Abstract

Background

   Mesenchymal stromal cell (MSC)-derived exosomes (MSC-Exo) have been
   recognized for their significant role in regulating macrophage
   polarization, a process crucial to the pathogenesis of abdominal aortic
   aneurysm (AAA). However, the therapeutic effects of MSC-Exo on AAA
   remain largely unexplored. Therefore, this study aimed to investigate
   the functional and mechanistic aspects of MSC-Exo in the progression of
   AAA.

Methods

   The MSC-derived exosomes were characterized using Transmission Electron
   Microscopy, Nanoparticle Tracking Analysis, and Western blotting. An
   experimental mouse model of AAA was established through the
   administration of angiotensin II (Ang II) in male apoe^−/− mice and
   calcium chloride (CaCl[2]) in male C57/B6 mice, with subsequent tail
   vein injection of exosomes to evaluate their efficacy against AAA.
   Macrophage polarization was assessed using immunofluorescence staining
   and WB analysis. Mechanistic analysis was performed using 4D Label-free
   Proteomics analysis.

Results

   We found that intravenous administration of MSC-Exo induced M2
   polarization of macrophages within an inflammatory environment,
   effectively impeding AAA development in Ang II or CaCl[2]-induced AAA
   model. The therapeutic efficacy of MSC-Exo treatment was dependent on
   the presence of macrophages. Mechanistically, MSC-Exo suppressed the
   levels of cluster of differentiation 74 (CD74), modulating macrophage
   polarization through the TSC2-mTOR-AKT pathway. These findings
   highlight the potential of MSC-Exo as a therapeutic strategy for AAA by
   modulating macrophage polarization.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s13287-024-03808-y.

   Keywords: Abdominal aortic aneurysm, Mesenchymal stromal cells,
   Exosomes, Macrophage polarization, Inflammation

Introduction

   Abdominal aortic aneurysm (AAA) is characterized by the localized
   dilation of the abdominal aorta, reaching or exceeding 50% of the
   normal arterial diameter [[45]1]. This condition poses a severe threat
   to life, as rupture of the aorta can lead to mortality rates as high as
   90% [[46]2]. Despite notable progress in surgical interventions for
   AAA, effective therapeutic agents remain limited, underscoring the
   imperative to enhance our comprehension of the underlying disease
   mechanisms.

   Inflammation assumes a pivotal role in the advancement of AAA, which is
   increasingly acknowledged as a fundamentally chronic inflammatory
   disorder [[47]3]. This inflammatory response arises from the
   accumulation and activation of various inflammatory cell populations,
   including macrophages [[48]4]. During the initial stages of AAA
   development, M1 macrophages contribute to disease progression by
   generating pro-inflammatory cytokines and matrix metalloproteinases,
   thereby fostering AAA formation. Subsequently, M2 macrophages
   demonstrate anti-inflammatory properties and contribute to matrix
   remodeling and tissue repair [[49]5]. Notably, investigations have
   revealed that Circular RNA Cdyl facilitates the development of AAA by
   promoting M1 macrophage polarization and instigating an M1-type
   inflammatory response [[50]6]. Moreover, the transition from
   pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages has
   been implicated in modulating vascular inflammation within AAA [[51]7].
   Accordingly, early intervention targeting macrophage polarization holds
   promise for impeding excessive AAA progression or rupture, rendering it
   a potential therapeutic target for AAA treatment.

   Mesenchymal stromal cells (MSCs) possess multipotent characteristics
   and exhibit regenerative and immunomodulatory functions [[52]8].
   Notably, in recent years, MSC-based therapy has demonstrated promising
   efficacy in various cardiovascular ailments. Numerous studies have
   substantiated the inhibitory effects of MSCs on AAA formation through
   their modulation of the inflammatory response and mitigation of aortic
   elastin degradation [[53]9, [54]10]. Moreover, emerging evidence
   underscores the capacity of MSC transplantation to promote M2
   macrophage polarization [[55]11]. The therapeutic benefits attributed
   to MSCs primarily arise from their paracrine effects, wherein they
   release various factors that contribute to tissue repair and immune
   regulation [[56]12]. MSC-derived exosomes (MSC-Exo), lipid bilayer
   extracellular vesicles secreted by MSCs, play a critical role in
   promoting the therapeutic effects of MSCs [[57]13]. Preclinical
   research suggests that MSC-Exo exhibit anti-inflammatory and
   immunomodulatory properties [[58]14, [59]15]. However, it is unknown
   whether MSC-Exo could protect against AAA by inducing macrophages
   polization towards an anti-inflammatory phenotype. Previous research
   has indicated that the TSC2-mTOR-AKT pathway, a crucial metabolic
   signaling pathway, plays a significant role in modulating macrophage
   polarization [[60]16]. In this study, we utilized an AngII and
   CaCl[2]-induced AAA model and found that MSC-Exo effectively attenuated
   AAA formation by regulating macrophage polarization. This was achieved
   through down-regulation of CD74, leading to the inhibition of PKM2
   expression and modulation of the TSC2-mTOR-AKT pathway.

