Autophagy Regulates the Effects of Adipose-derived Stem Cells Exosomes on Lipopolysaccharide- induced Pulmonary Microvascular Barrier Damage

Chichi Li The First A liated Hospital of Wenzhou Medical University Liqun Li The First A liated Hospital of Wenzhou Medical University Min Wang The First A liated Hospital of Wenzhou Medical University Wangjia Wang The First A liated Hospital of Wenzhou Medical University Yuping Li The First A liated Hospital of Wenzhou Medical University Dan Zhang (  zhangdan6250@yeah.net ) The First A liated Hospital of Wenzhou Medical University https://orcid.org/0000-0001-8174-1238


Background
Sepsis-induced acute lung injury (ALI) is a major cause of acute respiratory distress syndrome, which is a major contributor to high morbidity and mortality. Pulmonary microvascular leakage is one of the characteristics of blood-air barrier dysfunction in ALI [1]. In recent years, multiple experimental and clinical studies have been conducted to clarify the pathogenesis of ALI, and advances have been made in ALI treatment. However, few effective therapies have been developed to improve the outcome of ALI.
Stem cell-related treatments have been shown to be effective in treating injury and the repair of some organs. Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cell that have been identi ed as ideal candidates for cell-based therapies based on their relative abundance and easy accessibility [2]. In addition, some recent studies have shown that ADSCs have much stronger paracrine potential and tolerance under certain stress conditions than other types of stem cells [3,4]. Paracrine components, especially exosomes, have been shown to be vital contributors to the e cacy of stem cell paracrine signaling. Exosomes, which are small membraned vesicles (30-100 nm), originate from multivesicular bodies formed by the inward budding of the endosomal membrane. Exosomes carry complex biologically active components, including proteins, DNA, mRNA and lipids, among which miRNAs have been suggested to have an effective role in mediating exosome functions [5][6][7]. Our previous study showed that ADSCs protect against lipopolysaccharide (LPS)-induced pulmonary microvascular barrier damage [8]. However, the effect of ADSC-derived exosomes (ADSC-Exos) under this condition is still unknown.
Autophagy is a protein and organelle degradation pathway that is pivotal for maintaining cellular homeostasis and promoting survival in response to stress conditions. Recently, the relevance of autophagy to exosomes has been tested. Autophagy affects the production of exosomes, which may be attributed to the link between exosome biogenesis and autophagy via the endolysosomal pathway, and these two processes share common proteins [9][10][11]. In addition, autophagy is a major cellular degradation process that is capable of degrading various biological molecules, including proteins, lipids, and RNA, some of which are the bioactive components of exosomes [12,13]. In a previous study, we indeed found that autophagy regulated the release of certain growth factors from ADSCs in LPS-induced lung injury [8]. Based on the aforementioned ndings, we hypothesized that autophagy regulates exosome function by regulating the bioactive components in exosomes. The goals of this study were to examine the function of ADSC-Exos in LPS-induced pulmonary microvascular endothelial barrier injury and determine the role of autophagy in mediating the effects of ADSC-Exos.
Adipose-derived stem cell culture and treatment Human ADSCs, purchased from Cyagen Biosciences (Santa Clara, CA, USA), were cultured in DMEM. The primary cells were harvested when they had grown to approximately 80% con uence, and then the cells were plated on new culture dishes at approximately 6000 cells/cm 2 . To determine whether autophagy in uenced ADSC-Exo effects on LPS-induced microvascular barrier damage, we constructed ADSC lines with or without autophagy inhibition with an siRNA targeting ATG5. For siRNA transfection, 2 × 10 6 cells were transfected with 50 nM siATG5 using a siRNA transfection reagent system. After 36 h, the autophagy level of the cells was measured. Then, the cells were treated with interleukin (IL)-1β for 6h, and exosomes were collected according to the undermentioned experimental method. ADSCs siATG5 -Exos and ADSCs-Exos represent exosomes derived from ADSCs with and without autophagy inhibition, respectively.

