Mesenchymal stem cell–derived conditioned medium attenuate angiotensin II‐induced aortic aneurysm growth by modulating macrophage polarization

Abstract Mesenchymal stem cells (MSCs) exhibit therapeutic benefits on aortic aneurysm (AA); however, the molecular mechanisms are not fully understood. The current study aimed to investigate the therapeutic effects and potential mechanisms of murine bone marrow MSC (BM‐MSCs)–derived conditioned medium (MSCs‐CM) on angiotensin II (AngII)‐induced AA in apolipoprotein E‐deficient (apoE−/−) mice. Murine BM‐MSCs, MSCs‐CM or control medium were intravenously administrated into AngII‐induced AA in apoE−/− mice. Mice were sacrificed at 2 weeks after injection. BM‐MSCs and MSCs‐CM significantly attenuated matrix metalloproteinase (MMP)‐2 and MMP‐9 expression, aortic elastin degradation and AA growth at the site of AA. These treatments with BM‐MSCs and MSCs‐CM also decreased Ly6chigh monocytes in peripheral blood on day 7 and M1 macrophage infiltration in AA tissues on day 14, whereas they increased M2 macrophages. In addition, BM‐MSCs and MSCs‐CM reduced MCP‐1, IL‐1Ra and IL‐6 expression and increased IL‐10 expression in AA tissues. In vitro, peritoneal macrophages were co‐cultured with BM‐MSCs or fibroblasts as control in a transwell system. The mRNA and protein expression of M2 macrophage markers were evaluated. IL‐6 and IL‐1β were reduced, while IL‐10 was increased in the BM‐MSC systems. The mRNA and protein expression of M2 markers were up‐regulated in the BM‐MSC systems. Furthermore, high concentration of IGF1, VEGF and TGF‐β1 was detected in MSCs‐CM. Our results suggest that MSCs‐CM could prevent AA growth potentially through regulating macrophage polarization. These results may provide a new insight into the mechanisms of BM‐MSCs in the therapy of AA.


| INTRODUC TI ON
formation by increasing M2 macrophage polarization. 6 Elastin-derived peptides (EDPs) promoted a pro-inflammatory environment in aortic tissues and attenuated aortic dilation through induction of M1 polarization, and neutralization of EDPs. Furthermore, injection of M2-polarized macrophages reduced AA expansion. 7 According to a recent study, CD31 agonist P8RI induced M2 macrophage polarization and promoted the healing of experimental dissected AA in apo E −/− mice. 8 This evidence indicates that macrophage polarization might be a potential target for the treatment of AA.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) exert immunosuppressive effects and represent a promising treatment in regenerative medicine. Increasing evidence supports that MSCs provide the therapeutic potential to inhibit AA progression via paracrine effect. 9-12 BM-MSC-derived conditioned medium (MSCs-CM) is thought to be a potent secretory component of BM-MSCs containing specific cytokines, growth factors and small RNAs, which play an important role in MSC-mediated therapeutic effects. 13 Several recent studies have demonstrated that administration of MSCs-CM was as effective as BM-MSCs treatment itself in animal models of tissue injury and inflammatory diseases. [14][15][16][17] Recent studies have disclosed that BM-MSCs reduced inflammatory response and promoted tissue repair by inducing macrophage phenotype conversion from M1 to M2 in various disease models. [18][19][20][21][22] Previously, Hashizume and colleagues have reported that BM-MSCs attenuated AngII-induced AA growth in apoE −/− mice by surgically implanting BM-MSCs sheet on the surface of aortic adventitial. 9 Recently, our group have reported that intravenous administration of BM-MSCs prevented AngII-induced AA development in apoE −/− mice. 23,24 These experiments demonstrated the effects of BM-MSCs on the treatment of AA.
Nonetheless, the underlying mechanisms are not fully understood and the effects of MSCs-CM on the development of AA remain to be determined. Based on these previous studies, in the present study, we demonstrated that MSCs-CM could effectively alleviate AngIIinduced AA growth in apoE −/− mice compared with BM-MSCs, and these protective effects contributed at least partially to modulating macrophage polarization.