Methods

Animal experimental protocol

   Apoe^−/− and C57BL/6 mice (8–10 weeks old) were procured from the Model
   Animal Research Centre of Nanjing University. The mice were housed
   under a 12-hour dark/light cycle with controlled environmental
   conditions of temperature (22 ± 1℃) and humidity (65–70%). They were
   provided with standard laboratory diet and water ad libitum. The mice
   were anesthetized using isoflurane (1.5-2%) and cervical dislocation
   was employed for euthanizing the mice. The work has been reported in
   line with the ARRIVE guidelines 2.0.

Cell experimental protocol

   Peritoneal macrophages were exposed to lipopolysaccharide (LPS,
   Sigma-Aldrich) at a concentration of 100 ng/ml for 6 h to induce the M1
   phenotype. Following this, the culture medium was replaced with fresh
   medium. The cells were then incubated with either MSC-Exo at a
   concentration of 40 µg/ml or phosphate-buffered saline (PBS) for an
   additional 24 h. Untreated cells served as the negative control. After
   24 h, the supernatants were collected for ELISA assay, while the cells
   were harvested for immunofluorescence staining as well as RNA and
   protein extraction.

Exosome isolation and characterization

   When mesenchymal stem cells (MSCs) reached 70–80% confluency, the
   culture medium was replaced with medium containing 5% exosome-depleted
   fetal bovine serum (FBS). The cells were then cultured for 48 h.
   Exosomes were isolated using a differential centrifugation method: the
   cell culture supernatants were first centrifuged at 3,000 g for 25 min
   and then at 10,000 g for 1 h at 4 °C to remove cellular debris and
   shedding vesicles. Subsequently, the supernatant was centrifuged at
   100,000 g for 3 h at 4 °C, and the resulting pellet was resuspended in
   phosphate-buffered saline (PBS).

   The morphology of exosomes was visualized using a transmission electron
   microscope (JEM-1011, Japan). The concentration and size of the
   exosomes were assessed using the NanoSight NS300 system (Malvern, UK).
   Protein markers including CD63, CD9, and TSG101 were evaluated by
   western blot to confirm the presence of exosomes. The total protein
   concentration of the exosomes was quantified using the BCA assay
   (Thermo Scientific).

Statistical analysis

   GraphPad Prism (version 9.5) software was employed for all statistical
   analyses. Quantitative data are presented as mean ± standard deviation
   (SD). Comparisons between multiple groups were performed using one-way
   ANOVA, while comparisons between two groups were conducted using
   Student’s t-test. The D’Agostino-Pearson omnibus normality test or the
   Shapiro-Wilk normality test was utilized to assess whether the data
   followed a normal distribution. Statistical significance was considered
   at a P value < 0.05, and all tests were two-tailed. The significance
   levels were denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001,
   ****P < 0.0001; ns represented non-significant results.

   Specific experimental methods are detailed in the supplementary
   materials section of our study. Please refer to the supplementary
   materials for a comprehensive description of the experimental
   procedures.

Results

The characterization of MSC-Exo

   Exosomes were purified from MSC culture supernatants by gradient
   centrifugation and characterised by transmission electron microscopy,
   nanoparticle tracking analysis (NTA) and western blot analysis. First,
   transmission electron microscopy showed that the isolated vesicles had
   a bilayer membrane structure with a typical cup-shaped morphology
   (Fig. [61]1A). NTA showed that the size distribution of the vesicles
   ranged from 50 to 150 nm (Fig. [62]1B). Western blot analysis of the
   vesicles further confirmed the expression of exosomal markers such as
   CD63, CD9, and TSG101 (Fig. [63]1C). These data indicated that what we
   had isolated from MSC supernatants were indeed exosomes.

Fig. 1.

