore lung function through macrophage immunomodulation. American journal of respiratory and critical care medicine, 197(1), p. 104-116.

Witwer, K. W., Van Balkom, B. W., Bruno, S., Choo, A., Dominici, M., Gimona, M., Hill, A. F., De Kleijn, D., Koh, M. & Lai, R. C. (2019). Defining mesenchymal stromal cell (MSC)-derived small extracellular vesicles for therapeutic applications. Journal of extracellular vesicles, 8(1), p. 1609206.

Yan, Y., Jiang, W., Tan, Y., Zou, S., Zhang, H., Mao, F., Gong, A., Qian, H. & Xu, W. (2017). hucMSC exosome-derived GPX1 is required for the recovery of hepatic oxidant injury. Molecular Therapy, 25(2), p. 465-479.

Yang, J., Jin, J., Zhang, Z., Zhang, L. & Shen, C. (2013). Integration microarray and regulation datasets for chronic obstructive pulmonary disease. Eur Rev Med Pharmacol Sci, 17(14), p. 1923-1931.

Yang, J., Liu, X.-X., Fan, H., Tang, Q., Shou, Z.-X., Zuo, D.-M., Zou, Z., Xu, M., Chen, Q.-Y. & Peng, Y. (2015). Extracellular vesicles derived from bone marrow mesenchymal stem cells protect against experimental colitis via attenuating colon inflammation, oxidative stress and apoptosis. PloS one, 10(10), p.

Yao, H., Hwang, J.-w., Moscat, J., Diaz-Meco, M. T., Leitges, M., Kishore, N., Li, X. & Rahman, I. (2010). Protein kinase Cζ mediates cigarette smoke/aldehyde-and lipopolysaccharide-induced lung inflammation and histone modifications. Journal of Biological Chemistry, 285(8), p. 5405-5416.

Yao, J., Zheng, J., Cai, J., Zeng, K., Zhou, C., Zhang, J., Li, S., Li, H., Chen, L. & He, L. (2019). Extracellular vesicles derived from human umbilical cord mesenchymal stem cells alleviate rat hepatic ischemia-reperfusion injury by suppressing oxidative stress and neutrophil inflammatory response. The FASEB Journal, 33(2), p. 1695-1710.

Yu, B., Zhang, X. & Li, X. (2014). Exosomes derived from mesenchymal stem cells. Int J Mol Sci, 15(3), p. 4142-57.

Yu, D., Liu, X., Zhang, G., Ming, Z. & Wang, T. (2018). Isoliquiritigenin inhibits cigarette smoke-induced COPD by attenuating inflammation and oxidative stress via the regulation of the Nrf2 and NF-κB signaling pathways. Frontiers in pharmacology, 9, p. 1001.

Zeng, S. L., Wang, L. H., Li, P., Wang, W. & Yang, J. (2015). Mesenchymal stem cells abrogate experimental asthma by altering dendritic cell function. Mol Med Rep, 12(2), p. 2511-20.

Zhang, W.-G., He, L., Shi, X.-M., Wu, S.-S., Zhang, B., Mei, L., Xu, Y.-J., Zhang, Z.-X., Zhao, J.-P. & Zhang, H.-L. (2014). Regulation of transplanted mesenchymal stem cells by the lung progenitor niche in rats with chronic obstructive pulmonary disease. Respiratory research, 15(1), p. 33.

Zheng, H., Liu, Y., Huang, T., Fang, Z., Li, G. & He, S. (2009). Development and characterization of a rat model of chronic obstructive pulmonary disease (COPD) induced by sidestream cigarette smoke. Toxicol Lett, 189(3), p. 225-34.

Zhou, L., Liu, Y., Chen, X., Wang, S., Liu, H., Zhang, T., Zhang, Y., Xu, Q., Han, X. & Zhao, Y. (2018). Over-expression of nuclear factor-κB family genes and inflammatory molecules is related to chronic obstructive pulmonary disease. International journal of chronic obstructive pulmonary disease, 13, p. 2131.

Zhu, Y. g., Feng, X. m., Abbott, J., Fang, X. h., Hao, Q., Monsel, A., Qu, J. m., Matthay, M. A. & Lee, J. W. (2014). Human mesenchymal stem cell microvesicles for treatment of Escherichia coli endotoxin‐induced acute lung injury in mice. Stem cells, 32(1), p. 116-125.

Zulueta, A., Colombo, M., Peli, V., Falleni, M., Tosi, D., Ricciardi, M., Baisi, A., Bulfamante, G., Chiaramonte, R. & Caretti, A. (2018). Lung mesenchymal stem cells-derived extracellular vesicles attenuate the inflammatory profile of cystic fibrosis epithelial cells. Cellular signalling, 51, p. 110-118.

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Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Ameliorate Airway Inflammation in a Rat Model of chronic obstructive pulmonary disease (COPD)

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