Isolation of exosomes
For exosome isolation, a total exosome isolation kit was used according to the manufacturer's protocol. Brie y, ADSCs were washed with PBS several times and cultured in DMEM supplemented with 10% exosome-free fetal bovine serum. After reaching con uence, the cells were treated with DMEM containing 1 ng/ml recombinant human IL-1β and incubated for 24 h. The culture medium was collected and centrifuged at 300 × g for 15 min at 4°C, followed by centrifugation at 2500 × g for 30 min. The supernatant was then ltered and ultracentrifuged at 100,000 × g for 4 h at 4°C. Then, the pellets were overlaid on a 30% sucrose/D2O cushion and ultracentrifuged at 100, 000 × g for 1 h at 4°C. Finally, the extracted exosomes were collected and resuspended in 200 μl of PBS.
Human pulmonary microvascular endothelial cell culture and in vitro cell groupings Human pulmonary microvascular endothelial cells (PMVECs) (PromoCell, Heidelberg, Germany) were cultured in endothelial cell medium. The cells were detached and transferred to new dishes at a split ratio of 1:2 for further propagation until they grew to con uence (usually 3-5 d). PMVECs at passages 3 to 5 were selected for analysis. PMVECs were divided into four groups as follows: PMVECs, LPS-challenged PMVECs, and LPS-challenged PMVECs cultured with ADSCs-Exos or ADSCs siATG5 -Exos. To mimic LPSinduced lung microvascular injury, PMVECs were incubated in endothelial cell medium supplemented with 10% fetal bovine serum containing 100 ng/ml LPS, followed by the addition of 20 μg/ml exosomes in 100 μl of PBS. After 24 h, the cells were collected for further study.

Identi cation of ADSC-derived exosomes
According to previous reports [14], transmission electron microscopy was used to observe the doublelayer ultrastructure of puri ed ADSC-Exos. Nanoparticle tracking analysis was used to determine the average diameter and concentration of exosomes. The expression of the protein markers TSG101 and CD9 was measured by western blotting.

Protein preparation and immunoblotting
ADSCs or PMVECs were homogenized in RIPA lysis buffer, and then the homogenate was incubated on ice for 45 min and centrifuged at 4°C (12,000 g for 5 min). After determining the protein concentration, the protein was collected and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis at 120 V for 2 h. The proteins in the gels were transferred onto a polyvinylidene di uoride membrane, which was then incubated with speci c primary antibodies, followed by incubation with horseradish peroxidaseconjugated secondary antibody for 1 h. Finally, protein visualization was performed by using Pierce ECL western blotting Substrate and autoradiography. The following primary antibodies were used: anti-LC3B, anti-Beclin-1, anti-ATG5, anti-ZO-1, anti-claudin-5, anti-TSG101, anti-CD9, anti-Bcl-2, anti-Bax and anti-GAPDH. Quantity One 4.6 software was used to analyze the blots. The data were normalized to GAPDH and are expressed as the optical density (OD) integration.

Trans-endothelial permeability assay
PMVECs were cultured on the upper wells in a Transwell system, and FITC-dextran (1 mg/ml, MW 40,000) was added to the top of the wells and allowed to permeate through the PMVEC monolayer. After LPS treatment and ADSC-Exo culture for 6 h, the medium was collected from the lower compartments of the Transwell chambers and replaced with an equal volume of basal cell medium. The uorescence value of FITC-dextran in the medium was determined with a uorescence microplate reader (FLX800TBID, BioTek Instruments, Inc., Winooski, VT, USA) at an excitation wavelength of 492 nm and an emission wavelength of 520 nm.

Detection of PMVEC viability
We used the MTT assay to assess the viability of PMVECs. Each group was analyzed in triplicate at a density of 2000 cells/well. The cells were incubated with 5 mg/ml MTT during the last 4 h of LPS challenge. After removing the supernatant, 100 ml of dimethyl sulfoxide was added to each well, followed by 10 min of shaking to dissolve the crystals. The OD of each well was measured at 490 nm with a spectrophotometer. The experiment was repeated three times in each group.
A LIVE/DEAD viability/cytotoxicity kit was used to further measure cell viability. Brie y, the cells were cultured on sterile glass coverslips as con uent monolayers. Then, 20 ml of 2 mM ethidium homodimer (EthD)-1 was added to 10 ml of PBS and combined with 5 ml of a 4 mM calcein AM solution. The working solution, which contained 2 mM calcein AM and 4 mM EthD-1, was directly added to the cells.
After 15 min, the cells were examined using a confocal laser-scanning microscope.

Detection of apoptosis by ow cytometry
The Annexin V-FITC apoptosis detection kit and ow cytometry were used to determine the apoptosis rate according to the manufacturer's instructions. Brie y, PMVECs were digested with 0.25% trypsin and then rinsed twice with PBS. Then, the cells were resuspended in 1× binding buffer at a concentration of 1×10 6 cells/ml, and 100 µl of the resuspended cell solution was transferred to 5-ml culture tubes. Then, 5 µl of Annexin V-FITC and 5 µl of propidine iodide were added to the culture tubes. The resulting solution was incubated at room temperature in the dark for 15 min, after which 400 µl of 1× binding buffer was added.
The apoptosis rates were analyzed immediately by ow cytometry (BD Biosciences, San Jose, CA, USA).