   [64]Fig. 1
   [65]Open in a new tab

   The MSC-derived exosomes were characterized in this study. (A)
   Transmission electron microscopy was performed to visualize the
   morphology of MSC-derived exosomes, showing images at different scales
   (1 μm, 500 nm, and 200 nm). (B) Nanoparticle tracking analysis was used
   to determine the size distribution of MSC-derived exosomes. (C) Western
   blot analysis was conducted to confirm the presence of exosomal
   markers, including CD63, CD9, and TSG101, in the conditioned media
   (CM), MSC lysates, and MSC-derived exosomes. Full-length blots/gels are
   presented in Supplementary Fig. [66]1

MSC-Exo attenuates angiotensin II (AngII)-induced AAA formation and promotes
M2 macrophage polarization in Apoe^−/− mice

   To evaluate the impact of MSC-Exo on angiotensin II (AngII)-induced AAA
   formation, we investigated the duration of MSC-Exo presence in mice. In
   vivo imaging using DiR-labeled MSC-Exo revealed effective uptake and
   sustained presence in the mouse for up to 7 days (Fig. [67]2A, B).
   Thus, intravenous administration of MSC-Exo or phosphate-buffered
   saline (PBS) was performed every 7 days, starting from the day of AngII
   injection. Aortic tissue samples were collected on day 28 after AngII
   injection. Due to budget constraints, we used a total of 25 mice for
   the experiment. This sample size is sufficient to support our
   conclusions.25 male apoe^−/− mice were randomly assigned into three
   groups: control group, AngII + PBS group, and AngII + Exo group. The
   AngII + Exo group received intravenous injection of MSC-Exo (n = 10) at
   a dose of 3 µg/g, while the AngII + PBS group received the same dose of
   PBS (n = 10), both concurrent with AngII infusion (1000 ng/kg/min) for
   4 weeks. An additional 5 male apoe^−/− mice were injected with PBS and
   infused with saline for 4 weeks as controls. No AAA formation was
   observed in the control group following tail vein injection with PBS
   (Fig. [68]2C, D). In the AngII + Exo group, the incidence of AAA was
   50% (5/10), significantly lower than the AngII + PBS group (70%, 7/10)
   (Fig. [69]2D). Abdominal ultrasound examination further confirmed that
   the maximal diameter of the abdominal aorta in the AngII + PBS group
   was significantly higher compared to the control group(2.44 verus
   0.91 ± 0.4285 cm, 95%CI:0.6043 to 2.456), while intervention with
   MSC-Exo noticeably decreased the maximal diameter of the abdominal
   aorta in AngII-induced mice (2.44 verus 1.49 ± 0.3678 cm,95%CI:-1.726
   to -0.1737) (Fig. [70]2E). Moreover, histological analysis revealed
   reduced degradation of elastic fibers in the vessel wall of AngII + Exo
   mice compared to AngII + PBS mice (Fig. [71]2F). These findings
   demonstrate that MSC-Exo mitigated the development of AngII-induced
   AAA.

Fig. 2.

   [72]Fig. 2
   [73]Open in a new tab

   The effects of intravenous administration of MSC-Exo on AngII-induced
   AAA formation and macrophage polarization in mice. (A) In vivo imaging
   was performed after injecting DiR-labeled MSC-Exo into the mouse tail
   veins, showing fluorescence distribution. (B) The average radiant
   efficiency was calculated to quantify the signals from the labeled
   exosomes. (C) Macroscopic images of the aorta were captured in
   AngII-induced AAA mice treated with MSC-Exo or PBS. (D) The incidence
   of AAA was determined in AngII-induced mice treated with MSC-Exo or
   PBS. (E) Abdominal ultrasound imaging was conducted to measure the
   maximal abdominal aortic diameter in PBS- and MSC-Exo-treated
   AngII-induced AAA mice. (F) Histological analysis was performed using
   H&E and EVG staining of the abdominal aortas in PBS- and
   MSC-Exo-treated AngII-induced AAA mice (magnified photographs
   included). (G) Western blot analysis was conducted to assess the
   expression levels of iNOS and Arg1 in the abdominal aortas of PBS- and
   MSC-Exo-treated AngII-induced AAA mice, Full-length blots/gels are
   presented in Supplementary Fig. [74]2. (H) Immunofluorescence staining
   was performed in the abdominal aortas of PBS- and MSC-Exo-treated
   AngII-induced AAA mice, using CD206 (red), iNOS (green), and DAPI.
   (blue) (scale bars, 20 μm). *P < 0.05; **P < 0.01; ***P < 0.001;
   ****P < 0.0001

   In order to investigate the potential effects of MSC-derived exosomes
   (MSC-Exo) on macrophage polarization in AngII-induced AAA mice, we
   examined whether MSC-Exo could influence the phenotypes of macrophages
   towards M1 or M2 polarization. Western blot analysis demonstrated a
   significant decrease in the M1 marker iNOS expression and an increase
   in the M2 marker Arg1 expression in the AngII + Exo group compared to
   the AngII + PBS group (Fig. [75]2G). Additionally, immunofluorescence
   staining further confirmed a significant reduction in the M1 marker
   iNOS and an elevation in the M2 marker CD206 following MSC-Exo
   treatment (Fig. [76]2H). Collectively, these findings suggest that in
   AngII-induced AAA mice, MSC-Exo promotes the polarization of
   macrophages towards the M2 phenotype.