| Macrophages and BM-MSC culturing
Bone marrow MSCs (BM-MSCs) were isolated and cultured using standard protocols. 23 The femurs from eight-week-old apoE −/− mice were flushed with PBS to collect bone marrow cells. Cells were then cultured in T25 cell culture flasks in low glucose Dulbecco's modified Eagle's medium containing 15% FBS (Invitrogen) at 37°C with 5% CO 2 . After 4 days, culturing medium was changed to remove the non-adherent cells and subsequently at three-day intervals. The adherent cells were trypsinized with 0.25% trypsin-EDTA (Invitrogen) and passaged into new flasks for further expansion when the cells reached 80%-90% confluence; at passage 3-5, BM-MSCs were used for the detection of immunophenotype and multipotent differentiation potential. For murine embryonic fibroblast (MEF) isolation, uteri isolated from 13.5 days pregnant mice were collected and washed with PBS. The head and visceral tissue were removed from each embryo. The remainder of the bodies were washed in fresh PBS, minced with a pair of scissors, transferred into a 0.25-mmol/L trypsin/ 1mmol/L EDTA solution (3 mL per embryo) and incubated at 37°C for 20 minutes. After incubation, an additional 3 mL PBS was added and then the mixture was incubated at 37°C for 20 minutes. After trypsinization, an equal amount of medium (6 mL per embryo of DMEM containing 10% FBS) was added and aspirated with a pipette to achieve tissue dissociation. Cells were filtered through a mesh, seeded at 1 × 10 6 cells per 10-cm tissue culturing dish and incubated at 37°C in 5% CO 2 . To prepare peritoneal macrophages, apoE −/− mice were injected intraperitoneally with 1 mL of 3% thioglycollate medium (Sigma). After 3 days, cells were harvested from the peritoneal cavities by lavage.

| Preparation of bone marrow MSCs-CM
BM-MSCs of passage 2-3 were used to prepare the MSCs-CM. Upon 70%-80% of confluence, culture medium was removed and cells were washed with PBS before feeding with 12 mL of DMEM supplemented with 1% FBS and 1% penicillin/streptomycin. After 48 hours of incubation, the cell culture-conditioned medium was collected and was centrifuged (8000 g, 5 minutes at 4°C) and filtered through a 0.2-mm-pore size filter to remove cell debris. Then, the conditioned medium was concentrated using ultrafiltration with a cut-off of 10 kD (Millipore). The collected culture-conditioned medium was defined as MSCs-CM and was stored at −80°C before being used for the following experiments.

| Measurement of elastin content in aortic tissue
The elastin amounts of the part of aorta with maximum diameter harvested from apoE −/− mice were quantified using a Fastin elastin assay kit (Biocolor, County Antrim) as previously described. 23  F I G U R E 1 Schema of in vivo protocol. AA was induced by Ang II infusion for 2 wk in ApoE −/− mice; 1 × 10 6 BM-MSCs (in 0.2 mL saline) were transplanted via tail vein on day 0, and day 7. 0.2 mL MSCs-CM or 0.2 mL DMEM alone was injected via the tail vein on day 0, day 3, day 6, day 9 and day 12. Peripheral blood was collected on day 7 for flow cytometry. Instead of AngII administration, saline was used as sham. All mice were sacrificed, and AA tissue was evaluated on day 14

| Immunostaining
The part of aorta with maximum diameter were harvested on day 14 and embedded in Tissue-Tek OCT compound (SAKURA 4583, Sakura

| Aortic cytokine measurements
The part of aorta with maximum diameter were snap-frozen and analysed using a Proteome Profiler Mouse Cytokine Array Panel A Kit (R&D Systems; catalogue number ARY006). The tissues were processed according to the manufacturer's protocol. The densitometric volume was determined by spectrophotometry using Thermo Scientific software (Thermo Fisher Scientific). All the procedures were performed strictly according to the manufacturers' instructions.