F-actin labeling
We determined stress ber formation by measuring F-actin using a rhodamine-conjugated phalloidin molecular probe according to the manufacturer's instructions. Cells were treated with 100 ng/ml LPS and ADSC-Exos, xed with 3.7% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100, and nally stained with rhodamine-conjugated phalloidin. The nuclei were labeled with 4',6-diamidino-2phenylindole. The labeled cells were analyzed under a Nikon A1 R laser confocal microscope. We quanti ed F-actin levels by analyzing the percentage of cells containing stress bers in different groups.
Quanti cation of ve speci c miRNAs in exosomes using real-time RT-PCR Total RNA was isolated from exosomes using a GenElute Mammalian Total RNA Kit according to the manufacturer's instructions. cDNA synthesis was executed with PrimeScript reverse transcription reagent kit with gDNA eraser. Reverse-transcription was performed using One Step PrimeScript™ RT-PCR Kit. The sequences of the forward primers used are shown in Table 1. Statistical analysis Data were obtained from at least three separate experiments performed in triplicate. SPSS 13.0 software was used for data processing. The results are shown as the mean ± standard deviation (SD). Differences between groups were determined by one-way analysis of variance and post hoc Bonferroni corrections for multiple comparisons. A P-value < 0.05 was considered to be statistically signi cant.

Results
Effective inhibition of autophagy by siATG5 ATG5 is indispensable in both canonical and noncanonical autophagy. Through siATG5 treatment, we effectively reduced autophagy levels. Western blotting demonstrated that the expression of ATG5 was most effectively diminished in siATG5 439-transfected ADSCs (Fig. 1a), and so siATG5 439 was selected to inhibit autophagy in subsequent experiments. In addition to the expression of ATG5, the expression of LC3-II and Beclin-1, two other essential autophagy proteins, was markedly inhibited by siATG5 (Fig. 1b, c). Morphological assessment via transmission electron microscopy showed that autophagosomes were double-or multimembrane structures that engulfed cytoplasmic components (Fig. 1d). Statistically, the number of autophagosomes per mm 2 of cell cross section in the siATG5-treated group was signi cantly lower than that in the control group (Fig. 1e).

Isolation and characterization of ADSC-derived exosomes
Previous studies have shown that preconditioning mesenchymal stem cells with cytokines or speci c conditioned medium can enhance their paracrine functions, including the effects of exosomes on tissue injury and repair [15][16][17]. In this study, we preconditioned ADSCs for 6 h with IL-1β, one of the vital proin ammatory cytokines induced by LPS, and then collected ADSC-derived extracellular vesicles with an exosome extraction kit. Western blotting demonstrated the presence of the exosomal marker proteins TSG101 and CD9 in these vesicles (Fig. 2a). In addition, the isolated ADSC-derived extracellular vesicles ranged in size from 70-120 nm, as determined by nanoparticle tracking analysis, and IL-1β preconditioning promoted the production of these extracellular vesicles (Fig. 2b). Transmission electron microscopy analysis showed that isolated ADSC-derived extracellular vesicles had a typical cup-shaped morphology in both the control and IL-1β preconditioning groups (Fig. 2c). These ndings indicated that these vesicles ful lled the minimal experimental criteria of exosomes [18]. ADSC-EVs are therefore referred to as ADSC-Exos. We collected ADSC-Exos from IL-1β-preconditioned ADSCs for further experiments.
Autophagy inhibition reduced the protective effect of ADSC-Exos on the expression of tight junction-related proteins To further test the effect of autophagy on exosome function in LPS-induced pulmonary microvascular barrier damage, we extracted equal concentrations of exosomes from ADSCs in the presence or absence of autophagy inhibition. Then, these exosomes were added to PMVECs in the presence of LPS. We found that LPS inhibited the expression of ZO-1 and claudin-5, two critical tight junction-related proteins in PMVEC. ADSC-Exo treatment, however, signi cantly inhibited this change in PMVECs, and autophagy inhibition weakened the effect of ADSC-Exos on the expression of ZO-1 and claudin-5 (Fig. 3a, b).
Autophagy inhibition reduced the protective effect of ADSCderived exosomes on PMVEC apoptosis and viability PMVEC apoptosis has been used as one of the critical assessment indices for LPS-induced pulmonary microvascular barrier damage [19]. Flow cytometry showed that LPS markedly increased the percentage of endothelial cell apoptosis, which was effectively reduced by ADSC-Exos. Autophagy inhibition, however, signi cantly weakened the function of ADSC-Exos (Fig. 4a, b). In addition, we measured the expression of Bax and Bcl-2, which are classic pro-and antiapoptotic proteins, respectively. LPS promoted the expression of Bax and reduced the expression of Bcl-2; ADSC-Exos inhibited the expression of Bax and promoted that of Bcl-2 under LPS stimulation. Autophagy inhibition weakened these effects of ADSC-Exos (Fig. 4 c, d).
Cell viability was measured to further test the effect of autophagy on exosome function. A LIVE/DEAD viability/cytotoxicity kit was used to investigate cell viability. As shown, LPS treatment signi cantly increased the percentage of dead cells, which were characterized by PI staining of the nuclei. Exosome pretreatment markedly alleviated LPS-induced cell death. However, autophagy inhibition markedly weakened exosome-mediated abrogation of cell death (Fig. 5 a, b). In addition, the MTT assay was used to further test cell viability. LPS signi cantly reduced cell viability, which was apparently alleviated by ADSC-Exos. Autophagy inhibition reduced the effect of exosome (Fig. 5c).
Autophagy inhibition reduced the protective effect of ADSC-Exos on pulmonary microvascular permeability Microvascular permeability has been used as one of the representative indices to assess pulmonary microvascular barrier integrity [20]. In this study, we found that LPS stimulation for 6 h or 12 h increased microvascular endothelial cell permeability, which was signi cantly reduced by exosome treatment. Autophagy inhibition markedly weakened the effect of ADSC-Exos on LPS-induced microvascular permeability (Fig. 6).