MSC-Exo alleviates calcium chloride (CaCl[2])-induced AAA formation and
promotes M2 macrophage polarization in C57/B6 mice

   To further investigate the impact of MSC-derived exosomes (MSC-Exo) on
   AAA formation, we utilized the CaCl[2]-induced AAA model. 25 male
   C57/B6 mice were randomly assigned into three groups: control group,
   CaCl[2] + PBS group, and CaCl[2] + Exo group.The CaCl[2] + Exo group
   mice were intravenously injected with MSC-Exo (n = 10) at a dose of
   3 µg/g and the CaCl[2] + PBS group injected the same amount of PBS
   (n = 10) and subsequently administered CaCl[2]. Four weeks later, the
   mice were sacrificed for analysis. An additional 5 male WT mice
   received intravenous injection of PBS and saline infusion as controls.
   In vivo imaging revealed the uptake of DiR-labeled MSC-Exo by the
   CaCl[2]-induced AAA mice (Fig. [77]3A, B). No evidence of AAA formation
   or significant morphological changes in the abdominal aorta were
   observed in the control group (Fig. [78]3C, D, F). The incidence of AAA
   in the CaCl[2] + Exo group was 50% (5/10), significantly lower than
   that in the CaCl[2] + PBS group (80%, 8/10) (Fig. [79]3D).
   Additionally, the maximal diameter of the abdominal aorta in
   CaCl[2] + Exo mice was significantly smaller compared to CaCl[2] + PBS
   mice (1.47verus 2.38 ± 0.319 cm,95%CI: 0.2399 to 1.580)(Fig. [80]3E).
   Consistent with the AngII-induced AAA model, CaCl[2] + Exo mice
   exhibited reduced elastic fiber degradation in the vessel wall compared
   to CaCl[2] + PBS mice (Fig. [81]3F). These findings demonstrate that
   MSC-Exo intervention alleviated AAA formation in the CaCl[2]-induced
   AAA model when compared to CaCl[2] + PBS mice.

Fig. 3.

   [82]Fig. 3
   [83]Open in a new tab

   The effects of intravenous administration of MSC-Exo on CaCl[2]-induced
   AAA formation and macrophage polarization in mice. (A) Mouse in vivo
   imaging was performed after injecting DiR-labeled MSC-Exo, displaying
   the fluorescence distribution. (B) The average radiant efficiency was
   quantified to assess the signals from the labeled exosomes. (C)
   Macroscopic images of the aorta were captured in CaCl[2]-induced AAA
   mice treated with MSC-Exo or PBS. (D) The incidence of AAA was
   determined in CaCl[2]-induced mice treated with MSC-Exo or PBS. (E)
   Abdominal ultrasound imaging was conducted to measure the maximal
   abdominal aortic diameter in PBS- and MSC-Exo-treated CaCl[2]-induced
   AAA mice. (F) Histological analysis was performed using H&E and EVG
   staining of the abdominal aortas in PBS- and MSC-Exo-treated
   CaCl[2]-induced AAA mice (including magnified photographs). (G) Western
   blot analysis was conducted to evaluate the expression levels of iNOS
   and Arg1 in the abdominal aortas of PBS- and MSC-Exo-treated
   CaCl[2]-induced AAA mice, full-length blots/gels are presented in
   Supplementary Fig. [84]3. (H) Immunofluorescence staining was performed
   in the abdominal aortas of PBS- and MSC-Exo-treated CaCl[2]-induced AAA
   mice, employing CD206 (red), iNOS (green), and DAPI. (blue) (scale
   bars, 20 μm). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

   Subsequently, we examined the effects of MSC-Exo on macrophage
   polarization in CaCl[2]-induced AAA mice. Through western blot analysis
   (Fig. [85]3G) and immunofluorescence staining (Fig. [86]3H) of M1 and
   M2 protein expression, it was observed that the M1 marker expression
   was significantly reduced, whereas the M2 marker expression was
   increased following MSC-Exo intervention. These findings collectively
   indicate that MSC-Exo promotes M2 macrophage polarization in
   CaCl[2]-induced AAA mice.