| Direct or transwell co-culture
For direct co-culture experiments, 2 × 10 5 macrophages were washed with PBS and 2 × 10 5 BM-MSCs or 2 × 10 5 MEFs were plated directly in six-well plate and cultured for 48 hours. For transwell co-culturing, 1 × 10 5 peritoneal macrophages were seeded into a six-well plate. The next day, the 0.4-mm-pore size Corning transwell inserts (Sigma-Aldrich) containing 2 × 10 5 BM-MSCs or 2 × 10 5 MEFs were placed into the six-well plate with the macrophages that were initially seeded.

| Western blotting
The Western blotting analysis was performed as previously de-

| Statistics analysis
The statistical significance of differences between groups was calculated by chi-square test or unpaired t test, as appropriate, using GraphPad Prism 5.0 for Windows. The data are expressed as the mean ± SEM. Values were considered significantly different when P < .01.

| MSCs-CM attenuated AngII-induced aortic aneurysm growth
No deaths were observed in any of the mice in this study. The morphology of the aorta (maximum aortic diameter) in the saline, control medium, BM-MSCs and MSCs-CM groups are shown in Figure 2A. Typical AA could be observed in the control medium.
The mean of the maximum aortic diameter from the control medium group (2.626 ± 0.05 mm) was much larger than that from the BM-MSCs group (1. 62 ± 0.06 mm) and the MSCs-CM group (1.528 ± 0.13 mm; Figure 2B). These data indicated that both BM-MSCs and MSCs-CM could attenuate AngII-induced AA progression.

| BM-MSCs and MSCs-CM prevented AngII-induced aortic elastin degradation and MMPs expression
Representative images of elastic lamellae using EVG staining from four groups are shown in Figure 2C.

| BM-MSCs and MSCs-CM decreased subpopulation of CD45 + CD11b + Ly6c high monocytes
In the preliminary experiments, we observed that the subpopulation of CD45 + CD11b + Ly6c high monocytes in peripheral blood increased from Day 1 and peaked on Day 7 during AngII infusion (data not shown). In the present study, we measured the CD45 + CD11b + Ly6c high subpopulation of monocytes in peripheral blood on day 7. We found that mean percentage of inflammation-related monocytes (CD45 + CD11b + Ly6c high ) among the total leucocytes was significantly decreased in the peripheral blood in the BM-MSCs (7.567% ± 1.3) and MSCs-CM (7.323% ± 0.82) group compared with the control medium group (17.98% ± 0.92; Figure 3).

| BM-MSCs and MSCs-CM increased M2 macrophage and decreased M1 macrophage in aortic aneurysm
To further clarify the mechanisms associated with the effects of  Figure 4A,B).

| BM-MSCs and MSCs-CM inhibited expression of pro-inflammatory cytokines in AA tissues
Cytokines expression in the AA tissues was determined using the   (Table 2).

| D ISCUSS I ON
In the current study, we provide the first evidence that MSCs-CM

| CON CLUS IONS
Taken together, our results demonstrate that MSCs-CM could effectively alleviate AngII-induced AA growth in apoE −/− mice, and these protective effects contributed at least partially to modulating M1/M2 macrophage polarization. These findings may provide a novel insight into the mechanisms of BM-MSCs based treatment for AA. MSCs-CM could be a more optimized option for treating AA and resolving the clinical application disadvantages associated with BM-MSCs transplantation. We believe that MSCs-CM may serve as a basis of a novel cell-free therapeutic approach for AA.

ACK N OWLED G EM ENTS
This work was financially supported by the Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
XMZ and XMF designed the experiment and interpreted results.
YZZ and ZC drafted manuscript. YZZ, CZ, YW, QYW and YZ performed experiments. XBL, JML, XMF and XMZ made critical revision to manuscript. All authors have read and approved the final manuscript. XMZ, and XMF are co-corresponding authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.