Autophagy inhibition reduced the effect of ADSC-Exos on the LPS-induced formation of stress bers in PMVECs
Previous studies have shown that LPS induces F-actin polymerization to form contractile actin bundles and stress bers. The contraction of stress bers leads to the formation of intercellular gaps that increase the permeability of the endothelial barrier [21,22]. To test whether LPS-induced stress ber formation could be regulated by exosomes, we incubated endothelial cells with exosomes from ADSCs with or without autophagy inhibition under LPS stimulation. As shown, LPS signi cantly increased the formation of actin stress bers, and this effect was signi cantly inhibited by exosomes. However, autophagy inhibition reduced the effect of exosomes on stress ber formation (Fig. 7a). We further quanti ed the percentage of cells containing stress bers in the different groups. LPS treatment markedly increased the proportion of cells containing stress bers, which was effectively decreased by ADSC-Exo treatment. However, autophagy inhibition reduced the effect of ADSC-Exos on the formation of stress bers (Fig.   7b).

Autophagy affected the expression of speci c miRNAs from ADSC-Exos
To test the effect of autophagy on bioactive components transferred by ADSC-Exos, we detected the expression changes of some miRNAs (let-7-a-1, miR-21a, miR-143, miR-145a and miR-451a) those have been found in ADSC-Exos [23]. We measured the expression pro le of aforementioned miRNAs in ADSC-Exos with or without autophagy inhibition. IL-1β treatment increased the expression of miR-21a and decreased that of let-7-a-1, miR-143 and miR-145a, but did not affect the expression of miR-451a. Interestingly, autophagy inhibition weakened the expression of all these miRNAs under IL-1β stimulation (Fig. 8).