Systemic depletion of macrophages reduces the efficacy of MSC-Exo treatment

   To explore the role of macrophages in the efficacy of MSC-derived
   exosomes (MSC-Exo), we depleted macrophages using clodronate liposomes.
   Flow cytometry analysis confirmed a significant reduction in macrophage
   populations in the spleen, blood, and aorta upon clodronate liposome
   treatment (Supplementary material online, Figure [87]S1). In
   AngII-induced AAA mice, the diameter of AAAs showed a significant
   increase in the group of mice treated with exosomes and clodronate
   liposomes compared to the group treated with exosomes alone(2.41verus
   1.44 ± 0.4009 cm,95%CI: 0.04558 to 1.894).(Fig. [88]4A) .Similar
   results were observed in the CaCl[2]-induced AAA model(2.16verus
   1.22 ± 0.3421 cm,95%CI: 0.1512 to 1.729) (Fig. [89]4B). These findings
   indicate that the depletion of macrophages impairs the protective
   efficacy of MSC-Exo, suggesting that macrophages play a crucial role in
   mediating the beneficial effects of MSC-Exo against AAA formation.

Fig. 4.

   [90]Fig. 4
   [91]Open in a new tab

   Systemic depletion of macrophages reduces the efficacy of MSC-Exo
   treatment. (A) Macroscopic images of the aorta and maximal abdominal
   aortic diamete were assessed in AngII-induced AAA mice. (B) Similarly,
   macroscopic images of the aorta and maximal abdominal aortic diamete in
   CaCl[2]-induced AAA mice (per group n = 5) ***P < 0.001;
   ****P < 0.0001; ns, not significant

MSC-Exo promoted macrophage conversion to M2 phenotype in inflammatory
environment in vitro

   In order to further investigate the impact of MSC-derived exosomes
   (MSC-Exo) on macrophage polarization, we treated peritoneal macrophages
   with MSC-Exo. To assess the internalization of MSC-Exo by macrophages,
   PKH67-labeled MSC-Exo was incubated with peritoneal macrophages for
   6 h. Immunofluorescence images demonstrated efficient uptake of
   PKH67-labeled MSC-Exo by peritoneal macrophages (Fig. [92]5A).
   Lipopolysaccharide (LPS) was then added to the culture medium to induce
   an inflammatory environment, followed by the addition of MSC-Exo to the
   LPS-stimulated macrophages. After 24 h, the concentrations of
   pro-inflammatory and anti-inflammatory cytokines in the supernatants,
   as well as the protein levels of iNOS and Arg1, were assessed.
   Immunofluorescence staining revealed that MSC-Exo inhibited LPS-induced
   iNOS production and enhanced the upregulation of Arg1 (Fig. [93]5B).
   Western blot analysis demonstrated consistent changes with the
   immunofluorescence staining (Fig. [94]5C). Furthermore, the levels of
   pro-inflammatory cytokines IL-1β and IL-6, as well as anti-inflammatory
   cytokines IL-10 and TGF-β, were measured in the supernatants
   (Fig. [95]5D). The levels of these inflammatory cytokines were elevated
   in the supernatants of the LPS group compared to the control group.
   However, MSC-Exo attenuated the LPS-induced increase in
   pro-inflammatory cytokines (IL-1β and IL-6) and promoted the
   upregulation of anti-inflammatory cytokines (IL-10 and TGF-β).
   Collectively, these results indicate that MSC-Exo facilitated the
   conversion of macrophages to the M2 phenotype.

Fig. 5.

   [96]Fig. 5
   [97]Open in a new tab

   MSC-Exo promoted the conversion of macrophages to M2 phenotype in
   inflammatory environment in vitro. (A) Fluorescence imaging showing the
   uptake of PKH67-labeled exosomes (green) by macrophages, with DAPI
   staining for nuclei (blue). The “Dye only” control represents
   macrophages stained with PKH67 alone, while “Not stained” indicates
   macrophages without fluorescence labeling. Scale bar = 50 μm. (B)
   Immunofluorescence images depicting the expression of iNOS and Arg1 in
   LPS-stimulated macrophages after 24 h of culture with MSC-Exo or PBS.
   n = 3, Scale bar = 50 μm. (C) Western blot analysis and statistical
   analysis of iNOS and Arg1 protein levels in LPS-stimulated cells
   cultured with MSC-Exo or PBS. (n = 3, Full-length blots/gels are
   presented in Supplementary Fig. [98]5). (D) Concentration of cytokines
   associated with M1 markers (IL-1β and IL-6) and M2 markers (IL-10 and
   TGF-β) in the supernatants of LPS-stimulated cells cultured with
   MSC-Exo or PBS. n = 5. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns,
   not significant

CD74 was a candidate effector of MSC-Exo mediated macrophage polarization

   To elucidate the mechanisms underlying the regulation of macrophage
   polarization by MSC-derived exosomes (MSC-Exo), LPS-stimulated
   peritoneal macrophages were treated with PBS or MSC-Exo. Label-free
   proteomics analysis was conducted to identify differentially expressed
   proteins. The volcano plot (Fig. [99]6C) and heat map (Fig. [100]6B)
   demonstrated 78 differentially expressed proteins in the LPS + Exo
   group compared to the LPS + PBS group, consisting of 50 up-regulated
   proteins and 28 down-regulated proteins. Notably, the top 6 most
   significantly up-regulated proteins were Jpt1, Ppp1r2, S100a13,
   Psme3ip1, Ccnh, and Dnase2 (Figure [101]S2A). Conversely, the top 6
   most significantly down-regulated proteins were Rsad2, Tgln1, Clec4e,
   Fxyd5, Klra2, and CD74 (Figure [102]S3A). Subsequently, siRNAs
   targeting the 6 most significantly up-regulated proteins were applied
   to LPS-stimulated peritoneal macrophages cultured with MSC-Exo.
   However, their knockdown did not affect the effect of MSC-Exo on
   macrophage polarization, suggesting that these proteins are not direct
   targets of MSC-Exo for macrophage polarization (Figure [103]S2B). On
   the other hand, the effects of the 6 most significantly down-regulated
   proteins on macrophage polarization were investigated. qRT-PCR results
   confirmed that knockdown of CD74, among these proteins, exhibited a
   similar effect as MSC-Exo, promoting the conversion of macrophages to
   the M2 phenotype under an inflammatory environment, while the other 5
   down-regulated proteins did not show the same effect (Figure [104]S3B).
   Additional, tissue immunofluorescence staining revealed a notable
   increase in CD74 expression in the abdominal aorta tissues of mice
   treated with AngII and CaCl2, compared to the control group. However,
   following exosome treatment, there was a significant decrease in CD74
   expression(Figure [105]S4). These data indicated that CD74 may be a
   candidate effector of MSC-Exo mediated macrophage polarization.

Fig. 6.

   [106]Fig. 6
   [107]Open in a new tab

   CD74 is involved in MSC-Exo mediated macrophage polarization in vitro.
   (A) Volcano plot comparing the differentially expressed proteins
   between the LPS+Exo group and the LPS group, with red indicating
   up-regulated proteins and green indicating down-regulated proteins. (B)
   Heat map displaying the differential expression of the top 50
   up-regulated and top 28 down-regulated proteins between the LPS+Exo
   group and the LPS group. （C）Representative immunofluorescence images
   illustrating the expression of iNOS and Arg1 in LPS-stimulated
   peritoneal macrophages cultured with MSC-Exo or CD74 siRNA. iNOS is
   shown in red, Arg1 in green, and DAPI staining for nuclei in blue.
   Scale bars = 50 μm. (D-E) Western blot analysis (D) and statistical
   analysis (E) of CD74, iNOS, and Arg1 protein levels in LPS-stimulated
   cells cultured with MSC-Exo or CD74 siRNA(Full-length blots/gels are
   presented in Supplementary Figure [108]6). (F) Concentration of
   cytokines associated with M1 markers (IL-1β and IL-6) and M2 markers
   (IL-10 and TGF-β) in the supernatants of LPS-stimulated macrophages
   cultured with MSC-Exo or CD74 siRNA. (per group n = 5). *P < 0.05;
   **P < 0.01; ***P < 0.001; ****P < 0.0001

   To validate the previous findings at the protein level,
   immunofluorescence staining was performed and demonstrated that the M1
   marker iNOS was significantly reduced while the M2 marker Arg1 was
   elevated after MSC-Exo treatment. Similarly, CD74 siRNA recapitulated
   the effect of MSC-Exo on macrophage polarization (Fig. [109]6C).
   Western blot analysis further confirmed that CD74 siRNA effectively
   reduced CD74 protein expression and replicated the effect of MSC-Exo on
   promoting macrophage polarization towards the M2 phenotype
   (Fig. [110]6D-E). Additionally, the levels of pro-inflammatory
   cytokines IL-1β and IL-6, as well as anti-inflammatory cytokines IL-10
   and TGF-β, were measured in the supernatants (Fig. [111]6F). The LPS
   group exhibited increased levels of these inflammatory cytokines
   compared to the control group. However, both MSC-Exo and CD74 siRNA
   inhibited the LPS-induced increase in pro-inflammatory cytokines (IL-1β
   and IL-6) and enhanced the upregulation of anti-inflammatory cytokines
   (IL-10 and TGF-β). These results collectively suggest that CD74 is a
   potential effector involved in MSC-Exo-mediated macrophage
   polarization.