Discussion
In this study, we found that ADSC-Exos protect against LPS-induced pulmonary microvascular barrier damage by alleviating apoptosis and reducing the loss of the tight junction-related proteins ZO-1 and claudin-5. Autophagy is one of the essential regulators of the protective effect of exosomes by affecting the expression pro les of at least the aforementioned ve speci c miRNAs within ADSC-Exos.
Exosomes are one of the pivotal components of stem cell paracrine and have been shown to be much more effective in some organ injuries and repairs than direct stem cell differentiation. Under normal conditions, most cells can secrete exosomes; however, pathogens or other stress stimuli may promote exosome secretion and/or alter exosomal contents [24][25][26]. Hypoxic preconditioning enhanced the protective effect of bone marrow stromal cells-derived exosomes against acute myocardial infarction [27]. Ischemic preconditioning can potentiate the protective effect of marrow stromal cells-derived exosomes on endotoxin-induced acute lung injury [28]. In addition, LPS pretreatment not only induced exosome secretion by macrophages but also enhanced the effects of macrophage-derived exosomes on the proliferation and activation of hepatic stellate cells [29]. Similarly, in the present study, we showed that preconditioning with IL-1β, one of the key proin ammatory factors induced by LPS, promoted the production of ADSC-Exos and affected the expression of miRNAs in exosomes. Based on these studies, IL-1β preconditioning is a viable option to enhance exosome functions and protect against LPS-induced lung injury.
In the present study, we found that exosome treatment signi cantly reduced endothelial cell apoptosis, which is one of the classic characteristics of LPS-induced endothelial barrier damage. Our ndings are consistent with those of previous studies. The administration of exosomes to staurosporine-treated Chinese hamster ovary cells effectively alleviated apoptosis and enhanced cellular viability [30]. In a skin lesion model, ADSC-Exos inhibited HaCaT cell apoptosis and promoted cell proliferation to accelerate cutaneous wound healing [31]. However, exosomes released from different types of cells have different biological effects, and different stress stimuli may trigger different functions in homologous exosomes. Some researchers found that tumor-derived exosomes carrying immunosuppressive factors can induce apoptosis in activated CD8 + cells and NK cells to suppress immunotherapy e cacy [32]. These ndings suggest that the effects of exosomes on target cell apoptosis are not uniform and that the origin and condition may be key regulatory factors.
The bioactivity of exosomes is ultimately attributed to their protein and nucleic acid components.
miRNAs are the most numerous cargo molecules in exosomes; they are selectively sorted into exosomes and transferred to recipient cells, where they mediate some target mRNAs and cell functions. In the present study, we found that stimulation with IL-1β increased the expression of miR-21a and decreased that of let-7-a-1, miR-143 and miR145a, but did not affect the expression of miR-451a. These ndings hinted more than one miRNA participate in regulating exosomes effects on alleviating LPS-induced endothelial barrier damage. Although many previous studies have highlighted the pivotal effects of miRNAs on exosomes, many of these studies focused on the function of speci c miRNAs. Our nding suggests that exosome functions are likely to be due to the cooperative effects of various miRNAs. It is essential for us to implement further studies to clarify the relevance and crosstalk among at least aforementioned four speci c miRNAs that are altered in ADSC-Exos under IL-1β conditions.
Autophagy, which is a lysosomal-dependent degradation and recycling pathway, has traditionally been suggested to maintain protein, lipid and organelle homeostasis. Recently, autophagy has been identi ed as one of the vital mediators of exosome biogenesis and function. We found that the same concentration of exosomes collected from ADSCs in the presence or absence of autophagy inhibition had different protective effects on LPS-induced pulmonary microvascular endothelial barrier damage. Autophagy inhibition partly weakened the protective effect of ADSC-Exos, as indicated by increases in the apoptosis rate and stress ber formation but reduced expression of tight junction-related proteins in endothelial cells. These data provide extremely strong evidence to suggest that autophagy can affect exosome functions. Our ndings are consistent with those of previous studies. Autophagy regulation modulates the effect of retinal astrocyte-derived exosomes on the proliferation and migration of endothelial cells [33]. On the one hand, autophagy shares molecular machinery with exosome biogenesis, and there is substantial crosstalk between these two processes [34]. In addition to its traditional roles in maintaining protein, lipid and organelle homeostasis, increasing evidence indicates that autophagy can impact RNA homeostasis. Autophagy can degrade RNA, RNA-binding proteins and ribonucleoprotein complexes beyond its other degradative capabilities [35,36]. In the present study, we found that autophagy inhibition lowered the expression of let-7-a-1, miR-21a, miR-143, miR145a and miR-451a under IL-1β stimulation in ADSC-Exos. This result is likely to explain why autophagy affects exosome functions. Further studies are essential to classify the mechanism by which autophagy mediates exosomal miRNA expression.

Conclusion
In conclusion, we showed that ADSC-Exos were bene cial in maintaining pulmonary microvascular barrier integrity. Autophagy inhibition affected the expression levels of let-7-a-1, miR-21a, miR-143, miR145a and miR-451a and mediated the protective effects of ADSC-Exos on LPS-induced damage to the lung microvascular endothelial barrier. These results provide new insights into the roles and mechanisms mediating ADSC-Exos in LPS-induced acute lung injury and suggest that the regulation of autophagy might be a potential strategy for modulating the treatment e cacy of ADSC-Exos in lung injury.       The permeability of PMVECs was measured through a Transwell assay. LPS increased the permeability of endothelial cells, and this effect was signi cantly alleviated by ADSC-Exo treatment. Autophagy inhibition weakened the protective effect of ADSC-Exos against LPS-induced endothelial permeability.
The experiment was repeated three times.

Figure 7
The effect of ADSC-Exos with or without autophagy inhibition on stress ber formation. a Representative uorescent images showing stress bers labeled with F-actin staining. Nuclei were stained with DAPI. b Statistical analysis of the percentage of cells containing stress bers in each experimental group. The results are expressed as the mean ± SD of three independent experiments.