Inhibition of CD74 activated the TSC2-mTOR -AKT pathway

   To gain further insights into the regulatory mechanisms of CD74 on
   macrophage phenotype, bioinformatics analysis was conducted. Gene
   ontology analysis (Fig. [112]7A) and KEGG pathway enrichment analysis
   (Fig. [113]7B-C) revealed that the differentially expressed proteins
   were mainly involved in metabolic pathways. Notably, the TSC2-mTOR-AKT
   signaling pathway has been implicated in the regulation of macrophage
   polarization^17. Therefore, we investigated the effects of MSC-Exo and
   CD74 siRNA on TSC2 and its downstream signaling pathways using
   peritoneal macrophages (Fig. [114]7D). In vitro experiments
   demonstrated that MSC-Exo treatment in an inflammatory environment
   resulted in significant upregulation of TSC2 and Akt levels, while Rheb
   and mTOR expression levels were downregulated. Similarly, treatment
   with CD74 siRNA activated the TSC2-mTOR-AKT pathway. These findings
   suggest that CD74 is involved in the modulation of the TSC2-mTOR -AKT
   signaling cascade.

Fig. 7.

   [115]Fig. 7
   [116]Open in a new tab

   CD74 was involved in the TSC2-mTOR-AKT signalling cascades in
   peritoneal macrophages. (A) The names of significantly enriched Gene
   Ontology terms from the Gene Ontology analysis.(B-C) KEGG pathway
   enrichment analysis results showing the top 20 pathways with their
   enrichment scores. The rich factor represents the degree of enrichment
   of the differentially expressed proteins, and the p-value is indicated
   by a color scale. (D) Representative images of western blots and
   statistical analysis demonstrating the TSC2-Mtor-AKT signaling pathway
   in LPS-stimulated peritoneal macrophages treated with MSC-Exo or CD74
   siRNA. (per group n = 3, Full-length blots/gels are presented in
   Supplementary Fig. [117]7) *P < 0.05; **P < 0.01; ***P < 0.001;
   ****P < 0.0001

CD74 regulated macrophage polarization by interacting with PKM2

   In both mice and humans, the classical activation of M1 macrophages
   relies primarily on glycolytic metabolism [[118]17]. Therefore, to
   further elucidate the mechanism by which CD74 modulates macrophage
   phenotypes, we focused on pyruvate kinase M (PKM), a key enzyme
   involved in glycolytic metabolism. To determine whether PKM1 or PKM2 is
   a target of CD74 in macrophages, the expression levels of PKM1 and PKM2
   were measured by western blot in peritoneal macrophages treated with
   MSC-Exo or CD74 siRNA. The results revealed that PKM2 protein levels
   were significantly reduced, while PKM1 protein levels did not show
   significant changes in peritoneal macrophages treated with MSC-Exo or
   CD74 siRNA (Fig. [119]8A). Subsequently, the impact of PKM2 knockdown
   on macrophage phenotypes was investigated. Western blot analysis
   demonstrated that iNOS levels were significantly down-regulated and
   Arg1 levels were up-regulated after transfection with PKM2 siRNA in
   peritoneal macrophages (Fig. [120]8B). Additionally, the effects of
   PKM2 siRNA on TSC2-mTOR-AKT signaling pathways were investigated. In
   vitro experiments revealed that PKM2 siRNA exhibited similar regulatory
   effects on the TSC2-mTOR-AKT pathways as MSC-Exo in peritoneal
   macrophages (Fig. [121]8C) .These findings suggest that PKM2 is
   involved in CD74-mediated macrophage polarization by targeting the
   TSC2-mTOR-AKT signaling cascades.

Fig. 8.

   [122]Fig. 8
   [123]Open in a new tab

   CD74 modulates peritoneal macrophages phenotype through targeting PKM2.
   (A) Representative images of western blots and statistical analysis for
   protein levels of PKM1 and PKM2 in LPS-stimulated peritoneal
   macrophages treated with MSC-Exo or CD74 siRNA. (B) Western blot
   analysis and statistical analysis of PKM2, iNOS, and Arg1 protein
   levels in LPS-stimulated peritoneal macrophages cultured with MSC-Exo
   or PKM2 siRNA. (C) Representative images of western blots and
   statistical analysis for the TSC2-mTOR-AKT signaling pathway in
   LPS-stimulated peritoneal macrophages treated with MSC-Exo or PKM2
   siRNA. (D) Co-immunoprecipitation assays demonstrating the association
   of CD74 and PKM2 in peritoneal macrophages. (per group n = 3,
   Full-length blots/gels are presented in Supplementary Fig. [124]8)

   To investigate how CD74 affects the expression of PKM2, experiments
   were carried out using peritoneal macrophages (Fig. [125]8D). The
   immunoprecipitation (Co-IP) experiments revealed a significant
   interaction between CD74 and PKM2. These findings strongly suggest that
   CD74 modulates the expression of PKM2 by interacting with PKM2.

Discussion

   This study confirms the protective effects of MSC-Exo against AngII or
   CaCl[2]-induced AAA formation and their ability to promote macrophage
   polarization towards the M2 phenotype. We further demonstrate that CD74
   plays a crucial role in mediating MSC-Exo-induced macrophage
   polarization. Moreover, we uncover that CD74 regulates the downstream
   TSC2-mTOR-AKT pathway through its interaction with PKM2, thereby
   modulating macrophage polarization. These findings provide valuable
   insights into the therapeutic effects of MSC-Exo on AAA and highlight
   the critical involvement of CD74 in this process.

   Macrophages have been shown to have both pathogenic and reparative
   roles in AAA, contributing to extracellular matrix remodeling,
   inflammation promotion and resolution, and tissue repair responses
   [[126]3]. In our study, we discovered the critical involvement of
   macrophages in mediating the effects of MSC-Exo through a macrophage
   depletion model. In AAA formation, two distinct subgroups of
   macrophages, namely pro-inflammatory M1 macrophages and
   anti-inflammatory M2 macrophages, play significant roles [[127]18]. Our
   study, using both an AAA mouse model and in vitro experiments,
   demonstrated that MSC-Exo effectively switched macrophages from an M1
   to an M2 phenotype under inflammatory conditions. This phenotypic shift
   attenuated the inflammatory response and promoted tissue repair,
   ultimately inhibiting AAA development. Through protein mass
   spectrometry analysis and subsequent PCR validation, we identified CD74
   as a potential effector involved in the immunomodulatory effects of
   MSC-Exo. Notably, there are currently no available studies
   investigating the relationship between CD74 and AAA, nor have the roles
   of CD74 in the immunomodulatory effects of MSC-Exo been explored. Our
   research provides novel insights by demonstrating, for the first time,
   that CD74 expression is negatively regulated by MSC-Exo in peritoneal
   macrophages, leading to the switching of macrophages towards the M2
   phenotype.

   Mechanically, bioinformatics analysis revealed that differentially
   expressed proteins in MSC-Exo-treated peritoneal macrophages were
   primarily enriched in metabolic pathways. Notably, PKM2, a key enzyme
   involved in glycolysis, has been implicated in the regulation of matrix
   metalloproteinase-9 (MMP9) activity and expression [[128]19], which
   plays a critical role in AAA formation [[129]20]. Therefore, we further
   investigated the association between CD74 and PKM2. Our data confirmed
   that inhibiting CD74 reduced PKM2 expression, and it was observed that
   CD74 could interact with PKM2. Additionally, inhibiting PKM2 resulted
   in a shift of macrophages from an M1 to an M2 phenotype, mimicking the
   beneficial effects seen with CD74 siRNA in vivo. Furthermore, our
   investigation revealed that CD74 and PKM2 impact the TSC2-mTOR-AKT
   pathway, with the potential to influence macrophage polarization
   mediated by MSC-Exo.

   This study has several limitations that warrant further investigation.
   Firstly, additional studies are needed to explore the regulation of
   macrophage polarization by MSC-Exo through artificial manipulation of
   CD74 activation or inactivation at the cellular level. Secondly, the
   specific role of CD74 in macrophages in the pathogenesis of AAA is not
   fully understood and requires further investigation using cell
   type-specific knockouts and relevant physiological models. Lastly, our
   study focused on male mice, limiting the generalizability of the
   findings to female populations. Future studies should include both male
   and female subjects to assess any potential sex differences in the
   observed effects.

   In summary, our study demonstrates that MSC-Exo protect against AAA
   formation by inducing a shift in macrophage polarization towards an
   anti-inflammatory phenotype. CD74 plays a crucial role in this process
   by modulating the PKM2-regulated TSC2-mTOR-AKT signaling pathway. These
   findings provide important insights into the therapeutic potential of
   MSC-Exo in AAA and highlight the significance of CD74 in regulating
   macrophage polarization. In future, more research using diverse models
   and conditions, along with corroboration through clinical studies or in
   vitro experiments using human samples, is needed to enhance the
   applicability of our findings.

Conclusions

   Our research indicate that MSC-Exo protect against abdominal aortic
   aneurysm formation through CD74 modulation of macrophage polarization
   in mice.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [130]Supplementary Material 1^ (711.2KB, docx)
   [131]Supplementary Material 2^ (864.2KB, xlsx)
   [132]Supplementary Material 3^ (1.2MB, docx)

Acknowledgements